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Abstract
The present study was undertaken to verify whether induction of senescence could be sufficient to reverse drug resistance and, if so, to determine the underlying mechanism(s). Our findings indicated that co-treatment of drug-resistant neuroblastoma cells with doxorubicin, at sublethal concentrations, in combination with the pan-caspase inhibitor, Q-VD-OPH, elicited a strong reduction of cell viability that occurred in a caspase-independent manner. This was accompanied by the appearance of a senescence phenotype, as evidenced by increased p21/WAF1 expression and senescence-associated β-galactosidase activity. Experiments using specific inhibitors of major cellular proteases other than caspases have shown that inhibition of cathepsin L, but not proteasome or cathepsin B, was responsible for the senescence-initiated reversal of drug resistance. This phenomenon appeared to be general because it was valid for other drugs and drug-resistant cell lines. A nonchemical approach, through cell transfection with cathepsin L small interfering RNA, also strongly reversed drug resistance. Further investigation of the underlying mechanism revealed that cathepsin L inhibition resulted in the alteration of intracellular drug distribution. In addition, in vitro experiments have demonstrated that p21/WAF1 is a substrate for cathepsin L, suggesting that inhibition of this enzyme may result in p21/WAF1 stabilization and its increased accumulation. All together, these findings suggest that cathepsin L inhibition in drug-resistant cells facilitates induction of senescence and reversal of drug resistance. This may represent the basis for a novel function of cathepsin L as a cell survival molecule responsible for initiation of resistance to chemotherapy.
... In the context of cancerogenesis, secreted Cts contribute to the tumor ECM degradation and remodeling, while intracellular cathepsins are pivotal components of signaling pathways, which can enhance cancer cell growth and inflammation [12,13]. Also, Cts are engaged in response to anticancer therapy within the tumor microenvironment, and they can have crucial roles in the development of resistance phenomena to the therapeutics [14][15][16]. Cys138-His276-Asn300 triad at the active side is colored (model created with SWISS-MODEL and PyMOL). (c) Scheme Cts maturation as the function of endolysosomal pH. ...
... As a result of this phenomenon, CTSL knock-down in ovarian cancer cells SCOV3 resulted in increased apoptosis induced by paclitaxel (the most common drug in the management of ovarian cancer) [16]. Also, Cts L played a role in inhibiting the cell senescence process in different tumor cell lines, providing an additional mechanism of protease-mediated drug target elimination (trapping a drug in lysosomes) and drug resistance [15,138,195]. Treatment of drug-resistant neuroblastoma cells with doxorubicin and pan-caspase inhibitor (Q-VD-OPH) significantly decreased cell viability and senescence phenotype, as evidenced by an increased p21/WAF1 expression, senescence-associated β-galactosidase activity, and cell growth arrest. Experiments have established that this phenomenon was the result of CtsL inhibition [15]. ...
... Treatment of drug-resistant neuroblastoma cells with doxorubicin and pan-caspase inhibitor (Q-VD-OPH) significantly decreased cell viability and senescence phenotype, as evidenced by an increased p21/WAF1 expression, senescence-associated β-galactosidase activity, and cell growth arrest. Experiments have established that this phenomenon was the result of CtsL inhibition [15]. ...
Cysteine cathepsins are lysosomal enzymes belonging to the papain family. Their expression is misregulated in a wide variety of tumors, and ample data prove their involvement in cancer progression, angiogenesis, metastasis, and in the occurrence of drug resistance. However, while their overexpression is usually associated with highly aggressive tumor phenotypes, their mechanistic role in cancer progression is still to be determined to develop new therapeutic strategies. In this review, we highlight the literature related to the role of the cysteine cathepsins in cancer biology, with particular emphasis on their input into tumor biology.
... Zheng et al. postulated that iCL and CatL inhibition through siRNA led to an accumulation of topoisomerase II alpha expression in a multi-drug resistance protein 1 (MRP1)-independent manner. Alternatively, since the inhibition of CatL through iCL or siRNA forces cells to undergo senescence, as is evidenced through the up-regulation of the expression of the CatL substrate and the cell cycle inhibitor p21/WAF1 [194], it is possible that the induction of senescence through CatL inhibition may be sufficient to reverse drug resistance [194]. ...
... Zheng et al. postulated that iCL and CatL inhibition through siRNA led to an accumulation of topoisomerase II alpha expression in a multi-drug resistance protein 1 (MRP1)-independent manner. Alternatively, since the inhibition of CatL through iCL or siRNA forces cells to undergo senescence, as is evidenced through the up-regulation of the expression of the CatL substrate and the cell cycle inhibitor p21/WAF1 [194], it is possible that the induction of senescence through CatL inhibition may be sufficient to reverse drug resistance [194]. ...
The upregulation of proteolytic enzymes has been demonstrated to promote primary tumor development and metastatic bone cancer. The secreted proteases increase tumor growth and angiogenesis, and potentiate neoplastic cell dissemination. This article reviews the role and mechanisms of cathepsins in normal physiology, cancer, bone remodeling, and the tumor–bone interface, with a specific focus on cathepsins B, D, H, G, L, and K. In this review, we highlight the role of cathepsins in primary bone cancer (i.e., osteosarcoma (OS)), as well as metastatic breast (BCa) and prostate (PCa) cancer. In addition, we discuss the clinical utility and therapeutic potential of cathepsin-targeted treatments in primary and secondary bone cancers.
... For example, inactivation of the p21 gene prevents senescence in human fibroblasts (25), whereas ectopic overexpression of p21 causes premature senescence (26). In addition, there is ample evidence implicating the involvement of LRF in regulating senescence in other cell types (8,(27)(28)(29). ...
... A role of increased p53 expression in sensitization of non-small cell lung cancer to chemotherapeutic agents has been reported previously (17). Furthermore, induction of senescence, through inhibition of cathepsin L and stabilization of p21, reduces drug resistance in neuroblastoma, osteosarcoma and leukemia cell lines (29). In light of our findings, LRF appears to be a key determinant in chondrosarcoma survival and could be a potential target for new therapeutic approach. ...
Chondrosarcoma is a form of malignant skeletal tumor of cartilaginous origin. The non-malignant form of the disease is termed chondroma. Correctly distinguishing between the two forms is essential for making therapeutic decisions. However, due to their similar histological appearances and the lack of a reliable diagnostic marker, it is often difficult to distinguish benign tumors from low-grade chondrosarcoma. Therefore, it is necessary to search for a potential marker that has diagnostic and prognostic values in chondrosarcoma. In this study, we demonstrated by immunohistochemistry that elevated leukemia/lymphoma-related factor (LRF) expression was associated with increased malignancy in human chondrosarcoma tissue microarrays. Moreover, siRNA depletion of LRF drastically reduced proliferation of chondrosarcoma cell lines and effectively induced senescence in these cells. This could be attributed to the observation that LRF-depleted cells were arrested at the G(1) phase, and had increased p53 and p21 expression. Moreover, LRF depletion not only drastically reduces the cellular migration and invasion potentials of chondrosarcoma cells but also sensitized these cells to the apoptosis-inducing chemotherapeutic agent doxorubicin. We conclude that LRF is a survival factor in chondrosarcomas and its expression correlates with tumor malignancy and chemoresistance. Our data implicate the potential role of LRF as both a diagnostic marker and therapeutic target for chondrosarcomas.
... For example, inactivation of the p21 gene prevents senescence in human fibroblasts (25), whereas ectopic overexpression of p21 causes premature senescence (26). In addition, there is ample evidence implicating the involvement of LRF in regulating senescence in other cell types (8,(27)(28)(29). ...
... A role of increased p53 expression in sensitization of non-small cell lung cancer to chemotherapeutic agents has been reported previously (17). Furthermore, induction of senescence, through inhibition of cathepsin L and stabilization of p21, reduces drug resistance in neuroblastoma, osteosarcoma and leukemia cell lines (29). In light of our findings, LRF appears to be a key determinant in chondrosarcoma survival and could be a potential target for new therapeutic approach. ...
Chondrosarcoma is a form of malignant skeletal tumor of cartilaginous origin. The non-malignant form of the disease is termed chondroma. Correctly distinguishing between the two forms is essential for making therapeutic decisions. However, due to their similar histological appearances and the lack of a reliable diagnostic marker, it is often difficult to distinguish benign tumors from low grade chondrosarcoma. Therefore, it is necessary to search for a potential marker that has diagnostic and prognostic values in chondrosarcoma. Here, we demonstrated by immunohistochemistry that elevated LRF expression was associated with increased malignancy in human chondrosarcoma tissue microarrays. Moreover, siRNA-depletion of LRF drastically reduced proliferation of chondrosarcoma cell lines and effectively induced senescence in these cells. This could be attributed to the observation that LRF-depleted cells were arrested at the G1 phase, and had increased p53 and p21 expression. Moreover, LRF-depletion not only drastically reduces the cellular migration and invasion potentials of chondrosarcoma cells, but also sensitized these cells to the apoptosis-inducing chemotherapeutic agent doxorubicin. We conclude that LRF is a survival factor in chondrosarcomas and its expression correlates with tumor malignancy and chemo-resistance. Our data implicate the potential role of LRF as both a diagnostic marker and therapeutic target for chondrosarcomas.
... "GO:0071276: cellular response to cadmium ion" was identified as enriched pathway for genes that show different expression levels between AML subgroups and normal bone marrow (BM) [32]. The mechanisms of cellular responses involving resistance to chemotherapy in cancer have been demonstrated in several studies [33][34][35]. Cheng et al. [33] suggested that drug efficacy can be enhanced by Asplatin through altering the cellular response. ...
Azacitidine, a DNA methylation inhibitor, is employed for the treatment of acute myeloid leukemia (AML). However, drug resistance remains a major challenge for effective azacitidine chemotherapy, though several studies have attempted to uncover the mechanisms of azacitidine resistance. With the aim to identify the mechanisms underlying acquired azacitidine resistance in cancer cell lines, we developed a computational strategy that can identify differentially regulated gene networks between drug-sensitive and -resistant cell lines by extending the existing method, differentially coexpressed gene sets (DiffCoEx). The technique specifically focuses on cell line-specific gene network analysis. We applied our method to gene networks specific to azacitidine sensitivity and identified differentially regulated gene networks between azacitidine-sensitive and -resistant cell lines. The molecular interplay between the metallothionein gene family, C19orf33, ELF3, GRB7, IL18, NRN1, and RBM47 were identified as differentially regulated gene network in drug resistant cell lines. The biological mechanisms associated with azacitidine and AML for the markers in the identified networks were verified through the literature. Our results suggest that controlling the identified genes (e.g., the metallothionein gene family) and “cellular response”-related pathways (“cellular response to zinc ion”, “cellular response to copper ion”, and “cellular response to cadmium ion”, where the enriched functional-related genes are MT2A, MT1F, MT1G, and MT1E) may provide crucial clues to address azacitidine resistance in patients with AML. We expect that our strategy will be a useful tool to uncover patient-specific molecular interplay that provides crucial clues for precision medicine in not only gastric cancer but also complex diseases.
... Regarding cancer development, secreted caths play a role in the degradation and remodeling of the tumor extracellular matrix (ECM), while intracellular caths serve as crucial components of signaling pathways that can promote cell growth and inflammation [6,7]. Furthermore, caths are involved in response to anticancer therapy within the tumor microenvironment and can play pivotal roles in the development of resistance to therapeutic interventions [8][9][10][11][12]. ...
Cathepsins (Caths) are lysosomal proteases that participate in various physiological and pathological processes. Accumulating evidence suggests that caths play a multifaceted role in cancer progression and radiotherapy resistance responses. Their proteolytic activity influences the tumor's response to radiation by affecting oxygenation, nutrient availability, and immune cell infiltration within the tumor microenvironment. Cathepsin-mediated DNA repair mechanisms can promote radioresistance in cancer cells, limiting the efficacy of radiotherapy. Additionally, caths have been associated with the activation of prosurvival signaling pathways, such as PI3K/Akt and NF-κB, which can confer resistance to radiation-induced cell death. However, the effectiveness of radiotherapy can be limited by intrinsic or acquired resistance mechanisms in cancer cells. In this study, the regulation and expression of cathepsin B (cath B) in the colon carcinoma cell line (caco-2) before and after exposure to radiation were investigated. Cells were exposed to escalating ionizing radiation doses (2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy). Analysis of protein expression, in vitro labeling using activity-based probes DCG04, and cath B pull-down revealed a radiation-induced up-regulation of cathepsin B in a dose-independent manner. Proteolytic inhibition of cathepsin B by cathepsin B specific inhibitor CA074 has increased the cytotoxic effect and cell death due to ionizing irradiation treatment in caco-2 cells. Similar results were also obtained after cathepsin B knockout by CRISPR CAS9. Furthermore, upon exposure to radiation treatment, the inhibition of cath B led to a significant upregulation in the expression of the proapoptotic protein BAX, while it induced a significant reduction in the expression of the antiapoptotic protein BCL-2. These results showed that cathepsin B could contribute to ionizing radiation resistance, and the abolishment of cathepsin B, either by inhibition of its proteolytic activity or expression, has increased the caco-2 cells susceptibility to ionizing irradiation.
... Previous studies have demonstrated that the expression of ctsl was upregulated in several cancers; therefore, supporting its use as a therapeutic target for cancer (Rudzińska et al., 2019). There is evidence that ctsl may be central in drug resistance development (Zheng et al., 2004). Further research has shown that by inhibiting ctsl, development of resistance to cancer drugs such as doxorubicin, a chemotherapy medication used to treat cancer, can be reversed, and prevented. ...
The sex of both humans and Danio rerio has previously been shown to affect the way individuals respond to drug exposure. Genes which allow identification of sex in juvenile zebrafish show potential to reveal these confounding variables between sex in toxicological and preclinical trials but the link between these is so far missing. These sex-specific, early expressed genes where expression is not altered by drug exposure must be carefully selected for this purpose. We aimed to discover genes which can be used in pharmaceutical trials and environmental toxicology studies to uncover sex-related variations in gene expression with drug application using the model organism Danio rerio. Previously published early sex determining genes from King et al. were evaluated as well as additional genes selected from our zebrafish Next-generation sequencing (NGS) data which are known from previously published works not to be susceptible to changes in expression with drug exposure. NGS revealed a further ten female-specific genes (vtg1, cyp17a1, cyp19a1a, igf3, ftz-f1, gdf9, foxl2a, Nr0b1, ipo4, lhcgr) and five male related candidate genes (FKBP5, apobb1, hbaa1, dmrt1, spata6) which are also expressed in juvenile zebrafish, 28 days post fertilisation (dpf). Following this, a literature review was performed to classify which of these early-expressed sex specific genes are already known to be affected by drug exposure in order to determine candidate genes to be used in pharmaceutical trials or environmental toxicology testing studies. Discovery of these early sex-determining genes in Danio rerio will allow identification of sex-related responses to drug testing to improve sex-specific healthcare and the medical treatment of human patients.
... Cathepsin L (CTSL) is a cysteine protease belonging to papain-like family and is associated with cancer migration and malignancy [90][91][92]. CTSL can induce chemoresistance via preventing cell senescence [93]. In PTX-resistant lung cancer cells, CTSL undergoes upregulation that subsequently, mediates mesenchymal phenotype of cancer cells via EMT induction [94]. ...
Epithelial-to-mesenchymal transition (EMT) mechanism is responsible for metastasis and migration of cancer cells to neighboring cells and tissues. Morphologically, epithelial cells are transformed to mesenchymal cells, and at molecular level, E-cadherin undergoes down-regulation, while an increase occurs in N-cadherin and vimentin levels. Increasing evidence demonstrates role of EMT in mediating drug resistance of cancer cells. On the other hand, paclitaxel (PTX) and docetaxel (DTX) are two chemotherapeutic agents belonging to taxene family, capable of inducing cell cycle arrest in cancer cells via preventing microtubule depolymerization. Aggressive behavior of cancer cells resulted from EMT-mediated metastasis can lead to PTX and DTX resistance. Upstream mediators of EMT such as ZEB1/2, TGF-β, microRNAs, and so on are involved in regulating response of cancer cells to PTX and DTX. Tumor-suppressing factors inhibit EMT to promote PTX and DTX sensitivity of cancer cells. Furthermore, three different strategies including using anti-tumor compounds, gene therapy and delivery.
... The Cts specific contribution regarding phenomena of chemoresistance occurrence is presently at issue. CtsB and L overexpression was linked to the inactivation of drug increase and, most importantly, with trafficking of lysosome increase to the membrane of plasma and lysosomalcargo secretion [14], and consequently, knock-down of CTSL in cancer of ovarian cells SCOV3 led to apoptosis increase with induction of paclitaxel which is the most prevalent drug in ovarian cancer treatment [15], Moreover, Cts L contributed to the inhibition of the process of cell senescence in various lines of tumor cell, contributing a further protease-mediated drug target eradication mechanism (confining a drug within lysosomes) and resistance of drug [16][17][18]. Within this framework, Cts L inhibition led to increase of accumulation of Doxorubicin cell and a further convenient nuclear distribution of drug in the cells in spite of the expression of P-gp [18]. ...
Cysteine cathepsins are defined as lysosomal enzymes which are member of the papain family. Cysteine cathepsins (Cts) prevalently exist in whole organisms varying from prokaryotes to mammals and possess in their active site greatly conserved residue of cysteine. Cts are engaged in the digestion of cellular protein, activation of zymogen, and remodeling of extracellular matrix (ECM). Host cells are entered by SARS-CoV-2 via endocytosis. Cathepsin L and phosphatidylinositol 3-phosphate 5-kinase are crucial in terms of the endocytosis by cleaving the spike protein, which permits viral membrane fusion with endosomal membrane, and succeeded by the releasing of viral genome to the host cell. Thereby, inhibition of cathepsin L may be advantageous in terms of decreasing infection caused by SARS-CoV-2. Coordinate inhibition of multiple Cts and lysosomal function by different drugs and biological agents might be of value for some purposes such as parasite or viral infections and anti-neoplastic applications. It has been found that Zn ²⁺ deficiency or dysregulation leads to an exaggerated activity of Cysteine cathepsin increasing the autoimmune/inflammatory response. At this purpose Zn ²⁺ metal can be safely combined with a drug that increases the anti-proteolytic effect of endogenous Zn ²⁺ lowering the excessive activity of some CysCts. Biguanide derivatives complex with Zn ²⁺ have been found to be promising inhibitors of CysCts protease reactions. Molecular docking studies of Cathepsin L Inhibited by Metformin-Zn+2 complex have been performed showing two strong key interactions ( Cys-25&His-163) and an extra H-bond with Asp-163 compared to the co-crystallized Zn ⁺² (PDB ID 4axl).
... However, based on the considerations above, inhibition of cysteine cathepsins also is hypothesized to be a strategy for chemosensitization or chemopotentiation in a standard or targeted chemotherapy regimen. Exemplarily, a combination of doxorubicin and the fluoromethylketone-based cathepsin L inhibitor 1a enabled the induction of senescence in various murine and human drug-resistent cancer cell lines (Zheng et al., 2004). More evidence for chemosensitizing effects due to cathepsin inhibition has been provided by the same group more recently by showing that the cathepsin L-selective aldehyde inhibitor iCL (compound 11) led to a reversal of resistance to doxorubicin in human neuroblastoma and osteosarcoma cells in vitro and in nude mice xenografted with doxorubicinresistant human neuroblastoma cells in vivo (Zheng et al., 2009). ...
Papain-like cysteine proteases bear an enormous potential as drug discovery targets for both infectious and systemic human diseases. The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging. After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge toward their involvement in tumor progression, with a special emphasis on their role in therapy response. In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.
Background
Melanoma central nervous system (CNS) metastasis remains a leading cause of patient mortality, and the underlying pathological mechanism has not been fully elucidated, leading to a lack of effective therapeutic strategies.
Materials and Methods
In this study, we conducted an integrated analysis of single‐cell transcriptomic data related to melanoma brain metastasis (MBM) and leptomeningeal metastasis (LMM). We focused on differences of subset composition and molecular expression of monocytes in blood, primary tumor, brain metastases, and leptomeningeal metastases.
Results
Significant differences were observed among monocytes in blood, primary tumor, and different CNS metastatic tissues, particularly in terms of subset differentiation and gene expression patterns. Subsequent analysis revealed the upregulation of cell proportions of six monocyte subsets in brain metastasis and leptomeningeal metastasis. Based on differential gene analysis, four of these subsets exhibited increased expression of factors promoting tumor migration and survival, including AREG⁺ monocytes (AREG, EREG, THBS1), FABP5⁺ monocytes (SPP1, CCL2, CTSL), and CXCL3⁺ monocytes (CXCL3, IL8, IL1B). The proportions of TPSB2⁺ monocytes (IL32, CCL5) were notably elevated in melanoma leptomeningeal metastasis tissues. Pathway analysis indicated the activation of signaling pathways such as NOD‐like receptors, NFκB, and Toll‐like receptors in these metastasis‐related subsets.
Conclusion
Our findings elucidate that AREG⁺, FABP5⁺ and CXCL3⁺ monocytes are associated with brain metastasis and TPSB2⁺ monocytes are associated with leptomeningeal metastasis in melanoma, which may be contribute to the development of therapeutic strategies focusing on monocytes or cytokines for melanoma CNS metastasis.
Cathepsin L (CatL) is a lysosomal cysteine protease whose activity has been related to several human pathologies. However, although preclinical trials using CatL inhibitors were promising, clinical trials have been unsuccessful up to now. We are presenting a study of two designed dipeptidyl keto Michael acceptor potential inhibitors of CatL with either a keto vinyl ester or a keto vinyl sulfone (KVS) warhead. The compounds were synthesized and experimentally assayed in vitro, and their inhibition molecular mechanism was explored based on molecular dynamics simulations at the density functional theory/molecular mechanics level. The results confirm that both compounds inhibit CatL in the nanomolar range and show a time-dependent inhibition. Interestingly, despite both presenting almost equivalent equilibrium constants for the reversible formation of the noncovalent enzyme/inhibitor complex, differences are observed in the chemical step corresponding to the enzyme–inhibitor covalent bond formation, results that are mirrored by the computer simulations. Theoretically determined kinetic and thermodynamic results, which are in very good agreement with the experiments, afford a detailed explanation of the relevance of the different structural features of both compounds having a significant impact on enzyme inhibition. The unprecedented binding interactions of both inhibitors in the P1′ site of CatL represent valuable information for the design of inhibitors. In particular, the peptidyl KVS can be used as a starting lead compound in the development of drugs with medical applications for the treatment of cancerous pathologies since sulfone warheads have previously shown promising cell stability compared to other functions such as carboxylic esters. Future improvements can be guided by the atomistic description of the enzyme–inhibitor interactions established along the inhibition reaction derived from computer simulations.
Lung cancer as the leading cause of cancer related mortality is always one of the main global health challenges. Despite the recent progresses in therapeutic methods, the mortality rate is still significantly high among lung cancer patients. A wide range of therapeutic methods including chemotherapy, radiotherapy, and surgery are used to treat lung cancer. Doxorubicin (DOX) and Paclitaxel (TXL) are widely used as the first-line chemotherapeutic drugs in lung cancer. However, there is a significant high percentage of DOX/TXL resistance in lung cancer patients, which leads to tumor recurrence and metastasis. Considering, the side effects of these drugs in normal tissues, it is required to clarify the molecular mechanisms of DOX/TXL resistance to introduce the efficient prognostic and therapeutic markers in lung cancer. MicroRNAs (miRNAs) have key roles in regulation of different pathophysiological processes including cell division, apoptosis, migration, and drug resistance. MiRNA deregulations are widely associated with chemo resistance in various cancers. Therefore, considering the importance of miRNAs in chemotherapy response, in the present review, we discussed the role of miRNAs in regulation of DOX/TXL response in lung cancer patients. It has been reported that miRNAs mainly induced DOX/TXL sensitivity in lung tumor cells by the regulation of signaling pathways, autophagy, transcription factors, and apoptosis. This review can be an effective step in introducing miRNAs as the non-invasive prognostic markers to predict DOX/TXL response in lung cancer patients.
In this issue of Cancer Cell, Wang et al. reveal that chemoresistant muscle-invasive bladder cancer is associated with partial squamous differentiation. Targeting of Cathepsin H overcomes this chemotherapy-induced semi-squamatization and promotes terminal squamous differentiation and tumor suppression.
Cisplatin-based chemotherapy remains the primary treatment for unresectable and metastatic muscle-invasive bladder cancers (MIBCs). However, tumors frequently develop chemoresistance. Here, we established a primary and orthotopic MIBC mouse model with gene-edited organoids to recapitulate the full course of chemotherapy in patients. We found that partial squamous differentiation, called semi-squamatization, is associated with acquired chemoresistance in both mice and human MIBCs. Multi-omics analyses showed that cathepsin H (CTSH) is correlated with chemoresistance and semi-squamatization. Cathepsin inhibition by E64 treatment induces full squamous differentiation and pyroptosis, and thus specifically restrains chemoresistant MIBCs. Mechanistically, E64 treatment activates the tumor necrosis factor pathway, which is required for the terminal differentiation and pyroptosis of chemoresistant MIBC cells. Our study revealed that semi-squamatization is a type of lineage plasticity associated with chemoresistance, suggesting that differentiation via targeting of CTSH is a potential therapeutic strategy for the treatment of chemoresistant MIBCs.
Cysteine cathepsins are defined as lysosomal enzymes that are members of the papain family. Cysteine cathepsins (Cts) prevalently exist in whole organisms, varying from prokaryotes to mammals, and possess greatly conserved cysteine residues in their active sites. Cts are engaged in the digestion of cellular proteins, activation of zymogens, and remodeling of the extracellular matrix (ECM). Host cells are entered by SARS-CoV-2 via endocytosis. Cathepsin L and phosphatidylinositol 3-phosphate 5-kinase are crucial in endocytosis by cleaving the spike protein, which permits viral membrane fusion with the endosomal membrane and succeeds in the release of the viral genome to the host cell. Therefore, inhibition of cathepsin L may be advantageous in terms of decreasing infection caused by SARS-CoV-2. Coordinate inhibition of multiple Cts and lysosomal function by different drugs and biological agents might be of value for some purposes, such as a parasite or viral infections and antineoplastic applications. Zn ²⁺ deficiency or dysregulation leads to exaggerated cysteine cathepsin activity, increasing the autoimmune/inflammatory response. For this purpose, Zn ²⁺ metal can be safely combined with a drug that increases the anti-proteolytic effect of endogenous Zn ²⁺ , lowering the excessive activity of some CysCts. Biguanide derivative complexes with Zn ²⁺ have been found to be promising inhibitors of CysCts protease reactions. Molecular docking studies of cathepsin L inhibited by the metformin-Zn+2 complex have been performed, showing two strong key interactions (Cys-25&His-163) and an extra H-bond with Asp-163 compared to cocrystallized Zn ⁺² (PDB ID 4axl).
Cathepsins (CTS) are mainly lysosomal acid hydrolases extensively involved in the prognosis of different diseases, and having a distinct role in tumor progression by regulating cell proliferation, autophagy, angiogenesis, invasion, and metastasis. As all these processes conjunctively lead to cancer progression, their site-specific regulation might be beneficial for cancer treatment. CTS regulate activation of the proteolytic cascade and protein turnover, while extracellular CTS is involved in promoting extracellular matrix degradation and angiogenesis, thereby stimulating invasion and metastasis. Despite cancer regulation, the involvement of CTS in cellular adaptation toward chemotherapy and radiotherapy augments their therapeutic potential. However, lysosomal permeabilization mediated cytosolic translocation of CTS induces programmed cell death. This complex behavior of CTS generates the need to discuss the different aspects of CTS associated with cancer regulation. In this review, we mainly focused on the significance of each cathepsin in cancer signaling and their targeting which would provide noteworthy information in the context of cancer biology and therapeutics.
Cathepsin L (CTSL), a cysteine protease, is closely related to tumor occurrence, development, and metastasis, and possibly regulates cancer cell resistance to chemotherapy. miRNAs, especially the miR-200 family, have been implicated in drug-resistant tumors. In this study we explored the relationship of CTSL, micRNA-200c and drug resistance, and the potential regulatory mechanisms in human lung cancer A549 cells and A549/TAX cells in vitro. A549/TAX cells were paclitaxel-resistant A549 cells overexpressing CTSL and characterized by epithelial-mesenchymal transition (EMT). We showed that micRNA-200c and CTSL were reciprocally linked in a feedback loop in these cancer cells. Overexpression of micRNA-200c in A549/TAX cells decreased the expression of CTSL, and enhanced their sensitivity to paclitaxel and suppressed EMT, whereas knockdown of micRNA-200c in A549 cells significantly increased the expression of CTSL, and decreased their sensitivity to paclitaxel and induced EMT. Overexpression of CTSL in A549 cells significantly decreased the expression of micRNA-200c, and reduced their sensitivity to paclitaxel and induced EMT, but these effects were reversed by micRNA-200c, whereas knockdown of CTSL in A549/TAX cells attenuated paclitaxel resistance and remarkably inhibited EMT, but the inhibition of micRNA-200c could reverse these effects. Therefore, micRNA-200c may be involved in regulating paclitaxel resistance through CTSL-mediated EMT in A549 cells, and CTSL and micRNA-200c are reciprocally linked in a feedback loop.
Cancer cured in mice! Sounds hilarious to a nonacademic naive public, but for cancer researchers, every bit of information about cancer from worms, flies, and mice to human is immensely valuable. For a cancer biologist, it is not important to know in which living species on earth the cancer was cured, but how it was cured. We have learned a lot about the intricacy of signaling molecules and the underlying mechanisms involved in oncogenesis and cancer treatment. In this chapter, we review two important cellular properties, which are lost during cancer progression. These two properties, namely senescence and apoptosis, act as strong natural barriers to cancer. Vast majority of literature suggest that chemotherapeutics stop cancer progression by inducing apoptosis or cell death. In addition, the current literature also suggests that restoration of senescence in cancer cells by chemotherapeutic agents results in inhibition of cancer progression and in some cases even tumor regression.
Normal somatic cells invariably enter a state of permanent growth arrest and functional decline after a finite number of divisions (1). This phenomenon, termed cellular senescence, is believed to be a contributing factor in aging. As we age, the organisms’ ability to withstand the erosive effects of internal and environmental stress diminishes, rendering us vulnerable to a variety of chronic illnesses including cancer, arthritis, diabetes, heart, and neurodegenerative diseases (2-5). Conventional wisdom holds that the body’s decreased resistance to stress during aging may result from the decreased efficiency of normal maintenance and repair mechanisms. The accumulation of damage overtime may either signal for the stimulation of antistress defense, allowing somatic cells to survive longer but enhancing the risk of becoming cancerous, or to a cellular surrender leading to senescence. Accordingly, senescence induction may be considered as a barrier for cancer and thus should be exploited for the treatment of this disease. However, the respective benefits and risks of targeting senescence in cancer cells to treat tumors or in somatic cells to prevent cancer onset must be defined. For this, it is critical to define the relationship between senescence and cancer and characterize the molecular events eliciting these unique cellular stress responses.
Recent studies have demonstrated the capacity of the human organism to prevent the growth of potentially carcinogenic cells, paralyzing them. This antitumor mechanism, which acts as a brake on the malignant process, was already known in lab studies "in vitro" but has now also been verified "in vivo" in mice and in tissue samples from cancer patients. This mechanism is known as cellular senescence and is defined as an emergency defense system for cells on the way to becoming cancerous, i.e., a response to the stimulation of an oncogene. These cells are sentenced to "life imprisonment", impeding the progression of premalignant lesions. This review aims to describe this mechanism and present an update of the evidence on this phenomenon in the setting of oral cancer and precancer.
The presence of glioma stem cells (GSCs) in tumor is relevant for glioma treatment resistance. This study assessed whether knockdown of Cathepsin L can influence GSC growth, tumor radiosensitivity, and clinical outcome. Protein levels of Cathepsin L and stem cell markers (CD133 and Nestin) were analyzed in samples from 90 gliomas of different WHO grades and 6 normal brain tissues by immunohistochemistry. Two glioma stem cell lines with overexpressed Cathepsin L were stably transfected with Cathepsin L short hairpin RNA expression vectors. The effect of Cathepsin L inhibition on radiosensitivity, self-renewal, stemness, DNA damage, and apoptosis were evaluated. In addition, an intracranial animal model and subcutaneous tumor xenografts in nude mice were used to assess tumor response to Cathepsin L inhibition in vivo. Our results proved that expression of Cathepsin L and CD133, but not of Nestin, correlated with malignant grades of glioma tissues. GSCs with high Cathepsin L and CD133 co-expression were extraordinarily radioresistant. Cathepsin L inhibition with radiotherapy significantly reduced GSC growth, promoted apoptosis, and improved radiosensitivity. Knockdown of Cathepsin L resulted in a dramatic reduction of CD133 expression, as well as the decreased phosphorylation of DNA repair checkpoint proteins (ATM and DNA-PKcs). Furthermore, combination of Cathepsin L inhibition and radiotherapy potently blocked tumor growth and decreased blood vessel formation in vivo. Taken together, these findings suggest the Cathepsin L as a promising therapeutic target for clinical therapy in GBM patients.
Proteolytic enzymes may serve as promising targets for novel therapeutic treatment strategies seeking to impede cancer progression and metastasis. One such enzyme is cathepsin L (CTSL), a lysosomal cysteine protease. CTSL upregulation, a common occurrence in a variety of human cancers, has been widely correlated with metastatic aggressiveness and poor patient prognosis. In addition, CTSL has been implicated to contribute to cancer associated osteolysis; a debilitating morbidity affecting both life expectancy and the quality of life. In this review we highlight the mechanisms by which CTSL contributes to tumor progression and dissemination and discuss the therapeutic utility of CTSL intervention strategies aimed at impeding metastatic progression and bone resorption.
Copyright © 2015. Published by Elsevier Inc.
Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro.
Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy.
Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G2/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2.
Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro.
Stress-Induced Premature Senescence (SIPS), a senescence-like state achieved as a result of a variety of
stresses, is associated with relative inability to undergo apoptosis. Sirtuins are a family of histone deacetylases
known primarily for their implication in the aging process; however their role in cancer, an aging associated disease, is still poorly understood. In the present study, we investigated whether alterations in sirtuins expression may be associated with aggressive tumor behavior. We have compared expression profiles of sirtuins between drug sensitive and resistant cancer cells MCF7, SaOS-2, A2780 and HL-60. Expression levels of Sirtuin 1-6 varied among cell lines; however Sirtuin-7 was significantly reduced in all chemoresistant cells tested. Knockdown of Sirt7 expression in human breast MCF7 cell line by RNAi induced senescence-associated β-galactosidase activity, reduced cell proliferation rate, induced drug resistance and increased cell migration, suggesting that this gene may play an active role in regulating cancer cell response to stress. Thus, Sirt7 may represent a compelling target for anti-cancer interventions.
Embryonic signaling pathways, in particular those mediated by Wnt and TGF beta, are known to play key roles in tumor progression through the induction of epithelial-mesenchymal transition (EMT). Their simultaneous targeting could therefore represent a desirable anti-cancer strategy. Based on recent findings that both Wnt and TGF beta associated pathways are regulated by Hippo signaling in mammalian cells, we reasoned that targeting the latter would be more effective in inhibiting EMT. In a search for such inhibitors, we identified a small molecule (C19) with remarkable inhibitory activity not only against Hippo, but also against Wnt and TGF beta pathways. C19 inhibited cancer cell migration, proliferation, and resistance to doxorubicin in vitro, and exerted strong anti-tumor activity in a mouse tumor model. Mechanistically, C19 induced GSK 3 beta mediated degradation of the Hippo transducer TAZ, through activation of the Hippo kinases Mst/Lats and the tumor suppressor kinase AMPK upstream of the degradation complex. Overall, this study identified C19 as a multi-EMT pathway inhibitor with a unique mechanism of action. The findings that both AMPK and Mst/Lats mediate the anti-tumor activity of C19 shed light on a potential cross-talk between metabolic and organ size control pathways in regulating cancer progression. By simultaneously targeting these two pathways, C19 may represent a new type of agents to suppress cancer progression and/or its recurrence.
Irinotecan (CPT-11) is topoisomerase I inhibitor used in the treatment of disseminated colorectal cancer. In colon cancer cells it induces DNA damage which leads to cytotoxicity with ensuing apoptosis or premature senescence. Despite its clinical use and efficiency in malignant colonocytes, its effects in normal colonic cells are relatively underexplored. In this work we report that CPT-11 induces dose-dependent cytotoxicity which results in apoptosis and premature senescence whose occurrence nevertheless varies in relation to the type of exposed cells. In normal colonic epithelial cells (NCM) the prevailing type of response is apoptosis whereas in normal colonic fibroblasts (NCF) it is premature senescence. Further analyses showed that CPT-11 induced in both types of cells DNA damage and activated stress response pathways including p53 and p16 but with varying activity of stress kinase p38 and selected stress-associated microRNAs. Epithelial cells upregulated the expression of p53, which was subsequently specifically phosphorylated, massively activated p38 and initiated mitochondrial, caspase-dependent apoptosis. These events occurred in the presence of moderately increased expression of miR-34a only. Conversely, in colonic fibroblasts p38 was only moderately activated, p53 as well as p16 expressions were upregulated in the presence of increased expression of miR-34a, miR-128a and miR-449a. Caspase-dependent apoptosis was found only in a minority of treated cells and the premature senescence phenotype was prevailing. Specific inhibition further proved that p53-dependent as well as independent mechanisms might be responsible for these cell type-specific differences.
Cellular senescence is considered as a tumor suppressive mechanism. Recent evidence indicates however that senescent cells secrete various growth factors and cytokines, some of which may paradoxically promote cancer progression. This phenomenon termed senescence-associated secretory phenotype (SASP) must be inhibited in order for anti-proliferative agents to be effective. The present study was designed to determine whether the β-catenin destruction complex (BCDC), known to integrate the action of various growth factors and cytokines, would represent a suitable target to inhibit the activity of SASP components. For this, we carried out experiments to determine the effect of drug-induced senescence on secretion of SASP, β-catenin transactivation, and the relationship between these processes. Moreover, genetic and pharmacological approaches were used to define the implication of BCDC in mediating the effects of SASP components on cell migration and resistance to drugs. The findings indicate that drug-induced senescence was associated with expression of various Wnt ligands in addition to previously known SASP components. Beta catenin transactivation and expression of genes implicated in epithelial-mesenchymal transition (EMT) also increased in response to drug-induced SASP. These effects were prevented by Pyrvinium, a recently described activator of BCDC. Pyrvinium also suppressed the effects of SASP on cell migration and resistance to doxorubicin. Together, these findings provide insights on the potential role of BCDC in mediating the effects of drug-induced SASP on cancer cell invasion and resistance to therapy, and suggest that targeting this pathway may represent an effective approach to enhance the activity of current and prospective anti-cancer therapeutics.
Protease inhibitors (PIs) encompass a large group of proteins that regulate the hydrolytic activity of proteolytic enzymes and play important physiological roles in all the living organisms. The protease/PI balance is necessary for cellular homeostasis, though, when such balance is broken, pathological conditions like cancer development are induced. The study of PIs as anti-cancer agents has been an important subject of research since more than three decades ago. There are a variety of mechanisms by which PIs perform their effects which depend on two main aspects: the nature, structure and functions of PI and, the interaction with a complex microenvironment that differs, even between cancers originated on the same source. There are many promissory cases, as well as, failures and paradoxical examples of PIs in the treatment of cancer which must be taken into consideration to propouse PI as possible part of the pharmaceutilcal strategies against cancer. In the following chapter the recent knowledge regarding signaling pathways involved in the anti-cancer effect of different classes of PIs, as well as their main features and the current perspectives in regard with their application as anti-cancer drugs will be discussed.
Despite the expansion of knowledge about neuroblastoma (NB) in recent years, the therapeutic outcome for children with a high-risk NB has not significantly improved. Therefore, more effective therapies are needed. This might be achieved by aiming future efforts at recently proposed but not yet developed targets for NB therapy. In this review, we discuss the recently proposed molecular targets that are in clinical trials and, in particular, those that are not yet explored in the clinic. We focus on the selection of these molecular targets for which promising in vitro and in vivo results have been obtained by silencing/inhibiting them. In addition, these selected targets are involved at least in one of the NB tumorigenic processes: proliferation, anti-apoptosis, angiogenesis and/or metastasis. In particular, we will review a recently proposed target, the microtubule-associated proteins (MAPs) encoded by doublecortin-like kinase gene (DCLK1). DCLK1-derived MAPs are crucial for proliferation and survival of neuroblasts and are highly expressed not only in NB but also in other tumours such as gliomas. Additionally, we will discuss neuropeptide Y, its Y2 receptor and cathepsin L as examples of targets to decrease angiogenesis and metastasis of NB. Furthermore, we will review the micro-RNAs that have been proposed as therapeutic targets for NB. Detailed investigation of these not yet developed targets as well as exploration of multi-target approaches might be the key to a more effective NB therapy, i.e. increasing specificity, reducing toxicity and avoiding long-term side effects.
Tumor recurrence after chemotherapy or radiation remains a major obstacle to successful cancer treatment. A subset of cancer cells, termed cancer stem cells, can elude conventional treatments and eventually regenerate a tumor that is more aggressive. Despite the large number of studies, molecular events that govern the emergence of aggressive therapy-resistant cells with stem cell properties after chemotherapy are poorly defined. The present study provides evidence for the rare escape of tumor cells from drug-induced cell death, after an intermediate stay in a non-cycling senescent stage followed by unstable multiplication characterized by spontaneous cell death. However, some cells appear to escape and generate stable colonies with an aggressive tumor stem cell-like phenotype. These cells displayed higher CD133 and Oct-4 expression. Notably, the drug-selected cells that contained low levels of reactive oxygen species (ROS) also showed an increase in antioxidant enzymes. Consistent with this in vitro experimental data, we observed lower levels of ROS in breast tumors obtained after neoadjuvant chemotherapy compared with samples that did not receive preoperative chemotherapy. These latter tissues also expressed enhanced levels of ROS defenses with enhanced expression of superoxide dismutase. Higher levels of Oct-4 and CD133 were also observed in tumors obtained after neoadjuvant chemotherapy. Further studies provided evidence for the stabilization of Nrf2 due to reduced 26 S proteasome activity and increased p21 association as the driving signaling event that contributes to the transition from a high ROS quiescent state to a low ROS proliferating stage in drug-induced tumor stem cell enrichment.
Prostate cancer is a disease of the old and with increasing life expectancy, its incidence will continue to increase in the future. Control of prostate cancer has involved androgen ablation as a routine form of therapy. However, after an initial response, therapy-resistant clones can appear and result in cancer progression and metastasis with high mortality. The precise mechanisms for the development of androgen resistance are yet uncertain. It appears to be multi-factorial and relates not only to newly acquired genomic capabilities of the cancer cells but also to their interaction with their microenvironment.
Overcoming cellular senescence is essential for oncogenesis. Although it seems to be a protective response for normal cells to avoid malignant transformation, senescence can on the other hand promote tumour progression. Interaction of senescent cancer cells with their microenvironment may be the key link to survival or regression of neoplastic cells. Hence, there is speculation that senescence may be a useful new target for therapy in the future.
We review the role of senescence in prostate cancer and the effect of tumour microenvironment on androgen resistance.
Cathepsin L is a lysosomal cysteine proteinase primarily devoted to the metabolic turnover of intracellular proteins. However, accumulating evidence suggests that this endopeptidase might also be implicated in the regulation of other important biological functions, including bone resorption in normal and pathological conditions. These findings support the concept that cathepsin L, in concert with other proteolytic enzymes involved in bone remodeling processes, could contribute to facilitate bone metastasis formation. In support of this hypothesis, recent studies indicate that cathepsin L can foster this process by triggering multiple mechanisms which, in part, differ from those of the major cysteine proteinase of osteoclasts, namely cathepsin K. Therefore, cathepsin L can be regarded as an additional target in the treatment of patients with metastatic bone disease. This review discusses the clinical and therapeutic implications related to these findings.
Increased neovasculature and resistance to chemotherapy are hallmarks of aggressive cancer; therefore, the development of approaches to simultaneously inhibit these two processes is highly desirable. Previous findings from our laboratory have demonstrated that cathepsin L plays a key role in the development of drug resistance in cancer, and that its inhibition reversed this phenomenon. The goal of the present study was to determine whether targeting cathepsin L would inhibit angiogenesis. For this, the effects of a specific cathepsin L inhibitor, Napsul-Ile-Trp-CHO (NSITC), were tested in vitro on endothelial cell proliferation and interaction with the extracellular matrix, and also in vivo, by measuring its effect on angiogenesis in the chick chorioallantoic membrane (CAM) and mouse matrigel models. The results indicated that NSITC readily inhibits the proliferation of endothelial cells by inducing cell cycle arrest at the G(0)/G(1) phase, and suppresses cell adhesion to different substrates. Investigation of the underlying mechanism(s) indicated that NSITC was able to reduce expression of the adhesion molecule alphaVbeta3 integrin, inhibit cathepsin L-mediated degradation of the extracellular matrix, and disrupt secretion of the pro-angiogenic factors fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). NSITC demonstrated potent efficacy in inhibiting growth factor- and tumor mediated-angiogenesis in the CAM and mouse matrigel models of angiogenesis. The anti-angiogenic effects of NSITC resulted in inhibition of tumor growth in the CAM and in nude mouse xenograft models. Together, these findings provide evidence that cathepsin L plays an important role in angiogenesis and suggest that NSITC represents a potential drug for the treatment of aggressive cancer.
To define a functional role for the endosomal/lysosomal cysteine protease cathepsin L (Ctsl) during squamous carcinogenesis, we generated mice harboring a constitutive Ctsl deficiency in addition to epithelial expression of the human papillomavirus type 16 oncogenes (human cytokeratin 14 (K14)-HPV16). We found enhanced tumor progression and metastasis in the absence of Ctsl. As tumor progression in K14-HPV16 mice is dependent on inflammation and angiogenesis, we examined immune cell infiltration and vascularization without finding any effect of the Ctsl genotype. In contrast, keratinocyte-specific transgenic expression of cathepsin V, the human orthologue of mouse Ctsl, in otherwise Ctsl-deficient K14-HPV16 mice restored the phenotype observed in the control HPV16 skin. To better understand this phenotype at the molecular level, we measured several oncogenic signal transduction pathways in primary keratinocytes on stimulation with keratinocyte-conditioned cell culture medium. We found increased activation of protein kinase B/Akt and mitogen-activated protein kinase pathways in protease-deficient cells, especially if treated with media conditioned by Ctsl-deficient keratinocytes. Similarly, the level of active GTP-Ras was increased in Ctsl-deficient epidermis. We conclude that Ctsl is critical for the termination of growth factor signaling in the endosomal/lysosomal compartment of keratinocytes and, therefore, functions as an anti-tumor protease.
Introducción. En el desarrollo de daño secundario tras lesión medular están implicadas diversas caspasas. La terapia anti-caspasas ha utilizado como diana una sola caspasa que ha sido investigada en una gran variedad de estudios tanto in-vitro como in-vivo. Estos estudios han examinado el efecto neuroprotector del Q-VD-PPh, un inhibidor pan-caspasa, en un modelo de lesión medular en rata. Material y métodos. Se dividieron 30 ratas Wistar en tres grupos de 10 ratas cada uno: una lesión medular traumática (con un trauma de 40 g-cm) se realizó a nivel torácico grupo control (grupo 1), grupo trauma control (grupo 2) y el grupo de ratas tratadas con QVD- OPh (grupo 3) se realizó a nivel torácico (T8-T10) mediante la técnica de caída de peso. La respuesta a la lesión y los efectos neuroprotectores de Q-VD-OPh se valoraron mediante el examen histopatológico y la técnica de TUNEL 24 horas y 5 días tras el traumatismo. Se usó la prueba del plano inclinado de Rivlin y Tator y una versión modificada de la escala de Tarlov para valorar el resultado funcional de las ratas 24 horas, 3 días y 5 días tras la lesión. Resultado. Veinticuatro horas tras la lesión, el estudio histopatológico de las secciones obtenidas del grupo 2 revelaron hemorragia, necrosis, trombos vasculares y edema. Las secciones obtenidos del grupo 3 mostraron hallazgos similares en ese momento. 24 horas tras la lesión el número de células apoptóticas fue 4.47 ± 0.35 en el grupo 2 y 1.58 ± 0.33 en el grupo 3. Cinco días tras la lesión el número medio de células apoptóticas fue de 4.35 ± 0.47 en el grupo 2 y de 1.25 ± 0.34 en el grupo 3. De esta forma el número de células TUNEL positivas en la médula dañada se redujo de forma considerable con el tratamiento con Q-VD-OPh. La función neurológica (tanto con el plano inclinado como con las escalas motoras) fueron significativamente mejores en el grupo de ratas tratadas mediante Q-VD-OPh que en el grupo control. Conclusión. Los marcados efectos antiapoptóticos de la Q-VD-OPh debido a la inhibición de todas las caspasas hace que sea un agente prometedor.
Various caspases have been implicated in the development of secondary damage after spinal cord injury (SCI). Anticaspase therapy that targets only one caspase has been investigated in a variety of in vitro and in vivo studies. This study examined the neuroprotective effects of Q-VD-OPh, a pan-caspase inhibitor, in a rat model of SCI.
Thirty Wistar albino rats were divided into 3 groups of 10 each: the sham-operated controls (group 1), the trauma-created controls (group 2), and the QVD- OPh-treated rats (group 3). An SCI (a trauma of 40 g-cm) was produced at the thoracic level (T8-T10) by the weight-drop technique. The response to injury and the neuroprotective effects of Q-VD-OPh were investigated by histopathologic examination and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) 24 hours and 5 days after trauma. The inclined plane technique of Rivlin and Tator and a modified version of Tarlov's grading scale were used to assess the functional status of the rats 24 hours, 3 days, and 5 days after injury.
Twenty-four hours after trauma, light microscopic examination of a specimen taken from group 2 rats revealed hemorrhage, necrosis, vascular thrombi, and edema. Group 3 tissue samples showed similar features at that time. Twenty-four hours after trauma, the mean apoptotic cell number was 4.47 +/- 0.35 cells in group 2 and 1.58 +/- 0.33 in group 3. Five days after injury, the mean apoptotic cell count was 4.35 +/- 0.47 in group 2 and 1.25 +/- 0.34 in group 3. Thus the number of TUNEL-positive cells in an injured spinal cord was greatly reduced by treatment with Q-VDOPh. The neurologic function scores (both the inclined plane performance and motor grading scores) were significantly better in the Q-VD-OPh-treated group than in the trauma-created control group.
The marked antiapoptotic properties of Q-VD-OPh due to the inhibition of all caspases render it a promising novel agent. A therapeutic strategy using Q-VD-OPh may eventually lead to the effective treatment of SCI in humans.
Cathepsin L, a cysteine protease, is considered to be a potential therapeutic target in cancer treatment. Proteases are involved in the development and progression of cancer. Inhibition of activity of specific proteases may slow down cancer progression. In this review, we evaluate recent studies on the inhibition of cathepsin L in cancer. The effects of cathepsin L inhibition as a monotherapy on apoptosis and angiogenesis in cancer are ambiguous. Cathepsin L inhibition seems to reduce invasion and metastasis, but there is concern that selective cathepsin L inhibition induces compensatory activity by other cathepsins. The combination of cathepsin L inhibition with conventional chemotherapy seems to be more promising and has yielded more consistent results. Future research should be focused on the mechanisms and effects of this combination therapy.
The first aim of this study was to diagnose more aggressive and potentially recurrent meningiomas using an in vitro embryonic chick heart invasiveness assay in which lysosomal enzyme cathepsin B was used as the invasiveness marker. The second aim was to confirm if cathepsin B and/or cathepsin L and their endogenous inhibitors were also prognostic parameters in the clinical study of 119 patients with meningioma.
Primary meningioma cultured spheroids were "confronted" with embryonic chick heart spheroids in vitro, and cathepsin B was used as molecular marker to immunolabel the invasive tumor cells. In vitro invasion assays of the malignant meningioma cells were used to assess the invasive potential related to the cysteine cathepsins. As to the second aim, the possible association of cathepsin B along with selected molecular markers, cathepsin L, and endogenous cysteine protease inhibitors (stefins A and B and cystatin C) with meningioma malignancy was determined using enzyme-linked immunosorbent assays in tumor homogenates. Univariate and multivariate analyses were used to compare these parameters with established biological markers of meningioma recurrence in 119 patients with meningiomas.
The more invasive tumors, which characteristically overgrew the normal tissue, were identified even within a group of histologically benign meningiomas. More intensive staining of cathepsin B in these tumors was not only found at the tumor front, but also in the invading pseudopodia of a single migrating tumor cells. Matrigel invasion of malignant meningioma cells was significantly altered by modulating cathepsin B activity and by stefin B silencing. In the clinical samples of meningioma, the levels of cathepsins B and L, stefin B, and cystatin C were highest in the tumors of higher histological grades, whereas stefin A and progesterone receptor were the only markers that were significantly increased and decreased, respectively, in WHO Grade III lesions. With respect to the prognosis of relapse, cathepsin L (p = 0.035), stefin B (p = 0.007), cystatin C (p = 0.008), and progesterone receptor (p = 0.049) levels were significant, whereas cathepsin B was not a prognosticator. As expected, WHO grade, age, and Simpson grade (complete tumor resection) were prognostic, with Simpson grade only relevant in the short term (up to 90 months) but not in longer-term follow-up. Of note, the impact of all these parameters was lost in multivariate analysis, due to overwhelming prognostic impact of stefin B (p = 0.039).
The data indicate that the cysteine cathepsins and their inhibitors are involved in a process related to early meningioma recurrence, regardless of their histological classification. Of note, the known tumor invasiveness marker cathepsin B, measured in whole-tumor homogenates, was not prognostic, in contrast to its endogenous inhibitor stefin B, which was highly significant and the only independent prognostic factor to predict meningioma relapse in multivariate analysis and reported herein for the first time. Stefin B inhibition of local invasion was confirmed by in vitro invasion assay, although its other functions cannot be excluded.
Cancer stem cells are known for their inherent resistance to therapy. Here we investigated whether normal stem cells with acquired resistance to stress can be used to identify novel markers of cancer stem cells. For this, we generated a human embryonic stem cell line resistant to Trichostatin A and analyzed changes in its gene expression. The resistant cells over-expressed various genes associated with tumor aggressiveness, many of which are also expressed in the CD133+ glioma cancer stem cells. These findings suggest that stress-resistant stem cells generated in vitro may be useful for the discovery of novel markers of cancer stem cells.
Tumors of the nervous system are among the most common and most chemoresistant neoplasms of childhood and adolescence. Malignant tumors of the brain collectively account for 21% of all cancers and 24% of all cancer-related deaths in this age group. Neuroblastoma, a peripheral nervous system tumor, is the most common extracranial solid tumor of childhood, and 65% of children with this tumor have only a 10 or 15% chance of living 5 years beyond the time of initial diagnosis. Novel pharmacological approaches to nervous system tumors are urgently needed. This review presents the role of and current challenges to pharmacotherapy of malignant tumors of the nervous system during childhood and adolescence and discusses novel approaches aimed at overcoming these challenges.
Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.
Anticancer drug-induced tumor suppression may involve mechanisms of protection against neoplastic transformation that are normally latent in mammalian cells and consist in a genetic program implemented during anti-tumoral defense. This defense program results in the self elimination of cells harboring potentially dangerous mutations by triggering cell death through apoptosis and/or autophagy or in the execution of a program that leads to a permanent growth arrest known as senescence. These responses are considered crucial tumor suppressive mechanisms and their study appears to be essential to develop therapeutical procedures based on the enhancement of the different responses. This review summarizes fundamental knowledge on the underlying mechanisms able to limit excessive or aberrant cellular proliferation and on the prognostic value of both apoptosis and senescence detection. In addition, interesting evidence showing that different drugs induce senescence or cell death depending on the genetic features of the tumor cells as well as on the integrity of the relative pathways is reported.
Overexpression and enhanced activity of insulin-like growth factor-I receptor (IGF-IR) in diverse tumor types make it an attractive target for cancer therapy. BMS-536924 is a potent small molecule inhibitor of IGF-IR, which shows antitumor activity in multiple tumor models, including sarcoma. To facilitate the development of IGF-IR inhibitors as cancer therapy, identification of biomarkers for selecting patients most likely to derive clinical benefit is needed. To do so, 28 sarcoma and neuroblastoma cell lines were screened for in vitro response to BMS-536924 to identify sensitive and resistant cell lines. Notably, Ewing's sarcoma, rhabdomyosarcoma, and neuroblastoma are more responsive to BMS-536924, suggesting these specific subtypes may represent potential targeted patient subpopulations for the IGF-IR inhibitor. Gene expression and protein profiling were performed on these cell lines, and candidate biomarkers correlating with intrinsic and/or acquired resistance to BMS-536924 were identified. IGF-I, IGF-II, and IGF-IR were highly expressed in sensitive cell lines, whereas IGFBP-3 and IGFBP-6 were highly expressed in resistant lines. Overexpression of epidermal growth factor receptor (EGFR) and its ligands in resistant cell lines may represent one possible resistance mechanism by the adaptation of IGF-IR-independent growth using alternative signaling pathways. Based on cross-talk between IGF-IR and EGFR pathways, combination studies to target both pathways were performed, and enhanced inhibitory activities were observed. These results provide a strategy for testing combinations of IGF-IR inhibitors with other targeted therapies in clinical studies to achieve improved patient outcomes. Further exploration of mechanisms for intrinsic and acquired drug resistance by these preclinical studies may lead to more rationally designed drugs that target multiple pathways for enhanced antitumor efficacy.
Previous studies have suggested that the production of cathepsin S (CatS), a cysteine protease, was specifically induced in radiation-induced rat mammary tumors. In this study, we further investigate the mechanism by which CatS is induced by radiation and its function. Radiation induced production of CatS at both the mRNA and protein level, and increased its protease activity. In addition, these radiation induced changes occurred in a dose and time-dependent fashion. Agents such as bleomycin, As(2)O(3) and H(2)O(2), which produce reactive oxygen species (ROS), also induced CatS expression; however, other agents that damage DNA such as taxol and cisplatin did not. Additionally, treatment of the cells with the ROS scavengers, N-acetylcysteine and catalase, inhibited the radiation induced CatS expression. Furthermore, radiation-induced ROS was also involved in IFN-gamma production, which was responsible for radiation-mediated CatS expression. Moreover, electrophoretic mobility shift assay (EMSA) data obtained using an IFN-stimulated response element (ISRE) oligonucleotide revealed that IFN regulatory factor-1 (IRF1) was the critical transcriptional mediator of IFN-gamma-dependent CatS production after radiation. Finally, CatS overespression was found to induce radioresistance; however, knockdown of CatS resulted in the suppression of radioresistance. Taken together, the results of this study indicate that radiation induced CatS expression via ROS-IFN-gamma pathways, and that this increased expression may be involved in radioresistance.
Cathepsin L is a lysosomal enzyme thought to play a key role in malignant transformation. Recent work from our laboratory has demonstrated that this enzyme may also regulate cancer cell resistance to chemotherapy. The present study was undertaken to define the relevance of targeting cathepsin L in the suppression of drug resistance in vitro and in vivo and also to understand the mechanism(s) of its action. In vitro experiments indicated that cancer cell adaptation to increased amounts of doxorubicin over time was prevented in the presence of a cathepsin L inhibitor, suggesting that inhibition of this enzyme not only reverses but also prevents the development of drug resistance. The combination of the cathepsin L inhibitor with doxorubicin also strongly suppressed the proliferation of drug-resistant tumors in nude mice. An investigation of the underlying mechanism(s) led to the finding that the active form of this enzyme shuttles between the cytoplasm and nucleus. As a result, its inhibition stabilizes and enhances the availability of cytoplasmic and nuclear protein drug targets including estrogen receptor-alpha, Bcr-Abl, topoisomerase-IIalpha, histone deacetylase 1, and the androgen receptor. In support of this, the cellular response to doxorubicin, tamoxifen, imatinib, trichostatin A, and flutamide increased in the presence of the cathepsin L inhibitor. Together, these findings provided evidence for the potential role of cathepsin L as a target to suppress cancer resistance to chemotherapy and uncovered a novel mechanism by which protease inhibition-mediated drug target stabilization may enhance cellular visibility and, thus, susceptibility to anticancer agents.
The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.
Cytotoxic drugs used in chemotherapy of leukemias and solid tumors cause apoptosis in target cells. In lymphoid cells the CD95 (APO-1/Fas)/CD95 ligand (CD95-L) system is a key regulator of apoptosis. Here we describe that doxorbicin induces apoptosis via the CD95/CD95-L system in human leukemia T-cell lines. Doxorubicin-induced apoptosis was completely blocked by inhibition of gene expression and protein synthesis. Also, doxorbicin strongly stimulates CD95-L messenger RNA expression in vitro at concentrations relevant for therapy in vivo. CEM and jurkat cells resistant to CD95-mediated apoptosis were also resistant to doxorbicin-induced apoptosis . Furthermore, doxorbicin-induced apoptosis was inhibited by blocking F(ab')2 anti-APO-1 (anti-CD95) antibody fragments. Expression of CD95-L mRNA and protein in vitro was also stimulated by other cytotoxic drugs such as methotrexate. The finding that apoptosis caused by anticancer drugs may be mediated via the CD95 system provides a new molecular insight into resistance and sensitivity toward chemotherapy in malignancies.
The U-A10 cell line, a doxorubicin-selected variant of human U-937 myeloid leukemia cells, exhibits a redistribution of anthracyclines into a expanded vesicular compartment. The acidic nature of this compartment was confirmed by vital staining with a pH sensitive dye, LysoSensor yellow/blue DND-160. Identification of the vesicular compartment was performed by immunofluorescence analysis. Staining for the LAMP-1 and LAMP-2 antigens showed that the vesicles are enlarged lysosomes that are eccentrically placed near the nucleus of U-A10 cells. By contrast, the expression of the multidrug resistance-associated protein and the P-glycoprotein was observed predominately on the plasma membrane of the drug-resistant cells. The accumulation of daunorubicin into cellular compartments was quantified using radiolabeled drug. Exposing cells to 3[H]-daunorubicin and then isolating intact nuclei showed that nuclei from U-A10 cells accumulated twofold to threefold less anthracycline than nuclei from U-937 cells. However, when nuclei were isolated first and then exposed to 3[H]-daunorubicin, little difference in net nuclear drug accumulation was detected. Cytoplasts prepared from U-A10 and U-937 cells were exposed to 3[H]-daunorubicin to measure cytoplasmic drug accumulation. At external daunorubicin concentrations of 100 ng/mL or higher, cytoplasts from U-A10 cells accumulated significantly more daunorubicin than cytoplasts from U-937 cells. Moreover, studies with the lysosomotropic agent chloroquine showed that U-A10 cells accumulated twofold more chloroquine and showed twofold enhanced sensitivity to this agent as compared with parental U-937 cells. Fluorescence microscopy showed that chloroquine affects vesicular anthracycline sequestration in U-A10 cells with an associated increase in daunorubicin nuclear fluorescence. Although chloroquine did not alter anthracycline cytotoxicity in parental cells, it restored daunorubicin and doxorubicin sensitivity to U-A10 cells. Taken together, these studies demonstrate that U-A10 cells exhibit a redistribution of the lysosomal compartment. The trapping of drug into an expanded acidic vesicular compartment results in decreased nuclear drug accumulation and decreased cytotoxicity. Lysosomotropic agents, such as chloroquine, warrant further study as modulators of this acquired drug-resistance phenotype.
Cytotoxic drugs commonly used in chemotherapy of leukemia and solid tumors have been shown to primarily act by inducing apoptosis in sensitive target cells. Apoptosis may involve activation of death-inducing ligand/receptor systems such as CD95 (APO-1/Fas). Treatment with anticancer drugs such as doxorubicin, methotrexate, cytarabine, etoposide and cisplatin at therapeutic concentrations leads to induction of CD95-ligand (CD95-L). CD95-L can mediate cell death in an autocrine/paracrine manner by crosslinking CD95 receptor (CD95). Interfering with CD95-ligand/receptor interaction by antagonistic antibodies to the receptor reduces sensitivity to drug-mediated apoptosis in some cell systems. In addition, treatment with cytotoxic drugs may result in upregulation of CD95, thereby increasing the sensitivity to the CD95 death signal. Apoptosis depends on activation of caspases. Deficient activation of the CD95 system was found in drug-resistant cells. In addition, CD95-resistant and doxorubicin-resistant cells displayed cross-resistance for induction of cell death. Thus, intact apoptosis pathways such as the CD95 system may play a role in determining sensitivity or resistance of tumor cells to chemotherapy.
Upon binding of their ligands, death receptors belonging to the tumor necrosis factor (TNF) receptor family initiate a signaling pathway leading to the activation of caspases and ultimately apoptosis. TNF, however, in parallel elicits survival signals, protecting many cell types from cell death that can only be induced by combined treatment with TNF and inhibitors of protein synthesis. Here, we report that in NIH3T3 cells, apoptosis in response TNF and cycloheximide is not inhibited by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD. fmk). Moreover, treatment with zVAD.fmk sensitizes the cells to the cytotoxic action of TNF. Sensitization was also achieved by overexpression of a dominant-negative mutant of Fas-associated death domain protein and, to a lesser extent, by specific inhibition of caspase-8. A similar, but weaker sensitization of zVAD.fmk to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) or anti-CD95 antibody was demonstrated. The unexpected cell death in response to TNF and caspase inhibition occurs despite the activation of nuclear factor kappaB and c-Jun N-terminal kinases. The mode of cell death shows several signs of apoptosis including DNA fragmentation, although activation of caspase-3 was excluded. TNF/zVAD.fmk-induced cell death is preceded by an accumulation of cells in the G(2)/M phase of the cell cycle, indicating an important role of cell cycle progression. This hypothesis is further strengthened by the observation that arresting the cells in the G(1) phase of the cell cycle inhibited TNF/zVAD.fmk-induced cell death, whereas blocking them in the G(2)/M phase augmented it.
Apoptosis of SK-HEP-1 human hepatoma cells induced by treatment with ginsenoside Rh2 (G-Rh2) is associated with rapid and selective activation of cyclin A-associated cyclin-dependent kinase 2 (Cdk2). Here, we show that in apoptotic cells, the Cdk inhibitory protein p21(WAF1/CIP1), which is associated with the cyclin A-Cdk2 complex, undergoes selective proteolytic cleavage. In contrast, another Cdk inhibitory protein, p27(KIP1), which is associated with cyclin A-Cdk2 and cyclin E-Cdk2 complexes, remained unaltered during apoptosis. Ectopic overexpression of p21(WAF1/CIP1) suppressed apoptosis as well as cyclin A-Cdk2 activity induced by treatment of SK-HEP-1 cells with G-Rh2. The suppressive effects of p21(WAF1/CIP1) were much higher in the cells transfected with p21D112N, an expression vector that encodes a p21(WAF1/CIP1) mutant resistant to caspase 3 cleavage. Overexpression of cyclin A in SK-HEP-1 cells dramatically up-regulated cyclin A-Cdk2 activity and accordingly enhances apoptosis induced by treatment with G-Rh2. These up-regulating effects were blocked by coexpression of a dominant negative allele of cdk2. Furthermore, olomoucine, a specific inhibitor of Cdks, also blocked G-Rh2-induced apoptosis. These data suggest that the induction of apoptosis in human hepatoma cells treated with G-Rh2 occurs by a mechanism that involves the activation of cyclin A-Cdk2 by caspase 3-mediated cleavage of p21(WAF1/CIP1).
Caspases, a family of cysteine proteases, play a central role in the pathways leading to apoptosis. Recently, it has been reported that a broad spectrum inhibitor of caspases, the tripeptide Z-VAD-fmk, induced a switch from apoptosis to necrosis in dexamethasone-treated B lymphocytes and thymocytes. As such a cell death conversion could increase the efficiency of radiation therapy and in order to identify the caspases involved in this cell death transition, we investigated the effects of caspase-3-related proteases inhibition in irradiated MOLT-4 cells. Cells were pretreated with Ac-DEVD-CHO, an inhibitor of caspase-3-like activity, and submitted to X-rays at doses ranging from 1 to 4 Gy. Our results show that the inhibition of caspase-3-like activity prevents completely the appearance of the classical hallmarks of apoptosis such as internucleosomal DNA fragmentation or hypodiploid particles formation and partially the externalization of phosphatidylserine. However, this was not accompanied by any persistent increase in cell survival. Instead, irradiated cells treated by this inhibitor exhibited characteristics of a necrotic cell death. Therefore, functional caspase-3-subfamily not only appears as key proteases in the execution of the apoptotic process, but their activity may also influence the type of cell death following an exposure to ionizing radiation.
Lysosomal cysteine proteinases of the papain family are involved in lysosomal bulk proteolysis, major histocompatibility complex class II mediated antigen presentation, prohormone processing, and extracellular matrix remodeling. Cathepsin L (CTSL) is a ubiquitously expressed major representative of the papain-like family of cysteine proteinases. To investigate CTSL in vivo functions, the gene was inactivated by gene targeting in embryonic stem cells. CTSL-deficient mice develop periodic hair loss and epidermal hyperplasia, acanthosis, and hyperkeratosis. The hair loss is due to alterations of hair follicle morphogenesis and cycling, dilatation of hair follicle canals, and disturbed club hair formation. Hyperproliferation of hair follicle epithelial cells and basal epidermal keratinocytes-both of ectodermal origin-are the primary characteristics underlying the mutant phenotype. Pathological inflammatory responses have been excluded as a putative cause of the skin and hair disorder. The phenotype of CTSL-deficient mice is reminiscent of the spontaneous mouse mutant furless (fs). Analyses of the ctsl gene of fs mice revealed a G149R mutation inactivating the proteinase activity. CTSL is the first lysosomal proteinase shown to be essential for epidermal homeostasis and regular hair follicle morphogenesis and cycling.
We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.
Caspase-8 is a proximal effector protein of the tumor necrosis factor receptor family death pathway. In the present human postmortem study, we observed a significantly higher percentage of dopaminergic (DA) substantia nigra pars compacta neurons that displayed caspase-8 activation in Parkinson's disease (PD) patients compared with controls. In an in vivo experimental PD model, namely subchronically 1,2,3,6-tetrahydropyridine-treated mice, we also show that caspase-8 is indeed activated after exposure to this toxin early in the course of cell demise, suggesting that caspase-8 activation precedes and is not the consequence of cell death. However, cotreatment of 1-methyl-4-phenylpyridinium-intoxicated primary DA cultures with broad-spectrum and specific caspase-8 inhibitors did not result in neuroprotection but seemed to trigger a switch from apoptosis to necrosis. We propose that this effect is related to ATP depletion and suggest that the use of caspase inhibitors in pathologies linked to intracellular energy depletion, such as PD, should be cautiously evaluated.
In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.
The transcription factor Stat3 has been reported to play a key role in protecting cells against apoptosis by up-regulating expression of the anti-apoptotic gene BCl-xL. This investigation analyzes the relationship between the development of resistance to doxorubicin-mediated apoptosis in neuroblastoma cells (SKN-SH) and activation of the Stat3 signaling pathway.
A drug-resistant cell line (SKN-SH/Dox6) was generated by continuous exposure to incremental concentrations of doxorubicin. Specific antibodies were utilized for Western blots and confocal microscopy to determine the nuclear localization of activated Stat3.
Doxorubicin-mediated DNA fragmentation was inhibited and caspase-3 activity decreased in SKN-SH/Dox6 cells. Up-regulation of Stat3 phosphorylation and Bcl-xL expression, increased nuclear translocation of phospho-Stat3, and binding to DNA occurred only in resistant SKN-SH/Dox6 cells. The expression of Bcl-xL was inhibited by AG490, an inhibitor of the JAK/Stat3 signaling pathway, suggesting that the regulation of Bcl-xL and Stat3 involved a common mechanism. Activation of Stat3 in SKN-SH/Dox6 cells was contingent upon stimulation evoked by ligands secreted by the drug-resistant cells. Evidence to support this hypothesis was provided by experiments in which doxorubicin-sensitive SKN-SH cells were preincubated with conditioned media obtained from doxorubicin-resistant SKN-SH/Dox6 cells. This treatment increased Stat3 activation. It also rendered SKN-SH cells resistant to doxorubicin as demonstrated by a sharp decrease in doxorubicin-induced DNA degradation and cytotoxic potency.
These findings suggest that the resistance of SKN-SH/Dox6 cells to doxorubicin may be mediated by anti-apoptotic factor(s) that are synthesized and secreted by tumor cells in response to cytotoxic agents.
From their discovery in the first half of the 20th century, lysosomal cysteine proteases have come a long way: from being the enzymes non-selectively degrading proteins in lysosomes to being those responsible for a number of important cellular processes. Some of the features and roles of their structures, specificity, regulation and physiology are discussed.
Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterizedCe-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential
role forcpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP)
was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and
becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the
eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in
further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis
and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization
of a related cathepsin L in the filarial nematodeOnchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.
Treatment with chemotherapy or radiation is not invariably cytotoxic to all tumor cells. Some of the cells that survive treatment recover and resume proliferation, whereas others undergo permanent growth arrest. To understand the nature of treatment-induced terminal growth arrest, colon carcinoma cells were exposed to doxorubicin, and surviving cells were separated into proliferating and growth-arrested populations. Only growth-arrested cells displayed phenotypic markers of cell senescence and failed to form colonies. Gene expression was compared between senescent and proliferating fractions of drug-treated cells by using cDNA microarray hybridization and reverse transcription-PCR. Drug-induced senescence was associated with inhibition of genes involved in cell proliferation and with coinduction of multiple intracellular and secreted growth inhibitors. Several tumor suppressors and other genes that are down-regulated in carcinogenesis were up-regulated in senescent tumor cells. Induction of most growth inhibitors was delayed but not abolished in cells with homozygous knockout of p53, in agreement with only limited p53 dependence of drug-induced terminal growth arrest. On the other hand, senescent cells overexpressed secreted proteins with antiapoptotic, mitogenic, and angiogenic activities, suggesting that drug-induced senescence is associated with paracrine tumor-promoting effects. About one-third of the genes up-regulated in senescent cells and almost all of the down-regulated genes showed decreased or delayed changes in p21(Waf1/Cip1/Sdi1)-deficient cells, indicating that p21 is a major mediator of the effects of p53 on gene expression. Elucidation of molecular changes in tumor cells that undergo drug-induced senescence suggests potential strategies for diagnostics and therapeutic modulation of this antiproliferative response in cancer treatment.
Dilated cardiomyopathy is a frequent cause of heart failure and is associated with high mortality. Progressive remodeling of the myocardium leads to increased dimensions of heart chambers. The role of intracellular proteolysis in the progressive remodeling that underlies dilated cardiomyopathy has not received much attention yet. Here, we report that the lysosomal cysteine peptidase cathepsin L (CTSL) is critical for cardiac morphology and function. One-year-old CTSL-deficient mice show significant ventricular and atrial enlargement that is associated with a comparatively small increase in relative heart weight. Interstitial fibrosis and pleomorphic nuclei were found in the myocardium of the knockout mice. By electron microscopy, CTSL-deficient cardiomyocytes contained multiple large and apparently fused lysosomes characterized by storage of electron-dense heterogeneous material. Accordingly, the assessment of left ventricular function by echocardiography revealed severely impaired myocardial contraction in the CTSL-deficient mice. In addition, echocardiographic and electrocardiographic findings to some degree point to left ventricular hypertrophy that most likely represents an adaptive response to cardiac impairment. The histomorphological and functional alterations of CTSL-deficient hearts result in valve insufficiencies. Furthermore, abnormal heart rhythms, like supraventricular tachycardia, ventricular extrasystoles, and first-degree atrioventricular block, were detected in the CTSL-deficient mice.
Cathepsins B and L are widely expressed cysteine proteases implicated in both intracellular proteolysis and extracellular matrix remodeling. However, specific roles remain to be validated in vivo. Here we show that combined deficiency of cathepsins B and L in mice is lethal during the second to fourth week of life. Cathepsin B(-/-)/L(-/-) mice reveal a degree of brain atrophy not previously seen in mice. This is because of massive apoptosis of select neurons in the cerebral cortex and the cerebellar Purkinje and granule cell layers. Neurodegeneration is accompanied by pronounced reactive astrocytosis and is preceded by an accumulation of ultrastructurally and biochemically unique lysosomal bodies in large cortical neurons and by axonal enlargements. Our data demonstrate a pivotal role for cathepsins B and L in maintenance of the central nervous system.
One of the mechanisms by which chemotherapeutics destroy cancer cells is by inducing apoptosis. Apoptosis can be activated through several different signalling pathways, but these all appear to converge at a single event - mitochondrial membrane permeabilization (MMP). This 'point-of-no-return' in the cell death program is a complex process that is regulated by the composition of the mitochondrial membrane and pre-mitochondrial signal-transduction events. MMP is subject to a complex regulation, and local alterations in the composition of mitochondrial membranes, as well as alterations in pre-mitochondrial signal-transducing events, can determine chemotherapy resistance in cancer cells. Detecting MMP might thus be useful for detecting chemotherapy responses in vivo. Several cytotoxic drugs induce MMP by a direct action on mitochondria. This type of agents can enforce death in cells in which upstream signals normally leading to apoptosis have been disabled. Cytotoxic components acting on mitochondria can specifically target proteins from the Bcl-2 family, the peripheral benzodiazepin receptor, or the adenine nucleotide translocase, and/or act by virtue of their physicochemical properties as steroid analogues, cationic ampholytes, redox-active compounds or photosensitizers. Some compounds acting on mitochondria can overcome the cytoprotective effect of Bcl-2-like proteins. Several agents which are already used in anti-cancer chemotherapy can induce MMP, and new drugs specifically designed to target mitochondria are being developed.
The inhibition of apoptosis is generally believed to be a major determinant of resistance to chemotherapy. However, recent findings have shown that caspase inhibitors do not protect cancer cells from death by cytotoxic agents, but may switch drug-induced apoptosis to an alternative 'default death'. The primary goals of this study were to determine the major characteristics of the 'default death' and the mechanism by which this switch is activated. For this purpose, we first investigated putative cell death modes induced by doxorubicin. Molecular markers associated with these death modes were utilized to identify the default death resulting from the inhibition of apoptosis. Our findings demonstrated that doxorubicin induced at least three distinct types of cell death, senescence, apoptosis and a type of necrosis, which were concentration dependent. Specific molecular markers such as p21/WAF1, activated caspase-3 and activated Akt were associated with these death modes. The pan-caspase inhibitor (Q-VD-OPH) greatly reduced doxorubicin-induced caspase-3 activation but did not protect cells against drug toxicity. The combination of doxorubicin and Q-VD-OPH caused an increased expression of p21/WAF1 and senescence -associated -beta-galactosidase activity, but did not alter Akt activation. Collectively, these findings suggest that the inhibition of apoptosis may lead to an increased expression of cell cycle inhibitors and cellular senescence.
The U-A10 cell line, a doxorubicin-selected variant of human U-937 myeloid leukemia cells, exhibits a redistribution of anthracyclines into a expanded vesicular compartment. The acidic nature of this compartment was confirmed by vital staining with a pH sensitive dye, LysoSensor yellow/blue DND-160. Identification of the vesicular compartment was performed by immunofluorescence analysis. Staining for the LAMP-1 and LAMP-2 antigens showed that the vesicles are enlarged lysosomes that are eccentrically placed near the nucleus of U-A10 cells. By contrast, the expression of the multidrug resistance-associated protein and the P-glycoprotein was observed predominately on the plasma membrane of the drug-resistant cells. The accumulation of daunorubicin into cellular compartments was quantified using radiolabeled drug. Exposing cells to 3[H]-daunorubicin and then isolating intact nuclei showed that nuclei from U-A10 cells accumulated twofold to threefold less anthracycline than nuclei from U-937 cells. However, when nuclei were isolated first and then exposed to 3[H]-daunorubicin, little difference in net nuclear drug accumulation was detected. Cytoplasts prepared from U-A10 and U-937 cells were exposed to 3[H]-daunorubicin to measure cytoplasmic drug accumulation. At external daunorubicin concentrations of 100 ng/mL or higher, cytoplasts from U-A10 cells accumulated significantly more daunorubicin than cytoplasts from U-937 cells. Moreover, studies with the lysosomotropic agent chloroquine showed that U-A10 cells accumulated twofold more chloroquine and showed twofold enhanced sensitivity to this agent as compared with parental U-937 cells. Fluorescence microscopy showed that chloroquine affects vesicular anthracycline sequestration in U-A10 cells with an associated increase in daunorubicin nuclear fluorescence. Although chloroquine did not alter anthracycline cytotoxicity in parental cells, it restored daunorubicin and doxorubicin sensitivity to U-A10 cells. Taken together, these studies demonstrate that U-A10 cells exhibit a redistribution of the lysosomal compartment. The trapping of drug into an expanded acidic vesicular compartment results in decreased nuclear drug accumulation and decreased cytotoxicity. Lysosomotropic agents, such as chloroquine, warrant further study as modulators of this acquired drug-resistance phenotype.
Sequestration of drugs in intracellular vesicles has been associated with multidrug-resistance (MDR), but it is not clear why vesicular drug accumulation, which depends upon intracellular pH gradients, should be associated with MDR. Using a human uterine sarcoma cell line (MES-SA) and a doxorubicin (DOX)-resistant variant cell line (Dx-5), which expresses p-glycoprotein (PGP), we have addressed the relationship between multidrug resistance, vesicular acidification, and vesicular drug accumulation. Consistent with a pH-dependent mechanism of vesicular drug accumulation, studies of living cells vitally labeled with multiple probes indicate that DOX and daunorubicin (DNR) predominately accumulate in lysosomes, whose lumenal pH was measured at < 4.5, but are not detected in endosomes, whose pH was measured at 5.9. However, vesicular DOX accumulation is more pronounced in the drug-sensitive MES-SA cells and minimal in Dx5 cells even when cellular levels of DOX are increased by verapamil treatment. While lysosomal accumulation of DOX correlated well with pharmacologically induced differences in lysosome pH in MES-SA cells, lysosomal accumulation was minimal in Dx5 cells regardless of lysosomal pH. We found no differences in the pH of either endosomes or lysosomes between MES-SA and Dx5 cells, suggesting that, in contrast to other MDR cell systems, the drug-resistant Dx5 cells are refractory to pH-dependent vesicular drug accumulation. These studies demonstrate that altered endomembrane pH regulation is not a necessary consequence of cell transformation, and that vesicular sequestration of drugs is not a necessary characteristic of MDR. J. Cell. Physiol. 184:263–274, 2000. © 2000 Wiley-Liss, Inc.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1994. Includes bibliographical references (leaves 226-254).
Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.
Although the primary cellular targets of many anticancer agents have been identified, less is known about the processes leading to the selective cell death of cancer cells or the molecular basis of drug resistance. p53-deficient mouse embryonic fibroblasts were used to examine systematically the requirement for p53 in cellular sensitivity and resistance to a diverse group of anticancer agents. These results demonstrate that an oncogene, specifically the adenovirus E1A gene, can sensitize fibroblasts to apoptosis induced by ionizing radiation, 5-fluorouracil, etoposide, and adriamycin. Furthermore, the p53 tumor suppressor is required for efficient execution of the death program. These data reinforce the notion that the cytotoxic action of many anticancer agents involves processes subsequent to the interaction between drug and cellular target and indicate that divergent stimuli can activate a common cell death program. Consequently, the involvement of p53 in the apoptotic response suggests a mechanism whereby tumor cells can acquire cross-resistance to anticancer agents.
A widely held tenet of present day oncology is that tumor cells treated with anticancer agents die from apoptosis, and that cells resistant to apoptosis are resistant to cancer treatment. We suggest, in this review, that this tenet may need to be reexamined for human tumors of nonhematological origin, for two principal reasons: (a) cell killing has often been assessed in short term assays that are more influenced by the rate, than the overall level, of cell killing. This has tended to underestimate cell killing for cells not susceptible to apoptosis or having mutant p53; and (b) conclusions from experiments with normal cells transformed with dominant oncogenes have often been extrapolated to tumor cells. This does not take into account the fact that tumor cells have invariably undergone selection to an apoptotically resistant phenotype. In this review, we examine the impact of these two factors with particular emphasis on the influence of mutations in p53 on the sensitivity of tumor cells to DNA-damaging agents. We find that because wild-type p53 predisposes cells to a more rapid rate of cell death after DNA damage, particularly with normal or minimally transformed cells, that short-term assays have led to the conclusion that mutations in p53 confer resistance to genotoxic agents. On the other hand, if clonogenic survival is used to assess killing in cells derived from actual solid human tumors, then apoptosis and the genes controlling it, such as p53 and bcl-2, appear to play little or no role in the sensitivity of these cells to killing by anticancer drugs and radiation.
PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.
The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.
Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by degeneration of dopamine-containing neurons in the midbrain. In cases of familial PD, mutations that lead to failure of the ubiquitin–proteasome system (UPS) have been identified. These genetic abnormalities do not occur in sporadic PD, but we propose that impairment of the UPS could also contribute to neurodegeneration in this disorder. We discuss evidence that failure of the UPS is a common aetiopathogenic factor that underlies the development of familial and sporadic PD, an idea that might help to explain clinical and pathological differences and similarities in these disorders.
We examined the role of caspases in the early programmed cell death (PCD) of motoneurons (MNs) in the chick embryo cervical cord between embryonic day (E) 4 and E5. An increase in caspase-3-like activity in MNs was observed at E4.5. Treatment with an inhibitor of caspase-3-like activity, Ac-DEVD-CHO, for 12 h blocked this increase and revealed that caspase-3-like activity is mainly responsible for DNA fragmentation and the nuclear changes during PCD but not for degenerative changes in the cytoplasm. When a more broad-spectrum caspase inhibitor was used (bocaspartyl (OMe)-fluoromethyl ketone, BAF), the appearance of degenerative changes in the cytoplasm was delayed by at least 12 h. However, following treatment with either Ac-DEVD-CHO or BAF for 24 h, the number of surviving healthy MNs did not differ from controls, indicating a normal occurrence of PCD despite the inhibition of caspases. These results suggest that caspase cascades that occur upstream of and are independent of the activation of caspase-3-like activity are responsible for the degenerative changes in the cytoplasm of dying cervical MNs. These data also suggest that, although one function of caspases may be to facilitate the kinetics of PCD, caspases are nonetheless dispensable for at least some forms of normal neuronal PCD in vivo.
Cell death induced by etoposide in the human lymphoma cell line U-937 GTB was characterized. Activity of caspases -3, -8 and -9 was measured by spectrophotometric detection of specific cleavage products, DNA fragmentation by TdT-mediated dUTP nick end-labelling (TUNEL), and apoptotic morphology by conventional staining and microscopy, as well as by a novel method-the microculture kinetics (MiCK) assay. Synthesis of protein and DNA during exposure was monitored by incorporation of radioactive leucine and thymidine, respectively. The effects of caspase inhibitors on total viability, as well as early and late morphological changes were studied. Etoposide rapidly induced apoptosis, dependent on caspase-3 and -8, but inhibition of these caspases did not prevent major cell death, but promoted a switch in late morphology. The novel MiCK assay added valuable information on early morphological events during cell death. Hence, this study provides support for caspase-8-mediated apoptosis in U-937 GTB when exposed to etoposide. General caspase inhibition switches cell death to one with a different morphology.
Even though the pan-caspase inhibitor zVAD has been widely used as an anti-apoptotic agent, inefficient prevention or even enhancement of cell death has been reported in certain cells. To further investigate its effects on cell death, three different cell types were exposed to various apoptotic stimuli in the presence or absence of zVAD. In Jurkat cells, zVAD protected against cell death induced by tumor necrosis factor (TNF), sodium nitroprusside (SNP) and etoposide, whereas in L929 cells cell death was increased. In RAW246.7 macrophages, zVAD showed similar effects as in L929 cells. However, unlike L929 cells, in which the cell death by TNF is known to be necrosis, RAW246.7 cells manifested features of apoptosis such as chromatin condensation and nuclear fragmentation. Induction of cell death by zVAD in lipopolysaccharide (LPS)-activated RAW246.7 cells also showed the same features as those observed in SNP- and etoposide-treated cells. Initiation of an apoptotic process by zVAD not only disputes the sole role of caspases in apoptosis but also suggests an anti-apoptotic function of certain caspase(s). Death of LPS-activated macrophages may be controlled by an anti-apoptotic caspase.
p21(Waf1/Cip1/Sdi1) is best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, but p21 also interacts with many other regulators of transcription or signal transduction. p21 induction, which is mediated by p53 and by p53-independent mechanisms, is essential for the onset of cell cycle arrest in damage response and cell senescence. The effects of p21 knockout in mice and its expression patterns in human cancer are consistent with a role for p21 as both a tumour suppressor and an oncogene. Several functions of p21 are likely to promote carcinogenesis and tumour progression. These include endoreduplication and abnormal mitosis that develop in tumour cells after release from p21-induced growth arrest, the ability of p21 to inhibit apoptosis through several different mechanisms, and its ability to stimulate transcription of secreted factors with mitogenic and anti-apoptotic activities. The latter effects of p21 show close resemblance to paracrine activities of senescent cells and to tumour-promoting functions of stromal fibroblasts. Therapeutic strategies targeting the oncogenic consequences of p21 expression may provide a new approach to chemoprevention and treatment of cancer.
Inhibition of the program of apoptosis has been reported to have little or no effect on clonogenic survival after treatment with drugs or radiation in several tumor cell lines. A decrease in apoptosis is compensated in such cell lines by an increase in the fractions of cells that undergo permanent growth arrest with phenotypic features of cell senescence, or die through the process of mitotic catastrophe. Most of the tested tumor cell lines have retained the capacity of normal cells to undergo accelerated senescence after treatment with DNA-interactive drugs, ionizing radiation, or cytostatic agents. p53 and p21(Waf1/Cip1/Sdi1) act as positive regulators of treatment-induced senescence, but they are not required for this response in tumor cells. The senescent phenotype distinguishes tumor cells that survived drug exposure but lost the ability to form colonies from those that recover and proliferate after treatment. Although senescent cells do not proliferate, they are metabolically active and may produce secreted proteins with potential tumor-promoting activities. The expression of such proteins is mediated at least in part by the induction of p21(Waf1/Cip1/Sdi1). The other anti-proliferative response of tumor cells is mitotic catastrophe, a form of cell death that results from abnormal mitosis and leads to the formation of interphase cells with multiple micronuclei. Mitotic catastrophe is induced by different classes of cytotoxic agents, but the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. Mitotic catastrophe can also develop as a consequence of aberrant reentry of tumor cells into cell cycle after prolonged growth arrest. Elucidation of the factors that regulate different aspects of treatment-induced senescence and mitotic catastrophe should assist in improving the efficacy and decreasing side effects of cancer therapy.
We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4(+) T-cell deficiency. Also, we recently reported that nkt (now Ctsl(nkt)) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4(+) T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctsl(nkt)/Ctsl(nkt) mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2-/- Ctsl(nkt)/Ctsl(nkt) double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation.
Cancer cells often develop resistance to chemotherapy or irradiation through mutations in the p53 tumour-suppressor gene, which prevent apoptosis induction in response to cellular damage. Death receptors — members of the tumour-necrosis factor receptor (TNFR) superfamily — signal apoptosis independently of p53. Decoy receptors, by contrast, are a non-signalling subset of the TNFR superfamily that attenuate death-receptor function. Agents that are designed to activate death receptors (or block decoy receptors) might therefore be used to kill tumour cells that are resistant to conventional cancer therapies.
The targeted degradation of key regulatory proteins is an essential element of cell cycle control. The proteasome plays a central role in the degradation of such proteins and has therefore become an important therapeutic target for diseases involving cell proliferation, notably cancer. This review summarizes numerous studies demonstrating that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents both and The potent and selective proteasome inhibitor, PS-341, is particularly promising from a therapeutic perspective, and it is the only such inhibitor that has progressed to clinical trials. Preliminary data indicate that the drug is well tolerated by patients with cancer, and further trials are underway to assess the safety and efficacy of proteasome inhibition in hematologic and solid tumors, both as a monotherapy and in combination with other chemotherapeutics.
The ubiquitin-proteasome system (UPS) plays a vital role in directing molecules to the 26S proteasome for degradation as well as other locales in the cell. Disrupting UPS function can lead to the aggregation of mutant or misfolded proteins, which disrupts normal cellular activity in diverse ways. Here we discuss how UPS dysfunction might contribute to a variety of neurological problems.
From an improved understanding of apoptosis mechanisms, multiple novel strategies have emerged for restoring apoptosis sensitivity in cancer, and clinical testing of several of these approaches is underway. Much remains to be learned about how best to exploit these new potential therapies, matching genetic lesion in cancers to the optimal agent. It remains to be determined to what extent toxicity to normal tissues will limit application of apoptosis-based therapies for cancer treatment. Cancer cells, however, may be relatively more dependent on apoptosis suppression than normal cells, due to aberrations in protooncogene activity and cell cycle checkpoint control, wandering of cancer cells from their normal sites of trophic support, and hypoxic conditions caused by inadequate vascular supply. Exploiting this differential dependence on apoptosis-suppressing mechanisms offers hope that improved clinical outcomes may not be far from realization.
If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells
- I B Roninson
- E V Broude
Roninson, I. B., Broude, E. V., and Chang, B. D. If not apoptosis, then what?
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A biomarker that identifies senescent human cells in culture and in aging skin in vivo
- G P Dimri
- X Lee
- G Basile
- M Acosta
- G Scott
- C Roskelley
- E E Medrano
- M Linskens
- I Rubelj
- O Pereira-Smith
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Linskens, M., Rubelj, I., and Pereira-Smith, O. A biomarker that identifies senescent
human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA, 92:
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Lysosomal cysteine proteases: facts and opportunities
- V Turk
- B Turk
- D Turk
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Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route
- V Stoka
- B Turk
- S L Schendel
- T H Kim
- T Cirman
- S J Snipas
- L M Ellerby
- D Bredesen
- H Freeze
- M Abrahamson
- D Bromme
- S Krajewski
- J C Reed
- X M Yin
- V Turk
- G S Salvesen
Stoka, V., Turk, B., Schendel, S. L., Kim, T. H., Cirman, T., Snipas, S. J., Ellerby,
L. M., Bredesen, D., Freeze, H., Abrahamson, M., Bromme, D., Krajewski, S., Reed,
J. C., Yin, X. M., Turk, V., and Salvesen, G. S. Lysosomal protease pathways to
apoptosis. Cleavage of bid, not pro-caspases, is the most likely route. J. Biol. Chem.,
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Mitochondrial control of cell death
- G Kroemer
- J Reed