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Chemosensitization of Pancreatic Cancer by Inhibition of the 26S Proteasome
Abstract
Pancreatic cancer is extremely resistant to the induction of apoptosis by chemotherapies; agents that regulate sensitivity to apoptosis may lead to chemosensitization of pancreatic cancer.
MIA-PaCa-2 human pancreatic cancer cells were treated in vitro with the 26S-proteasome inhibitor PS-341. Levels of the apoptosis-regulating proteins (BCL-2, BAK, and BAX) were determined by Western blotting. The effect of PS-341 on BCL-2 gene transcription was examined using a BCL-2 promoter/luciferase reporter construct. The chemosensitizing effect of PS-341 was determined by measurement of the cytotoxic effect of gemcitabine in the presence of PS-341 (10-1000 nM) using the MTT assay. A corresponding in vivo experiment using tumor xenografts in athymic mice was also performed.
PS-341 decreased BCL-2, without effect on BAX or BAK. The downregulation of BCL-2 by PS-341 appears to be transcriptionally mediated. PS-341 induced apoptosis at high does (1000 nM) and increased the cytotoxicity of gemcitabine at low doses (10-100 nM). Xenograft growth was inhibited 59% by gemcitabine; the addition of PS-341 increased growth inhibition to 75%.
Inhibition of the 26S proteasome disrupts the cellular content of key regulators of cell cycle progression and apoptotic control leading to increased sensitivity to standard chemotherapeutic agents, such as gemcitabine, in pancreatic cancer. Combination therapy may lead to better response rates.
... In sum, a decreased proteasomal activity causes a deregulation of signaling pathways, leading to an increased cell proliferation resulting in the development of cancer. However, numerous studies show that proteasomal activity is enhanced in cancer cells [75][76][77][78][79][80][81][82][83][84][85][86][87][88][89] representing an obvious discrepancy. A plethora of point mutations in cancer genomes lead to a very high number of misfolded proteins [90]. ...
... Proteasomal activity seems to be an important factor in cancerogenesis, since proteasomal activity is altered in many cancer types (Table 3) and the use of proteasome activators and inhibitors as anticancer therapeutics showed promising results [100,164,165]. In most cancer types, proteasomal activity is elevated [75][76][77][78][79][80][81][82][83][84][85][86][87][88][89]. Until now, the reason for this increase is unknown. ...
... Consequently, an association between primary cilia and cancer is very likely. Altered proteasomal activity is an often observed feature in cancer cells [75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][98][99][100][101][102][103] and it was demonstrated that ciliary proteins control proteasomal activity [50,122,123,126]. Previously, it was suggested that the dysfunction of the cilia-controlled proteasome is only one contributory factor of the ciliopathic pathology [122]. ...
The primary cilium is an essential structure for the mediation of numerous signaling pathways involved in the coordination and regulation of cellular processes essential for the development and maintenance of health. Consequently, ciliary dysfunction results in severe human diseases called ciliopathies. Since many of the cilia-mediated signaling pathways are oncogenic pathways, cilia are linked to cancer. Recent studies demonstrate the existence of a cilia-regulated proteasome and that this proteasome is involved in cancer development via the progression of oncogenic, cilia-mediated signaling. This review article investigates the association between primary cilia and cancer with particular emphasis on the role of the cilia-regulated proteasome.
... For the chemotherapy treatment of the scaffolds, the chemotherapeutic agent gemcitabine (GEM, Sigma-Aldrich, UK) was selected. GEM is used extensively for treatment of pancreatic cancer both in vivo 12,14,56,59,[68][69][70][71][72][73][74] and in vitro 12,14,19,34,35,51,52,75 studies. ...
... 76 These concentrations were selected based on dosages used in previously published PDAC in vitro studies. 10,33,35,68,72 Thereaer, the scaffolds were characterised 24 h and 17 days aer treatment with sectioning, staining and advanced imaging. These time points were selected to study the immediate responses to GEM 33,34,36 and to mimic in vivo treatment regimes. ...
Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly and aggressive disease with a very low survival rate. This is partly due to the resistance of the disease to currently available treatment options. Herein, we report for the first time the use of a novel polyurethane scaffold based PDAC model for screening the short and relatively long term (1 and 17 days post-treatment) responses of chemotherapy, radiotherapy and their combination. We show a dose dependent cell viability reduction and apoptosis induction for both chemotherapy and radiotherapy. Furthermore, we observe a change in the impact of the treatment depending on the time-frame, especially for radiation for which the PDAC scaffolds showed resistance after 1 day but responded more 17 days post-treatment. This is the first study to report a viable PDAC culture in a scaffold for more than 2 months and the first to perform long-term (17 days) post-treatment observations in vitro. This is particularly important as a longer time-frame is much closer to animal studies and to patient treatment regimes, highlighting that our scaffold system has great potential to be used as an animal free model for screening of PDAC.
... The effect of gemcitabine is impaired by extrinsic and/or intrinsic resistance [5]. Bortezomib, a dipeptide boronic acid derivative, has been shown to reverse resistance and sensitize pancreatic cancer cells to the gemcitabine therapy when administered in a schedule-dependent manner [3,6,7]. Bortezomib potently and reversibly inhibits the multicatalytic intracellular 26S proteasome (at the chymotrypsin-like enzyme site of the 19S particle) that is responsible for selective and non-lysosomal proteolysis of polyubiquitin-tagged proteins, resulting in the release of recyclable short peptides. ...
... Bortezomib potently and reversibly inhibits the multicatalytic intracellular 26S proteasome (at the chymotrypsin-like enzyme site of the 19S particle) that is responsible for selective and non-lysosomal proteolysis of polyubiquitin-tagged proteins, resulting in the release of recyclable short peptides. This inhibition affects many key proteins of signaling cascades and cell cycle regulatory proteins, including those involved in cell proliferation, regulation of apoptosis, and gene transcription [6]. ...
Pancreatic cancer has extremely poor prognosis with an 85% mortality rate that results from aggressive and asymptomatic growth, high metastatic potential, and rapid development of resistance to already ineffective chemotherapy. In this study, plasmonic hollow gold nanoshells (GNS) coated with PEGylated thermosensitive lipids were prepared as an efficient platform to ratiometrically co-deliver two drugs, bortezomib and gemcitabine (GNS-L/GB), for combinational chemotherapy and photothermal therapy of pancreatic cancer. Bortezomib was loaded within the lipid bilayers, while gemcitabine was loaded into the hydrophilic interior of the porous GNS via an ammonium sulfate-driven pH gradient method. Physicochemical characterizations and biological studies of GNS-L/GB were performed, with the latter using cytotoxicity assays, cellular uptake and apoptosis assays, live/dead assays, and western blot analysis of pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). The nanoshells showed remotely controllable drug release when exposed to near-infrared laser for site-specific delivery. GNS-L/GB showed synergistic cytotoxicity and improved internalization by cancer cells. High-powered near-infrared continuous wave laser (λ=808nm) effectively killed cancer cells via the photothermal effect of GNS-L/GB, irrespective of cell type in a power density-, time-, and GNS dose-dependent manner. These results suggest that this method can provide a novel approach to achieve synergistic combinational chemotherapy and photothermal therapy, even with resistant pancreatic cancer.
... Preclinical data suggest that bortezomib enhances the activity of gemcitabine. 27 In conjunction with our findings, a larger-scale study is currently underway at our centre to evaluate the efficacy of bortezomib in combination with GC as first-line therapy (ChiCTR2000035916). This phase 2, randomized clinical trial enrolled not only ICC patients but also patients with GBC as our further research showed PTEN deficiency could be tested in about 60% of patients with GBC and also facilitate the therapeutic vulnerability to bortezomib. ...
Introduction
Intrahepatic cholangiocarcinoma (ICC) is characterized by a dismal prognosis with limited therapeutic alternatives. To explore phosphatase and tension homolog (PTEN) as a biomarker for proteasome inhibition in ICC, we conducted a phase II trial to assess the second‐line efficacy of bortezomib in PTEN‐deficient advanced ICC patients.
Methods
A total of 130 patients with advanced ICC in our centre were screened by PTEN immunohistochemical staining between 1 July 2017, and 31 December 2021, and 16 patients were ultimately enrolled and treated with single‐agent bortezomib 1.3 mg/m² on days 1, 4, 8 and 11 of a 21‐day cycle. The primary endpoint was the objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors v1.1.
Results
The median follow‐up was 6.55 months (95% confidence interval [CI]: 0.7–19.9 months). Among the 16 enrolled patients, the ORR was 18.75% (3/16) and the disease control rate was 43.75% (7/16). The median progress‐free survival was 2.95 months (95% CI: 2.1–5.1 months) and the median overall survival (mOS) was 7.2 months (95% CI: 0.7–21.6 months) in the intent‐to‐treat‐patients. Treatment‐related adverse events of any grade were reported in 16 patients, with thrombopenia being the most common toxicity. Patients with PTEN staining scores of 0 were more likely to benefit from bortezomib than those with staining scores > 0.
Conclusions
Bortezomib yielded an encouraging objective response and a favourable OS as a second‐line agent in PTEN‐deficient ICC patients. Our findings suggest bortezomib as a promising therapeutic option for patients with PTEN‐deficient ICC.
Highlights
There is a limited strategy for the second‐line option of intrahepatic cholangiocarcinoma (ICC).
This investigator‐initiated phase 2 study evaluated bortezomib in ICC patients with phosphatase and tension homology deficiency.
The overall response rate was 18.75% and the overall survival was 7.2 months in the intent‐to‐treat cohort.
These results justify further developing bortezomib in ICC patients with PTEN deficiency.
... Future studies are needed to explore other potential biomarkers predictive of bortezomib response in a larger cohort. Preclinical data suggest that bortezomib enhances the activity of gemcitabine [24]. ...
Background: Intrahepatic cholangiocarcinoma (ICC) is characterized by a dismal prognosis with limited therapeutic options. To explore phosphatase and tension homology deleted on chromosome ten (PTEN) as a biomarker for proteasome inhibition in ICC, we conducted a phase II trial to assess the second line efficacy of bortezomib in PTEN-deficient advanced ICC patients.
Methods: Between July 1, 2017, and June 30, 2021, a total of 130 patients with advanced ICC were screened by PTEN immunohistochemical staining and 16 patients were enrolled. Patients with PTEN deficiency who had progressed after gemcitabine combined cisplatin received single-agent bortezomib 1.3 mg/m2 on days 1, 4, 8, and 11 of a 21-day cycle. The primary endpoint was objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors v1.1.
Results: The median time of follow up was 4.63 months (95% CI: 0.7~17.2 months). Among the 16 enrolled patients, 13 of them had completed treatment with bortezomib at least 2 cycles and been evaluated. The ORR was 23% (3/13) and disease control rate was 54% (7/13). The median progress-free survival (mPFS) was 2.95 months (95% CI: 2.1~5.1 months) and the median overall survival (mOS) was 7.2 (95% CI: 0.7~21.6 months) months in the intent-to-treat patients. Treatment-related adverse events of any grade were reported in 16 patients, with thrombopenia being the most common toxicity. Patients with PTEN staining score of 0 were more likely to benefit from bortezomib than those with staining score > 0.
Conclusions: Bortezomib yielded encouraging objective response and a favorable overall survival as a second-line therapy in PTEN-deficient ICC patients. Our findings suggest bortezomib as a promising treatment option in selected ICC patients with PTEN deficiency.
Trial Registration: ClinicalTrials.gov (NCT03345303).
... Proteasomal inhibition: more than twenty years ago, Bold et al. [664,665] proposed proteasomal inhibitors to sensitize PDAC to gemcitabine. In this regard, there is evidence supporting proteasomal inhibitors for gemcitabine resistance [666][667][668][669][670][671]. ...
Simple Summary
PDAC is one of the most malignant tumors and its treatment, whether surgery or chemotherapy, has shown poor results. Resistance to gemcitabine and other chemotherapeutic drugs is an essential factor in this failure. This review analyzes the molecular causes of gemcitabine resistance and discusses the possibilities of new approaches aimed at decreasing, delaying or even reversing chemoresistance in pancreatic cancer.
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with a poor prognosis and inadequate response to treatment. Many factors contribute to this therapeutic failure: lack of symptoms until the tumor reaches an advanced stage, leading to late diagnosis; early lymphatic and hematic spread; advanced age of patients; important development of a pro-tumoral and hyperfibrotic stroma; high genetic and metabolic heterogeneity; poor vascular supply; a highly acidic matrix; extreme hypoxia; and early development of resistance to the available therapeutic options. In most cases, the disease is silent for a long time, andwhen it does become symptomatic, it is too late for ablative surgery; this is one of the major reasons explaining the short survival associated with the disease. Even when surgery is possible, relapsesare frequent, andthe causes of this devastating picture are the low efficacy ofand early resistance to all known chemotherapeutic treatments. Thus, it is imperative to analyze the roots of this resistance in order to improve the benefits of therapy. PDAC chemoresistance is the final product of different, but to some extent, interconnected factors. Surgery, being the most adequate treatment for pancreatic cancer and the only one that in a few selected cases can achieve longer survival, is only possible in less than 20% of patients. Thus, the treatment burden relies on chemotherapy in mostcases. While the FOLFIRINOX scheme has a slightly longer overall survival, it also produces many more adverse eventsso that gemcitabine is still considered the first choice for treatment, especially in combination with other compounds/agents. This review discusses the multiple causes of gemcitabine resistance in PDAC.
... Also in other tumor models significant tumor responses have been observed with proteasome inhibitors. For example, pancreatic tumor xenografts were responsive to PS-341 used in combination with gemcitabine or in combination with irinotecan [38,39]. Recently is has been shown in a model of adult Tcell leukemia that therapy with PS-341 in combination with a humanized antibody against interleukin-2 receptor alpha (HAT) was associated with a complete remission in a proportion of treated mice, whereas only a partial response was observed in mice treated with HAT alone [40]. ...
... It is already applied to the clinical therapy of hematologic neoplasms. Preclinical studies of BTZ in solid tumors showed that the drug could induce many cancer cell apoptosis and sensitize them to other anticancer agents [22][23][24]. Shin et al. demonstrated that BTZ could inhibit tumor adaptation to hypoxia by repressing HIF-1α [25]. We used Western blot analysis and found that BTZ enhances SiHa and HeLa cells' radiation sensitivity to IR by inhibiting HIF-1α expression, which meant that BTZ could reverse hypoxia-induced radiation resistance. ...
Objective:
This study aimed to investigate the radiosensitivity of bortezomib to cervical cancer and the possible underlying mechanism.
Methods:
HeLa and SiHa cell lines with or without hypoxia treatment were divided into control, radiation alone, bortezomib alone, and radiotherapy plus bortezomib groups. CCK8 assay, clone formation assay, flow cytometry, and immunofluorescence test were used to measure cell proliferation, colony formation, apoptosis, and DNA double-strand break (DSB). Western blot analysis was performed to detect the expression of HIF-1α, PARP-1, and caspase-3, -8, and -9.
Result:
Statistical analysis of data revealed that bortezomib at nanomolar level exerted a radiosensitization effect on both cervical cancer cell lines in normoxia or hypoxia. Western blot analysis showed that the drug could inhibit hypoxia-related HIF-1α expression to increase apoptosis-related caspase-3, -8, and -9 activation and DNA DSB-related PARP-1 cleavage.
Conclusions:
Radiotherapy sensitization of bortezomib on cervical cancer cell lines had a drug-dose relation, and sensitization in hypoxia was more remarkable than in normoxia. Bortezomib may be a potential radiotherapy sensitization drug for cervical cancer.
... Combining proteasome inhibitors with CPT-11 has the significant potential to overcome the chemotherapy resistance in colorectal cancer cell lines (11). Other experimental data have consistently demonstrated the ability of bortezomib to sensitize a variety of cancer cells to the apoptotic effects of diverse chemotherapeutic agents (12)(13)(14). In this study we investigated the in vitro antitumor activity of bortezomib in human gastric cancer cell lines and its combination antitumor effects with docetaxel and cisplatin, which are standard drugs in the treatment of advanced gastric cancers. ...
The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of tumor cells. Bortezomib is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome and has prominent effects in vitro and in vivo against several solid tumors. We examined the anti-proliferative and apoptotic effects of bortezomib in three gastric cancer cell lines (SNU638, MUGC-3 and MKN-28), along with its antitumor combination effects with other chemotherapeutic agents. Tumor cell growth inhibition and apoptosis was measured by MTT assay and FACS analysis, respectively. Apoptosis- and cell cycle-associated protein expression levels were measured by Western blot assay. Bortezomib induced the suppression of tumor cell growth and apoptosis in a dose-dependent manner with an inhibitory dose (ID) 50 of approximately 0.5 μg/ml in all gastric cancer cell lines tested. Further combination treatment with cisplatin and docetaxel, in particular with docetaxel displayed dramatically increased tumor cell growth suppression in all three gastric cancer cell lines, as compared to single drug treatment alone. This was concomitant with the induction patterns of apoptotic cells. Bortezomib treatment increased the Bax protein expression. Moreover, combination treatment of bortezomib plus docetaxel resulted in a dramatic increase in the Bax expression. In contrast, Bcl-2 expression was decreased by combination treatment with bortezomib plus docetaxel in SNU638 cells. Finally, bortezomib, docetaxel and to a greater degree bortezomib plus docetaxel increased the expression levels of p27 proteins even without influencing p53 expression levels. Bortezomib has profound effects on tumor cell growth inhibition and induction of apoptosis in human gastric cancer cells, suggesting that bortezomib may be an effective therapeutic drug for patients with gastric cancer. Further combination studies with other chemotherapeutic drugs, in particular docetaxel showing more tumor cell growth inhibition and apoptosis suggest that combining bortezomib with docetaxel might be more effective for displaying tumor cell growth inhibitory effects in gastric cancer cells through regulation of Bcl-2, Bax and p27 proteins in vitro.
... którzy byli poddani przynajmniej jednemu cyklowi leczenia, a od niedawna do leczenia pacjentów z nowo rozpoznanym szpiczakiem plazmocytowym [42]. Należy jednak podkreślić, iż bortezomib indukuje apoptozę nie tylko komórek nowotworowych szpiczaka plazmocytowego [21], ale również raka trzustki [7,46], stercza, jajnika [17] oraz raka głowy i szyi [52]. Trwają badania nad zastosowaniem tego inhibitora w różnego typu chłoniakach oraz nowotworach złośliwych, takich jak czerniak złośliwy, mięsak, niedrobnokomórkowy rak oskrzela, rak stercza, jajnika, piersi, płuc, nerek, okrężnicy, żołądka i nowotwór centralnego układu nerwowego [53]. ...
Proteasomes are multisubunit enzyme complexes. They contain three enzymatic active sites which are termed chymotrypsin-like, trypsin-like, and caspase-like. The elementary function of the proteasomes is degradation of damaged proteins. Proteasome inhibition leads to accumulation of damaged protein, which leads to caspase activation and cell death. This relationship is used in cancer therapy. Bortezomib is the first proteasome inhibitor approved by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma. Carfilzomib belongs to the second generation of drugs, which was approved by the US FDA in 2012. Currently in the study phase there are four new inhibitors: ixazomib (MLN9780/MLN2238), delanzomib (CEP-18770), oprozomib (ONX0912/PR-047) and marizomib (NPI-0052).
... Among its cell survival-associated molecular targets, the proteasome is known to play a critical role in the activation of the transcription factor, NFnB (14), which is constitutively activated in most human pancreatic cancer cell lines and primary tumors (9). In vitro and in vivo studies by our laboratory and others have demonstrated that bortezomib has antitumor activity in pancreatic cancer models (34,35) and that it sensitizes some pancreatic cancer cells to apoptosis induced by taxanes in vitro via a mechanism that involves inhibition of NFnB (36). The observation that NFnB inhibitors sensitize tumor cells to conventional chemotherapeutic agents has been reported extensively (reviewed in Refs. ...
Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome. We examined the antitumor activity of combination therapy with bortezomib + docetaxel in two human pancreatic cancer cell lines (MiaPaCa-2 and L3.6pl) selected for their divergent responses to bortezomib alone. Bortezomib blocked docetaxel-induced apoptosis in the MiaPaCa-2 cells and failed to enhance docetaxel-induced apoptosis in L3.6pl cells in vitro but did interact positively with docetaxel to inhibit clonogenic survival. These effects were associated with decreased accumulation of cells in M phase, stabilization of the cyclin-dependent kinase inhibitors, p21 and p27, and inhibition of cdk2 and cdc2 activities. In orthotopic xenografts, combination therapy produced significant reductions in tumor weight and volume in both models associated with accumulation of p21, inhibition of proliferation, and increased apoptosis. Combination therapy also reduced tumor microvessel densities, effects that were associated with reductions in tumor cell production of vascular endothelial growth factor and increased levels of apoptosis in tumor-associated endothelial cells. Together, our results suggest that bortezomib enhances the antitumoral activity of taxanes by enforcing cell growth arrest and inhibiting angiogenesis.
... The transcription factor is now free to become translocated to the nucleus where it binds to specific DNA elements and activates the transcription of NF-B-dependent genes (40). In vivo models of ovarian cancer, colorectal cancer and pancreatic cancer have shown that NF-B inhibition increases the efficacy of anticancer drugs (41)(42)(43)(44). It is thought that NF-B inhibition prevents tumours from becoming resistant to chemotherapeutic agents. ...
... Additionally, Teicher et al., demonstrate that using bortezomib, either alone or in conjunction with other chemotherapeutics decreases the process of metastases in a mouse model for lung metastasis(Teicher et al., 1999). Also, it has been reported that bortezomib increases the sensitivity of prostate cancer xenografts to radiotherapy(Pervan et al., 2001), of pancreatic cancer xenografts to chemotherapy(Richard et al., 2001;Shah et al., 2001) and of colon tumor xenografts to both radiation and chemotherapy(Cusack et al., 2001;Suzanne et al., 2001). Several molecular mechanisms for the mode of action of bortezomib in enhancing the response of tumor cells to chemotherapy may be implicated beside its role in blocking NFκB. ...
... This pathway plays a vital role in degrading regulatory proteins that govern cell cycle control, transcription factor activation, apoptosis, and cell trafficking, including p53, p21, p27, NF-jB, and bcl-2 [10][11][12][13]. Preclinical observations have suggested that inhibitors of the ubiquitin-proteasome pathway, agents like lactacystin and the calpain inhibitor ALLN and ALLM, and bortezomib can act through multiple mechanisms to arrest tumour growth, tumour spread, and angiogenesis [5,6,14,15]. Phase I trials have confirmed tolerability of the drug and have suggested possible clinical activity [7,16]. ...
In addition to its efficacy in the treatment of multiple myeloma, the proteasome inhibitor bortezomib appears to be active against a variety of other haematological malignancies. In a phase II trial, bortezomib, given twice weekly for 2 weeks of a 3 week cycle to patients with relapsed, refractory or untreated indolent non-Hodgkin's lymphoma (NHL) or mantle cell lymphoma (MCL), produced durable responses in 2/7 MCL patients and stable disease in five. Six of eight patients with follicular NHL achieved a durable response (1 CR, 5 PR). In another study, bortezomib was administered to patients with relapsed or refractory indolent or aggressive B-cell lymphoma. Durable responses were seen in 7/12 MCL patients, including three CR. This apparent activity in MCL is particularly encouraging, given its poor prognosis. These early trial results demonstrate that further clinical testing of bortezomib and additional exploration of the multiple biologic effects of proteasome inhibition are warranted in lymphoproliferative malignancies.
... Given the increasing role of chemotherapy in biliary tract cancers, further combination studies with bortezomib should be considered. Preclinical data suggest that bortezomib enhances the activity of multiple chemotherapy agents including gemcitabine, 5fluorouracil, and irinotecan, providing at least additive cell killing 24,[42][43][44][45] . This may be due to downregulation of NFkB, a known candidate mediator of chemotherapy resistance 45 . ...
Background
Patients with advanced biliary tract cancers have limited therapeutic options. Preclinical data suggest proteasome inhibition may be an effective therapeutic strategy. We thus evaluated the clinical efficacy of bortezomib in advanced biliary tract cancers.
Patients and Methods
Patients with locally advanced or metastatic cholangiocarcinoma or gallbladder adenocarcinoma who had received 0-2 prior therapies received bortezomib 1.3 mg/m2 days 1, 4, 8, and 11 of a 21-day cycle. The primary endpoint was objective response rate. A Simon two-stage design was employed (null response rate of < 5% and response rate of ≥ 20% of interest).
Results
Twenty patients enrolled: bile duct/gallbladder cancer (14/6), prior treatments 0/1/2 (10/6/3). The trial was discontinued early due to lack of confirmed partial responses. No unanticipated adverse events were noted. There was one unconfirmed partial response. Ten patients achieved stable disease as best response. Median time to progression was 5.8 months (95% CI 0.7-77.6 months). Median survival was 9 months (95% CI 4.6-18.5 months). The 6-month and 1-year survival rates were 70% and 38%. There was no difference in survival based on primary disease site.
Conclusions
Single agent bortezomib does not result in objective responses in biliary tract cancers. However, the rate of stable disease and time to progression benchmark is encouraging. Further development of bortezomib in combination with other therapies in this disease setting should be considered.
... One feature that chemotherapeutic treatments of cancer have in common is their activation of NF-κB, which mediates cell survival. In recent years, combinatorial approach has been used to overcome this problem by the application of rational chemotherapeutic combinations, such as combination of NF-κB inhibitors and chemotherapy drugs (36). In our study, 6shogaol was used in combination with gemcitabine to test its efficacy against pancreatic cancer. ...
Advanced pancreatic cancer still has a poor prognosis, even with the approval of several drugs, such as gemcitabine. Therefore, developing effective and safe antitumor agents is urgently needed. 6-Shogaol, a phenol extracted from ginger, has been linked to suppression of proliferation and survival of cancer with different mechanisms. In the present study, we investigated whether 6-shogaol could suppress pancreatic cancer progress and potentiate pancreatic cancer to gemcitabine treatment in vitro and in vivo. We found that 6-shogaol prevented the activation of toll like receptor 4 (TLR4)/NF-κB signaling. The modulation of NF-κB signaling by 6-shogaol was ascertained by electrophoretic mobility shift assay and western blot analysis. The suppression of NF-κB signaling and key cell survival regulators including COX-2, cyclinD1, survivin, cIAP-1, XIAP, Bcl-2, and MMP-9 brought the anti-proliferation effects in pancreatic cancer cells and sensitized them to gemcitabine treatment. Furthermore, in a pancreatic cancer xenograft model, we found a decreased proliferation index (Ki-67) and increased apoptosis by TUNEL staining in 6-shogaol treated tumors. It was also shown that 6-shogaol combined with gemcitabine treatment was more effective than drug alone, consistent with the downregulation of NF-κB activity along with its target genes COX-2, cyclinD1, survivin, cIAP-1, and XIAP. Overall, our results suggest that 6-shogaol can inhibit the growth of human pancreatic tumors and sensitize them to gemcitabine by suppressing of TLR4/NF-κB-mediated inflammatory pathways linked to tumorigenesis.
... This pathway plays a vital role in degrading regulatory proteins that govern cell cycle control, transcription factor activation, apoptosis, and cell trafficking, including p53, p21, p27, NF-jB, and bcl-2 [10][11][12][13]. Preclinical observations have suggested that inhibitors of the ubiquitin-proteasome pathway, agents like lactacystin and the calpain inhibitor ALLN and ALLM, and bortezomib can act through multiple mechanisms to arrest tumour growth, tumour spread, and angiogenesis [5,6,14,15]. Phase I trials have confirmed tolerability of the drug and have suggested possible clinical activity [7,16]. ...
In addition to its efficacy in the treatment of multiple myeloma, the proteasome inhibitor bortezomib appears to be active against a variety of other haematological malignancies. In a phase II trial, bortezomib, given twice weekly for 2 weeks of a 3 week cycle to patients with relapsed, refractory or untreated indolent non-Hodgkin's lymphoma (NHL) or mantle cell lymphoma (MCL), produced durable responses in 2/7 MCL patients and stable disease in five. Six of eight patients with follicular NHL achieved a durable response (1 CR, 5 PR). In another study, bortezomib was administered to patients with relapsed or refractory indolent or aggressive B-cell lymphoma. Durable responses were seen in 7/12 MCL patients, including three CR. This apparent activity in MCL is particularly encouraging, given its poor prognosis. These early trial results demonstrate that further clinical testing of bortezomib and additional exploration of the multiple biologic effects of proteasome inhibition are warranted in lymphoproliferative malignancies.
The use of molecular oxygen as the terminal oxidant in transition metal catalyzed oxidative process is an appealing and challenging task in organic synthetic chemistry. Here, we report a Ni‐catalyzed hydroxylarylation of unactivated alkenes enabled by a β‐ diketone ligand with high efficiency and excellent regioselectivity employing molecular oxygen as the oxidant and hydroxyl source. This reaction features mild conditions, broad substrate scope and incredible heterocycle compatibility, providing a variety of β ‐hydroxylamides, γ ‐hydroxylamides, β ‐aminoalcohols, γ ‐aminoalcohols, and 1,3‐diols in high yields. The synthetic value of this methodology was demonstrated by the efficient synthesis of two bioactive compounds, (±)‐3′‐methoxyl citreochlorol and tea catechin metabolites M4.
The use of molecular oxygen as the terminal oxidant in transition metal catalyzed oxidative process is an appealing and challenging task in organic synthetic chemistry. Here, we report a Ni‐catalyzed hydroxylarylation of unactivated alkenes enabled by a β‐diketone ligand with high efficiency and excellent regioselectivity employing molecular oxygen as the oxidant and hydroxyl source. This reaction features mild conditions, broad substrate scope and incredible heterocycle compatibility, providing a variety of β‐hydroxylamides, γ‐hydroxylamides, β‐aminoalcohols, γ‐aminoalcohols, and 1,3‐diols in high yields. The synthetic value of this methodology was demonstrated by the efficient synthesis of two bioactive compounds, (±)‐3′‐methoxyl citreochlorol and tea catechin metabolites M4.
BORON İN ENDUSTRY AND HEALTH
Background
The ubiquitin-proteasome system (UPS) is critical in cellular protein degradation and widely involved in the regulations of cancer hallmarks. Targeting the UPS pathway has emerged as a promising novel treatment in hematological malignancies and solid tumors.
Objective
This review mainly focuses on the preclinical results of proteasome inhibitors in solid tumors.
Methods
We analyzed the published articles associated with the anticancer results of proteasome inhibitors alone or combination chemotherapy in solid tumors. Important data presented in abstract form were also discussed in this review.
Results/Conclusion
Proteasome inhibitors, such as bortezomib and carfilzomib, are highly effective in treating solid tumors. The anticancer efficacy is not limited to affect the proteasomal inhibition-associated signaling pathways but also widely involves the signaling pathways related to cell cycle, apoptosis, and epithelial-mesenchymal transition (EMT). In addition, proteasome inhibitors overcome the conventional chemo-resistance of standard chemotherapeutics by inhibiting signaling pathways, such as NF-κB or PI3K/Akt. Combination chemotherapy of proteasome inhibitors and standard chemotherapeutics are widely investigated in multiple relapsed or chemo-resistant solid tumor types, such as breast cancer and pancreatic cancer. The proteasome inhibitors re-sensitize the standard chemotherapeutic regimens and induce synergistic anticancer effects. The development of novel proteasome inhibitors and delivery systems also improves the proteasome inhibitors’ anticancer efficacy in solid tumors. This review summarizes the current preclinical results of proteasome inhibitors in solid tumors and reveals the potential anticancer mechanisms.
Within the past decade, knowledge of the molecular basis of inflammatory bowel disease (IBD), including Crohn disease (CD) and ulcerative colitis, has advanced owing to the explosive growth of research involving the human genome. Furthermore, a shared interest between molecular biologists and clinical researchers has contributed to an emerging understanding of IBD. Nucleotide-binding oligomerization domain 2 (NOD2) belongs to an apoptotic regulatory family of genes and has been linked to CD. In addition, research into nuclear factor kappa B (NF-kappa B), the proteasome, interleukins, and tumor necrosis factor alpha has improved our understanding of IBD. Our understanding of these molecules and other scientific discoveries offers hope for new diagnostic tests and therapeutic agents. In the future, genetic markers will predict disease susceptibility, therapeutic responsiveness, and long-term sequelae of modern therapeutics. Also on the horizon, molecular markers promise to define disease heterogeneity, thereby providing a rational basis for patient-specific therapies. The molecular discoveries that are changing our views of IBD will affect the clinician, the laboratory professional, and the patient.
Les gliomes malins, tumeurs du système nerveux central les plus fréquentes ont un pronostic particulièrement sombre. Parmi les traitements standards de ce type de tumeurs, la radiothérapie occupe une place prépondérante mais son utilisation est limitée en raison de la mauvaise tolérance du tissu cérébral normal. Une des stratégies visant à améliorer l'efficacité de la radiothérapie consiste en l'administration concomitante d'un agent radiosensibilisant. Les inhibiteurs de protéasome sont une nouvelle classe d'agents anticancéreux et le bortézomib (Velcade®), chef de file de cette nouvelle classe, possède une AMM dans le traitement du myélome multiple. Les objectifs de nos travaux sont d'évaluer le potentiel radiosensibilisant du bortézomib sur deux modèles de gliome malin humain xénogreffés chez la souris nude. Nous avons apporté une attention particulière à l'impact du fractionnement du bortézomib et de la radiothérapie sur l'efficacité antitumorale de l'association. Nos résultats montrent que le bortézomib est un radiosensibilisant puissant sur les deux modèles de gliome malin utilisés mais que ce pouvoir est perdu lorsque les deux modalités thérapeutiques sont délivrées selon des doses et des schémas d'administration pertinents au regard de la clinique. En condition de traitement fractionné, nos études montrent également que le bortézomib induit une diminution de l'apoptose radio-induit, probablement en lien avec une perte de son activité inhibitrice du protéasome au fil des injections. Enfin, la place du facteur de transcription NF-kB dans la réponse à la radiothérapie fractionnée ou non pour le type tumoral que nous avons étudié est discutée. En conclusion, nos travaux insistent sur la nécessité de réaliser des études précliniques dans des conditions les plus proches possibles de la situation clinique, afin d?apporter des éléments plus prédictifs de la réponse chez l'Homme.
Proteasomal system plays an important role in protein turnover, which is essential for homeostasis of cells. Besides degradation of oxidized proteins, it is involved in the regulation of many different signaling pathways. These pathways include mainly cell differentiation, proliferation, apoptosis, transcriptional activation and angiogenesis. Thus, proteasomal system is a crucial target for treatment of several diseases including neurodegenerative diseases, cystic fibrosis, atherosclerosis, autoimmune diseases, diabetes and cancer. Over the last fifteen years, proteasome inhibitors have been tested to highlight their mechanisms of action and used in the clinic to treat different types of cancer. Proteasome inhibitors are mainly used in combinational therapy along with classical chemo-radiotherapy. Several studies have proved their significant effects but serious side effects such as peripheral neuropathy, limits their use in required effective doses.
Recent studies focus on peripheral neuropathy as the primary side effect of proteasome inhibitors. Therefore, it is important to delineate the underlying mechanisms of peripheral neuropathy and develop new inhibitors according to obtained data. This review will detail the role of proteasome inhibition in cancer therapy and development of peripheral neuropathy as a side effect. Additionally, new approaches to prevent treatment-limiting side effects will be discussed in order to help researchers in developing effective strategies to overcome side effects of proteasome inhibitors.
The Hedgehog signalling pathway is evolutionarily highly conserved and essential for embryonic development of invertebrates and vertebrates. Consequently, impaired Hedgehog signalling results in very severe human diseases, ranging from holoprosencephaly to Pallister-Hall syndrome. Due to this great importance for human health, the focus of numerous research groups is placed on the investigation of the detailed mechanisms underlying Hedgehog signalling. Today, it is known that tiny cell protrusions, known as primary cilia, are necessary to mediate Hedgehog signalling in vertebrates. Although the Hedgehog pathway is one of the best studied signalling pathways, many questions remain. One of these questions is: How do primary cilia control Hedgehog signalling in vertebrates? Recently, it was shown that primary cilia regulate a special kind of proteasome which is essential for proper Hedgehog signalling. This review article will cover this novel cilia-proteasome association in embryonic Hedgehog signalling and discuss the possibilities provided by future investigations on this topic.
Programmed cell death is an energy-dependent process of cellular elimination that is necessary in all stages of life, including development, tissue homeostasis, and aging in multicellular organisms. Work by Wyllie, Kerr, and Currie identified the large phenotypic changes in the morphology of cells undergoing programmed cell death such as nuclear condensation and the dismantling of cellular material into membrane-enclosed packages; they termed these changes apoptosis. In 1972 they published a seminal paper that correctly predicted the physiologic and pathophysiologic relevance of their findings (1), and Wyllie went on to explore some of the biochemical characteristics that are signs of apoptosis, describing the cleavage of DNA into discrete nucleosomal multimers (DNA ladders) (2). In the 1970s Horvitz and colleagues described the loss of 131 cells during the development of the nematode Caenorhabetitis elegans. Mutagenesis studies subsequently implicated three genes in this process (ced-3, ced-4, and egl-1) that when inactivated caused the retention of the normally eliminated cells (3,4). A fourth gene (ced-9) was identified that promoted cell survival, in that its loss resulted in the death of more cells than normal (5). These proapoptotic gene products, EGL-1, CED-3, and CED-4, along with their antiapoptotic counterpart, CED-9, proved to be components of an evolutionarily conserved, biochemical cascade responsible for cellular elimination.
The Phase I development of the proteasome inhibitor, bortezomib (formerly known as PS-341), in patients with advanced cancer began in 1998. Since that time clinical trials have studied both single agent bortezomib and bortezomib in combination with other chemotherapy agents. The side effect profile that has emerged from dose escalation trials suggests manageable toxicities. Gastrointestinal side effects such as diarrhea and nausea were dose-related and peripheral sensory neuropathy was dose-limiting in some patients with solid tumors. Indications of efficacy in multiple myeloma (including a durable complete remission), non-Hodgkin’s lymphoma, and selected solid tumors were also reported. The recommended dose and schedule for Phase II development was 1.3 mg/m2/ dose given twice weekly for 2 wk with 10 d rest.
The transcription factor nuclear factor kappa B (NF-κB) is a member of the Rel family of proteins that plays an important role in a variety of cellular response mechanisms including immunity, inflammation, cell growth, and apoptosis. Recent evidence suggests that this transcription factor is induced following exposure of cancer cells to apoptotic stimuli such as tumor necrosis factor (TNF), chemotherapy, and irradiation. The activation of NF-κ involves the proteasome-dependent degradation of an inhibitor of NF-κB and results in a cascade of events leading to the suppression of apoptosis. This mechanism of inducible cancer therapy resistance may be overcome by inhibitors of NF-κ activation. The preliminary studies that led to the development of novel cancer therapies that combine chemotherapy and proteasome inhibition are discussed.
Multiple myeloma is incurable, and novel therapies are urgently needed. The proteasome inhibitor bortezomib targets both the tumor cell and the bone marrow microenvironment to overcome drug resistance in preclinical in vitro and in vivo models of human multiple myeloma. A recently completed phase II trial of bortezomib demonstrated durable responses and associated clinical benefit in patients with relapsed refractory myeloma, providing the basis for its Food and Drug Administration approval. Ongoing studies are evaluating bortezomib, alone and in combination, to treat patients earlier in the disease course.
The proteasome is a multicatalytic enzyme complex responsible for the majority of protein degradation in cells. Traditionally believed to be a mere recycler of damaged or misfolded proteins, the proteasome’s importance in modulating cell cycle and survival pathways is now recognized. By degrading regulatory proteins, the proteasome helps to remove signals that promote transcription, cell growth, angiogenesis, and cell adhesion. Preclinical studies have found that agents that inhibit the proteasome’s activity will promote cell cycle arrest and induce apoptosis in tumor cells, thus establishing the proteasome as a promising potential target for anticancer therapy (1). This evidence has led to the examination of these compounds for the treatment of human cancer, and one such proteasome inhibitor, bortezomib (VELCADE TM , formerly PS-341, LDP-341, MLN341), has entered human clinical trials.
Within the past decade, knowledge of the molecular basis of inflammatory bowel disease (M), including Crohn disease (CD) and ulcerative colitis, has advanced owing to the explosive growth of research involving the human genome. Furthermore, a shared interest between molecular biologists and clinical researchers has contributed to an emerging understanding of IBD. Nucleotide-binding oligomerization domain 2 (NOD2) belongs to an apoptotic regulatory family of genes and has been linked to CD. In addition, research into nuclear factor kappa B (NF-kappa B), the proteasome, interleukins, and tumor necrosis factor alpha has improved our understanding of IBD. Our understanding of these molecules and other scientific discoveries offers hope for new diagnostic tests and therapeutic agents. In the future, genetic markers will predict disease susceptibility, therapeutic responsiveness, and long-term sequelae of modem therapeutics. Also on the horizon, molecular markers promise to define disease heterogeneity, thereby providing a rational basis for patient-specific therapies. The molecular discoveries that are changing our views of IBD will affect the clinician, the laboratory professional, and the patient.
The nuclear factor NF-kappa B is an inducible transcription factor activating expression of genes the products of which play a key role in cardiovascular pathologies, carcinogenesis, inflammatory and viral diseases. The review discribes the processes inducing NF-kappa B activation and determines the components of a signal cascade that could constitute targets for NF-kappa B inhibition. The low-molecular-weight inhibitors of the NF-kappa B factor described systematically, and their properties and molecular action mechanism are considered.
Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-kappaB (NF-kappaB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-kappaB pathway. Nuclear extracts from these cell lines were tested for active NF-kappaB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-kappaB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-kappaB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 mumol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-kappaB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 +/- 0.8% and 48 +/- 1.6% in vehicle-treated cells to 27.7 +/- 0.32% and 34.3 +/- 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-kappaB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IkappaBalpha construct, which led to the inhibition of both constitutive and radiation-induced NF-kappaB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 +/- 0.8% in parental MeWo cells to 32.9 +/- 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-kappaB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-kappaB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-kappaB may be one of the pathways responsible for the radioresistance of melanoma cells and that strategies for inhibiting its influence may be useful in restoring the radioresponse of melanomas.
Since last years, the ubiquitin-proteasome concept has emerged as a new and exciting strategy in targeted anticancer therapy. Bortezomib (formerly known as PS341) remains the first selective proteasome inhibitor that has demonstrated both significant preclinical activity in several tumor models and a significant efficacy in patients with refractory or relapsed multiple myeloma, resulting in an accelerated approval in US and Europe in such a setting. The major biological effect of bortezomib consists upon the inhibition of the nuclear transcription factor NKκB, providing an inhibition of the growth tumor cells, an induction of apoptosis, an inhibition of angiogenesis and cellular adhesion. The better understanding of the role of the proteasome in the regulation of tumor cell growth has led to the development of new therapeutic approaches, notably in patients with multiple myeloma but also seems a promising novel therapeutic approach in other hematologic malignancies and solid tumors. This review provides a summary of the rational using proteasome inhibitors and an update on the available and ongoing clinical studies involved in human malignancies.
Pancreatic cancer is a common, highly lethal disease that is rising in incidence. Chemotherapy based on 5-fluorouracil (5-FU) has been shown to prolong survival in advanced pancreatic cancer. Gemcitabine improves major symptoms and survival outcomes compared with bolus 5-FU. Many novel small molecules are being widely and actively researched. These compounds are based on classical mechanisms of action as well as biological therapies targeting novel cellular survival pathways, and include fluoropyrimidines, nucleoside cytidine analogues, platinum analogues, topoisomerase-inhibitors, antimicrotubule agents, proteasome inhibitors, vitamin D analogues, arachidonic acid pathway inhibitors, histone deacytylator inhibitors, farnesyltransferase inhibitors and epidermal growth factor receptor therapies. Adjuvant chemotherapy has also demonstrated the best survival outcomes following resection compared to other adjuvant or neo-adjuvant strategies such as radiation-based treatments. These benefits are superimposed on the dramatic increase in resectability rates and reduction in post-operative mortality achieved by centralisation of treatment in high-volume speciality centres. Newer 'small-molecule' drugs as well as the latest 'large-molecule' biological agents hold considerable promise for the future. Real advances are anticipated over the next five years but are dependent on large randomised controlled trials for success.
The first three clinical studies of bortezomib tested regimens of differing dose-intensities: once weekly for 4 wk on a 6 wk cycle (least intensive), twice weekly for 2 wk of a 3 wk cycle, and twice weekly for 4 wk of a 6 wk cycle (most intensive). From these studies, the intermediate-intensity regimen has been advanced, because it is the best tolerated but still achieves a high level of proteasome activity. Patients on this regimen treated with 1.0–1.50 mg/m2bortezomib had a reduction in proteasome activity to about 40% of baseline but recovered most activity within the 72 h period between doses. Doselimiting toxicities for this regimen were peripheral sensory neuropathy (PSN) and diarrhea. Patients with preexisting damage from prior neurotoxic chemotherapy may be more likely to develop PSN, and this possibility is being investigated in ongoing trials. Diarrhea is also adequately managed with loperamide. Notably, hematologic events in the early phase I trials were uncommon—thrombocytopenia was not dose-limiting; febrile neutropenia was rare; and hepatic, renal, and cardiotoxicity have not been noted. Given this favorable side effect profile, bortezomib may be particularly effective in combination-treatment regimens. In preclinical studies, bortezomib has shown at least an additive effect with CPT-11, gemcitabine, and docetaxel, and trials are in progress to determine the optimum dosing schedules for these combinations. In these ongoing trials, no unexpected or additive toxicities have been observed yet.
Bortezomib (Velcade) is a boronic acid dipeptide, proteasome inhibitor drug that its antimyeloma activity in treatment of patients with multiple myeloma (MM) has been approved by FDA. Studies show that consumption of daily diet rich in polyphenols affect Bortezomib's bioavailability and reduce its antitumor activity. In the present study we made an attempt to investigate the interactions between Bortezomib and some common polyphenols to examine whether these polyphenols interact with the drug. For this purpose, interactions between four dietary polyphenols including: Emodin, Rhein, Epigallocatechingallate (EGCG) and Quercetin with methyl boronic acid, Bortezomib's substitute, were studied using 1H and 11B-NMR spectroscopy. The results suggest that the vicinal diols in the structure of the two polyphenols (EGCG and Quercetin) interact with the boronic acid moiety of Bortezomib and convert the active triangular boronic acid to an inactive tetrahedral boronate through direct chemical interaction, and this conversion abolishes the antimyeloma activity of Bortezomib. In conclusion, our study suggests that the consumption of dietary sources enriched in EGCG and Quecetin during treatment with Bortezomib in MM patients should be considered.
Anaemia appears to play an important role in left ventricular (LV) enlargement in chronic kidney disease patients. The objective of this study was to evaluate LV echocardiography changes during anaemia correction with recombinant human erythropoietin (rHu-Epo) in chronic haemodialysis patients (HD pts) with signs of anaemia and LV hypertrophy (LVH). The study included 20 HD pts aged 39,6 +/- 5,3 yrs, with the same condition of HD treatment, anaemia and echocardiographically LVH verified. At the beginning of the rHu-Epo treatment haemoglobin (Hb) level was < 90 g/L and the target Hb level was 110 g/L. Echocardiography was performed at the beginning (baseline) and after six months of rHu-Epo treatment. LVH was defined as LV mass index >100 g/m2 in women and >131 g/m2 in men. We observed significant reduction of LV mass index (LVMI) (mean 26,4%, p=0.008), as well as LV volumen. There was a significant negative correlation between Hb level and LVMI with predictive LVMI reduction of 2,317 g/m2 for each 1g/L rising of mean Hb level. The results of the study confirm the importance of early anaemia correction in haemodialysis patients aimed to improve LV parameters.
The majority of intracellular proteins undergo degradation through the ubiquitin-proteasome pathway. The proteasome pathway has a role in regulating cell proliferation, differentiation, survival and apoptosis. The naturally occurring proteasome inhibitor lactacystin was the first proteasome inhibitor noted to induce apoptosis in vitro. Compared with first-generation proteasome inhibitors, bortezomib (PS-341), a dipeptide boronic acid, has exhibited higher potency and specificity, and has been approved for the treatment of relapsed or refractory myeloma. However, there are some patients who do not respond to therapy or who respond briefly and then relapse. It is becoming increasingly clear that myeloma cells respond to the stress caused by proteasome inhibitors (bortezomib) via rapidly up-regulating pathways that suppress apoptosis, thus attenuating its antitumour activity. The delineation of these molecular pathways and mechanisms to circumvent them are needed to allow this important class of agents to remain vital in the armamentarium of the management of multiple myeloma and other malignancies.
Although approved for over a decade, the clinical utility of proteasome inhibitors (PIs) remains largely restricted to the treatment of patients with multiple myeloma (MM) and mantle cell lymphoma (MCL). This has fueled interest in understanding mechanisms of resistance to their antineoplastic actions, leading to the development of new and improved PIs (e.g., carfilzomib, ixazomib, marizomib) and rational combinations with other novel classes of targeted agents. With respect to the latter, histone deacetylase inhibitors (HDACIs) represent one of the most extensively studied classes of agents. PIs and HDACIs interact at multiple levels to trigger synergistic cell killing in a variety of tumor types through multiple mechanisms, including induction of oxidative stress and DNA damage, PI-mediated inhibition of the cytoprotective NF-κB pathway activated by HDACIs, and promotion of proteotoxic stress through simultaneous proteasome inhibition and disruption of aggresome formation and chaperone proteins, leading to the accumulation of misfolded proteins. Clinically, this combination may be closest to regulatory approval in MM, but represents a promising avenue of investigation in MCL, a relatively uncommon but challenging disease that has been the focus of much recent attention given Food and Drug Administration approvals in 2013 for the immunomodulatory drug lenalidomide and the first-in-class Bruton’s tyrosine kinase inhibitor, ibrutinib. In this chapter, we discuss the mechanisms of action of and interactions between PIs and HDACIs with an MCL focus and review the relevant preclinical and clinical data.
Proteasome inhibition is a new approach under investigation for cancer therapy. Protein degradation by the proteasome is a fundamental metabolic process. Inhibition of the proteasome has been shown to result in cell-cycle arrest and programmed cell death.
Bortezomib (Velcade®; formerly PS-341, Millennium Pharmaceuticals, Inc, Cambridge, Massachusetts) is the first proteasome inhibitor to enter clinical trials and is approved for the treatment of multiple myeloma in patients who have received at least two prior therapies and progressed on their last therapy. It is also being investigated for the treatment of a range of other cancers, both solid and hematologic. In preclinical studies, bortezomib causes apoptosis in cancer cells and seems to enhance the cytotoxicity of other conventional tumoricidal agents in human tumor cell lines, murine tumor models, and human xenograft models. In addition to targeting cancer cells directly, there is growing evidence that proteasome inhibition interferes with protective interactions between cancer cells and the bone marrow and may also act to prevent tumor-associated angiogenesis.
Preliminary data suggest that bortezomib inhibits proteasome activity in a dose-dependent and reversible manner, and has manageable toxicities. A phase II trial in relapsed and refractory multiple myeloma patients indicated that patients experienced substantial anticancer activity when treated with bortezomib monotherapy.
A perfect cancer treatment in a world of perfectly treatable cancers would target only the unique features of malignant cells and leave normal cells untouched. In the real world of real cancers, cancer-specific alterations of common pathways have been identified that can offer opportunities for the development of targeted drugs. A recent successful example is STI 571, which has been used to target the deregulated tyrosine-kinase bcr-abl in chronic myelogenous leukemia (CML) (1). This is recognized as a key translocation in this disease. Unfortunately, most cancers have many mutations (2), and several molecules may have to be targeted for success. Furthermore, recent data provide strong evidence that this process is not exclusively determined by mutations of the genome but is also driven by the tumor microenvironment and that, in principle, the process is reversible (3).
Biliary cancer and pancreatic cancer are considered to be difficult diseases to cure. Although complete resection provides the only means of curing these cancers, the rate of resectability is not high. Therefore, chemotherapy is often selected in patients with advanced unresectable biliary-pancreatic cancer. Many combination chemotherapy regimens have been applied in clinical trials. However, the survival time is not satisfactory. On the other hand, most chemotherapeutic agents induce anti-apoptotic transcriptional factor nuclear factor kappa b (NF-κB) activation, and agent-induced NF-κB activation is deeply involved in the onset of chemoresistance. Recently, novel approaches to potentiating chemosensitivity in cases of biliary-pancreatic cancer using NF-κB inhibitors with cytotoxic agents have been reported, most of which comprise translational research, although some clinical trials have also been conducted. Nevertheless, to date, there is no breakthrough chemotherapy regimen for these diseases. As some reports show promising data, combination chemotherapy consisting of a NF-κB inhibitor with chemotherapeutic agents seems to improve chemosensitivity and prolong the survival time of biliary-pancreatic cancer patients.
The aim of this study was to develop a drug delivery system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles for an efficient and targeted action of the proteasome inhibitor bortezomib against pancreatic cancer cells. The PLGA nanoparticles were formulated with a poloxamer, and further surface-modified with transferrin for tumor targeting. The nanoparticles were characterized as polymer carriers of bortezomib, and the cellular uptake and growth inhibitory effects were evaluated in pancreatic cells. Cellular internalization of nanoparticles was observed in normal and cancer cells, but with higher uptake by cancer cells. The sustained release of the loaded bortezomib from PLGA nanoparticles showed cytotoxic effects against pancreatic normal and cancer cells. Noteworthy differential cytotoxicity was attained by transferrin surface-modified PLGA nanoparticles since significant cell growth inhibition by delivered bortezomib was only observed in cancer cells. These findings demonstrate that the ligand transferrin enhanced the targeted delivery of bortezomib-loaded PLGA nanoparticles to pancreatic cancer cells. These in vitro results highlight the transferrin surface-modified PLGA nanoparticles as a promising system for targeted delivery of anticancer drugs.
The proteasome inhibitor bortezomib (PS-341) has displayed significant efficiency against pancreatic cancer cells. However, the underlying mechanisms are not fully understood. Here, we tested if ceramide production was involved in the bortezomib's effect.
Two transformed pancreatic cancer cell lines (PANC-1 and Mia) and the primary pancreatic cancer cells were used. Cell death was analyzed by MTT viability assay and trypan blue staining. Cell apoptosis was analyzed by Histone DNA-ELISA assay and Annexin V FACS. Western blots were used to test signal protein changes. The cellular ceramide level after bortezomib treatment was also determined.
In cultured pancreatic cancer cells, bortezomib increased cellular ceramide production to promote cell apoptosis. The ceramide de novo synthase inhibitor fumonisin B1 (F-B1) suppressed bortezomib-induced ceramide production and apoptosis, while exogenously added C6-ceramide facilitated bortezomib-induced pancreatic cancer cell death. Meanwhile, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the inhibitor of glucosylceramide synthetase as well as the sphingosine kinase 1 inhibitors (SKI-II and SKI-IV), facilitated bortezomib-induced ceramide production and subsequent cell apoptosis. Further, bortezomib-induced pro-apoptotic c-Jun N-terminal kinase (JNK) activation was also associated with ceramide production. JNK activation by bortezomib was suppressed by F-B1, but was enhanced by SKI-II and PDMP in pancreatic cancer cells. Finally, C6-ceramide, SKI-II, and PDMP dramatically enhanced bortezomib-induced cytotoxicity in primary cultured pancreatic cancer cells.
We found that bortezomib-induced apoptosis was associated with ceramide production in primary and transformed pancreatic cancer cells.
Tumors of the upper gastrointestinal tract (GI) are not common but represent a significant clinical challenge. Treatment of
locally advanced and metastatic tumors of the upper GI tract, liver and pancreas has met with limited success using standard
cytotoxic agents. The development of agents that block signal transduction pathways, cell cycle proteins, proteosomal degredation,
angiogenesis and immunologic-based approaches are being tested in this group of tumors. These studies have better defined
the toxicity profiles of these agents but have resulted in limited successful clinical responses. The pre-clinical and preliminary
clinical trial results will be reviewed and some of the problems with targeted therapy in the upper GI tract cancer population
will be discussed.
The Bcl-2 protein is a suppressor of programmed cell death that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of cell death. Expression of Bax in Saccharomyces cerevisiae as a membrane-bound fusion protein results in a lethal phenotype that is suppressible by co-expression of Bcl-2. Functional analysis of deletion mutants of human Bcl-2 in yeast demonstrated the presence of at least three conserved domains that are required to suppress Bax-mediated cytotoxicity, termed domains A (amino acids 11-33), B (amino acids 138-154), and C (amino acids 188-196). In vitro binding experiments using GST-Bcl-2 fusion proteins demonstrated that Bcl-2(delta B) and Bcl-2(delta C) deletion mutants had a markedly impaired ability to heterodimerize with Bax but retained the ability to homodimerize with wild-type Bcl-2. In contrast, Bcl-2(delta A) and an NH2-terminal deletion mutant Bcl-2(delta 1-82) retained Bax binding activity in vitro but failed to suppress Bax-mediated cytotoxicity in yeast. Sequences downstream of domain C in the region 197-218 also were shown to be required for Bax-binding in vitro and anti-death function in yeast. Analysis of Bcl-2/Bcl-2 homodimerization using both in vitro binding assays as well as a yeast two-hybrid method provided evidence in support of a head-to-tail model for Bcl-2/Bcl-2 homodimerization and revealed that sequences within the NH2-terminal A domain interact with a structure that requires the presence of both the carboxyl B and C domains in combination. In addition to further delineating structural features within Bcl-2 that are required for homo-dimerization, the findings reported here support the hypothesis that Bcl-2 promotes cell survival by binding directly to Bax but suggest that ability to bind Bax can be insufficient for anti-cell death function.
Activation of the inducible transcription factor NF-kappa B involves removal of the inhibitory subunit I kappa B-alpha from a latent cytoplasmic complex. It has been reported that I kappa B-alpha is subject to both phosphorylation and proteolysis in the process of NF-kappa B activation. In this study, we present evidence that the multicatalytic cytosolic protease (proteasome) is involved in the degradation of I kappa B-alpha. Micromolar amounts of the peptide Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), a specific inhibitor of the chymotrypsin-like activity of the proteasome, prevented activation of NF-kappa B in response to tumor necrosis factor-alpha (TNF) and okadaic acid (OA) through inhibition of I kappa B-alpha degradation. The m-calpain inhibitor Cbz-Leu-leucinal was ineffective. In the presence of PSI, a newly phosphorylated form of I kappa B-alpha accumulated in TNF- and OA-stimulated cells. However, the covalent modification of I kappa B-alpha was not sufficient for activation of NF-kappa B: no substantial NF-kappa B DNA binding activity appeared in cells because the newly phosphorylated form of I kappa B-alpha was still tightly bound to p65 NF-kappa B. Pyrrolidinedithiocarbamate, an antioxidant inhibitor of NF-kappa B activation which did not interfere with proteasome activities, prevented de novo phosphorylation of I kappa B-alpha as well as its subsequent degradation. This suggests that phosphorylation of I kappa B-alpha is equally necessary for the activation of NF-kappa B.(ABSTRACT TRUNCATED AT 250 WORDS)
Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. We found that the proteasome-specific inhibitor lactacystin (LC) induced expression of the cell cycle inhibitor p21WAF1/CIP1 in human cancer cells regardless of their p53 status. Both wild-type (wt) p53 and p21 protein levels increased by two hours in wt p53 containing cells, whereas mutant (mt) p53 levels decreased and the increase in p21 levels was delayed to 6 hr following inhibition of proteolysis by LC in mt p53 expressing cells. We found that wt but not mt p53 expressing cells increased p21 mRNA and p21-promoter reporter levels following LC exposure, suggesting transcriptional induction of p21. Inhibition of protein synthesis by cycloheximide demonstrated increased p21 protein half-life in the presence of LC in mutant p53 containing cells. p21 induction was correlated with the cytostatic effects of LC. The results suggest that p21 protein expression could be increased by transcriptional mechanisms as well as inhibition of proteolysis by LC.
Degradation by the 26S proteasome of specific proteins that have been targeted by the ubiquitin pathway is the major intracellular non-lysosomal proteolytic mechanism and is involved in a broad range of processes, such as cell cycle progression, antigen presentation and control of gene expression. Recent work, reviewed here, has shown that this pathway is often the target of cancer-related deregulation and can underlie processes, such as oncogenic transformation, tumour progression, escape from immune surveillance and drug resistance.
The bcl-2 proto-oncogene and the p53 tumor suppressor gene are important determinants of tumor cell susceptibility to apoptosis. bcl-2 and mutant p53 proteins inhibit apoptosis in vitro and can provide prognostic information in certain tumor types. We analyzed bcl-2 and p53 expression in archival pancreatic (n = 35) and ampullary (n = 6) adenocarcinomas, resected for cure, and their relationship to overall survival. Patients were treated with 5-fluorouracil and irradiation either pre- (n = 21) or postoperatively (n = 15); 5 patients received surgery alone. Using specific monoclonal antibodies, cytoplasmic bcl-2 and nuclear p53 proteins were detected in 22 of 40 (55%) and 20 of 37 (54%) tumors, respectively. No relationship was found between bcl-2 and p53 expression. Neither bcl-2 nor p53 correlated with histological response to preoperative chemoradiation. Lymph node involvement predicted poor overall survival (P = 0.02). A trend toward improved survival was seen in well-differentiated (P = 0.08) tumors and in those with increased bcl-2 expression (P = 0.06). p53 expression was not related to clinical outcome. In a multivariate analysis, nodal status was the single most important predictor of overall survival. Of note, the combined variable of bcl-2 expression and histological grade was a stronger prognostic variable than nodal status alone. Unlike nodal status, these features can potentially be evaluated in preoperative biopsy specimens.
Ubiquitin-proteasome-dependent protein processing appears to be an essential component in the control of radiation-induced apoptosis in human lymphocytes. This control is altered in chronic lymphocytic leukaemia (CLL), compared to that of normal human lymphocytes which mainly showed high apoptotic values after irradiation, but in some cases no sensitivity was observed. Interestingly, lactacystin activated the apoptotic pathway in both radio-resistant and sensitive CLL cells, at doses which had no effect in normal cells where significantly higher concentrations were required. Therefore the resistance of some CLL cells to apoptosis initiation by radiation does not correlate to observed increased sensitivity to lactacystin. The nuclear level of the transcription factor NF-kappaB or the cytoplasmic level of IkappaBalpha remained unaltered upon irradiation or lactacystin CLL cells treatment, suggesting that the activity of the other factors involved in apoptotic death control were altered through proteasomal inhibition. These results strongly suggest an essential role of the ubiquitin system in apoptotic cell death control in CLL lymphocytes. The inhibition of proteasome-ubiquitin-dependent processing could be a discriminatory apoptotic stimulus between normal versus malignant lymphocytes and therefore might potentially be of use in this specific human pathology.
The ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth. Dysregulating the degradation of such proteins should have profound effects on tumor growth and cause cells to undergo apoptosis. To test this hypothesis, we developed a novel series of proteasome inhibitors, exemplified by PS-341, which we describe here. As determined by the National Cancer Institute in vitro screen, PS-341 has substantial cytotoxicity against a broad range of human tumor cells, including prostate cancer cell lines. The PC-3 prostate cell line was, therefore, chosen to further examine the antitumor activity of PS-341. In vitro, PS-341 elicits proteasome inhibition, leading to an increase in the intracellular levels of specific proteins, including the cyclin-dependent kinase inhibitor, p21. Moreover, exposure of such cells to PS-341 caused them to accumulate in the G2-M phase of the cell cycle and subsequently undergo apoptosis, as indicated by nuclear condensation and poly(ADP-ribose) polymerase cleavage. Following weekly i.v. treatment of PS-341 to mice bearing the PC-3 tumor, a significant decrease (60%) in tumor burden was observed in vivo. Direct injection of PS-341 into the tumor also caused a substantial (70%) decrease in tumor volume with 40% of the drug-treated mice having no detectable tumors at the end of the study. Studies also revealed that i.v. administration of PS-341 resulted in a rapid and widespread distribution of PS-341, with highest levels identified in the liver and gastrointestinal tract and lowest levels in the skin and muscle. Modest levels were found in the prostate, whereas there was no apparent penetration of the central nervous system. An assay to follow the biological activity of the PS-341 was established and used to determine temporal drug activity as well as its ability to penetrate tissues. As such, PS-341 was shown to penetrate PC-3 tumors and inhibit intracellular proteasome activity 1.0 h after i.v. dosing. These data illustrate that PS-341 not only reaches its biological target but has a direct effect on its biochemical target, the proteasome. Importantly, the data show that inhibition of this target site by PS-341 results in reduced tumor growth in murine tumor models. Together, the results highlight that the proteasome is a novel biochemical target and that inhibitors such as PS-341 represent a unique class of antitumor agents. PS-341 is currently under clinical evaluation for advanced cancers.
Antitumor effects of the proapoptotic Bax gene have been evaluated in vitro and in vivo by a binary adenovirus system expressing the human Bax gene. Overexpression of the Bax gene in cultured cell lines from human lung carcinoma results in caspase activation, apoptosis induction, and cell growth suppression. Intratumoral injection of adenovirus vector expressing the Bar gene suppressed growth of human lung cancer xenografts established in nude mice. Histological examination of tumors from mice treated with the Bax gene demonstrated high levels of Bax expression and extensive apoptosis in tumors. In comparison with the treatment by an adenoviral vector expressing human p53, the Bax gene can effectively suppress tumor growth in both p53-sensitive and p53-resistant human lung carcinoma cell lines. Toxicity was not detected in liver and other systems in animals treated intralesionally with the Bax gene. Therefore, our results suggest that the Bar gene may be useful in cancer treatment.
The mechanisms controlling programmed cell death (PCD) during early B cell development are not well understood. Members of both the Bcl-2 family of apoptosis-related proteins and the nuclear factor-kappa B/Rel (NF-kappaB/Rel) family of transcription factors are expressed differentially during B cell development. To date, however, no direct interactions between these two families have been demonstrated. The FL5.12 cell line represents a model for progenitor B cell development. Such cells reproducibly undergo PCD upon IL-3 withdrawal. The signal to enter the apoptotic pathway is mediated by a shift in the ratio of Bcl-2:Bax. While bax levels remain constant, bcl-2 transcription rate, steady-state mRNA, and protein levels decrease. Analysis of the bcl-2 promoter reveals 3 kappaB sites functionally able to bind kappaB factors from FL5.12 nuclear extracts. Cotransfection studies demonstrate that NF-kappaB factors can repress bcl-2 transcription and that site-directed mutagenesis of the kappaB motifs abolishes this repression. These studies suggest that NF-kappaB mediates PCD in pro-B cells through transcriptional repression of the survival gene bcl-2, thus shifting the bcl-2:bax ratio in favor of death-promoting complexes.
As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor-kappaB (NFkappaB), releasing cytochrome c from their mitochondria, and ultimately dying. Further supporting an apoptotic mechanism, expression of the antiapoptotic Bcl-2 and Bcl-x proteins was increased following H/R. In this model, adenoviral-mediated transduction of lkappaB expression inhibited NFkappaB activation and significantly accelerated cytochrome c release and caspase-dependent neuronal death. At the same time, expression of mutated lkappaB prevented the increased expression of endogenous Bcl-2 and Bcl-x. In the presence of mutated lkappaB, singular overexpression of only Bcl-2 by adenoviral-mediated transduction significantly inhibited cytochrome c release, caspase-3-like activation, and cell death in response to H/R. These findings suggest a pathway where NFkappaB activation induces overexpression of Bcl-2 and Bcl-x, which function to prevent apoptotic cell death following H/R treatments.
As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor-κB (NFκB), releasing cytochrome c from their mitochondria, and ultimately dying. Further supporting an apoptotic mechanism, expression of the antiapoptotic Bcl-2 and Bcl-x proteins was increased following H/R. In this model, adenoviral-mediated transduction of lκB expression inhibited NFκB activation and significantly accelerated cytochrome c release and caspase-dependent neuronal death. At the same time, expression of mutated lκB prevented the increased expression of endogenous Bcl-2 and Bcl-x. In the presence of mutated lκB, singular overexpression of only Bcl-2 by adenoviral-mediated transduction significantly inhibited cytochrome c release, caspase-3-like activation, and cell death in response to H/R. These findings suggest a pathway where NFκB activation induces overexpression of Bcl-2 and Bcl-x, which function to prevent apoptotic cell death following H/R treatments.
Background: Gemcitabine is a new nucleoside analogue that produces a clinical response in 30% of patients with unresectable pancreatic carcinoma. The cytotoxic effects of many chemotherapeutic agents occur through induction of programmed cell death (apoptosis), which is controlled by the bcl-2 gene family. We determined whether induction of apoptosis by gemcitabine in pancreatic carcinoma is associated with cellular Bcl-2 content.
Methods: Four pancreatic carcinoma cell lines (MIA-PaCa-2, AsPC-1, Panc-1, and Panc-48) were screened by Western blotting for Bcl-2 protein expression. Dose-response relationships for the cytotoxic effects of gemcitabine were determined using methylthiotetrazole assays, and induction of apoptosis was confirmed by fluorescence-activated cell sorting analysis. MIA-PaCa-2 cells transfected with human bcl-2 were also analyzed for gemcitabine-induced apoptosis.
Results: Pancreatic cancer cell lines expressed varying amounts of Bcl-2, and the 50% lethal dose for gemcitabine-induced apoptosis was correlated with Bcl-2 content. Furthermore, Bcl-2 overexpression was associated with a significant increase in the 50% lethal dose for gemcitabine-induced apoptosis.
Conclusions: Cellular Bcl-2 content was directly correlated with the cytotoxicity of gemcitabine in pancreatic carcinoma. Therefore, routine immunohistochemical analyses may be useful in predicting gemcitabine efficacy, and patients who would likely not benefit could be spared gemcitabine administration. Furthermore, the effectiveness of gemcitabine and other chemotherapeutic agents may be increased by gene therapy-mediated alteration of bcl-2 gene family members.
Thesis (Ph. D.)--Harvard University, 1997. Includes bibliographical references.
Rapid degradation of specific proteins by ubiquitin/proteaseome-dependent pathways is a component of many cellular regulatory mechanisms. Recent work has shown that protein ubiquitination and deubiquitination are both mediated by large families of enzymes and that proteolysis can be modulated by alterations of the proteasome itself. The complexity of the ubiquitin system is reflected in the broad range of processes it regulates; these include key steps in cell cycle progression, processing of foreign proteins for presentation by class I MHC molecules, and the control of cell proliferation.
The bax gene promoter region contains four motifs with homology to consensus p53-binding sites. In cotransfection assays using p53-deficient tumor cell lines, wild-type but not mutant p53 expression plasmids transactivated a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase. In addition, wild-type p53 transactivated reporter gene constructs containing a heterologous minimal promoter and a 39-bp region from the bax gene promoter in which the p53-binding site consensus sequences reside. Introduction of mutations into the consensus p53-binding site sequences abolished p53 responsiveness of reporter gene plasmids. Wild-type but not mutant p53 protein bound to oligonucleotides corresponding to this region of the bax promoter, based on gel retardation assays. Taken together, the results suggest that bax is a p53 primary-response gene, presumably involved in a p53-regulated pathway for induction of apoptosis.
Mammalian cells contain two distinct proteolytic pathways that are involved in different aspects of protein breakdown. Proteins that enter the cell from the extracellular milieu (such as receptor-mediated endocytosed proteins) are degraded in lysosomes. Lysosomal degradation of intracellular proteins occurs mostly under stressed conditions. Nonlysosomal mechanisms are responsible for the highly selective turnover of intracellular proteins that occurs under basal metabolic conditions, but also for some aspects of degradation of intracellular proteins under stress. An important nonlysosomal proteolytic pathway is the ubiquitin system in which proteins are degraded by a 26s protease complex following conjugation by multiple molecules of ubiquitin. The “catalytic core” of the complex is a 20s protease complex also known as the proteasome. Three recent papers, describing three apparently independent biological processes, highlight the role of the ubiquitin-proteasome system as a major, however selective, proteolytic and regulatory pathway. Using specific inhibitors to the proteasome, Rock et al. (1994) demonstrate a role for this protease in the degradation of the major bulk of cellular proteins, but also in specific processing and subsequent presentation of major histocompatibility complex (MHC) class l-restricted antigens. A previous study by the same researchers (Michalek et al., 1993) showed that antigen processing requires the ubiquitin-activating enzyme, El, the first enzyme in the ubiquitin pathway cascade. Thus, it appears that antigen processing is both ubiquitin dependent and proteasome dependent. Palombella et al. (1994) show that maturation of ~105 NF-~6 precursor into the active ~50 subunit of the transcriptional activator also proceeds in a ubiquitin- and proteasomedependent manner. Furthermore, inhibitors to the proteasome block degradation of h&a and thus prevent tumor necrosis factor a (TNFa)-induced activation of mature NFKB and its entry into the nucleus. The two studies clearly demonstrate that the ubiquitin-proteasome system is involved not only in complete destruction of its protein substrates, but also in limited proteolysis and posttranslational processing in which biologically active peptides or fragments are generated. Treier et al. (1994) show that the unstable c-Jut% but not the stable v-Jun, is multiubiquitinated and degraded. The escape of the oncogenic v-Jun from ubiquitin-dependent degradation suggests a novel route to malignant transformation. Presented here is a review of the components, mechanisms of action, and cellular physiology of the ubiquitin-proteasome pathway.
The American Cancer Society's Department of Epidemiology and Statistics reports its 30th annual compilation of cancer incidence, survival, and mortality data for the United States and around the world.
Tumor necrosis factor α (TNF-α) signaling gives rise to a number of events, including activation of transcription factor NF-κB
and programmed cell death (apoptosis). Previous studies of TNF-α signaling have suggested that these two events occur independently.
The sensitivity and kinetics of TNF-α-induced apoptosis are shown to be enhanced in a number of cell types expressing a dominant-negative
IκBα (IκBαM). These findings suggest that a negative feedback mechanism results from TNF-α signaling in which NF-κB activation
suppresses the signals for cell death.
Studies on mice deficient in nuclear factor kappa B (NF-kappaB) subunits have shown that this transcription factor is important for lymphocyte responses to antigens and cytokine-inducible gene expression. In particular, the RelA (p65) subunit is required for induction of tumor necrosis factor-alpha (TNF-alpha)-dependent genes. Treatment of RelA-deficient (RelA-/-) mouse fibroblasts and macrophages with TNF-alpha resulted in a significant reduction in viability, whereas RelA+/+ cells were unaffected. Cytotoxicity to both cell types was mediated by TNF receptor 1. Reintroduction of RelA into RelA-/- fibroblasts resulted in enhanced survival, demonstrating that the presence of RelA is required for protection from TNF-alpha. These results have implications for the treatment of inflammatory and proliferative diseases.
The objectives of this study were to (1) assess the role of the 26S proteasome complex in regulating the expression of the inducible isoform of nitric oxide synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) in a model of chronic granulomatous colitis in vivo and (2) determine the role of the proteasome in regulating the inflammatory response observed in this model of chronic gut inflammation. The selective proteasome inhibitor MG-341 (0.3 mg/kg) was administered by gavage beginning immediately before the induction of colitis and continuing daily thereafter for the entire 14-day experimental period. We found that chronic proteasome inhibition using MG-341 significantly attenuated the peptidoglycan/polysaccharide (PG/PS)-induced up-regulation of iNOS in the colon and spleen and the consequent increase in plasma levels of nitrate and nitrite. Furthermore, we found that the proteasome inhibitor suppressed the up-regulation of the adhesion molecule VCAM-1 in the colon. We also found that MG-341 attenuated PG/PS-induced increases in macroscopic colonic inflammation, bowel wall thickness, colonic dry weight and colonic MPO activity. Treatment with MG-341 also significantly reduced PG/PS-induced increases in macroscopic spleen inflammation, spleen weight and spleen MPO activity. We conclude that the 26S proteasome complex plays an important role in regulating the PG/PS-induced up-regulation of iNOS and VCAM-1 in vivo and appears to be important in regulating colonic and splenic inflammation.
Potent and selective dipeptidyl boronic acid proteasome inhibitors are described. As compared to peptidyl aldehyde compounds, boronic acids in this series display dramatically enhanced potency. Compounds such as 15 are promising new therapeutics for treatment of cancer and inflammatory diseases.
Pancreatic adenocarcinoma is a leading cause of adult cancer mortality in the United States. Recent studies have revealed that point mutation of the K-ras oncogene is a common event in pancreatic cancer, and oncogenesis mediated by Ras may also involve activation of Rel/nuclear factor (NF)-kappa B transcription factors. Furthermore, the c-rel member of Rel/NF-kappa B transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kappa B family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kappa B transcription factors are activated in pancreatic cancer. Immunohistochemical analysis, Western blot and Northern blot analysis, electrophoretic mobility shift assays, and chloramphenicol acetyltransferase assays were performed to determine RelA activity in human pancreatic adenocarcinomas and normal tissues and nontumorigenic or tumorigenic cell lines. RelA, the p65 subunit of NF-kappa B, was constitutively activated in approximately 67% (16 of 24) of pancreatic adenocarcinomas but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines but not in nontumorigenic Syrian golden hamster cell lines. I kappa B alpha, a previously identified NF-kappa B-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant-negative mutants of I kappa B alpha, raf, and MEKK1. This is the first report demonstrating constitutive activation of RelA in nonlymphoid human cancer. These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathway involving Ras and mitogen-activated protein kinases in pancreatic tumor cells. Constitutive RelA activity may play a key role in pancreatic tumorigenesis through activation of its downstream target genes.
It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease.
The anticancer activity of the boronic acid dipeptide proteasome inhibitor PS-341 was examined in vitro and in vivo. PS-341 was a potent cytotoxic agent toward MCF-7 human breast carcinoma cells in culture, producing an IC90 of 0.05 microM on 24 h of exposure to the drug. In the EMT-6 tumor cell survival assay, PS-341 was equally cytotoxic administered p.o. or by i.p. injection up to a dose of 2 mg/kg. PS-341 was also toxic to the bone marrow colony-forming unit-granulocyte macrophage. PS-341 increased the tumor cell killing of radiation therapy, cyclophosphamide, and cisplatin in the EMT-6/Parent tumor, but was not able to overcome the in vivo resistance of the EMT-6/CTX and EMT-6/CDDP tumors. In the tumor growth delay assay, PS-341 administered p.o. had antitumor activity against the Lewis lung carcinoma, both primary and metastatic disease. In combination, regimens with 5-fluorouracil, cisplatin, Taxol and adriamycin, PS-341 seemed to produce primarily additive tumor growth delays against the s.c. tumor and was highly effective against disease metastatic to the lungs. The proteasome is an interesting new target for cancer therapy, and the proteasome inhibitor PS-341 warrants continued investigation in cancer therapy.
Ubiquitin-mediated proteolysis controls intracellular levels of various cell cycle regulatory proteins, and its inhibition has been shown to induce apoptosis in proliferating cells. In the present study, we examined induction of apoptosis in oral squamous cell carcinoma (OSCC) cells by treatment with specific proteasome inhibitors, carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal and lactacystin. In all three OSCC cell lines examined, apoptotic changes such as apoptotic body formation and DNA fragmentation were observed at various degrees after 24 h of the carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal or lactacystin treatment. HSC2 cells showed the most prominent apoptotic changes among the cell lines examined and demonstrated the highest level of accumulation of p27Kip1 protein after the treatment with proteasome inhibitor. Reduced expressions of cyclin D1 and phospho pRb were also observed after the treatment with proteasome inhibitor. Moreover, 12 h of treatment with the proteasome inhibitor inhibited cdk2/cyclin E kinase activity and increased the ratio of the cell cycle population at the G1 phase. The proteasome inhibitor led to inhibition of cell cycle progression. In addition, activation of CPP32 and reduced expression of Bcl-2 were observed. Because apoptosis induced by the proteasome inhibitor was inhibited by treatment with antisense p27Kip1 oligonucleotide, accumulation of the p27Kip1 protein might play an important role in the apoptosis induced by proteasome inhibitor. The present results suggest that inhibition of proteasome function may be used as a possible target of novel therapy for OSCC.
The ubiquitin proteasome pathway is a highly conserved intracellular pathway for the degradation of proteins. Many of the short-lived regulatory proteins which govern cell division, growth, activation, signaling and transcription are substrates that are temporally degraded by the proteasome. In recent years, new and selective inhibitors of the proteasome have been employed in cell culture systems to examine the anti-tumor potential of these agents. This review covers the chemistry of selected proteasome inhibitors, possible mechanisms of action in cell culture and the in vivo examination of proteasome inhibitors in murine and human xenograft tumor models in mice. One inhibitor, PS-341, has recently entered Phase I clinical trials in cancer patients with advanced disease to further test the potential of this approach.
A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.
A characteristic feature of apoptotic cell death is the activation of a cascade of cytoplasmic proteases that results in the cleavage of a limited number of target proteins. A central role in these proteolytic events has been assigned to members of the capase family. However, the use of low molecular weight proteasomal inhibitors has also demonstrated that protein degradation or processing by the ubiquitin-proteasome system of the cell has a decisive impact on cell survival and death as well, depending on the cell type and/or the proliferative status of the cells studied. Treatment of proliferating cells with proteasome inhibitors leads to cell death, potentially involving an internal signalling conflict between accumulating levels of the cdk inhibitor p27Kip1 and c-myc. In contrast, in terminally differentiated cells the same compounds have the opposite effect of blocking apoptosis, possibly by preventing proteasome-mediated degradation of a capase inhibitor. In this review the role of proteasome-mediated proteolysis in the dying cell is discussed and apparently conflicting results are integrated into a working hypothesis which functionally locates the proteasome upstream of capase3-like enzymes.
Intimate tales of asso-ciation and dissociation
- I M Verma
- J K Stevenson
- E M Schwartz
- D V Antwerp
- S Miyamoto
- Nfb
- Ib
Verma, I. M., Stevenson, J. K., Schwartz, E. M., Antwerp, D. V., and Miyamoto, S. Rel/NFB/IB family: Intimate tales of asso-ciation and dissociation. Genes Dev. 9: 2723, 1996.
Proteasome inhibitors: A novel class of potent and effective antitumor agents
- Adams
Structure-function analysis of bcl-2 protein
- Hanada
The nuclear factor-kappaB RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells
- Wang
The ubiquitin-proteasome proteolytic pathway
- Ciechanover
BCL-2 and p53 expression in resectable pancreatic adenocarcinomas: Association with clinical outcome
- Sinicrope