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Rhizoma Paridis saponins suppresses vasculogenic mimicry formation and metastasis in osteosarcoma through regulating miR-520d-3p/MIG-7 axis
Abstract
Osteosarcoma (OS) is a highly metastatic bone cancer that usually affects children. Rhizoma Paridis saponins (RPS) have been identified to show a broad-spectrum anti-tumor activity. Our previous study has identified vasculogenic mimicry (VM) as an indicator of poor prognosis for OS. Rhizoma Paridis ethanol extract exhibits potent anti-OS property. However, the anti-metastatic effect of RPS on OS and the detailed mechanisms remain unknown. RPS was characterized by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) analysis. The anti-OS, anti-metastasis and anti-VM activities of RPS were investigated using in vitro biological assays and a xenograft mouse model. Western blot, qRT-PCR, ELISA, Phalloidin staining and immunohistochemistry assays were conducted to investigate the molecular mechanism of RPS. A total of 34 phytochemicals from RPS were identified by LC/Q-TOF/MS. RPS dose-dependently suppressed the OS cell proliferation, metastasis and VM formation in vitro and in vivo. Mechanically, we found that RPS downregulated migration-inducing gene 7 (MIG-7) expression, resulting in inhibition of the PI3K/MMPs/Ln-5γ2 pathway and cell protrusion formation. Additionally, we confirmed that RPS downregulated MIG-7 by upregulating miR-520d-3p expression. Our results suggests that RPS inhibits the VM formation and metastasis of OS by modulating the miR-520d-3p/MIG-7 signaling axis.
... These results suggest that RPS inhibits the development of esophageal cancer by promoting apoptosis and cell cycle arrest and inhibiting the COX-2 pathway (Yan et al., 2015). RPS exerts antitumor effects in osteosarcoma by inhibiting tumor cell proliferation, metastasis, and vasculogenic mimicry (Yao et al., 2022). In addition, in an experimental study involving a mouse model of cancer pain, it was found that RPS can increase 5-HT and β-EP in the brain by inhibiting inflammatory pain caused by oxidative damage, suggesting that RPS has a good therapeutic effect on cancer pain and provides a new tool in the treatment of cancer (Wang G. et al., 2018). ...
Cancer is a major threat to human health, with high mortality and a low cure rate, continuously challenging public health worldwide. Extensive clinical application of traditional Chinese medicine (TCM) for patients with poor outcomes of radiotherapy and chemotherapy provides a new direction in anticancer therapy. Anticancer mechanisms of the active ingredients in TCM have also been extensively studied in the medical field. As a type of TCM against cancer, Rhizoma Paridis (Chinese name: Chonglou) has important antitumor effects in clinical application. The main active ingredients of Rhizoma Paridis (e.g., total saponins, polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII) have shown strong antitumor activities in various cancers, such as breast cancer, lung cancer, colorectal cancer, hepatocellular carcinoma (HCC), and gastric cancer. Rhizoma Paridis also has low concentrations of certain other active ingredients with antitumor effects, such as saponins polyphyllin E, polyphyllin H, Paris polyphylla-22, gracillin, and formosanin-C. Many researchers have studied the anticancer mechanism of Rhizoma Paridis and its active ingredients. This review article describes research progress regarding the molecular mechanism and antitumor effects of the active ingredients in Rhizoma Paridis, suggesting that various active ingredients in Rhizoma Paridis may be potentially therapeutic against cancer.
Introduction:
Vasculogenic mimicry (VM) alludes to the ability of cancer cells to organize on three-dimensional channel-like structures to obtain nutrients and oxygen. This mechanism confers an aggressive phenotype, metastatic potential, and resistance to chemotherapy resulting in a poor prognosis. Recent studies have been focused on the identification of microRNAs (miRNAs) that regulate the VM representing potential therapeutic targets in cancer.
Areas covered:
An overview of the roles of miRNAs on VM development and their functional relationships with tumor microenvironment. The functions of cancer stem-like cells in VM, and resistance to therapy are also discussed. Moreover, the modulation of VM by natural compounds is explored. The clinical significance of deregulated miRNAs as potential therapeutic targets in tumors showing VM is further highlighted.
Expert opinion:
The miRNAs are regulators of protein-encoding genes involved in VM; however, their specific expression signatures with clinical value in large cohorts of patients have not been established yet. We considered that genomic profiling of miRNAs could be useful to define some hallmarks of tumors such as stemness, drug resistance, and VM in cancer patients. However, additional studies are needed to establish the relevant role of miRNAs as effective therapeutic targets in tumors that have developed VM.
Osteosarcoma (OS) is a primary malignant bone tumor and is prevalent in children, adolescents, and elderly individuals. It has the characteristics of high invasion and metastasis. Neoadjuvant chemotherapy combined with surgical resection is the most commonly used treatment for OS. However, the efficacy of OS is considerably diminished by chemotherapy resistance. In recent years, noncoding RNAs (ncRNAs), including microRNAs, long noncoding RNAs, and circular RNAs, are hot topics in the field of chemotherapy resistance research. Several studies have demonstrated that ncRNAs are substantially associated with chemoresistance in OS. Thus, the present study overviews the abnormally expressed ncRNAs in OS and the molecular mechanisms involved in chemoresistance, with an emphasis on their function in promoting or inhibiting chemoresistance. ncRNAs are expected to become potential therapeutic targets for overcoming drug resistance and predictive biomarkers in OS, which are of great significance for enhancing the therapeutic effect and improving the prognosis.
As the most common and aggressive malignant form of skin cancer, melanoma has a poor prognosis in its late stage. MicroRNA (miR)-520d-3p has been reported as a key modulator that regulates the development of different types of cancer, but its role in melanoma remains unclear. The purpose of this study was to investigate the role and mechanism of miR-520d-3p in melanoma. The expression of anti-silencing function 1B histone chaperone (ASF1B) and miR-520d-3p in melanoma tissues and cells was detected by reverse transcription-quantitative polymerase chain reaction. The interaction between ASF1B and miR-520d-3p was verified by luciferase activity detection. Cell counting kit-8, bromodeoxyuridine, fluorescein isothiocyanate, and cell adhesion assays were performed to detect cell viability, proliferation, apoptosis, and adhesion in melanoma cells. ASF1B expression was evidently increased, whereas miR-520d-3p level was downregulated in melanoma tissues and cells. Overexpression of ASF1B enhanced cell growth and adhesion and hampered cell apoptosis in melanoma cells. Furthermore, miR-520d-3p suppressed the tumorigenic effects of melanoma cells. Moreover, miR-520d-3p suppressed the expression of ASF1B to suppress melanoma tumorigenesis. In conclusion, we have found out that miR-520d-3p suppressed melanoma tumorigenesis by inhibiting ASF1B, which could be a promising target for melanoma therapy.
Rhizoma Paridis, the rhizome of liliaceous plants Paris polyphylla, is one of the most commonly used herbal drugs in China. Phytochemical and pharmacological studies have shown that steroid saponins were the major effective ingredients of Rhizoma Paridis to exert antitumor, anti-inflammatory, hemostasis, and antifibrosis functions. In this review, we discussed the chemical structures of steroid saponins and their related biological activity and mechanisms in cellular and animal models, aiming to provide a reference for future comprehensive exploitation and development of saponins.
Osteosarcoma (OS) is a rare form of cancer with high death rate but is one of the most frequent forms of bone cancer in children and adolescents. MiRNAs are small endogenous RNAs that regulate gene expression post-transcriptionally. The discovery of miRNAs could allow us to obtain an earlier diagnosis, predict prognosis and chemoresistance, and lead to the discovery of new treatments in different types of tumors, including OS. Despite the fact that there is currently only one clinical trial being carried out on a single miRNA for solid tumors, it is very probable that the number of clinical trials including miRNAs as prognostic and diagnostic biomarkers, as well as potential therapeutic targets, will increase in the near future. This review summarizes the different miRNAs related to OS and their possible therapeutic application.
The actin cytoskeleton plays a crucial role in many cellular processes while its reorganization is important in maintaining cell homeostasis. However, in the case of cancer cells, actin and ABPs (actin-binding proteins) are involved in all stages of carcinogenesis. Literature has reported that ABPs such as SATB1 (special AT-rich binding protein 1), WASP (Wiskott-Aldrich syndrome protein), nesprin, and villin take part in the initial step of carcinogenesis by regulating oncogene expression. Additionally, changes in actin localization promote cell proliferation by inhibiting apoptosis (SATB1). In turn, migration and invasion of cancer cells are based on the formation of actin-rich protrusions (Arp2/3 complex, filamin A, fascin, α-actinin, and cofilin). Importantly, more and more scientists suggest that microfilaments together with the associated proteins mediate tumor vascularization. Hence, the presented article aims to summarize literature reports in the context of the potential role of actin and ABPs in all steps of carcinogenesis.
Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents, but its pathogenesis has been difficult to establish because of its well-known heterogeneous nature. OS has been associated with genetic and cytogenetic abnormalities, which include function-impairing mutations in tumor suppressors and the activation of oncogenes. OS tumorigenesis has been linked to alterations of several genes characterized by a high level of genetic instability and recurrent DNA amplifications and deletions. MicroRNAs (miRNAs), 18-25-nucleotide noncoding RNAs, are critical for various biological processes like differentiation, cell growth and cell death. Dysregulation of miRNA expression leads to phenotypic and genotypic changes in cells, which leads to cancer. Studies on miRNAs have initiated a significant effect in both diagnosis and treatment of cancer. This review focuses on the current knowledge of clinical applications of miRNAs for the better diagnosis and management of OS.
Distinct from classical tumor angiogenesis, vasculogenic mimicry (VM) provides a blood supply for tumor cells independent of endothelial cells. VM has two distinct types, namely tubular type and patterned matrix type. VM is associated with high tumor grade, tumor progression, invasion, metastasis, and poor prognosis in patients with malignant tumors. Herein, we discuss the recent studies on the role of VM in tumor progression and the diverse mechanisms and signaling pathways that regulate VM in tumors. Furthermore, we also summarize the latest findings of non-coding RNAs, such as lncRNAs and miRNAs in VM formation. In addition, we review application of molecular imaging technologies in detection of VM in malignant tumors. Increasing evidence suggests that VM is significantly associated with poor overall survival in patients with malignant tumors and could be a potential therapeutic target.
Evaluating the expression levels of miR-378a-5p both in a large melanoma patient cohort from The Cancer Genome Atlas database and in melanoma patients from our Institute, we found that miR-378a-5p is upregulated in metastatic melanoma specimens. miR-378a-5p expression was also increased in melanoma cells resistant to target therapy, and decreased in response to drug treatment. We also demonstrated that overexpression of miR-378a-5p enhances in vitro cell invasion and migration, and facilitates the ability of melanoma cells to form de novo vasculogenic structures. While performing downstream targeting studies, we confirmed the ability of miR-378a-5p to modulate the expression of known target genes, such as SUFU, FUS-1, and KLF9. Luciferase-3′UTR experiments also identified STAMBP and HOXD10 as new miR-378a-5p target genes. MMP2 and uPAR, two HOXD10 target genes, were positively regulated by miR-378a-5p. Genetic and pharmacologic approaches inhibiting uPAR expression and activity evidenced that the in vitro tumor-promoting functions of miR-378a-5p, were in part mediated by uPAR. Of note miR-378a-5p was also able to increase VEGF, as well as in vitro and in vivo angiogenesis. Finally, genetic and pharmacologic modulation of Bcl-2 evidenced Bcl-2 ability to regulate miR-378a-5p expression. In conclusion, to the best of our knowledge, this is the first study demonstrating that miR-378a-5p acts as an oncogenic microRNA in melanoma.
Purpose
Polyphyllin VI, a main active saponin isolated from traditional medicinal plant Paris polyphylla, has exhibited antitumor activities in several cancer cell lines. In the present study, we investigated the antitumor effect of Polyphyllin VI against human osteosarcoma cells (U2OS) and the underlying molecular mechanisms.
Methods
The U2OS cell lines were used to determine the antiproliferative effect of Polyphyllin VI by CCK8 assay. Cell cycle was analyzed by flow cytometry. The Polyphyllin VI-induced apoptosis was determined by Annexin V-APC/7-AAD apoptosis detection kit and JC-1 staining. Meanwhile, the autophagy was determined by acridine orange staining. The apoptosis and autophagy-related proteins were monitored by Western blot assay. Subsequently, intracellular hydrogen peroxide (H2O2) and the activation of ROS/JNK pathway were detected.
Results
Polyphyllin VI could potently inhibit cell proliferation by causing G2/M phase arrest. Polyphyllin VI induced mitochondria-mediated apoptosis with the upregulation of proapoptotic proteins Bax and poly ADP-ribose polymerase, and downregulation of antiapoptotic protein Bcl-2 in U2OS cells. Concomitantly, Polyphyllin VI provoked autophagy with the upregulation of critical Atg proteins and accumulation of LC3B-II. Intracellular H2O2 production was triggered upon exposure to Polyphyllin VI, which could be blocked by ROS scavenger. Polyphyllin VI dramatically promoted JNK phosphorylation, whereas it decreased the levels of phospho-p38 and ERK.
Conclusion
Our results reveal that Polyphyllin VI may effectively induce apoptosis and autophagy to suppress cell growth via ROS/JNK activation in U2OS cells, suggesting that Polyphyllin VI is a potential drug candidate for the treatment of osteosarcomas.
Quercetin has potential pharmacological values in various carcinomas including oral squamous cell carcinoma (OSCC). Moreover, the anti-tumor effect of quercetin is correlated with WNT/β-catenin pathway and miRNA dysregulation. In the present study, we aimed to further investigate whether quercetin can exert its anti-tumor function by regulating miR-22 together with miR-22 downstream pathway WNT1/β-catenin in OSCC. The results of Cell Counting Kit-8 (CCK-8) and flow cytometry analyses showed that quercetin treatment and miR-22 overexpression resulted in the reduction of cell viability and the increase of cell apoptotic rate in OSCC. WNT1 was a target of miR-22, which was confirmed by bioinformatics, luciferase reporter and RNA immunoprecipitation (RIP) assays. RT-qPCR assay showed that quercetin promoted miR-22 expression and suppressed WNT1 and β-catenin expression in OSCC cells, whereas this effect was abrogated by miR-22 inhibitor. Moreover, miR-22 depletion weakened quercetin-mediated viability inhibition and apoptosis increase in OSCC cells. Quercetin inhibited the growth of OSCC xenograft tumors by inducing miR-22 expression and repressing WNT1/β-catenin pathway in vivo. Taken together, quercetin hampered OSCC tumorigenesis by regulating miR-22/WNT1/β-catenin pathway in OSCC, providing a deep insight into the molecular targets of quercetin in the treatment of OSCC. Keywords: Quercetin, microRNA-22, WNT1/β-catenin pathway, Oral squamous cell carcinoma
Frequent local recurrence and metastasis are generally involved in human liposarcoma, but the management is a challenge. There is an urgent need for improved effective therapy. In the present study, we reported that SBF-1, a steroidal glycoside, inhibited the growth of cultured highly malignant human liposarcoma SW872-S cells in vitro and in vivo. SBF-1 down-regulated the phosphorylation of protein kinase B (AKT) and thus reduced cell adhesion to fibronectin and laminin. Then we found that SBF-1 inhibited the expression of oxysterol binding protein (OSBP) in SW872-S cells, indicating that OSBP may be involved in malignant liposarcoma cell survival. Cancer cell growth and AKT phosphorylation were inhibited significantly upon knockdown of OSBP in SW872-S cells in vitro. Taken together, these results suggest that SBF-1 causes an apparent loss of OSBP function in SW872-S cells, resulting in growth inhibition. Based on our findings, OSBP serves as a potential therapeutic target for human liposarcoma.
Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells and is related to the metastasis and poor prognosis of many cancer types, including hepatocellular carcinoma (HCC). Thus far, no effective drugs have been developed to target VM. In this study, patients with liver cancer exhibited reduced VM in tumor tissues after treatment with Rhizoma Paridis. Polyphyllin I (PPI), which is the main component of Rhizoma Paridis, inhibited VM formation in HCC lines and transplanted hepatocellular carcinoma cells. Molecular mechanism analysis showed that PPI impaired VM formation by blocking the PI3k-Akt-Twist1-VE-cadherin pathway. PPI also displayed dual effects on Twist1 by inhibiting the transcriptional activation of the Twist1 promoter and interfering with the ability of Twist1 to bind to the promoter of VE-cadherin, resulting in VM blocking. This study is the first to report on the clinical application of the VM inhibitor. Results may contribute to the development of novel anti-VM drugs in clinical therapeutics.
The aberrant expression of cancerous inhibitor of protein phosphatase 2A (CIP2A) indicates poor prognosis and promotes EMT and metastasis. EMT, a crucial cellular process that occurs during cancer progression and metastasis, has been reported to promote drug resistance in several previous studies. Consequently, ongoing research has been focused on exploring therapeutic options for preventing EMT to delay or reverse drug resistance. Polyphyllin I (PPI) is a natural component extracted from Paris polyphylla that displays anti-cancer properties. In the present study, we investigated whether PPI can be used in the cisplatin (DDP)-resistant human gastric cancer cell line SGC7901/DDP. The results demonstrated that PPI treatment significantly inhibited cell proliferation, invasion and EMT. TGF-β1 is known to promote EMT-induced metastasis in numerous tumor types. PPI inhibited the invasion of TGFβ1-induced SGC7901/DDP cells in vitro. PPI also increased the mRNA and protein expression levels of E-cadherin but decreased the expression levels of vimentin. Further examination of the mechanism revealed that the CIP2A/PP2A/Akt pathway is partially involved in this regulation of EMT-related biomarkers and invasion. Furthermore, xenograft tests also confirmed the antitumor effects of PPI in vivo. We propose that PPI could be developed as a candidate drug for treating cancer invasion and migration.
Cholangiocarcinoma (CCA) is the cancer of bile duct with high mortality rate particularly in Thailand. The clinical efficacy of the standard chemotherapeutics remains unsatisfactory, and therefore, discovery and development of the new alternative drugs with high efficacy and tolerability is needed. The aim of the study was to investigate cytotoxic activity as well as the underlying mechanisms through which atracylodin and β-eudesmol exert their activities on CCA cell growth inhibition, cell cycle arrest, and cell apoptosis. Effects of the compounds on cell cytotoxicity, cell cycle arrest, and cell apoptosis were analyzed using MTT assay, BD CycletestTM Plus DNA kit, and FITC Annexin V Apoptosis Detection Kit I, respectively. The cytotoxic activities of both compounds were concentration- and time-dependent. The IC50 [mean (SD)] of atractylodin and β-eudesmol were 41.66 (2.51) and 39.33 (1.15) μg/ml respectively. Both promoted cell cycle arrest at G1 phase, and induced cell apoptosis through activation of caspase-3/7. The highest activity was observed at 48 hours of exposure. Results suggest that these mechanisms are at least in part, explain the cell cytotoxic and anti-CCA activity of atractylodin and β-eudesmol shown in vitro and in vivo models.
Lung cancer is one of the most common causes of death in the world. Rhizoma paridis saponins (RPS) have been found to show inhibition of pulmonary adenoma in previous research. However, the detailed mechanisms of RPS from a holistic view have not been established. In this study, Lewis pulmonary adenoma mice were successfully established to analyze the pathways involved in RPS intervening tumor formation and progression. As a result, RPS inhibited levels of cytokines or receptors such as VEGFD, VEGFR3, RAGE, IL6R, IL17BR, and CXCL16 which were regarded as the initiators induced tumor cell proliferation, adhesion, angiogenesis, and invasion. Meanwhile, RPS raised the content of SOD and CAT enzymes and thereby inhibited the aberrantly active NF-κB, and phosphorylation of PI3K/Akt and MAPK (including p38, Erk1/2, and JNK) signaling pathways. Soon after, RPS changed mRNA expression of nuclear factors containing NF-κB, HIF-1A, STAT3, and Jun, and consequentially suppressed the expression of angiogenesis, lymphangiogenesis, adhesion, inflammation, and invasion enzymes. In conclusion, this research provided a holistic view to understand the multi-target antitumor mechanisms of RPS which promoted the application of RPS in the future.
Aggressive cancer cells gain robust tumor vascular mimicry (VM) capability which promotes tumor growth and metastasis. VE-cadherin is aberrantly overexpressed in vasculogenic cancer cells and regarded as a master gene of tumor VM. Although micro RNAs (miRNAs) play an important role in modulating tumor angiogenesis and cancer metastasis, the miRNA which targets VE-cadherin expression in cancer cells to inhibit tumor cell-mediated VM is enigmatic. In this study, we found that miR-27b levels are negatively co-related to VE-cadherin expression in ovarian cancer cells and tumor cell-mediated VM, and demonstrated that miR-27b could bind to the 3'-untranslated region (3'-UTR) of VE-cadherin mRNA. Overexpression of miR-27b in aggressive ovarian cancer cell lines Hey1B and ES2 significantly diminished intracellular VE-cadherin expression; convincingly, the inhibitory effect of miR-27b could be reversed by miR-27b specific inhibitor. Intriguingly, miR-27b not only effectively suppressed ovarian cancer cell migration and invasion, but also markedly inhibited formation of ovarian cancer cells-mediated capillary-like structure in vitro and suppressed generation of functional tumor blood vessels in mice. Together, our study suggests that miR-27b functions as a new inhibitor of ovarian cancer cell-mediated VM through suppression of VE-cadherin expression, providing a new potential drug candidate for anti-tumor VM and anti-ovarian cancer therapy.
Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents, but its pathogenesis has been difficult to establish because of its well-known heterogeneous nature. OS has been associated with genetic and cytogenetic abnormalities, which include function-impairing mutations in tumor suppressors and the activation of oncogenes. OS tumorigenesis has been linked to alterations of several genes characterized by a high level of genetic instability and recurrent DNA amplifications and deletions. MicroRNAs (miRNAs), 18–25-nucleotide noncoding RNAs, are critical for various biological processes like differentiation, cell growth and cell death. Dysregulation of miRNA expression leads to phenotypic and genotypic changes in cells, which leads to cancer. Studies on miRNAs have initiated a significant effect in both diagnosis and treatment of cancer. This review focuses on the current knowledge of clinical applications of miRNAs for the better diagnosis and management of OS.
Vasculogenic mimicry (VM) is a brand-new tumour vascular paradigm independent of angiogenesis that describes the specific capacity of aggressive cancer cells to form vessel-like networks that provide adequate blood supply for tumour growth. A variety of molecule mechanisms and signal pathways participate in VM induction. Additionally, cancer stem cell and epithelial-mesenchymal transitions are also shown to be implicated in VM formation. As a unique perfusion way, VM is associated with tumour invasion, metastasis and poor cancer patient prognosis. Due to VM's important effects on tumour progression, more VM-related strategies are being utilized for anticancer treatment. Here, with regard to the above aspects, we make a review of advanced research on VM in cancer.
© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
For the past 30 years, improvements in the survival of patients with osteosarcoma have been mostly incremental. Despite evidence of genomic instability and a high frequency of chromothripsis and kataegis, osteosarcomas carry few recurrent targetable mutations, and trials of targeted agents have been generally disappointing. Bone has a highly specialized immune environment and many immune signalling pathways are important in bone homeostasis. The success of the innate immune stimulant mifamurtide in the adjuvant treatment of non-metastatic osteosarcoma suggests that newer immune-based treatments, such as immune checkpoint inhibitors, may substantially improve disease outcome.
Context:
Saponins are active compounds in natural products. Many researchers have tried to find the method for knowing their concentration in herbs. Some methods, such as solid-liquid extraction and solvent extraction, have been developed. However, the extraction methods of the steroidal saponins from Paris polyphylla Smith var. yunnanensis (Liliaceae) are not fully researched.
Objective:
To establish a simple extraction method for the separation of steroidal saponins from the rhizomes of P. polyphylla Smith var. yunnanensis.
Materials and methods:
Macroporous adsorption resins were used for the separation of steroidal saponins. To select the most suitable resins, seven kinds of macroporous resins were selected in this study. The static adsorption and desorption tests on macroporous resins were determined. Also, we optimized the temperature and the ethanol concentration in the extraction method by the contents of five kinds of saponins. Then, we compared the extraction method with two other methods.
Results:
D101 resin demonstrated the best adsorption and desorption properties for steroidal saponins. Its adsorption data fits best to the Freundlich adsorption model. The contents of steroidal saponins in the product were 4.83-fold increased with recovery yields of 85.47%.
Discussion and conclusion:
The process achieved simple and effective enrichment and separation for steroidal saponins. The method provides a scientific basis for large-scale preparation of steroidal saponins from the Rhizoma Paridis and other plants.
Antiangiogenic therapy is one of the most significant advances in anticancer treatment. The benefits of antiangiogenic therapies of late-stage cancers have been investigated but are still too limited.
We used an ovarian cancer model to test the effect of short-term bevacizumab treatment on metastasis as measured by bioluminescence. Western blotting and CD34-PAS dual staining were performed to assess hypoxia-inducible transcription factor-1α (HIF-1α) expression and vasculogenic mimicry(VM) formation. Cell viability was examined by a CCK8 assay.
Bevacizumab demonstrated antitumor effects in models of ovarian cancer, but also accelerated metastasis together, with marked hypoxia and VM formation in mice receiving short-term therapy. Bevacizumab treatment did not affect SKOV3 cell viability and the amount of VM in three-dimensional culture.
These results suggest that antiangiogenic therapy may potentially influence the progression of metastatic disease, which has been linked to the hypoxic response and VM formation.
Expression of Migration inducting gene-7 (Mig-7) is limited to tumor cells and to date not found in normal tissues. Multiple tumor microenvironment factors, such as epidermal and hepatocyte growth factors, in concert with alphavbeta5 integrin ligation, induce Mig-7 mRNA expression. Gain or loss of Mig-7 protein studies shows that Mig-7 promotes invasion of colon and endometrial carcinoma cells. These data led us to hypothesize that targeting Mig-7 through various methods could decrease invasion, enhance monocyte cell killing of tumor cells, and inhibit disease progression. To begin testing this hypothesis, an in vitro chemoinvasion assay of endometrial carcinoma cells treated with Mig-7-specific or control antibodies was used. Mig-7 antibody significantly reduced invasion by >60% compared with controls. In another approach to test this hypothesis, an in vitro analysis of peptide-stimulated human peripheral blood monocyte cells and their killing of MCF-7 breast carcinoma cells was used. Mig-7 peptide treatment increased monocyte cell tumor necrosis factor expression and killing of MCF-7 cells 30-fold over no peptide stimulation and 3-fold over MUC-1 or control peptide treatments. Furthermore, stably expressing Mig-7-specific short hairpin RNA resulted in significantly reduced Mig-7 protein levels and early primary tumor growth in a xenograft nude mouse model. Reduced phosphorylation of ERK1/2, Akt, and S6 kinase as well as decreased membrane-type 1 matrix metalloproteinase activity were mechanisms through which Mig-7 protein caused these effects. Based on these collective data, Mig-7 expression could be a potential candidate for future targeted cancer therapies.
There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.
Vasculogenic mimicry (VM) describes the unique ability of highly aggressive melanoma tumor cells to express endothelial cell-associated genes (such as EphA2 and VE-cadherin) and form vasculogenic-like networks when cultured on a three-dimensional matrix. VM has been described in several types of aggressive tumors, including melanoma, prostate, breast, and ovarian carcinomas. However, the molecular underpinnings of this phenomenon remain somewhat elusive. In this study, we examined key molecular mechanisms underlying VM in aggressive human cutaneous and uveal melanoma. The data reveal that phosphoinositide 3-kinase (PI3K) is an important regulator of VM, specifically affecting membrane type 1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) activity, critical in the formation of vasculogenic-like networks. Using specific inhibitors of PI3K, melanoma VM was abrogated coincident with decreased MMP-2 and MT1-MMP activity. Furthermore, inhibition of PI3K blocked the cleavage of laminin 5 gamma 2 chain, resulting in decreased levels of the gamma 2' and gamma 2x promigratory fragments. Collectively, these results indicate that PI3K is an important regulator of melanoma VM directly affecting the cooperative interactions of MMP-2, MT1-MMP, and laminin 5 gamma 2 chain and, thus, the remodeling of the tumor cell microenvironment. PI3K may represent an excellent target for therapeutic intervention of a novel signaling cascade underlying VM.
Even in tumor centers using established protocols, the survival rate of patients with osteosarcoma has not improved significantly in recent years. Novel therapies are urgently needed as an adjunct to conventional treatment modalities, to reduce the dose and subsequent toxicity associated with current chemotherapy, improve local disease control, prevent development of metastases, and offer an alternative treatment for those tumors that are poorly responsive to chemotherapy. Anti-angiogenic therapy currently holds great potential in conjunction with conventional treatment modalities for osteosarcoma. Specifically, anti-angiogenic factors derived from cartilage, a natural barrier to osteosarcoma invasion, may have important therapeutic applications in osteosarcoma.
Ovarian cancer is a silent killer, and shows early extensive tumor invasion and peritoneal metastasis. The microcirculation of most tumors includes cooperation of pre-existing vessels, intussusceptive microvascular growth, postnatal vasculogenesis, glomeruloid angiogenesis and vasculogenic mimicry (VM). VM is critical for a tumor blood supply and is asscociated with aggressive features and metastasis. Our studies highlight the plasticity of aggressive human ovarian carcinoma cells and call into question the underlying significance of their ability to form VM in vitro induced by VEGF-a. These studies also show their clinicalpathological features of the cancers with human Paraffin-embedded tumor tissue samples. Results show that the process: VEGF-a-->EphA2-->MMPs-->VM is the main pathway for VM formation and VEGF-a appears to play an important role in the formation of VM based on our in vitro assays and clinical immunohistochemical analyses. VM-targeting strategies for ovarian cancer include anti-VEGF-a treatment, knocking down the EphA2 gene and using antibodies against human MMPs if the tumor is VM positive. This strategy may be of significant value in laying the foundation for a more explicit anti-tumor angiogenesis therapy.
Vasculogenic mimicry (VM) refers to a novel mode of tumor microcirculation, which provides an escape route for tumor metastasis, and thereby correlates with a poor prognosis. We previously reported MIG-7 plays a pivotal role in osteosarcoma (OS) VM. However, the precise mechanism of MIG-7 in regulating OS VM remains to be elucidated. The expression levels of miR-520d-3p and MIG-7 were measured in OS cell lines. The effects of the miR-520d-3p/MIG-7 axis were investigated by in vitro functional assays. An orthotopic xenograft model was established to assess the role of the miR-520d-3p/MIG-7 axis in OS cells in vivo. Phalloidin staining, western blot, immunohistochemistry, ELISA assays were carried out to explore the molecular events that were involved in the miR-520d-3p/MIG-7 axis-mediated VM formation. The miR-520d-3p expression level was inversely correlated with MIG-7 in these cell lines. miR-520d-3p overexpression suppressed the proliferation, migration, invasion, VM, and promotes the adhesion of OS cells in vitro. miR-520d-3p could directly bind to the 3'-UTR of MIG-7 and regulated MIG-7 expression, which led to impaired lamellipodia and filopodia formation and inactivation of the PI3K/MMPs/Ln-5γ2 signaling pathway. The anti-metastatic and anti-VM effects of miR-520d-3p were confirmed in vivo. Our findings suggest miR-520d-3p acts as a tumor suppressor by inhibiting VM formation in OS via targeting MIG-7.
High-mobility group box 1 (HMGB1), a highly conserved chromosome protein, is considered as a potential therapeutic target and novel biomarker because of its regulation in the proliferation and metastasis of Hepatocellular carcinoma (HCC). Calenduloside E (CE), a natural active product, has been reported to anti-cancer effect. However, the role and underlying molecular mechanism of CE in HCC is still unclear. The purpose of this study is to investigate the effects of CE on the proliferation and migration of HCC, and then explore the possible underlying molecular mechanism. HepG2 cells were treated with CE or transfected with HMGB1 shRNA plasmids, EdU and colony formation assays were used to detect cell proliferation ability. Wound healing and transwell assays were used to determine the role of CE in cell migration. The expression of Cyclins, PCNA, MMPs, HMGB1, N-cadherin, E-cadherin and phosphorylation of p38, ERK and JNK were all detected using Western blotting. Our results showed that CE inhibited HepG2 cells proliferation and migration in a dose dependent manner; reduced the expression levels of Cycins, PCNA, HMGB1, MMPs and N-cadherin; up-regulated E-cadherin expression; enhanced the phosphorylation of p38 and JNK signalling pathways. Blocking the activation of p38 and JNK obviously reversed CE-mediated inhibitory effects on HepG2 cell proliferation and migration; reversed CE-induced down-regulation of Cyclins, PCNA, MMPs, N-cadherin and HMGB1, as well as E-cadherin up-regulation. In conclusion, our study suggested that CE reduces the expression levels of Cyclins, MMPs and epithelial-mesenchymal transformation (EMT) through p38/JNK-HMGB1 signaling axis and then inhibits HepG2 cells proliferation and migration in HepG2 cells. This study provides a new perspective for the anti-tumour molecular mechanism of CE in HCC.
This article reviews the cause, clinical presentation, diagnostic methods, and management of osteosarcoma, the most common primary bone tumor and third most common cancer among children and adolescents. In the 1970s, the introduction of adjuvant chemotherapy following tumor resection improved overall 10-year survival from 30% to about 50% of patients. However, since that change in management strategy, the survival rate has since plateaued, with no improvement in overall 10-year survival since the 1990s. A better understanding of this disease is the first step to help improve these numbers.
Vasculogenic mimicry (VM) is a special type of vascular channel formed by tumor cells without endothelial cell participation. Migration-inducing gene 7 (MIG-7) plays an important role in regulating VM. In this study, immunohistochemical staining was used to detect MIG-7 in tissue specimens from 141 primary osteosarcoma patients, and the relationship between MIG-7 and VM was examined. Survival analysis were performed to evaluate the prognoses. MIG-7 knockdown osteosarcoma cells were used for cell proliferation, apoptosis, migration, invasiveness and VM formation assays. A spontaneously metastasizing cell line-derived orthotopic xenograft mouse model was established to evaluate the effect of MIG-7 knockdown on tumorigenesis, VM formation and lung metastasis. MIG-7 expression was associated with VM formation. There were significant differences in overall and metastasis-free survival between the MIG-7-positive and MIG-7-negative groups. The MIG-7 expression was shown to be an independent indicator of both overall and metastasis-free survival. In vitro knockdown of MIG-7 dramatically reduced migration, invasion and VM formation in osteosarcoma cells without any significant effect on cell proliferation and apoptosis. MIG-7 knockdown also exhibited potent antitumor, antimetastasis and anti-VM effects in the orthotopic mouse model of 143B osteosarcoma. Therefore, MIG-7 serves as an independent unfavorable prognostic indicator in osteosarcoma patients and MIG-7 is an important mediator of osteosarcoma VM formation.
Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE. PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10-60[Formula: see text][Formula: see text]g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.
Purpose:
This study aimed to determine the expression of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and endothelial tyrosine kinase receptor (Tie-2) in central nervous system (CNS) hemangioblastomas (HBs) and explore the correlation with peritumoral cyst formation.
Method:
A total of 22 paraffin-embedded specimens derived from 11 cyst-associated and 11 solid solitary CNS HBs were collected for streptavidin-peroxidase immunohistochemical staining. The expression levels of Ang-1, Ang-2 and Tie-2 in cystic and solid HBs were estimated and compared according to the integrated optical density (IOD) of staining in the sections. To further confirm the differential expression of Ang-1 and Ang-2 between cystic and solid HBs, an additional 9 frozen specimens from 5 cyst-associated and 4 solid solitary HBs were collected for Western blot analysis. Clinical histories and radiological records of the patients were reviewed retrospectively.
Results:
Compared to normal cerebellum tissue, Ang-1, Ang-2 and Tie-2 were prominently overexpressed in both the stromal cells and endothelial cells of CNS HBs. The expression of Ang-2 and the ratio of Ang-2/Ang-1 in the cystic group were significantly higher than those in the solid group. The intensity of Ang-2 expression in the cystic group was negatively correlated with age. There was no correlation between the expression level of the proteins and sex, tumor location, tumor volume or cyst volume.
Conclusions:
Ang/Tie signaling is upregulated in HBs and is likely associated with the process of intense vascularization and cyst formation. Higher Ang-2 levels and a higher Ang-2/Ang-1 ratio may contribute to cyst formation by increasing the vascular permeability. The decrease in Ang-2 expression with advanced age in cystic-associated HBs may be associated with the reduced growth rate of peritumoral cysts among elderly patients. Ang-2 shows great potential as an effective therapeutic target of symptomatic CNS HBs associated with cysts.
The present study was the first to examine the effect of microRNA-Let-7f (miR-Let-7f) inhibiting vasculogenic mimicry (VM) of human glioma cells. The postoperative survival time was significantly poor in VM-positive glioma patients compared with those without VM. Thus, it is reasonable to postulate that miR-Let-7f functions as a potent tumor suppressor by inhibiting glioma VM. However, the molecular mechanisms involved remain poorly clarified. Our preliminary studies revealed that miR-Let-7f suppressed VM by disturbing periostin (POSTN)-induced migration of glioma cells. Our results clearly demonstrated that inhibiting the pro-migratory function of POSTN by the overexpression of miR-Let-7f significantly reduced the formation of VM. Our findings suggest that miR-Let-7f may serve as a potential complementary therapeutic target in the anti‑angiogenesis treatment of gliomas via suppressing VM.
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related disease worldwide. Although HCC is mainly associated with viral hepatitis and alcoholic cirrhosis, numerous physiological and biochemical events are associated with HCC progression. The transcription factor Twist, which plays a key role in epithelial to mesenchymal transition, is reported to be associated with HCC. Overexpression of Twist causes various biochemical changes, such as increase of cell proliferation, reduction of apoptosis, cell cycle deregulation, generation of hepatic cancer stem cells, and in some cases, drug resistance. These changes result in various physiological changes, such as angiogenesis, cellular migration and invasion, and vasculogenic mimicry, which ultimately causes hepatocellular metastasis. Interestingly, targeting Twist via different strategies, especially small RNA technology, has shown promising therapeutic potential for future HCC treatment.
The basis for resistance to VEGF inhibition is not fully understood despite its clinical importance. In this study, we examined the adaptive response to VEGF-A inhibition by a loss-of-function analysis using plasmid-based shRNA. Tumor xenografts that initially responded to VEGF-A inhibition underwent an adaptation in vivo leading to acquired resistance. VEGF-A blockade in tumors was associated with HIF-1α expression and an increase in CD144+ vasculogenic mimicry (VM), leading to formation of channels displaying Tie-1 and MMP-2 upregulation. CD133+ and CD271+ melanoma stem-like cells (MSLC) accumulated in the perivascular niche. Tumor xenografts of melanoma cell populations that were intrinsically resistant to VEGF-A blockade did not exhibit any of these features, compared to non-target control counterparts. Thus, melanomas which are initially sensitive to VEGF-A blockade acquire adaptive resistance by adopting VM as an alternate angiogenic strategy, thereby enriching for deposition of MSLC in the perivascular niche through a HIF-1α-dependent process. Conversely, melanomas which are intrinsically resistant to VEGF-A blockade do not show any evidence of compensatory survival mechanisms that promote MSLC accumulation. Our work highlights the potential risk of anti-VEGF treatments owing to a selective pressure for an adaptive resistance mechanism that empowers the development of stem-like cancer cells, with implications for how to design combination therapies that can improve outcomes in patients.
Copyright © 2015, American Association for Cancer Research.
Aberrant expression of microRNAs (miRNAs) has been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the roles of miR-520d-3p on gastric cancer (GC) cell proliferation, migration, and invasion, and confirmed that this miRNA regulates EphA2 expression. The mRNA expression levels of miR-520d-3p and EphA2 in GC tissues and cell lines were evaluated. The clinical and prognostic significance of miR-520d-3p was assessed. The biological function of miR-520d-3p in GC cells was investigated using a methylthiazolyldiphenyl-tetrazolium bromide assay, cell cycle assay, transwell invasion assay, and wound-healing assay. miR-520d-3p expression was down-regulated and inversely correlated with the expression of EphA2 in GC tissues and cell lines. Lower expression of miR-520d-3p was associated with tumor invasion (P = 0.0357), lymph nodes metastasis (P = 0.0272), a higher clinical stage (P = 0.0041), and poorer overall survival (P = 0.0105). Luciferase assays revealed that miR-520d-3p inhibited EphA2 expression by targeting the 3'-untranslated region of EphA2 mRNA. Overexpression of miR-520d-3p dramatically inhibited the proliferation, cell cycle progression, invasion, and migration of GC cells, while down-regulation substantially promoted these properties. Moreover, c-Myc, CyclinD1, and matrix metalloproteinase-9 expression levels were down-regulated in miR-520d-3p mimic-transfected cells and up-regulated in miR-520d-3p inhibitor-transfected cells. Taken together, our data showed that miR-520d-3p appears to contribute to GC progression via the regulation of EphA2 and could serve as a novel prognostic and potential therapeutic marker.
Long-term outcome for patients with high-grade osteosarcoma has improved with the addition of systemic chemotherapy, but subsequent progress has been less marked. Modern, multiagent, dose-intensive chemotherapy in conjunction with surgery achieves a 5-year event-free survival of 60-70% in extremity localized, non-metastatic disease. A major, as yet unsolved, problem is the poor prognosis for metastatic relapse or recurrence, and for patients with axial disease. This article reviews the current state of the art of systemic osteosarcoma therapy by focusing on the experiences of cooperative osteosarcoma groups. Also, we shed light on questions and challenges posed by the aggressiveness of the tumor, and we consider potential future directions that may be critical to progress in the prognosis of high-grade osteosarcoma.
The purpose of this study was to investigate the functions of microRNA-9, which is a tissue-specific microRNA in central nervous system, in the vasculogenic mimicry (VM) of glioma cell lines in vitro and in vivo.Glioma cell lines U87MG, U251 and SHG44 were transfected with microRNA-9 mimic, microRNA-9 inhibitor or scramble sequences. The amount of microRNA-9 and Stathmin (STMN1) mRNA was determined by quantitative real-time PCR, and the protein expression of STMN1 was determined by western blot. Cell proliferation and apoptosis were assessed. The interactions between the 3'UTR of STMN1 and miR-9 was determined by luciferase reporter assay. The VM capacity in vitro was evaluated using VM formation assay, and the rescue experiment of STMN1 was carried out in U251 cells. The in vivo experiment was applied with animal models implanted with U87MG cells.MicroRNA-9 mimic transfection reduced proliferation and increased apoptosis in glioma cell lines (p < 0.05). MicroRNA-9 mimic up-regulated STMN1 mRNA levels but reduced its protein levels (p < 0.05), and luciferase activity of STMN1 was suppressed by microRNA-9 mimic transfection (p < 0.05). Furthermore, microRNA-9 mimic transfection suppressed tumor volume growth, as well as VM both in vitro and in vivo. The cell viability and microtube density were upregulated in U251 cells after STMN1 up-regulation (p < 0.05). STMN1 is a target of microRNA-9, and microRNA-9 could modulate cell proliferation, VM and tumor volume growth through controlling STMN1 expression. MicroRNA-9 and its targets may represent a novel panel of molecules for the development of glioma treatment.
Our previous studies have shown that epithelial-mesenchymal transition (EMT) may be involved in the vasculogenic mimicry (VM) formation in hepatocellular carcinoma. Here, we hypothesize that zinc finger E-box binding homeobox 1 (ZEB1) promotes VM formation in colorectal carcinoma (CRC) by inducing EMT. We identified VM in 39 (19.2%) out of 203 CRC patients. The presence of VM was associated with aggressive biological behavior and was an unfavorable prognostic indicator. By immunohistochemical analysis, we found that the VM-positive CRC samples showed increased ZEB1 expression compared with the VM-negative samples and the ZEB1 expression occurred concomitantly with features of EMT. In vitro, knockdown of ZEB1 in poorly differentiated HCT116 CRC cells destroyed the vessel-like structures in the 3-D culture, a property associated with VM formation. Knockdown of ZEB1 resulted in restoration of epithelial phenotypes and significantly inhibited the ability to migrate and invade. In addition, ZEB1 underexpression decreased the expression of vascular endothelial (VE)-cadherin and Flk-1, which are characteristics of endothelial cells. Taken together, our results suggest that ZEB1 can promote VM formation by inducing EMT in CRC and might represent an important target in CRC.
Unlabelled:
The up-regulation and nuclear relocation of epithelial-mesenchymal transition (EMT) regulator Twist1 have been implicated in the tumor invasion and metastasis of human hepatocellular carcinoma (HCC). The term vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. However, the relationship between Twist1 and VM formation is not clear. In this study, we explored HCC as a VM and EMT model in order to investigate the role of Twist1 in VM formation. We first examined the expression of Twist1 in human HCC samples and cell lines and found that Twist1 was frequently overexpressed in the nuclear relocation occurring in VM-positive HCCs (13/18 [72%]). Twist1 nuclear expression was likewise significantly associated with VM formation. Clinicopathological analysis revealed that both VM and Twist1 nuclear expressions present shorter survival durations than those without expression. We consistently demonstrated that an overexpression of Twist1 significantly enhanced cell motility, invasiveness, and VM formation in an HepG2 cell. Conversely, a knockdown of Twist1 by the short hairpin RNA approach remarkably reduced Bel7402 cell migration, invasion, and VM formation. Using chromatin immunoprecipitation, we also showed that Twist1 binds to the vascular endothelial (VE)-cadherin promoter and enhances its activity in a transactivation assay.
Conclusion:
The results of this study indicate that Twist1 induces HCC cell plasticity in VM cells more through the suppression of E-cadherin expression and the induction of VE-cadherin up-regulation than through the VM pattern in vivo and in a three-dimensional in vitro system. Our findings also demonstrate a novel cogitation in cancer stem-like cell differentiation and that related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.
Tissue sections from aggressive human intraocular (uveal) and metastatic cutaneous melanomas generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. The matrix that forms these loops or networks may be solid or hollow. Red blood cells have been detected within the hollow channel components of this patterned matrix histologically, and these vascular channel networks have been detected in human tumors angiographically. Endothelial cells were not identified within these matrix-embedded channels by light microscopy, by transmission electron microscopy, or by using an immunohistochemical panel of endothelial cell markers (Factor VIII-related antigen, Ulex, CD31, CD34, and KDR[Flk-1]). Highly invasive primary and metastatic human melanoma cells formed patterned solid and hollow matrix channels (seen in tissue sections of aggressive primary and metastatic human melanomas) in three-dimensional cultures containing Matrigel or dilute Type I collagen, without endothelial cells or fibroblasts. These tumor cell-generated patterned channels conducted dye, highlighting looping patterns visualized angiographically in human tumors. Neither normal melanocytes nor poorly invasive melanoma cells generated these patterned channels in vitro under identical culture conditions, even after the addition of conditioned medium from metastatic pattern-forming melanoma cells, soluble growth factors, or regimes of hypoxia. Highly invasive and metastatic human melanoma cells, but not poorly invasive melanoma cells, contracted and remodeled floating hydrated gels, providing a biomechanical explanation for the generation of microvessels in vitro. cDNA microarray analysis of highly invasive versus poorly invasive melanoma tumor cells confirmed a genetic reversion to a pluripotent embryonic-like genotype in the highly aggressive melanoma cells. These observations strongly suggest that aggressive melanoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis.
Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.
Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.
Cell migration plays a central role in a wide variety of biological events, including embryogenesis, inflammatory immune response, wound healing, or cancer invasion. Tight regulation of cell motility is a prerequisite for normal development and maintenance of an organism, and to avoid metastatic spread of tumor cells. An important determinant of migratory efficiency is the substrate over which a cell migrates. Laminin-5 (Ln-5) is an extracellular matrix component prominent in basement membranes and as such it is a substrate in direct contact with epithelial cells. Interestingly, Ln-5 has been shown to both stimulate and downregulate epithelial cell migration. In this article, we plan to give an overview on the different mechanisms cells employ to regulate their migratory behavior on Ln-5. We will discuss how proteolytic processing of Ln-5 acts as posttranslational modification that plays a major role in the regulation of cell migration. The different proteolytic Ln-5 species may bind to distinct cell surface receptors called integrins, which translate substrate binding into a specific cellular response that triggers cell motility. Furthermore, interaction between Ln-5-binding integrins and other transmembrane and cytoplasmic proteins increases complexity and may allow fine-tuning of cell migration in response to the cellular environment.
Although there has been considerable improvement in survival among children with osteosarcoma over the past 30 years, patients with metastatic disease fare very poorly. The best-case scenario for metastatic patients is a survival rate of 30% assuming complete resection of lung metastases without other disease. To achieve this optimal outcome, an aggressive surgical approach is recommended in which all metastatic disease is resected. This includes metastatic foci that are detected by imaging as well as those only identified by palpation at thoracotomy. Investigation into the biology of the metastatic process of osteosarcoma as well as in identification of molecular features that correlate with prognosis is very active and will likely yield important findings that will impact therapy in the future. An area of investigation that remains needed is the prospective evaluation of the surgical management of these patients with the goal of critically evaluating the impact of the various surgical strategies on patient outcome measures, such as disease-recurrence and survival.
In 1999, Maniotis reported that blood vessels of highly aggressive uveal melanomas are formed by tumor cells instead of endothelial cells. He termed this novel concept in tumor vascularization vasculogenic mimicry (VM). Since then, VM has been seen in several malignant tumor types such as breast cancer, liver cancer, glioma, ovarian cancer, melanoma, prostate cancer, and bidirectional differentiated malignant tumors. Laser scanning confocal angiography, electron microscopy, and three-dimensional cell culture have confirmed the existence of VM. The molecular mechanisms that underlie VM are not fully clear, but metalloproteinases via their cleavage of laminin, VE-cadherin by promoting adherence of the VM channel wall to tumor cells, tumor cell dedifferentiation, and tumor microenvironment have been shown to play a role in VM. Zhang and co-workers have proposed a three-stage phenomenon among VM channels, mosaic blood vessels, and endothelium-dependent blood vessels, wherein all three patterns participate in tumor blood supply. Therapeutic strategies that target endothelial cells have no effect on tumor cells that engage in VM. VM-targeting strategies include suppressing tyrosine kinase activity and using a knockout EphA2 gene, downregulating VE-cadherin, using antibodies against human MMPs and the laminin 5gamma2 chain, and using anti-PI3K therapy. We review here the current status of research on VM; discuss molecular mechanisms of VM, factors affecting VM formation, and its clinical significance; and explore the development of novel tumor-targeted treatments that are based on the biochemical and molecular events that regulate VM.
Molecular requirements for carcinoma cell interactions with the microenvironment are critical for disease progression but are poorly understood. Integrin alpha v beta 5, which senses the extracellular matrix, is important for carcinoma cell dissemination in vivo. alpha v beta 5 signaling induces Mig-7, a novel human gene product that is apparently carcinoma-specific. We hypothesized that Mig-7 expression facilitates tumor cell dissemination by increasing invasion and vasculogenic mimicry. Results show that embryonic cytotrophoblasts up-regulated Mig-7 expression before they acquired an invasive phenotype capable of pseudovasculogenesis. Mig-7 protein primarily co-localized with vasculogenic mimicry markers factor VIII-associated antigen, vascular endothelial-cadherin, and laminin 5 gamma 2 chain domain III fragment in lymph node metastases. Overexpression of Mig-7 increased gamma 2 chain domain III fragments known to contain epidermal growth factor (EGF)-like repeats that can activate EGF receptor. Interestingly, EGF also induced Mig-7 expression. Carcinoma cell adhesion to laminins was significantly reduced by Mig-7 expression. Remarkably, in two-dimensional and three-dimensional Matrigel cultures, Mig-7 expression caused invasion and vessel-like structures. Melanoma cells, which were previously characterized to invade aggressively and to undergo vasculogenic mimicry, expressed Mig-7. Taken together, these data suggest that Mig-7 expression allows cells to sense their environment, to invade, and to form vessel-like structures through a novel relationship with laminin 5 gamma 2 chain domain III fragments.
Throughout history, natural products have afforded a rich source of compounds that have found many applications in the fields of medicine, pharmacy and biology. Within the sphere of cancer, a number of important new commercialised drugs have been obtained from natural sources, by structural modification of natural compounds, or by the synthesis of new compounds, designed following a natural compound as model. The search for improved cytotoxic agents continues to be an important line in the discovery of modern anticancer drugs. The huge structural diversity of natural compounds and their bioactivity potential have meant that several products isolated from plants, marine flora and microorganisms can serve as "lead" compounds for improvement of their therapeutic potential by molecular modification. Additionally, semisynthesis processes of new compounds, obtained by molecular modification of the functional groups of lead compounds, are able to generate structural analogues with greater pharmacological activity and with fewer side effects. These processes, complemented with high-throughput screening protocols, combinatorial chemistry, computational chemistry and bioinformatics are able to afford compounds that are far more efficient than those currently used in clinical practice. Combinatorial biosynthesis is also applied for the modification of natural microbial products. Likewise, advances in genomics and the advent of biotechnology have improved both the discovery and production of new natural compounds.