Project description:<h4>Background</h4>Estrogen receptor (ER) ? acts as a tumor suppressor in malignant mesotheliomas.<h4>Methods</h4>Here we explored the anti-proliferative and anti-tumorigenic efficacy of the selective ER? agonist, KB9520, in human mesothelioma cell lines in vitro and in a mesothelioma mouse model in vivo.<h4>Results</h4>KB9520 showed significant anti-proliferative effect in ER? positive human malignant pleural mesothelioma cells in vitro. Selective activation of ER? with KB9520 sensitized the cells to treatment with cisplatin, resulting in enhanced growth inhibition and increased apoptosis. Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group. Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells.<h4>Conclusions</h4>Together, the data presented suggest that selective targeting of ER? may be an efficacious stand-alone treatment option and/or become an important add-on to existing malignant mesothelioma therapy.
Project description:The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear.The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference-mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity.Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent.RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.
Project description:Inflammation is a key mediator in the development of malignant mesothelioma, which has a dismal prognosis and poor therapeutic strategies. Curcumin, a naturally occurring polyphenol in turmeric, has been shown to possess anticarcinogenic properties through its anti-inflammatory effects. Inflammasomes, a component of inflammation, control the activation of caspase-1 leading to pyroptosis and processing of proinflammatory cytokines, interleukin (IL)-1? and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using in vitro models with mouse and human malignant mesothelioma cells, curcumin is shown to induce pyroptosis through activation of caspase-1 and increased release of high-mobility group box 1 (HMGB1) without processing of IL-1? and IL-18. Absence of IL-1? processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1? priming through inhibition of the NF-?B pathway. Furthermore, curcumin's cytotoxicity in malignant mesothelioma cells is demonstrated to be dependent on pyroptosis as inhibition of caspase-1 resulted in protection against curcumin-induced cell death. We also demonstrate that curcumin-mediated caspase-1 activation is oxidant dependent by using N-acetyl-L-cysteine (NAC) to inhibit pyroptosis. PCR array analysis using the human inflammasome template revealed that curcumin significantly downregulated levels of inflammasome-related gene expression involved in inflammation, e.g., NF-?B, toll-like receptors (TLR), and IL-1?. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation.
Project description:Malignant mesothelioma is an aggressive cancer with limited therapeutic options. Sialic acid-binding lectin isolated from Rana catesbeiana oocytes (cSBL) is a multifunctional protein with anti-cancer activity. The effects of pemetrexed, cisplatin, and cSBL were evaluated in mesothelioma and normal mesothelial cell lines. We evaluated cytotoxicity, apoptosis, caspase-3 cleavage and activation, cell proliferation, cell cycle arrest, and levels of cell cycle proteins in H28 cells treated with pemetrexed, cisplatin, and cSBL alone or in combination. Treatment with cSBL alone was cytotoxic to mesothelioma cells. The anti-cancer effect of cSBL was observed in a broader range of cell lines and exhibited greater cancer cell selectivity than pemetrexed or cisplatin. Combination treatment with pemetrexed + cSBL resulted in greater dose-dependent cytotoxicity than pemetrexed + cisplatin, the standard of care in mesothelioma. The synergistic effect of pemetrexed + cSBL was mediated by the cytostatic effect of pemetrexed and the cytotoxic effect of cSBL. It thus appears that cSBL has therapeutic potential for the treatment of mesothelioma.
Project description:Malignant pleural mesothelioma (MPM) is a rare, but aggressive tumor with dismal prognosis. Platinum-based chemotherapy is regularly used as part of multimodality therapy. The expression of metallothioneins (MT) has been identified as a reason for cisplatin resistance, which often leads to early therapy failure or relapse. Thus, knockdown of MT expression may improve response to cisplatin treatment. The MT gene- and protein expression of the MPM-cell lines MSTO-211H, NCI-H2052 and NCI-H2452 and the human fibroblast cell line MRC-5, as well as their sensitivity to cisplatin treatment have been evaluated. Knockdown of MT1A, 1B and 2A expression was induced by RNA interference. MT expression was measured using quantitative real-time PCR. An in vitro Assay based on enzyme activity was used to detect cell viability, necrosis and apoptosis before and after incubation with cisplatin. MT2A gene expression could be detected in all MPM cell lines, showing the highest expression in NCI-H2452 and NCI-H2052, whereas gene expression levels of MT1A and MT1B were low or absent. The immunohistochemically protein expression of MT-I/II reflect MT2A gene expression levels. Especially for MSTO-211H cell presenting low initial MT2A levels, a strong induction of MT2A expression could be observed during cisplatin treatment, indicating a cell line-specific and platin-dependent adaption mechanism. Additionally, a MT2A-dependent cellular evasion of apoptosis during cisplatin could be observed, leading to three different MT based phenotypes. MSTO-211H cells showed lower apoptosis rates at an increased expression level of MT2A after cisplatin treatment (from sixfold to fourfold). NCI-H2052 cells showed no changes in MT2A expression, while apoptosis rate is the highest (8-12-fold). NCI-H2452 cells showed neither changes in alteration rate of MT2A expression nor changes in apoptosis rates, indicating an MT2A-independent resistance mechanism. Knockdown of MT2A expression levels resulted in significantly induced apoptotic rates during cisplatin treatment with strongest induction of apoptosis in each of the MPM cell lines, but in different markedness. A therapeutic meaningful effect of MT2A knockdown and subsequent cisplatin treatment could be observed in MSTO-211H cells. The present study showed MT2A to be part of the underlying mechanism of cisplatin resistance in MPM. Especially in MSTO-211H cells we could demonstrate major effects by knockdown of MT2A expression, verifying our hypothesis of an MT driven resistance mechanism. We could prove the inhibition of MT2A as a powerful tool to boost response rates to cisplatin-based therapy in vitro. These data carry the potential to enhance the clinical outcome and management of MPM in the future.
Project description:<h4>Background</h4>Patients with malignant mesothelioma have a poor prognosis and only 40% respond to first line treatment; a combination of pemetrexed and cisplatin or carboplatin. We used primary malignant mesothelioma cells and an ex vivo chemosensitivity assay with future purpose to predict best choice of treatment. The clinical outcome of these patients might be predicted by measuring drug sensitivity.<h4>Methods</h4>Pleural effusions containing primary malignant mesothelioma cells were received from the diagnostic routine. We characterized and tested the chemosensitivity of 18 malignant samples and four benign samples from 16 different patients with pleural effusions. Cells were seeded in a 384-well plate for a robotized ex vivo testing of drug sensitivity to 32 different drugs. The primary cells were further characterized by immunocytochemistry to evaluate the proportion of malignant cells and to study the RRM1 and ERCC1 reactivity, two proteins associated with drug resistance.<h4>Results</h4>We observed great individual variability in the drug sensitivity. Primary cell isolates were affected by between one and ten drugs, and resistant to the remaining tested drugs. Actinomycin D and daunorubicin were the two drugs effective in most cases. Adjusting efficiency of individual drugs for varying proportion of tumor cells and to the average effect on benign cells correlated with effect of pemetrexed, cisplatin and survival time. General drug sensitivity, proportion of malignant cells and reactivity to RRM1 correlated to each other and to survival time of the patients.<h4>Conclusions</h4>The proportion of malignant cells and RRM1 reactivity in the pleural effusions correlate to drug sensitivity and survival time. The variability in response to the commonly used chemotherapies emphasizes the need for tests that indicate best individual choice of cytotoxic drugs. The efficiency of the obtained results should preferably be corrected for admixture of benign cells and effects of given drugs on benign cells.
Project description:Malignant mesothelioma (MM) is an aggressive and highly chemoresistant tumour. Although cisplatin is used in frontline therapy of this disease treatment remains palliative at best. The biochemical pathways activated by cisplatin and the mechanisms of resistance in mesothelioma cells are poorly understood. Overexpression of inhibitor of apoptosis proteins (IAPs) has been described in clinical mesothelioma tumours and proposed as therapeutic targets. In this study, we examined cisplatin-induced cell death pathways and IAPs in three mesothelioma-derived cell lines. Cisplatin induced cell death in mesothelioma cell lines was characterised by biochemical mechanisms classically associated with apoptosis including: mitochondrial depolarisation, phosphatidylserine translocation and caspase activation. Surprisingly mRNA expression of IAPs in mesothelioma was not upregulated relative to primary mesothelial cells except for survivin which was higher in the most resistant cell line. In contrast, protein expression of both XIAP and survivin was upregulated in all mesothelioma cells, consistent with post-translational regulation. Knockdown of either XIAP or survivin by RNAi did not affect the sensitivity to cisplatin in any of the cell lines. Survivin RNAi did, however, inhibit proliferation in the highest expressing cell line, ONE58. The pan-caspase inhibitor z-VAD and the more selective caspase 3/7 inhibitor z-DEVD had no effect upon the sensitivity of any of the cell lines to cisplatin indicating that caspase-independent pathways predominate. The findings of the present study provide insights into cisplatin-induced mechanisms in mesothelioma cells and show that alternative pathways are operating which may provide new options for targeting this extremely resistant tumour.
Project description:Malignant mesothelioma is an aggressive and lethal asbestos-related disease. Diagnosis of malignant mesothelioma is particularly challenging and is further complicated by the lack of disease subtype-specific markers. As a result, it is especially difficult to distinguish malignant mesothelioma from benign reactive mesothelial proliferations or reactive fibrosis. Additionally, mesothelioma diagnoses can be confounded by other anatomically related tumors that can invade the pleural or peritoneal cavities, collectively resulting in delayed diagnoses and greatly affecting patient management. High-throughput analyses have uncovered key genomic and epigenomic alterations driving malignant mesothelioma. These molecular features have the potential to better our understanding of malignant mesothelioma biology as well as to improve disease diagnosis and patient prognosis. Genomic approaches have been instrumental in identifying molecular events frequently occurring in mesothelioma. As such, we review the discoveries made using high-throughput technologies, including novel insights obtained from the analysis of the non-coding transcriptome, and the clinical potential of these genetic and epigenetic findings in mesothelioma. Furthermore, we aim to highlight the potential of these technologies in the future clinical applications of the novel molecular features in malignant mesothelioma.
Project description:Malignant mesothelioma is an aggressive cancer that is resistant to current therapy. The poor prognosis of mesothelioma has been associated with elevated Yes-associated protein (YAP) activity. In this study, we evaluated the effect of targeting YAP in mesothelioma. First, we comprehensively studied YAP activity in five mesothelioma cell lines (211H, H2052, H290, MS-1 and H2452) and one normal mesothelial cell line (LP9). We found decreased phospho-YAP to YAP protein ratio and consistently increased GTIIC reporter activity in 211H, H2052 and H290 compared to LP9. The same three cell lines (IC50 s < 1 ?M) were more sensitive than LP9 (IC50 = 3.5 ?M) to the YAP/TEAD inhibitor verteporfin. We also found that verteporfin significantly reduced YAP protein level, mRNA levels of YAP downstream genes and GTIIC reporter activity in the same three cell lines, indicating inhibition of YAP signaling by verteporfin. Verteporfin also impaired invasion and tumoursphere formation ability of H2052 and H290. To validate the effect of specific targeting YAP in mesothelioma cells, we down-regulated YAP by siRNA. We found siYAP significantly decreased YAP transcriptional activity and impaired invasion and tumoursphere formation ability of H2052 and H290. Furthermore, forced overexpression of YAP rescued GTIIC reporter activity and cell viability after siYAP targeting 3'UTR of YAP. Finally, we found concurrent immunohistochemistry staining of ROCK2 and YAP (P < 0.05). Inhibition of ROCK2 decreased GTIIC reporter activity in H2052 and 211H suggesting that Rho/ROCK signaling also contributed to YAP activation in mesothelioma cells. Our results indicate that YAP may be a potential therapeutic target in mesothelioma.
Project description:Y-box binding protein-1 (YB-1) is a multifunctional oncoprotein that has been shown to regulate proliferation, invasion and metastasis in a variety of cancer types. We previously demonstrated that YB-1 is overexpressed in mesothelioma cells and its knockdown significantly reduces tumour cell proliferation, migration, and invasion. However, the mechanisms driving these effects are unclear. Here, we utilised an unbiased RNA-seq approach to characterise the changes to gene expression caused by loss of YB-1 knockdown in three mesothelioma cell lines (MSTO-211H, VMC23 and REN cells). Bioinformatic analysis showed that YB-1 knockdown regulated 150 common genes that were enriched for regulators of mitosis, integrins and extracellular matrix organisation. However, each cell line also displayed unique gene expression signatures, that were differentially enriched for cell death or cell cycle control. Interestingly, deregulation of STAT3 and p53-pathways were a key differential between each cell line. Using flow cytometry, apoptosis assays and single-cell time-lapse imaging, we confirmed that MSTO-211H, VMC23 and REN cells underwent either increased cell death, G1 arrest or aberrant mitotic division, respectively. In conclusion, this data indicates that YB-1 knockdown affects a core set of genes in mesothelioma cells. Loss of YB-1 causes a cascade of events that leads to reduced mesothelioma proliferation, dependent on the underlying functionality of the STAT3/p53-pathways and the genetic landscape of the cell.