Human Mesothelioma Cell Research Articles

Organic extract of Geodia cydonium induces cell cycle block in human mesothelioma cells.

The serious side effects caused by chemotherapeutics and the development of cancer chemoresistance represent the most significant limitations in the treatment of cancer. Some alternative approaches have been developed in recent years, which are based on natural compounds, and have allowed important advances in cancer therapeutics. During the last 50 years, sponges have been considered a promising source of natural products from the marine environment, representing ~30% of all marine natural products. Among sponges, the Mediterranean species Geodia cydonium represents a potential source of these type of products with considerable biotechnological interest as pharmaceutical agents. The present study demonstrated the antiproliferative effect of an organic G. cydonium extract (GEOCYDO) against three human mesothelioma cell lines, MSTO-211H (MSTO), NCI-H2452 (NCI) and Ist-Mes2 (Mes2), which differ in their sensitivity (MSTO and NCI) and resistance (Mes2) to standard combined treatment with cisplatin and piroxicam. To this aim, the activity of the extract was evaluated by analyzing its effects on cell viability, cancer properties and cell cycle progression by means of colony formation assay, cell cycle analysis and protein expression analysis. The results revealed that in mesothelioma, this extract was able to reduce self-renewal, cell migration and it could induce cell cycle arrest in G0/G1 stage, thus blocking cell proliferation. In conclusion, to the best of our knowledge, the present results indicated for the first time that GEOCYDO can contain active compounds able to affect cell proliferation in mesothelioma, suggesting that it could be considered as a potential novel drug source for cancer treatment.

Dysregulation of microRNAs and tRNA-derived ncRNAs in mesothelial and mesothelioma cell lines after asbestiform fiber exposure

Experimental evidence demonstrated that fluoro-edenite (FE) can develop chronic respiratory diseases and elicit carcinogenic effects. Environmental exposure to FE fibers is correlated with malignant pleural mesothelioma (MPM). An early diagnosis of MPM, and a comprehensive health monitoring of the patients exposed to FE fibers are two clinical issues that may be solved by the identification of specific biomarkers. We reported the microRNA (miRNA) and transfer RNA-derived non coding RNA (tRNA-derived ncRNA) transcriptome in human normal mesothelial and malignant mesothelioma cell lines exposed or not exposed to several concentration FE fibers. Furthermore, an interactive mesothelioma-based network was derived by using NetME tool. In untreated condition, the expression of miRNAs and tRNA-derived ncRNAs in tumor cells was significantly different with respect to non-tumor samples. Moreover, interesting and significant changes were found after the exposure of both cells lines to FE fibers. The network-based pathway analysis showed several signaling and metabolic pathways potentially involved in the pathogenesis of MPM. From papers analyzed by NetME, it is clear that many miRNAs can positively or negatively influence various pathways involved in MPM. For the first time, the analysis of tRNA-derived ncRNAs molecules in the context of mesothelioma has been made by using in vitro systems. Further studies will be designed to test and validate their diagnostic potential in high-risk individuals' liquid biopsies.

H3K9me3 represses G6PD expression to suppress the pentose phosphate pathway and ROS production to promote human mesothelioma growth.

The role of glucose-6-phosphate dehydrogenase (G6PD) in human cancer is incompletely understood. In a metabolite screening, we observed that inhibition of H3K9 methylation suppressed aerobic glycolysis and enhances the PPP in human mesothelioma cells. Genome-wide screening identified G6PD as an H3K9me3 target gene whose expression is correlated with increased tumor cell apoptosis. Inhibition of aerobic glycolysis enzyme LDHA and G6PD had no significant effects on tumor cell survival. Ablation of G6PD had no significant effect on human mesothelioma and colon carcinoma xenograft growth in athymic mice. However, activation of G6PD with the G6PD-selective activator AG1 induced tumor cell death. AG1 increased tumor cell ROS production and the resultant extrinsic and intrinsic death pathway, mitochondrial processes, and unfolded protein response in tumor cells. Consistent with increased tumor cell death in vitro, AG1 suppressed human mesothelioma xenograft growth in a dose-dependent manner in vivo. Furthermore, AG1 treatment significantly increased tumor-bearing mouse survival in an intra-peritoneum xenograft athymic mouse model. Therefore, in human mesothelioma and colon carcinoma, G6PD is not essential for tumor growth. G6PD acts as a metabolic checkpoint to control metabolic flux towards the PPP to promote tumor cell apoptosis, and its expression is repressed by its promotor H3K9me3 deposition.

A Redox-Silent Analogue of Tocotrienol May Break the Homeostasis of Proteasomes in Human Malignant Mesothelioma Cells by Inhibiting STAT3 and NRF1.

6-O-Carboxypropyl-alpha-tocotrienol (α-T3E) is a multi-target redox-silent analogue of tocotrienol that exhibits cytotoxicity against many cancer cells, including malignant mesothelioma (MM) cells. α-T3E has several molecular targets to effectively induce cytotoxicity against MM cells; however, the mechanisms underlying this cytotoxicity remain unclear. In the present study, we demonstrated that the α-T3E-dependent disruption of the homeostasis of proteasomes strongly induced endoplasmic reticulum (ER) stress, which resulted in effective cytotoxicity against MM cells. The α-T3E-dependent disruption of the homeostasis of proteasomes depended on decreases in proteasome subunits via the inactivation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2 related factor-1 (NRF1), which inhibited protease activity, such as chymotrypsin-like activity, in proteasomes. The α-T3E-dependent inhibition of this activity also induced severe ER stress and ultimately resulted in effective cytotoxicity against MM cells with chemoresistance. The present results indicate that α-T3E acts as an effective anti-mesothelioma agent by disrupting the homeostasis of proteasomes through the simultaneous inactivation of STAT3 and NRF1.

Beta-carotene exerted anti-proliferative and apoptotic effect on malignant mesothelioma cells.

High blood levels of β-carotene and increased intake in the diets are inversely proportional to incidence of many cancer types. Antioxidant activity of β-carotene was proposed to be related with its antitumor effect. Despite this plant derivative substance being sought in many cancer types, the effectiveness of β-carotene against malignant mesothelioma remained unclear. Therefore, the present study aims to explore the impact of β-carotene on cell viability, apoptosis, and oxidative stress in mesothelioma cells. Human mesothelioma cell SPC212 were treated with β-carotene (3.125-200μM) for 24, 48, 72, and 96h. Cytotoxicity was measured with the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). Annexin-V/propidium iodide (PI) and caspase 3/7 biomarkers were used to identify apoptotic cells. Finally, the oxidative stress was evaluated with flow cytometry. The results of the measurements indicated a significant decline in viable mesothelioma cancer cell numbers upon β-carotene treatment in time- and concentration-dependent manner when compared to control cells. Furthermore, β-carotene treatment led to apoptosis induction according to both annexin V/PI and caspase 3/7 assays. Furthermore, β-carotene increased oxidative stress in SPC212 cells. These results show how β-carotene affects proliferative, apoptotic, and oxidative properties in SPC212 malignant pleural mesothelioma cells and provide useful insights into future studies.

3-Imino derivative-sulfahydantoins: Synthesis, in vitro antibacterial and cytotoxic activities and their DNA interactions

Sulfahydantoins are five-membered rings found in the structure of chemicals that exhibit antibacterial, anti-inflammatory, and anticonvulsant properties. They also activate serine protease enzymes that catalyze the hydrolysis of peptide bonds. Five 3-imino sulfahydantoin compounds were synthesized by using Strecker synthesis reaction with minor modifications. We used reflux of various aldehydes with excess sulfamide in 85% methanol in the presence of sodium cyanide. The spectroscopic properties of these compounds were studied in detail. Antibacterial activities of all synthesized new compounds against four Gram-positive (Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Streptococcus mutans) and four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella Enteritidis) bacteria were investigated by disc diffusion and microdilution method. pBR322 plasmid DNA binding abilities of compounds were investigated in vitro by agarose gel electrophoresis. In addition, the cytotoxic activities of the compounds against the human malignant pleural mesothelioma (SPC212) cell line were determined by the MTT method. The remarkable result in this study is that the synthesized compounds, especially 4b, 4d, and 4e, have significant biological activities. It has been demonstrated that these compounds, which cause DNA damage, also have an important antibacterial effect on both Gram-negative and Gram-positive bacteria when results compared with the control group antibiotics. Compound 4e exhibited the highest antibacterial potency against Streptococcus mutans (24.33 ± 0.57) from Gram-positive bacteria and Pseudomonas aeruginosa (24.66 ± 1.15) from Gram-negative bacteria. At the same time, MTT results determined that compounds 4b, 4d, and 4e showed cytotoxic activity against the SPC212 cells. In particular, compound 4b had a high cytotoxic effect, and the IC50 value was determined as 6.25 µM.

IL-27 Mediates PD-L1 Expression and Release by Human Mesothelioma Cells.

Simple SummaryMalignant mesothelioma (MM), a rare and aggressive tumor, is related to asbestos exposure, which mediates a chronic inflammatory process involving the cytokine IL-6. Recent studies indicate that the PD-1/PD-L1 immune-suppressive axis is a clinically relevant target for therapy. The expression of PD-L1 in tumor cells is generally a cytokine-mediated effect. Here we show that the IL-6-related cytokine IL-27 is able to enhance PD-L1 expression and soluble (s)PD-L1 release by cultured MM cells, whereas IL-6 is ineffective. In agreement with previous findings, we found high IL-6 levels in pleural exudates from 77 MM patients which correlated with worse survival. More importantly, we also found sPD-L1 and IL-27 in the same samples. sPD-L1 and IL-27 levels showed a moderate albeit significant correlation and association with worse survival, which suggested a potential effect of IL-27 on PD-L1-mediated immune resistance in MM.Malignant mesothelioma (MM) is a rare tumor with an unfavorable prognosis. MM genesis involves asbestos-mediated local inflammation, supported by several cytokines, including IL-6. Recent data showed that targeting PD-1/PD-L1 is an effective therapy in MM. Here, we investigated the effects of IL-6 trans-signaling and the IL-6-related cytokine IL-27 on human MM cells in vitro by Western blot analysis of STAT1/3 phosphorylation. The effects on PD-L1 expression were tested by qRT-PCR and flow-cytometry and the release of soluble (s)PD-L1 by ELISA. We also measured the concentrations of sPD-L1 and, by multiplexed immunoassay, IL-6 and IL-27 in pleural fluids obtained from 77 patients in relation to survival. IL-27 predominantly mediates STAT1 phosphorylation and increases PD-L1 gene and surface protein expression and sPD-L1 release by human MM cells in vitro. IL-6 has limited activity, whereas a sIL-6R/IL-6 chimeric protein mediates trans-signaling predominantly via STAT3 phosphorylation but has no effect on PD-L1 expression and release. IL-6, IL-27, and sPD-L1 are present in pleural fluids and show a negative correlation with overall survival, but only IL-27 shows a moderate albeit significant correlation with sPD-L1 levels. Altogether these data suggest a potential role of IL-27 in PD-L1-driven immune resistance in MM.

Abstract 1534: Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors

Abstract Mesothelin (MSLN) is a tumor-associated antigen over-expressed on the cell surface of various malignant tumor cells, including majority of mesothelioma and ovarian cancer. MSLN-directed therapy has been intensively studied in preclinical and clinical settings. However, the clinical efficacy and safety of mesothelin-targeted therapy remains to be demonstrated. To identify a safe and effective target for CAR-T cell therapy in solid tumors, we used artificial intelligent platform developed with public and private RNA transcription database to identify cancer-associated antigens caused by alternative spicing. One of interesting targets is an alternative spicing isoform from MSLN. Human MSLN transcript has at least three isoforms. The isoform 1 is the predominant transcript detected in normal and tumor tissues and has been a promising target for cancer immunotherapy. The isoform 2 is the minor transcript using alternatively spliced exons producing 8 additional amino acids insertion compared to isoform 1. The isoform 3 produces a truncated and soluble MSLN. Our data demonstrated that MSLN isoform 2 is specifically expressed in ovarian cancers but not normal tissues, confirmed by Q-PCR studies. Furthermore, we generated mouse hybridoma antibodies specifically targeting MSLN isoform 2. The antibody specificity was screened and confirmed by ELISA-based reaction to MSLN isoform 2-specific peptide and flow cytometry-based binding to 293T cells overexpressing MSLN isoform 2 but not isoform 1 proteins. We further detected the endogenous MSLN isoform 2 expression in human mesothelioma cell line NCI H226 and further confirmed MSLN isoform 2 expression on human primary mesothelioma and ovarian cancer tissue but not normal tissues by immunohistochemistry staining. To study if targeting MSLN isoform 2 with chimeric antigen receptor (CAR) can control the tumor growth, we made CAR retrovirus construct and showed that genetically modified allogenic gamma-delta T cells expressing CAR for MSLN isoform 2 can kill human mesothelioma cells in vitro and in vivo. In summary, we have demonstrated that MSLN isoform 2 is a tumor-specific antigen, which can be targeted for CAR-T cell therapy. Citation Format: Xiaohong Wang, Yibin Chen, Anupama Gopisetty, Leonardo Mirandola, Lucia Piccotti, Quynh Nguyen, Maurizio Chiriva-Internati. Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1534.

Abstract 1064: Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines

Abstract Despite the increasing knowledge of the biology of malignant pleural mesothelioma (MPM), patients with this diagnosis still have a poor prognosis with a 5-year survival rate of lower than 10%. The effectiveness demonstrated by Tumor Treating Fields (TTFields) in combination with chemotherapy in preclinical experiments and in MPM patients (NCT02397928) affirms TTFields as a promising therapeutic tool. Knowledge of the mechanism of action of TTFields and its interaction with chemotherapeutic agents may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients' thoracic cavity, before administration of therapeutic treatment, obtaining six cell lines from the 3 types of MPM cells: epithelioid CD473 and CD484, sarcomatoid CD60 and CD432, and biphasic CD487 and CD491. RNAseq analysis revealed that the transcriptional profiles of CD473 and CD60 cells were comparable with those derived from patient biopsies of the same histotype, representing clinically relevant MPM models. Antiproliferative effects induced by TTFields alone (1.12 V/cm; 150 KHz; for 96h duration) were studied in these MPM cell lines. Cell survival and cell cycle perturbations analyses during and after treatment were performed. Consistent with clinical observations, epithelioid MPM cells demonstrated greater sensitivity to TTFields than biphasic and sarcomatoid MPM cells. Cell cycle analysis revealed that the former cell type was blocked in G2M phase and was followed by formation of polyploid cells; while the latter cell types were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptotic cells were present in all treated samples, but while epithelioid and biphasic cell death was already observed during the first 24h of treatment, sarcomatoid cells needed to be treated for at least 24h before starting apoptotic pathways. The antiproliferative effect observed in epithelioid cells was persistent even after treatment cessation, whereas biphasic and sarcomatoid cells were able to reinitiate growth upon TTFields cessation. RNAseq experiments were performed in CD473 and CD60 cells at different times during TTFields application to explore transcriptional modifications that may explicate these varied MPM cell responses. Preliminary data suggested that TTFields induced a transcriptional response already detectable at earlier time points of treatment (8h; maximum effect at 24h). The number of differentially expressed genes was higher in CD473 relative to CD60 cells (1951 in CD473 and 336 in CD60 at 24h), involving several pathways, that need to be further investigated. These data demonstrate that TTFields induce specific effects on cell proliferation in varied subsets of mesothelioma cells and provide a mechanistic rationale for future therapies and combinations with other anticancer drugs for MPM treatment. Citation Format: Monica Lupi, Federica Mirimao, Nicolò Panini, Greta Piazza, Lara Paracchini, Laura Mannarino, Sergio Marchini, Federica Grosso, Serena Penpa, Antonio Maconi, Giovanni L. Ceresoli, Maurizio D'Incalci. Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1064.

Functional inhibition of heat shock protein 70 by VER-155008 suppresses pleural mesothelioma cell proliferation via an autophagy mechanism.

Background: Pleural mesothelioma, a devastating asbestos‐associated malignancy, urgently requires a novel effective therapy. Heat shock protein 70 (HSP70), which is synthesized in the cell response to protein damage, is expected to be a new target for antitumor treatment. In addition to its well‐known protein refolding function, HSP70 regulates cell proliferation through different pathways, including PI3K/AKT/mTOR, and autophagy in malignant cells. In this study, we attempted to clarify the effects of VER‐155008, an HSP70 inhibitor, on pleural mesothelioma. Methods: Human pleural mesothelioma cell lines 211H, H2452 and H28 were cultured with VER‐155008, and protein expression, cell proliferation, colony formation, cell cycle, synergistic effect with cisplatin, and autophagy induction were analyzed. Results: In mesothelioma cell lines, VER‐155008 (5.0 μM or more) inhibited cell growth and colony formation, accompanied by G1 cell cycle arrest. According to western blot analysis, VER‐155008 reduced p‐AKT expression. However, VER‐155008 failed to show a synergistic effect with cisplatin on cell growth. Mesothelioma cells transfected with the novel plasmid pMRX‐IP‐GFP‐LC3‐RFP‐LC3ΔG, which was developed for the quantitative and statistical estimation of macroautophagy, showed enhanced macroautophagy upon treatment with VER‐155008 and gefitinib which is an EGFR‐tyrosine kinase inhibitor. In addition, fetal bovine serum deprivation induced macroautophagy was further enhanced by VER‐155008. Conclusions: On the basis of these results, functional HSP70 inhibition by VER‐155008 suppressed cell growth in pleural mesothelioma cells, accompanied by enhanced macroautophagy. HSP70 inhibition is thus expected to become a new strategy for treating mesothelioma.Key points Significant findings of the study In pleural mesothelioma cells, inhibition of HSP70 function by VER‐155008 suppressed cell proliferation accompanied by induction of autophagy which was synergistically enhanced under the starvation condition, whereas gefitinib, an EGFR‐TKI, did not show the same synergistic effect in autophagy. What this study adds The inhibition of HSP70 induced autophagy and suppressed cell proliferation in mesothelioma cells.

Polyphenol Extract from Evening Primrose (Oenothera paradoxa) Inhibits Invasion Properties of Human Malignant Pleural Mesothelioma Cells.

Extracts from the defatted evening primrose (Oenothera paradoxa Hudziok) seeds are the source of a range of stable polyphenolic compounds, including ellagic acid, gallic acid, and catechin. Our studies evaluate, for the first time, the influence of evening primrose isopropanol extract (EPE) on malignant pleural mesothelioma (MPM) cells. MPM is rarely diagnosed, its high aggressiveness and frequently noted chemoresistance limit its treatment schemes and it is characterized by low prognostic features. Here, we demonstrate that EPE inhibited MPM growth in a dose-dependent manner in cells with increased invasion properties. Moreover, EPE treatment resulted in cell cycle arrest in the G2/M phase and increased apoptosis in invasive MPM cell lines. Additionally, EPE strongly limited invasion and MMP-7 secretion in MPM cancer cells. Our original data provide evidence about the potential anti-invasive effects of EPE in MPM therapy treatment.

Laser ablation inductively coupled plasma mass spectrometry as a novel clinical imaging tool to detect asbestos fibres in malignant mesothelioma.

Malignant pleural mesothelioma is an extremely aggressive and incurable malignancy associated with prior exposure to asbestos fibres. Difficulties remain in relation to early diagnosis, notably due to impeded identification of asbestos in lung tissue. This study describes a novel laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging approach to identify asbestos within mesothelioma models with clinical significance. Human mesothelioma cells were exposed to different types of asbestos fibres and prepared on plastic slides for LA-ICP-MS analysis. No further sample preparation was required prior to analysis, which was performed using an NWR Image 266 nm laser ablation system coupled to an Element XR sector-field ICP mass spectrometer, with a lateral resolution of 2 μm. Data was processed using LA-ICP-MS ImageTool v1.7 with the final graphic production made using DPlot software. Four different mineral fibres were successfully identified within the mesothelioma samples based on some of the most abundant elements that make up these fibres (Si, Mg and Fe). Using LA-ICP-MS as an imaging tool provided information on the spatial distribution of the fibres at cellular level, which is essential in asbestos detection within tissue samples. Based on the metal counts generated by the different types of asbestos, different fibres can be identified based on shape, size, and elemental composition. Detection of Ca was attempted but requires further optimisation. Detection of asbestos fibres in lung tissues is very useful, if not necessary, to complete the pathological dt9iagnosis of asbestos-related malignancies in the medicolegal field. For the first time, this study demonstrates the successful application of LA-ICP-MS imaging to identify asbestos fibres and other mineral fibres within mesothelioma samples. Ultimately, high-resolution, fast-speed LA-ICP-MS analysis has the potential to be integrated into clinical workflow to aid earlier detection and stratification of mesothelioma patient samples.

Mitochondrial ROS Induce Partial Dedifferentiation of Human Mesothelioma via Upregulation of NANOG.

The expression of pluripotency factors is a key regulator of tumor differentiation status and cancer stem cells. The purpose of this study was to examine the expression of pluripotency factors and differentiation status of human mesothelioma and the role of mitochondria in their regulation. We tested the expression of OCT4/POU5F1, NANOG, SOX2, PI3K-AKT pathway and BCL2 genes and proteins in 65 samples of human mesothelioma and 19 samples of normal mesothelium. Mitochondrial membrane potential, reactive oxygen species (ROS) generation and expression of pluripotency factors were also tested in human mesothelioma cell line. Human mesothelium and mesothelioma expressed SOX2, NANOG, PI3K and AKT genes and proteins and POU5F1 gene, whereby NANOG, SOX2 and phosphorylated (activated) AKT were upregulated in mesothelioma. NANOG protein expression was elevated in less differentiated samples of human mesothelioma. The expression of genes of PI3K-AKT pathway correlated with pluripotency factor genes. Mesothelioma cells had functional, but depolarized mitochondria with large capacity to generate ROS. Mitochondrial ROS upregulated NANOG and mitoTEMPO abrogated it. In conclusion, human mesothelioma displays enhanced expression of NANOG, SOX2 and phosphorylated AKT proteins, while elevated NANOG expression correlates with poor differentiation of human mesothelioma. Mitochondria of mesothelioma cells have a large capacity to form ROS and thereby upregulate NANOG, leading to dedifferentiation of mesothelioma.

Microwave-assisted ultra-fast synthesis of carbon quantum dots from linter: Fluorescence cancer imaging and human cell growth inhibition properties

The water dispersed-fluorescent carbon quantum dots (CDs) were synthesized using microwave-assisted hydrothermal process in the closed system from the waste cotton linter as a new carbon source. This method provided an ultra-fast, more effective, economical and easier synthesis for cancer-imaging applications compared to the other methods presented in literature. The morphological and optical properties of the hydrothermally produced carbon quantum dots were characterized by using FT-IR, transmission electron microscopy (TEM), SEM, EDX, UV–vis and fluorescent spectrophotometry techniques. An emission peak was observed at 420 nm when the CDs were excited at 376 nm. CDs were calculated to have an average particle diameter of 10.14 nm by TEM. In order to determine the usability of the CDs in cell imaging, two different concentrations (50 μL/mL and 100 μL/mL) of the CDs colloidal solution were applied to a human mesothelioma cell line, H2452, and human umbilical vein endothelial cells (HUVEC) for varying durations. While the fluorescent photos revealed that 2 h of exposure was sufficient for the cells to take the C-dots in and higher concentrations appeared brighter in the wavelengths studied (red channel (excitation/emission 586/646 nm), blue channel (390/446 nm) and the green channel (482/532 nm)), cell viability and proliferation assays indicated that the material was cytotoxic against the both cell lines and inhibited the cell growth in a time- and dose- dependent manner.

Demonstration of anti-tumour bystander killing with prodrug-preloaded suicide gene-engineered tumour cells: a potential improvement for cancer therapeutics

BackgroundTherapeutic approaches for cancer rely on careful consideration of finding the optimal way of delivering the pro-drug for cellular-based cancer treatment. Cell lines and cell cultures have been used in these studies to compare the in vitro and in vivo efficacy of autologous vs. allogeneic tumour cellular gene therapy. Here we have investigated and are reporting for the first time the effect of prodrug ganciclovir (GCV)-preloading (pre-treatment) in suicide gene therapy of cancer.MethodsThis study examines the effect of GCV-preloading (pre-treatment) on a range of tumour cell lines in conjunction with suicide gene therapy of cancer. To determine the efficacy of this modality, a series of in vitro and in vivo experiments were conducted using genetically modified and unmodified tumour cell lines.ResultsFollowing co-culture of herpes simplex virus thymidine kinase (HSV-TK) modified tumour cells and unmodified tumour cells both in vitro and in vivo, GCV-preloading (pre-treatment) of TK-modified human and mouse mesothelioma cells and ovarian tumour cells allowed them to mediate efficiently bystander killing of neighbouring unmodified tumour cells in vitro. In contrast, GCV-preloading of TK-modified human and mouse mesothelioma cells and ovarian tumour cells abolished their in vivo ability to induce bystander killing of unmodified tumour cells, although there was some tumour regression compared to control groups but this was not statistically significant. These results suggest that preloading TK modified tumour cells with GCV needs further study to define the most effective strategy for an in vivo application to retain their bystander killing potential after exposure to lethal doses of GCV in vitro.ConclusionsThis study highlights the promising possibility of improving the efficacy of pro-drug system to prevent any damage to the immune system and enhancing this type of suicide gene therapy of cancer, as well as the need for further studies to explore the discrepancies between in vitro and in vivo results.

Paclitaxel-loaded expansile nanoparticles improve survival following cytoreductive surgery in pleural mesothelioma xenografts

ObjectiveMalignant pleural mesothelioma is a lethal malignancy with poor survival and high local recurrence rates despite multimodal therapy with cytoreduction and chemoradiation. We evaluated the antitumor efficacy of a paclitaxel-loaded pH-responsive expansile nanoparticle (PTX-eNP) in 2 clinically relevant murine xenograft models of malignant pleural mesothelioma. MethodsLuciferase-transfected MSTO-211H human mesothelioma cells were injected into the thoracic cavity of immunodeficient Nu/J mice. Tumor burden was monitored by bioluminescent imaging. Animals were randomized into 2 models of disease treatment chemotherapy with PTX-eNPs alone delivered locally for early limited disease or cytoreductive surgery plus local PTX-eNP chemotherapy for advanced disease. Within each disease model, anti-tumor efficacy of PTX-eNP was compared against standard formulation paclitaxel and drug-empty nanoparticles. Influence on survival was calculated. Fluorescently labeled PTX-eNPs and immunohistochemistry evaluated in vivo drug localization to tumor. ResultsIntrathoracic injection of MSTO-211H resulted in large tumor deposits distributed within the pleural space of the murine thoracic cavity. Local multidose treatment with PTX-eNPs alone in limited stage disease more than doubled survival compared with drug-empty nanoparticles (P ≤ .0001) and standard formulation paclitaxel (P = .0004). In the model of advanced disease, local multidose treatment with PTX-eNPs following cytoreductive surgery also prolonged survival by 126% and 69.4% compared with drug-empty nanoparticles (P = .0018) and standard formulation paclitaxel (P = .03457), respectively. Immunohistology demonstrated PTX-eNP accumulation within tumor cells in vitro and in vivo. ConclusionsLocal delivery of paclitaxel via eNPs confers prolonged survival in a murine model of malignant pleural mesothelioma as single modality treatment for limited disease and in combination with cytoreductive surgery for advanced disease.

Cisplatin unleashes Toll-like receptor 3-mediated apoptosis through the downregulation of c-FLIP in malignant mesothelioma.

Toll-like receptor 3 (TLR3) is an immune receptor that behaves like a death receptor in tumor cells, thereby providing an original target for cancer therapy. The therapeutic potential of TLR3 targeting in malignant mesothelioma, an aggressive and incurable neoplasia of the pleura and peritoneum, has so far not been addressed. We investigated TLR3 expression and sensitivity of human mesothelioma cell lines to the synthetic dsRNA Poly(I:C), alone or in combination with cisplatin, the gold standard chemotherapy in mesothelioma. Activation of TLR3 by Poly(I:C) induced apoptosis of 4/8 TLR3-positive cell lines but not of TLR3-negative cell lines. The combined cisplatin/Poly(I:C) treatment enhanced apoptosis of 3/4 Poly(I:C)-sensitive cell lines and overcame resistance to Poly(I:C) or cisplatin alone in 2/4cell lines. Efficacy of the combined treatment relied on cisplatin-induced downregulation of c-FLIP, the main regulator of the extrinsic apoptotic pathway, leading to an enhanced caspase-8-mediated pathway. Of note, 6/6 primary cell samples isolated from patients with peritoneal mesothelioma expressed TLR3. Patient-derived cells were sensitive to Poly(I:C) alone while the combined cisplatin/Poly(I:C) treatment induced dramatic cell death. Our findings demonstrate that TLR3 targeting in combination with cisplatin presents an innovative therapeutic strategy in mesothelioma.

Baicalin Regulates Proliferation, Apoptosis, Migration, and Invasion in Mesothelioma.

BackgroundBaicalin, one of the main bioactive components extracted from the traditional Chinese medicine baical Skullcap root, has an anti-tumor activity which had been studied in several cancers. However, its role in human mesothelioma remains unknown. In this study, we investigated the anti-tumor mechanisms of baicalin in the mesothelioma cell line MESO924.Material/MethodsEffects of baicalin on mesothelioma were assessed by measuring cell viability, apoptosis, migration, invasion, inactivation of signaling intermediates, and cell-cycle alterations.ResultsBaicalin inhibited the proliferation, migration, and invasion of human mesothelioma cells and increased their apoptosis, all in a dose-dependent manner. Specifically, baicalin decreased the expression of p-EGFR, p-AKT, p-MAPK, p-S6, Bcl-2, and VEGF and increased the expression of Bax in mesothelioma cells. The suppressed mesothelioma cellular proliferation is due to the arrest of the S cell cycle by baicalin. Inhibition of the PI3K/AKT/mTOR signaling pathway by a PI3K/AKT/mTOR inhibitor augmented the anti-proliferation effects induced by baicalin. In addition, baicalin increased the sensitivity of MESO924 to the chemotherapeutic drugs doxorubicin, cisplatin, and pemetrexed.ConclusionsThese results highlight the roles of baicalin in inhibiting cell growth, migration, and invasion of mesothelioma cells while increasing apoptosis and sensitizing cells to chemotherapeutic agents through the PI3K/AKT/mTOR signaling pathway, which indicates that baicalin could be a useful drug for mesothelioma therapy.

Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin.

Malignant mesothelioma (MM) is an aggressive asbestos-linked neoplasm, characterized by dysregulation of signaling pathways. Due to intrinsic or acquired chemoresistance, MM treatment options remain limited. Calretinin is a Ca2+-binding protein expressed during MM tumorigenesis that activates the FAK signaling pathway, promoting invasion and epithelial-to-mesenchymal transition. Constitutive calretinin downregulation decreases MM cells’ growth and survival, and impairs tumor formation in vivo. In order to evaluate early molecular events occurring during calretinin downregulation, we generated a tightly controlled IPTG-inducible expression system to modulate calretinin levels in vitro. Calretinin downregulation significantly reduced viability and proliferation of MM cells, attenuated FAK signaling and reduced the invasive phenotype of surviving cells. Importantly, surviving cells showed a higher resistance to cisplatin due to increased Wnt signaling. This resistance was abrogated by the Wnt signaling pathway inhibitor 3289-8625. In various MM cell lines and regardless of calretinin expression levels, blocking of FAK signaling activated the Wnt signaling pathway and vice versa. Thus, blocking both pathways had the strongest impact on MM cell proliferation and survival. Chemoresistance mechanisms in MM cells have resulted in a failure of single-agent therapies. Targeting of multiple components of key signaling pathways, including Wnt signaling, might be the future method-of-choice to treat MM.

MA12.03 PARP Inhibitor Sensitivity Does Not Depend on BAP1 but Is Enhanced by Temozolomide in MGMT Deficient Human Mesothelioma Cells

BRCA1 associated protein 1 (BAP1), a nuclear deubiquitinase involved in DNA double-strand (DSB) break repair by homologous recombination (HR), is frequently mutated in mesotheliomas. Because poly (ADP-ribose) polymerase inhibitors (PARPIs) target PARP1 and PARP2 and induce synthetic lethality in BRCA1/2 mutant cancers deficient in HR, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesotheliomas. Ten patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression and function. Cellular sensitivity to two clinical PARPIs, olaparib and talazoparib were tested as single agents, and in combination with temozolomide. BAP1-deleted mesothelioma cellular models were generated by CRISPR/Cas9 and assessed for sensitivity to PARPIs. Because Schlafen 11 (SLFN11) and O6-methylguanine methyltransferase (MGMT) also drive response to temozolomide and PARPIs, we tested their expression and relationship with drug response. BAP1 inactivating mutations were present in eight of ten cell lines, with two harboring homozygous deletion. Cell lines exhibiting BAP1 expression also showed deubiquitinase activity (DUB). IC50 of olaparib and talazoparib plot classified them into sensitive or resistant population irrespective of BAP1 status (Figure 1). Although BAP1 knockout led to the loss of DUB activity, it did not increase the sensitivity of the cell ines to PARPI. Interestingly, cellular sensitivity to PARPI was increased by temozolomide in MGMT-negative and SLFN11-positive cell lines (Table 1).Table 1Summary reflecting combination study between talazoparib and temozolomide in different cell lines having varying MGMT and SLFN11 expression status.View Large Image Figure ViewerDownload Hi-res image Download (PPT) BAP1 status does not determine cellular sensitivity to PARPIs in patient-derived mesothelioma cell lines. In MGMT-deficient and SLFN11-positive cells, combination of PARPI and temozolomide is synergistic.