Pleural Mesothelioma Cell Lines Research Articles

Abstract 4711: Evaluation of effect of autophagy inhibition by SAR405, a selective Vps34 inhibitor, on proliferation of pleural mesothelioma cells

Abstract Pleural mesothelioma is a poor prognostic malignancy because the efficacy of therapies remains limited; thus, there is an urgent need to elucidate the pathophysiology leading to newer treatment modalities. Macroautophagy (hereafter referred to as autophagy) is an intracellular degradation system in which cytoplasmic components are degraded by the lysosome. Autophagy plays pivotal roles in various physiological processes, including adaptation to starvation, preimplantation embryonic development, elimination of intracellular pathogens, and regulation of innate and adaptive immunity. In cancer cells, autophagy may have the potential to function protectively for the cells and, alternatively, to work suppressively. It can promote tumor growth in established cancers through autophagy-mediated intracellular recycling, providing substrates for metabolism and maintain that the functional pool of mitochondria. On the other hand, it may lead to a cytotoxic effect called autophagy-dependent cell death. Several reports have demonstrated that autophagy has cytoprotective effects on pleural mesothelioma cell lines. In this study, we assessed effects of inhibition of autophagy using SAR405, which specifically inhibits Vps34 in an early step of autophagy, on pleural mesothelioma cells. Additionally, we investigated whether SAR405 could enhance the growth suppression induced by cisplatin in these cells. Mesothelioma cell lines, including NCI-H28 (H28), NCI-H2452 (H2452), and MSTO-211H (211H), were cultured with 0.1-50 μM SAR405 or 1.0-5.0 μM cisplatin for cell viability analysis using water-soluble tetrazolium salt-8, and cell cycle analysis. For the assessment of autophagy, a plasmid, pMRX-IP-GFP-LC3-RFP-LC3ΔG, was stably transfected into mesothelioma cells. Transfected cells generate GFP-LC3-RFP-LC3ΔG, and it is cleaved into GFP-LC3 and RFP-LC3ΔG by inherent ATG4. GFP-LC3 levels are decreased by autophagy activation, while RFP-LC3ΔG serves as an internal control. SAR405 dose-dependently suppressed cell viability, accompanied by a significantly increase in the proportions of cells in the G2/M phase. The IC50 of SAR405 at 72 hours was 11.5 μM for H28, 16.7 μM for H2452, and 14.9 μM for 211H. SAR405 inhibited autophagy 24 hours after treatment at 2.5 μM for H28 and 211H, and at 5.0 μM for H2452. Furthermore, cisplatin induced autophagy at 5.0 μM for H28, 0.5 μM for H2452, 1.0 μM for 211H. The combination of 2.5-10 µM SAR405 and 0.5-5.0 µM cisplatin exhibited additive or synergistic effects on cell growth inhibition in all plural mesothelioma cell lines. In conclusion, Vps34 inhibition by SAR405 suppressed cell growth in pleural mesothelioma cells, accompanied by autophagy inhibition. The combination of SAR405 and cisplatin resulted in a synergistic inhibition of cell proliferation. Citation Format: Yoshiki Kuwabara, Kosuke Sakai, Shigeru Ishi, Shin Yokosuka, Masatoshi Abe, Tomoyuki Takahashi, Yuichiro Kawano, Hiroaki Nishimura, Maiko Toda-Sasaki, Yumiko Kobayashi-Ogawa, Satoshi Kikuchi, Yusuke Hirata, Hiroyuki Kyoyama, Gaku Moriyama, Nobuyuki Koyama, Kazutsugu Uematsu. Evaluation of effect of autophagy inhibition by SAR405, a selective Vps34 inhibitor, on proliferation of pleural mesothelioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4711.

Abstract 1336: Verification of the effect of human allogeneic iPS cell-derived gene-engineered NK cells (eNK cells HLCN061) on mesothelioma

Abstract Malignant pleural mesothelioma is a malignant tumor that is caused by exposure to asbestos. Because it is a rare cancer, there are few drugs that can be used to treat it. Healios is developing iPSC-derived NK cells (eNK cells) (HLCN061) engineered with NKG2D, DAP10, IL-15, CD16, CCL19, and CCR2B genes for the treatment of refractory solid tumors. This study evaluated the antitumor effect of eNK cells against human malignant pleural mesothelioma cell lines.(Method) The antitumor effect of eNK cells on human malignant pleural mesothelioma cell lines in vitro was evaluated using the Viability/Cytotoxicity Multiplex Assay Kit (WST-8/LDH).In vivo, immunodeficient mice were subcutaneously inoculated with human malignant pleural mesothelioma cell lines. After subcutaneous tumor formation, eNK cells were inoculated into or near the tumor, the size of the subcutaneous tumor was measured, and the tumor volume was compared with that of an untreated group. After the study was completed, the tumors were removed and the tumors in the untreated group and the eNK cell-administered group were analyzed using RNA-seq.(Results) The antitumor effect of eNK cells against human malignant pleural mesothelioma cell lines was confirmed in vitro. In a mouse subcutaneous tumor model, the eNK cell treatment group suppressed tumor growth compared to the untreated group (p≦0.05). RNA-seq showed increased expression of granulysin, IL2RB, cathepsin W, etc. in subcutaneous tumors treated with eNK cells. Citation Format: Seiji Matsumoto, Yoshiko Fujita, Kumiko Goto, Yuka Sato, Fusako Nishigaki, Yasuyuki Higashi, Hironobu Kimura, Kouichi Tamura. Verification of the effect of human allogeneic iPS cell-derived gene-engineered NK cells (eNK cells HLCN061) on mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1336.

Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors.

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.

SPARC Is a Novel Positive Immunohistochemical Marker of Epithelioid Mesothelioma to Differentiate It From Lung Adenocarcinoma and/or Squamous Cell Carcinoma.

Epithelioid mesothelioma with a solid histologic pattern (solid epithelioid mesothelioma) is difficult to distinguish from a poorly differentiated squamous cell lung carcinoma and/or solid lung adenocarcinoma. Thus, immunohistochemical markers are essential for diagnosis; however, the sensitivity and specificity of pre-existing mesothelial markers are suboptimal, particularly for differentiation from squamous cell carcinoma. Using a cancer-dependency map, we analyzed gene expression data of pleural mesothelioma and non-small cell lung cancer cell lines (squamous cell carcinoma and adenocarcinoma) and identified secreted protein acidic and cysteine-rich (SPARC) as a promising candidate for the differential diagnosis of epithelioid mesothelioma from lung squamous cell carcinoma and/or lung adenocarcinoma. SPARC expression in mesothelioma and lung cancer cell lines was validated using reverse-transcription polymerase chain reaction, western blotting, and immunohistochemistry. Immunohistochemical staining was performed using anti-SPARC antibodies against solid epithelioid mesothelioma, solid lung adenocarcinoma, and poorly differentiated lung squamous cell carcinoma. SPARC positivity was seen in 42/45 (93.3%) of solid epithelioid mesothelioma, 2/40 (5%) solid lung adenocarcinoma, and 2/45 (4.5%) of lung squamous cell carcinomas. The sensitivity, specificity, and diagnostic accuracy for differentiating solid epithelioid mesothelioma from lung cancer (solid lung adenocarcinoma and poorly differentiated lung squamous cell carcinoma) were 93.3, 95.2, and 94.6%, respectively. In conclusion, SPARC is a novel mesothelial marker that can be used to differentiate epithelioid mesothelioma from squamous cell carcinoma and lung adenocarcinoma.

Small and Large Extracellular Vesicles Derived from Pleural Mesothelioma Cell Lines Offer Biomarker Potential

Pleural mesothelioma, previously known as malignant pleural mesothelioma, is an aggressive and fatal cancer of the pleura, with one of the poorest survival rates. Pleural mesothelioma is in urgent clinical need for biomarkers to aid early diagnosis, improve prognostication, and stratify patients for treatment. Extracellular vesicles (EVs) have great potential as biomarkers; however, there are limited studies to date on their role in pleural mesothelioma. We conducted a comprehensive proteomic analysis on different EV populations derived from five pleural mesothelioma cell lines and an immortalized control cell line. We characterized three subtypes of EVs (10 K, 18 K, and 100 K), and identified a total of 4054 unique proteins. Major differences were found in the cargo between the three EV subtypes. We show that 10 K EVs were enriched in mitochondrial components and metabolic processes, while 18 K and 100 K EVs were enriched in endoplasmic reticulum stress. We found 46 new cancer-associated proteins for pleural mesothelioma, and the presence of mesothelin and PD-L1/PD-L2 enriched in 100 K and 10 K EV, respectively. We demonstrate that different EV populations derived from pleural mesothelioma cells have unique cancer-specific proteomes and carry oncogenic cargo, which could offer a novel means to extract biomarkers of interest for pleural mesothelioma from liquid biopsies.

Effects of pharmacological primary cilium disturbance in the context of in vitro 2D and 3D malignant pleura mesothelioma

IntroductionPrimary cilium (PC) is a single non-motile antenna-like organelle composed of a microtubule core axon originating from the mother centriole of the centrosome. The PC is universal in all mammalian cells and protrudes to the extracellular environment receiving mechanochemical cues that it transmits in the cell. Aim: To investigate the role of PC in mesothelial malignancy in the context of two-dimensional (2D) and three-dimensional (3D) phenotypes. Materials and methodsThe effect of pharmacological deciliation [using ammonium sulphate (AS) or chloral hydrate (CH)] and PC elongation [using lithium chloride (LC)] on cell viability, adhesion, and migration (2D cultures) as well as in mesothelial sphere formation, spheroid invasion and collagen gel contraction (3D cultures) was investigated in benign mesothelial MeT-5A cells and in malignant pleural mesothelioma (MPM) cell lines, M14K (epithelioid) and MSTO (biphasic), and primary malignant pleural mesothelioma cells (pMPM). ResultsPharmacological deciliation or elongation of the PC significantly affected cell viability, adhesion, migration, spheroid formation, spheroid invasion and collagen gel contraction in MeT-5A, M14K, MSTO cell lines and in pMPM cells compared to controls (no drug treatment). ConclusionsOur findings indicate a pivotal role of the PC in functional phenotypes of benign mesothelial cells and MPM cells.

Mesothelioma and Small Cell Lung Cancer; Effects of Nigella Sativa Thymoquinone on Cell Lines

Background: Lung cancer and malignant mesothelioma are types of cancer with a poor prognosis and fatal. Small cell lung cancer is much more aggressive and survival shorter than non-small cell lung carcinoma. Mesothelioma is a rare malignant disease that commonly affects the pleura. Cisplatin is frequently used in chemotherapy protocols. Thymoquinone is a chemical with antineoplastic effects procured from the Nigella Sativa plant. It was aimed to investigate the effects of thymoquinone and cisplatin on small cell lung cancer and mesothelioma cell lines.
 Methodology: The study was done in the Cell Culture Laboratory of Gaziantep University. Cell lines of small cell lung cancer, malignant pleural mesothelioma and non-cancerous bronchial epithelium were used in the study. Cells were cultured in dimethyl sulfoxide. The effective doses of thymoquinone and cisplatin were calculated. Accordingly, which were detected doses of thymoquinone as 100 μM and cisplatin as 200 μM. The viability of cells were evaluated using 3-(4,5-dimethylthiazol-2-il) 2,5-diphenyl tetrazolium bromide test. Experiments were repeated 4 times at different times by the same team in the same laboratory. Statistical analysis of the study was done using the Chi-square test. The study was was accordance with international standards on cell lines in the laboratory.
 Results: Chemical treats were administering on all cell lines at doses of 100 μM and 200 μM. Thymoquinone at a dose of 100 mm; viability of cells were detected in 48% in mesothelioma, 44% in small cell lung cancer and 55% in noncancerous epithelium cell lines. Cisplatin at a dose of 200 μM; viability of cells were detected in 63% in mesothelioma, 48% in small cell lung cancer and 59% in noncancerous epithelium cell lines. There was no significant toxicity of dimethyl sulfoxide used as a chemical solvent when compared with physiological saline.
 Conclusions: Thymoquinone at a dose of 100 μM was more effective than cisplatin at a dose of 200 μM on both small cell lung cancer and malignant pleural mesothelioma cell lines. Cisplatin was more effective in small cell lung cancer than malignant pleural mesothelioma at a dose of 200 μM. The effects of thymoquinone were similar in both cancer cell lines.

EP07.02-002 Investigating the Role of MicroRNAs in Cell Growth and Cisplatin-Sensitivity of Malignant Pleural Mesothelioma

In recent years, in vitro studies, including our own, have shown that re-expression of tumour suppressive microRNAs can alter malignant pleural mesothelioma (MPM) cell line growth, and that dysregulation of microRNAs might contribute to the intrinsic resistance towards the standard therapeutic regimen of platinum/pemetrexed. Since our screening for predictive biomarkers has revealed several candidates, we have recently decided to expand our studies from the three previously analysed microRNAs to a systematic evaluation of a larger number of predictive and prognostic biomarker candidates.

Discrimination of Malignant Pleural Mesothelioma Cell Lines Using Amino Acid Metabolomics with HPLC.

Malignant pleural mesothelioma (MPM) is a malignancy closely associated with asbestos exposure. Although early diagnosis provides a chance of effective treatment and better prognosis, invasive biopsy and cytological procedure are required for definitive diagnosis. In this study, we developed a method to differentiate between MPM and control cell lines, named "amino acid metabolomics," consisting in the assessment of the balance of their amino acid levels in the cell culture medium. Culture media of MESO-1 (MPM cell line) and Met-5A (control) cells were used in this study to evaluate amino acid levels using HPLC, following the fluorescence derivatization method. The time-dependent changes in amino acid levels were visualized on the score plot following principal component analysis, and the results revealed differential changes in amino acid levels between the two cell culture supernatants. A discriminative model based on linear discriminant analysis could distinguish MPM and control cells.

Headspace Volatile Organic Compound Profiling of Pleural Mesothelioma and Lung Cancer Cell Lines as Translational Bridge for Breath Research.

IntroductionMalignant pleural mesothelioma (MPM) is a lethal cancer for which early-stage diagnosis remains a major challenge. Volatile organic compounds (VOCs) in breath proved to be potential biomarkers for MPM diagnosis, but translational studies are needed to elucidate which VOCs originate from the tumor itself and thus are specifically related to MPM cell metabolism.MethodsAn in vitro model was set-up to characterize the headspace VOC profiles of six MPM and two lung cancer cell lines using thermal desorption-gas chromatography-mass spectrometry. A comparative analysis was carried out to identify VOCs that could discriminate between MPM and lung cancer, as well as between the histological subtypes within MPM (epithelioid, sarcomatoid and biphasic).ResultsVOC profiles were identified capable of distinguishing MPM (subtypes) and lung cancer cells with high accuracy. Alkanes, aldehydes, ketones and alcohols represented many of the discriminating VOCs. Discrepancies with clinical findings were observed, supporting the need for studies examining breath and tumor cells of the same patients and studying metabolization and kinetics of in vitro discovered VOCs in a clinical setting.ConclusionWhile the relationship between in vitro and in vivo VOCs is yet to be established, both could complement each other in generating a clinically useful breath model for MPM.

Pleural mesothelioma: research of mechanisms involved in carcinogenesis. The role of CD10

The present study was conducted at Surgical Pathology Department of “Santo Spirito” hospital in Casale Monferrato (AL) and in collaboration with the Cellular Biology and Physiology laboratory located in the institution of DiSIT-Università del Piemonte Orientale. In the pathology facility a retrospective analysis on the immunohistochemical expression of CD10 marker in malignant pleural mesothelioma was delineated. For this purpose it was collected a sample of 63 pleural biopsies with a diagnosis of pleural mesothelioma come in the period of January 2015 and November 2020. In this cohort, CD10 expression was assessed from a prognostic perspective and in relation to morphologic aspects of the neoplasm. Results from previous analysis built the basis of the experiment conducted in the DiSIT facility. Two pleural mesothelioma cell lines, REN and MM98, got incubated with the cytokine TGF-β aiming to analyze the potential relationship between epithelial-to-mesenchymal transition (EMT) and CD10 cell expression pattern. Bioinformatic server Haddock 2.4 helped to predict and model potential cross-talk between EMT underlying pathways and CD10 signalling. Objectives: The present study aimed to assess prognostic role of CD10 immunohistochemical expression in a cohort of patients affected by pleural mesothelioma. Second goal was to analyze the relationship between CD10 biopsy expression, phenotypical and imunophenotypical features (referring in particular to PDL-1 expression) of the neoplasm. Results from this retrospective analysis established next aim of the study: induction of EMT in two mesothelioma cell lines, REN and MM98, and analysis of change in CD10 cellular pattern. Final goal was to predict a potential cross-talk between EMT related pathways and CD10 activity based on bioinformatic server Haddock 2.4.Methods: Immunohistochemical analysis of biopsies and cell cultures were processed on Ventana Benchmark immunostainer. Tools adopted in qRT-PCR of cell cultures were: Power Sybr Green Mastermix probes (Ambion Austin, TX, USA) and KiCqStart® SYBR® Green primers (Sigma-Aldrich); Real-Time PCR CFX384 detection system (Bio-Rad Laboratories, Hercules, CA, USA). Gene expressione was measured with ∆∆Ct method. Parametrization of CD10 immunohistochemical expression was accomplished with cell imaging software CellProfiler 3.1.9. Statistical analysis were ran with R software version 3.6.2. Results: CD10 showed to be a negative prognostic factor, positively associated to neoplastic grade, in the cohort of patients analyzed. Induction of EMT in REN and MM98 TGF-β treated cultures, augmented CD10 cytosolic expression against non-treated cultures. Specifically, a change in CD10 immunoexpression pattern, from membranous to cytosolic, was observed for EMT+ REN cell culture. This data agrees with prevalent CD10 cytosolic expression observed in mesothelioma with low differentiation, where differentiation represents a function of EMT. Furthermore, Haddock data highlighted a potential promotion of EMT by CD10 via a NF-kB interaction. CD10 could establish a positive feedback loop with EMT. Conclusion: CD10 represents a positive marker of neoplastic progression in pleural mesothelioma that, based on its expression level, can stratify survival. Experimental data showed that CD10 could be upregulated by EMT pathways. Bionformatic analysis also suggest that CD10 could interact with EMT establishing a positive feedback loop. Clarifying tumorigenicity of CD10 in mesothelioma is fundamental to define its possible therapeutic role.

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.

Selected Articles from This Issue

Medulloblastoma is a common and deadly pediatric malignancy whose lethality is compounded by a lack of targeted therapies, partially owing to the differential biologies of the four major molecular subtypes. The most prevalent subtype, Sonic Hedgehog medulloblastoma (SHH-MB), is driven by hyperactivation of the eponymous SHH pathway within MATH-1-expressing granule cell precursors in the developing brain. Therefore, efforts to develop molecular targeted therapies for SHH-MB have focused largely on downstream targets and effectors of the SHH pathway such as the proto-oncogene SOX9, which has been shown to be specifically overexpressed in SHH-MB but whose functional role in disease progression has yet to be defined. Here, Adolphe and colleagues demonstrate that loss of SOX9 does not significantly impact the course of SHH-MB disease development and progression, indicating a dispensable role for this proto-oncogene in SHH-MB despite its elevated expression in most cases. This finding may have significant implications for ongoing preclinical and translational work aimed at identifying suitable molecular drivers of SHH-MB for therapeutic development.Lymphangioleiomyomatosis is a rare neoplastic disease of unknown origin that is nearly exclusive to women, with therapies targeting mTOR as the recognized standard of care. However, there is substantial variability in disease presentation, progression, and therap. response. Here, Espin, Baiges and colleagues perform multi-omics characterization of patient-derived lymphangioleiomyomatosis cells and tissues to identify the molecular features and potential causes of disease heterogeneity. The data suggest that differential expression of certain transcription factors—such as YB1, RUNX1, IRF1, estrogen receptor, and progesterone receptor—as well as certain immunity factors may account for some of these features. Based on the molecular features identified herein, the authors also nominate a breast mesenchymal cell line and certain pleural mesothelioma cell lines as potential surrogate models of lymphangioleiomyomatosis, expanding on the biological basis of the disease. Taken together, the data provide a high-value resource for further exploration of the molecular drivers of lymphangioleiomyomatosis progression.Glioma patients are frequently treated with temozolomide, a DNA alkylating chemotherapeutic that is known to cross the blood-brain barrier. The isocitrate dehydrogenase (IDH) R132H mutation is a common molecular feature of gliomas and is generally associated with gliomagenesis, but also with a more robust response to temozolomide therapy. However, the molecular underpinnings of this observation are not defined. In this study, Zhan, Ma, and colleagues use a heterozygous knock-in approach to model the effects of IDH R132H/wild-type allele balance on glioma cell biology. They find that the heterozygous IDH genotype in tandem with glioma driver gene alterations in p53, RB, and ATRX predisposes glioma cells to pro-senescent features. This combination of molecular features was associated with increased DNA damage markers and potentiated the effects of temozolomide in this model. However, heterozygous IDH R132H mutation also predisposed astroglial cells toward a senescence-associated secretory phenotype, which is known to exert a pro-tumorigenic effect within a tumor-permissive microenvironment. These data offer a potential mechanistic explanation for how a single mutation within IDH may provide a pro-tumorigenic stimulus while also sensitizing glioma cells to alkylating chemotherapy with temozolomide.Potentiating the effects of genotoxic interventions such as chemo- or radiotherapy requires the suppression of DNA damage response pathways, such as non-homologous end joining (NHEJ). Blocking NHEJ with chemical inhibitors is one strategy, but none have been clinically approved for this application. Building upon prior efforts to develop chemical screening strategies for NHEJ inhibitors, Du and colleagues report a miniaturized screening protocol that can be carried out in 96-well plates. By assessing an FDA-approved drug library in this format, the authors identify several potential candidates that function to inhibit NHEJ at concentrations below their cytotoxic dose. One of these drugs, ompalisib, was shown to suppress the phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in response to chemo- or radiotherapy, a key step in the initiation of NHEJ to mediate their genotoxic effects. Overall, the authors advance a novel screening strategy to examine the effects of pharmacological interventions on DNA damage responses via NHEJ, and further nominate a candidate FDA-approved drug for development as a chemo- and radiosensitizer.

Synthetic Study Aiming at the Tricyclic Core of 12-epi-JBIR-23/24.

The synthetic study toward highly enantio- and diastereoselective synthesis of the tricyclic framework of 12-epi-JBIR-23/24, a natural product analogue showing inhibitory activity against four malignant pleural mesothelioma cell lines, is presented herein. In this synthesis, a rhodium-catalyzed asymmetric three-component Michael/aldol reaction introduces three consecutive tertiary carbon centers, while the unique epoxyquinol core motif is successfully forged via [3,3]-sigmatropic rearrangement of an allylic xanthate, vinylogous Pummerer rearrangement, and a selective mesylation/epoxidation cascade of a triol.

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.

Bex1 significantly contributes to the proliferation and invasiveness of malignant tumor cells.

Invasion has a significant role in cancer progression, including expansion to surrounding tissue and metastasis. Previously, we assessed the invasive ability of cancer cells using an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) method by which cancer cell invasion can be easily visualized. The present study examined multiple lung adenocarcinoma and malignant pleural mesothelioma (MPM) cell lines using the DL-CGH method and identified inherently invasive cell lines. Next, by comparing gene expression between invasive and non-invasive cells by cDNA microarray, the potential candidate gene brain-expressed x-linked protein 1 (Bex1) was identified to be involved in cancer invasion, as it was highly expressed in the invasive cell lines. Downregulation of Bex1 suppressed the invasion and proliferation of the invasive tumor cell lines. The findings of the present study suggested that Bex1 may promote metastasis in vivo and could be a potential oncogene and molecular therapeutic target in lung adenocarcinoma and MPM.

Screening of Pleural Mesothelioma Cell Lines for Kinase Activity May Identify New Mechanisms of Therapy Resistance in Patients Receiving Platin-Based Chemotherapy.

Background Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumor associated with a dismal prognosis. Multimodal therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons underlying the rather poor efficacy of platinum compounds remain largely unknown. Kinase activity might influence cellular response to these regimens. Materials and Methods For this exploratory study, we screened MPM cell lines (NCI-H2452, NCI-H2052, and MSTO-211H) differing in response to cisplatin and benign control fibroblasts (MRC-5) for overall phosphorylation patterns as well as kinase activity with respect to cellular response to cisplatin-based therapeutics. We analysed the cell lines for cellular kinases in a high-throughput manner using the highly innovative technique PamGene. Cell state analysis including apoptosis, necrosis, and cell viability was performed by using enzyme activity and fluorescent-based assays. Results Cisplatin alters cellular phosphorylation patterns affecting cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis. In cisplatin-responsive cell lines, phosphorylation of AKT1 and GSK3B was decreased but could not be influenced in cisplatin-resistant NCI-H2452 cells. Cisplatin-responsive cell lines showed increased phosphorylation levels of JNK1/2/3 but decreased phosphorylation in cisplatin-resistant NCI-H2452 cells. Conclusion Kinase phosphorylation and activity might play a crucial role in cellular response to cytostatic agents. Cisplatin influences phosphorylation patterns with distinct features in cisplatin-resistant cells. These alterations exert a significant impact on cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis of the respective tumor cells. Based on our results, the induction of p38 or JNK1/3, or inhibition of AKT1 by, for example, BIA-6, might offer a positive synergistic effect by induction of an apoptotic response to cisplatin-based treatment, thus potentially enhancing the clinical outcome of MPM patients.

MicroRNA-206 inhibits cell growth in malignant pleural mesothelioma

Objective To investigate the suppressive effect of miR-206 on malignant pleural mesothelioma (MPM) cell lines and mechanism. Methods A mimic of miR-206 was introduced into MPM cell lines H513, H2052 and H2373 to overexpress this microRNA. (1) miRNA-induced changes in cell growth were evaluated by crystal violet staining. (2) Genes associated with cell cycle homeostasis and apoptosis were examined by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). (3) By gene knockdown experiment and RT-qPCR, the potential mechanism of the suppressive effect was analyzed. Results (1) Cellular growth in H513, H2052, H2373 were significantly inhibited after overexpression of miR-206 at day 3 (570 nm: 0.406±0.196 vs. 0.682±0.042, 0.192±0.181 vs. 0.587±0.264, 0.178±0.056 vs. 0.794±0.276, P<0.05). (2) Gene expression levels of B cell lymphoma/leukemia-2 associated X protein (bax), p53, and p27 were all significantly increased (average fold change: 1.489±0.355, 1.298±0.336, 1.746±0.389, P<0.05) in miR-206-transfected MPM cells. Coordinately, gene expression levels of proliferating cell nuclear antigen (PCNA) and B cell lymphoma/leukemia-2 (bcl-2) were significantly decreased (average fold change: 1.489±0.355, 1.298±0.336, P<0.05). Significant attenuation of histone deacetylase (HDAC) activity was observed (average fold change: 0.984±0.271 vs. 0.442±0.149 vs. 0.520±0.193, P<0.05), and suppression of HDAC activity mediated by negative regulation of HDAC4 and 6 genes is an effect unique to miR-206. (3) All MPM cell lines tested with either HDAC4 or HDAC6 knockdown exhibited significant growth arrest by crystal violet staining (570 nm: 0.971±0.205 vs. 0.427±0.152 vs. 0.520±0.191, P<0.05), which was consist with suppression of HDAC, indicating HDAC4 and HDAC6 were significant effector molecules for the suppressive effect. Conclusion Overexpression of miR-206 acts as a tumor suppressor in MPM. Key words: Malignant pleural mesothelioma; MicroRNA; Apoptosis; Growth arrest; Histone deacetylase

Inhibiting crosstalk between MET signaling and mitochondrial dynamics and morphology: a novel therapeutic approach for lung cancer and mesothelioma

ABSTRACTThe receptor tyrosine kinase MET is frequently involved in malignant transformation and inhibiting its activity in MET-dependent cancers is associated with improved clinical outcomes. Emerging evidence also suggests that mitochondria play an essential role in tumorigenesis and Dynamin Related Protein (DRP1), a key component of the mitochondrial fission machinery, has emerged as an attractive therapeutic target. Here, we report that inhibiting MET activity with the tyrosine kinase inhibitor MGCD516 attenuates viability, migration, and invasion of non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) cell lines in vitro, and significantly retards tumor growth in vivo. Interestingly, MGCD516 treatment also results in altered mitochondrial morphology in these cell lines. Furthermore, inhibiting MET pharmacologically or knocking down its expression using siRNA, decreases DRP1 activity alluding to possible crosstalk between them in these two cancers. Consistently, a combination of MGCD516 and mdivi-1, a quinazolinone reported to inhibit mitochondrial fission, is more effective in attenuating proliferation of NSCLC and MPM cell lines than either drug alone. Considered together, the present study has uncovered a novel mechanism underlying mitochondrial regulation by MET that involves crosstalk with DRP1, and suggests that a combination therapy targeting both MET and DRP1 could be a novel strategy for NSCLC and MPM.

Berberine induces mitochondrial‑mediated apoptosis and protective autophagy in human malignant pleural mesothelioma NCI‑H2452 cells.

Increasing evidence shows that berberine has antitumor effects against a number of tumor cells. In the present study, we evaluated the effect of berberine on the proliferation of the human malignant pleural mesothelioma (MPM) cell line NCI‑H2452, and explored the therapeutic potential and underlying mechanisms of this agent. Our results showed that berberine inhibited the proliferation of NCI‑H2452 cells in a dose‑and time‑dependent manner and could induce apoptosis, possibly through a caspase‑9‑dependent intrinsic mitochondrial pathway. In addition, autophagy was induced by berberine, which was characterized by the accumulation of LC3‑II and decreased p62 expression. We used inhibitors of apoptosis and autophagy, and an inducer of autophagy, to evaluate the significance of autophagy in berberine‑induced cell death. The results demonstrated that apoptosis is the primary route through which berberine induces NCI‑H2452 cell death. Berberine‑induced autophagy may be an adaptive response to antitumor agents and have a protective role in MPM cells. Inhibition of autophagy enhanced berberine‑induced apoptosis. Therefore, inhibition of autophagy may be an effective treatment strategy in the management of MPM. In conclusion, berberine is a potent antitumor agent for treating MPM, and it induces mitochondrial‑mediated apoptosis and protective autophagy in human NCI‑H2452 MPM cells.