Non-malignant Mesothelial Cells Research Articles

Mineral fibre toxicity: expression of retinoblastoma (Rb) and phospho-retinoblastoma (pRb) protein in alveolar epithelial and mesothelial cell lines exposed to fluoro-edenite fibres

Several asbestos-like mineral fibres, including fluoro-edenite, may cause lung cancer and/or other lung diseases. However, biological and molecular mechanisms linked to cancer development after mineral fibre exposure have not been fully investigated. In the present study, human non-malignant mesothelial (MeT-5A) and human bronchoalveolar alveolar epithelial (A549) cell lines were incubated with rising concentrations of fluoro-edenite to evaluate the expression of retinoblastoma (Rb) protein, which has been demonstrated to play an important role in cell cycle control and tumour progression. Intriguingly, these results show that Rb expression was unchanged, while the level of the phosphorylated protein increased significantly in a dose-dependent manner, suggesting an involvement of this regulator protein in the pathogenesis of the lung diseases induced by mineral fibres. In conclusion, fluoro-edenite regulates the expression of phospho-retinoblastoma to trigger a network of signals strictly connected with cell proliferation and neoplastic cell transformation.

Latent TGF- β binding proteins (LTBPs) 1 and 3 differentially regulate transforming growth factor- β activity in malignant mesothelioma

Malignant mesothelioma is an aggressive cancer of the pleura with poor prognosis. There is a need to identify new biomarkers and therapeutic targets for this invasive and fatal disease. Transforming growth factor β (TGF-β) can promote mesothelioma tumorigenesis through multiple mechanisms. Latent TGF-β binding proteins (LTBPs) regulate TGF-β activation by targeting the growth factor into the extracellular matrix from where it can be released and activated. We investigated here the expression patterns of different LTBP isoforms in malignant mesothelioma tissues and in 2 established malignant mesothelioma cell lines. All LTBPs were expressed, but LTBP-3 was the main isoform in healthy pleura and in cultured nonmalignant mesothelial cells. We observed down-regulation of LTBP-3 expression in malignant mesothelioma, which was associated with high P-Smad2 levels indicative of TGF-β activity specifically in the tumor tissue. Small interfering RNA-mediated suppression of LTBP-3 expression in mesothelioma cells increased the secretion of TGF-β activity. Immunoreactivity of LTBP-1, on the other hand, was markedly strong in the tumor stroma, which showed significantly lower levels of P-Smad2. A strong negative correlation between LTBP-1 and P-Smad2 immunoreactivity was found, implying that LTBP-1 is not likely to contribute directly to the increased levels of TGF-β activity in malignant mesothelioma. Current results suggest that LTBPs 1 and 3 may have specific roles in malignant mesothelioma pathogenesis through the regulation of TGF-β activation in the tumor tissue and the structure of the tumor stroma.

The promyelocytic leukemia zinc-finger gene, PLZF, is frequently downregulated in malignant mesothelioma cells and contributes to cell survival

DNA copy number analysis was performed, using SNP mapping arrays, to fine map genomic imbalances in human malignant mesothelioma (MM) cell lines derived from primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. All 22 cell lines examined showed homozygous deletions of 9p21.3, centering at the CDKN2A/ARF and CDKN2B loci. Other commonly underrepresented segments included 1p36, 1p22, 3p21-22, 4q13, 4q34, 11q23, 13q12-13, 14q32, 15q15, 18q12 and 22q12, each observed in 55%–90% of cell lines. Focal deletions of 11q23 encompassed the transcriptional repressor gene PLZF (promyelocytic leukemia zinc finger), which was validated by analysis of genomic DNA using real-time PCR. Semi-quantitative RT-PCR and immunoblot analysis revealed that PLZF is greatly downregulated in MM cell lines compared to non-malignant mesothelial cells. Ectopic expression of PLZF in PLZF-deficient MM cells resulted in decreased cell viability, reduced colony formation, as well as increased apoptosis, the latter based on results of various cell death assays and the observation of increased cleavage of caspase 3, PARP and Mcl-1. These data indicate that deletions of PLZF are a common occurrence in MM and that downregulation of PLZF may contribute to MM pathogenesis by promoting cell survival.

The Plexin-A1 Receptor Activates Vascular Endothelial Growth Factor-Receptor 2 and Nuclear Factor-κB to Mediate Survival and Anchorage-Independent Growth of Malignant Mesothelioma Cells

The semaphorins and their receptors, the neuropilins and the plexins, are constituents of a complex regulatory system that controls axonal guidance. Moreover, many types of tumor cells express various members of semaphorins and receptors, but the biological activities within tumor mass and the signal transduction mechanism(s) they use are largely unknown. Here, we show that in asbestos-related malignant pleural mesothelioma (MPM), Semaphorin-6D (Sema6D) and its receptor plexin-A1 are frequently expressed and trigger a prosurvival program that promotes anchorage-independent growth of MPM cells. Interestingly, the same response is also controlled by the tyrosine kinase receptors of vascular endothelial growth factor (VEGF) through a nuclear factor-kappaB (NF-kappaB)-dependent pathway. We found that in MPM cells, plexin-A1 and VEGF-receptor 2 (VEGF-R2) are associated in a complex. Moreover, the presence of Sema6D promotes the tyrosine phosphorylation of VEGF-R2 in a plexin-A1-dependent manner. This is necessary for basal and Sema6D-induced NF-kappaB transcriptional activity, and NF-kappaB mediates tumor cell survival. Expression of Sema6D and plexin-A1 is induced by asbestos fibers and overexpression of plexin-A1 in nonmalignant mesothelial cells inhibits cell death after asbestos exposure. This work identifies a new biological function of semaphorins in cancer cells and suggests the involvement of an undescribed survival pathway during MPM tumorigenesis.

Anti-proliferative effects of simocyclinone D8 (SD8), a novel catalytic inhibitor of topoisomerase II

Simocyclinone D-8 (SD8), a semi-synthetic compound derived from yeast, has been shown to decrease the proliferation of MCF-7 breast cancer cells. It has been shown to be a potent bacterial DNA gyrase inhibitor, a homologue of human topoisomerase II (hTopoII). We tested SD8 activity alone and in combination with cisplatin against malignant mesothelioma (MM) and non-small cell cancer (NSCLC) cell lines. Inhibition of hTopoII supercoiling function by SD8 and a known hTopoII poison, etoposide, were done by in vitro assay using purified hTopoII and kinetoplast DNA as the substrate. The DNA products were analyzed by agarose gel electrophoresis after treatment with increasing concentrations of each drug. Mesothelioma cell lines (H2373, H2461 and H2596) and NSCLC cell lines (H2030, H460, and H2009) grown in RPMI with 10% calf serum were used. Non-malignant mesothelial cells, LP9, were grown in 1:1 ratio of MCDB:199E medium supplemented with 15% calf serum, 0.4 microg/mL hydrocortisone, and 15 ng/mL epidermal grown factor. Cell proliferation assays were performed in 96-well plates using the CCK-8 kit (Dojindo inc.). Cells were treated for 72 h with various SD8 concentrations and controls containing equal volume of the vehicle, DMSO. Treated cells were assayed for the induction of apoptosis with poly ADP-ribose polymerase-1 (PARP) cleavage assay. Biochemical assays revealed that the IC(50) for hTopoII inhibition was 100 microM for SD8 and 400 microM for etoposide. SD8 inhibited hTopoII function without inducing DNA cleavage events. SD8 inhibited the growth of NSCLC and Mesothelioma cells with IC(50) ranging from 75-125 microM. Furthermore, SD8 was not toxic to non-transformed primary mesothelial cell line, LP9 at the IC(50) doses. SD8 induced apoptosis in all cell lines tested. SD8 inhibits hTopoII in vitro without inducing DNA strands breaks and has significant activity against NSCLC and MM cell lines. While doses required for SD8 anticancer activity are unlikely to be achieved in vivo, chemical modifications to SD8 to increase its potency could lead to improved therapies for these diseases.

Malignant mesothelioma cells are rapidly sensitized to TRAIL-induced apoptosis by low-dose anisomycin via Bim

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) holds promise for the treatment of tumors; however, many tumors are resistant to TRAIL alone. We previously showed that resistant malignant mesothelioma cells are sensitized to TRAIL-induced apoptosis by diverse toxic insults including chemotherapy, irradiation, or protein translation inhibitors such as cycloheximide. In seeking nontoxic sensitizers for TRAIL, we tested the protein translation inhibitor anisomycin at subtoxic concentrations 10- to 100-fold below those reported to inhibit protein translation. At these low concentrations (25 ng/mL), anisomycin potently and rapidly sensitized mesothelioma cells to TRAIL-induced apoptosis. Moreover, such sensitization occurred in malignant but not in nonmalignant mesothelial cells. Sensitization by anisomycin was dependent on Bid, indicating a role for mitochondrial amplification in the apoptotic synergy with TRAIL signaling. Consistent with this, we found that anisomycin induces rapid accumulation of the BH3-only protein Bim; moreover, small interfering RNA knockdown of Bim inhibits anisomycin-induced sensitization. Bim accumulation seems not to be transcriptional; instead, it is associated with Bim phosphorylation and increased stability, both consistent with the activation of c-jun NH2-terminal kinase signals by anisomycin. Overall, our data indicate that the rapid and selective sensitization by anisomycin in mesothelioma cells is mediated by posttranslational potentiation of Bim, which primes the cells for apoptosis via the death receptor pathway. Such subtoxic approaches to sensitization may enhance the value of TRAIL in cancer therapy.

α-Tocopheryl Succinate Inhibits Malignant Mesothelioma byDisrupting the Fibroblast Growth Factor Autocrine Loop: MECHANISM AND THEROLE OF OXIDATIVESTRESS

We have studied the potential effect against human malignant mesotheliomas (MM) of alpha-tocopheryl succinate (alpha-TOS), a redox-silent vitamin E analog with strong pro-apoptotic and anti-cancer activity. alpha-TOS at sub-apoptotic levels inhibited proliferation of MM cell lines, while being nontoxic to nonmalignant mesothelial cells. Because MM cells are typified by a highly metastatic phenotype, we investigated the effect of alpha-TOS on genes playing a major role in MM progression. Of these, alpha-TOS down regulated fibroblast growth factor (FGF)-1 and, in particular, FGF-2 on the transcriptional level in MM cells, and this was not observed in their nonmalignant counterparts. FGF-2 short interfering RNA suppressed proliferation of MM cells. Down-regulation of FGF-2 was likely because of inhibition of the egr-1 transcription activity that was decreased in MM cells via oxidative stress induced by alpha-TOS, as evidenced by EPR spectroscopy, whereas nonmalignant cells did not show this response. Treatment of MM cells with egr-1 short interfering RNA suppressed proliferation, which was overridden by exogenously added recombinant FGF-1 and, in particular, FGF-2. An analog of coenzyme Q targeted to mitochondria and superoxide dismutase overrode inhibition of MM cell proliferation by alpha-TOS as well as alpha-TOS-induced inhibition of egr-1-dependent transactivation. Finally, alpha-TOS significantly suppressed experimental MM in immunocompromised mice. Our data suggest that alpha-TOS suppresses MM cell proliferation by disrupting the FGF-FGF receptor autocrine signaling loop by generating oxidative stress and point to the agent as a selective drug against thus far fatal mesotheliomas.

$alpha;-Tocopheryl succinate inhibits proliferation of mesothelioma cells by selective down-regulation of fibroblast growth factor receptors*1

α-Tocopheryl succinate (α-TOS), a redox-silent analogue of vitamin E , inhibits malignant mesotheliomas (MM) in a pre-clinical model. Here we investigated the underlying mechanism. Exposure of MM cells to α-TOS triggered apoptosis at higher and inhibited proliferation at lower concentrations, while this effect was not observed in non-malignant mesothelial cells. Sub-apoptotic doses of α-TOS caused down-regulation of fibroblast growth factor receptor-1 (FGFR1) selectively in MM cells, while the effect on FGFR2 was only marginal. FGF1 and FGF2 enhanced MM cell proliferation that was suppressed by α-TOS. Over-expression of E2F1 , a transcriptional factor of FGFR1, but not its dominant-negative counterpart, partially blocked the inhibitory activity of α-TOS on MM cell proliferation. Our data suggest a novel mechanism by which a clinically intriguing agent selectively suppresses proliferation of cancer cells, as shown here for the untreatable mesotheliomas.

α-Tocopheryl succinate inhibits proliferation of mesothelioma cells by selective down-regulation of fibroblast growth factor receptors

α-Tocopheryl succinate (α-TOS), a redox-silent analogue of vitamin E, inhibits malignant mesotheliomas (MM) in a pre-clinical model. Here we investigated the underlying mechanism. Exposure of MM cells to α-TOS triggered apoptosis at higher and inhibited proliferation at lower concentrations, while this effect was not observed in non-malignant mesothelial cells. Sub-apoptotic doses of α-TOS caused down-regulation of fibroblast growth factor receptor-1 (FGFR1) selectively in MM cells, while the effect on FGFR2 was only marginal. FGF1 and FGF2 enhanced MM cell proliferation that was suppressed by α-TOS. Over-expression of E2F1, a transcriptional factor of FGFR1, but not its dominant-negative counterpart, partially blocked the inhibitory activity of α-TOS on MM cell proliferation. Our data suggest a novel mechanism by which a clinically intriguing agent selectively suppresses proliferation of cancer cells, as shown here for the untreatable mesotheliomas.

Alpha-tocopheryl succinate and TRAIL selectively synergise in induction of apoptosis in human malignant mesothelioma cells.

Malignant mesothelioma (MM) is a fatal type of neoplasia with poor therapeutic prognosis, largely due to resistance to apoptosis. We investigated the apoptotic effect of α-tocopheryl succinate (α-TOS), a strong proapoptotic agent, in combination with the immunological apoptogen TNF-related apoptosis-inducing ligand (TRAIL) on both MM and nonmalignant mesothelial cells, since MM cells show low susceptibility to the clinically intriguing TRAIL. All MM cell lines tested were sensitive to α-TOS-induced apoptosis, and exerted high sensitivity to TRAIL in the presence of subapoptotic doses of the vitamin E analogue. Neither TRAIL or α-TOS alone or in combination caused apoptosis in nonmalignant mesothelial cells. Isobologram analysis of the cytotoxicity assays revealed a synergistic interaction between the two agents in MM cells and their antagonistic effect in nonmalignant mesothelial cells. TRAIL-induced apoptosis and its augmentation by α-TOS were inhibited by the caspase-8 inhibitor Z-IETD-FMK and the pan-caspase inhibitor Z-VAD-FMK. Activation of caspase-8 was required to induce apoptosis, which was amplified by α-TOS via cytochrome c release following Bid cleavage, with ensuing activation of caspase-9. Enhancement of TRAIL-induced apoptosis in MM cells by α-TOS was also associated with upregulation of the TRAIL cognate death receptors DR4 and DR5. Our results show that α-TOS and TRAIL act in synergism to kill MM cells via mitochondrial pathway, and are nontoxic to nonmalignant mesothelial cells. These findings are indicative of a novel strategy for treatment of thus far fatal MM.

Generation of reactive oxygen species by human mesothelioma cells.

Malignant mesothelioma cells contain elevated levels of manganese superoxide dismutase (MnSOD) and are highly resistant to oxidants compared to non-malignant mesothelial cells. Since the level of cellular free radicals may be important for cell survival, we hypothesized that the increase of MnSOD in the mitochondria of mesothelioma cells may alter the free radical levels of these organelles. First, MnSOD activity was compared to the activities of two constitutive mitochondrial enzymes; MnSOD activity was 20 times higher in the mesothelioma cells than in the mesothelial cells, whereas the activities of citrate synthase and cytochrome c oxidase did not differ significantly in the two cell lines. This indicates that the activity of MnSOD per mitochondrion was increased in the mesothelioma cells. Superoxide production was assayed in the isolated mitochondria of these cells using lucigenin chemiluminescence. Mitochondrial superoxide levels were significantly lower (72%) in the mesothelioma cells compared to the mesothelial cells. Oxidant production in intact cells, assayed by fluorimetry using 2′,7′-dichlorodihydrofluorescein as a fluorescent probe, did not differ significantly between these cells. We conclude that mitochondrial superoxide levels are lower in mesothelioma cells compared to nonmalignant mesothelial cells, and that this difference may be explained by higher MnSOD activity in the mitochondria of these cells. Oxidant production was not different in these cells, which may be due to the previously observed increase in H2O2-scavenging mechanisms of mesothelioma cells. © 1999 Cancer Research Campaign

Endogenous antioxidant enzymes and glutathione S-transferase in protection of mesothelioma cells against hydrogen peroxide and epirubicin toxicity.

We have previously shown that cultured malignant mesothelioma cells contain elevated manganese superoxide dismutase (MnSOD) mRNA levels and activities compared with non-malignant mesothelial cells. As many cytotoxic drugs generate both superoxide and hydrogen peroxide, we assessed the relative significance of catalase and the glutathione redox cycle, as well as glutathione S-transferase (GST), in protecting these cells against hydrogen peroxide and epirubicin toxicity. Mesothelioma cell lines containing high (M38K cells) and low (M14K cells) MnSOD, and non-malignant MeT-5A mesothelial cells were selected for the study. M38K cells were the most resistant of these three cell types to hydrogen peroxide (0.1-0.5 mM, 4 h) and epirubicin (0.1-0.5 microg ml(-1), 48 h) as judged by lactate dehydrogenase (LDH) release and by high-energy nucleotide (ATP, ADP, AMP) depletion. Total glutathione was higher in M38K cells (63.8 +/- 20.3 nnmol mg(-1) protein) than in M14K (25.2 +/- 8.2 nmol mg[-1]) or MeT-5A cells (23.5 +/- 4.5 nmol mg[-1]). Furthermore, GST specific activity was higher in M38K cells (111.3 +/- 15.8 U mg[-1]) than in M14K cells (77.4 +/- 6.6 U mg[-1]) or in MeT-5A cells (68.8 +/- 7.6 U mg[-1]). Western blotting indicated the presence of GST-pi in all these cells, the reactivity again being highest in M38K cells. Depletion of glutathione by buthionine sulphoximine and inhibition of catalase by aminotriazole enhanced hydrogen peroxide toxicity in all cell types, while only the depletion of glutathione increased epirubicin toxicity. We conclude that simultaneous induction of multiple antioxidant enzymes can occur in human mesothelioma cells. In addition to the high MnSOD activity, hydrogen peroxide scavenging antioxidant enzymes, glutathione and GST can partly explain the high hydrogen peroxide and epirubicin resistance of these cells in vitro.

EXPRESSION OF PLATELET-DERIVED GROWTH FACTOR (PDGF) AND PDGF RECEPTORS IN HUMAN MALIGNANT MESOTHELIOMAIN VITRO ANDIN VIVO

The expression of platelet-derived growth factor (PDGF) and PDGF receptors was studied in human normal and malignant mesothelial cells in vitro and in vivo. Staining with anti-cytokeratin and ME1 antibodies and ultrastructural analysis confirmed the mesothelial nature of the cell lines used to study PDGF and PDGF receptor expression in vitro. Using antibodies, mesothelioma cell lines were found to express PDGF and both the PDGF alpha- and the PDGF beta-receptor, whereas cultured normal mesothelial cells expressed PDGF and PDGF alpha-receptor. This PDGF and PDGF receptor staining pattern largely reflects the earlier described mRNA expression in these cell lines. The only exception was the immunocytochemical detection of PDGF alpha-receptors in the mesothelioma cell lines, which is different from the inability to detect alpha-receptor transcripts on Northern blots. Expression was also investigated in mesothelial cells in vivo. Expression of PDGF was observed in malignant mesothelioma cells on frozen tissue sections. In pleural effusions, a double immunofluorescence staining procedure for PDGF and epithelial membrane antigen (EMA) revealed PDGF expression by EMA-positive malignant mesothelioma cells. PDGF beta-receptors and occasionally PDGF alpha-receptors were detected in frozen tissue sections of malignant mesotheliomas, whereas mesothelioma cells in effusions showed faint expression of only the PDGF beta-receptor. In contrast, in effusions containing non-malignant mesothelial cells, only a very low level of PDGF alpha-receptor could be detected. Taken together, these results indicate that the pattern of PDGF and PDGF receptor expression in mesothelial cells in vivo largely corresponds to expression of PDGF and its receptors in vitro. Malignant mesothelioma cell lines thus constitute a good model system for studies on the role of PDGF in this malignancy. Furthermore, the data reported in this paper are consistent with the idea that an autocrine growth stimulatory effect of PDGF via PDGF receptors may play a role in the pathogenesis of malignant mesothelioma.

EXPRESSION OF PLATELET‐DERIVED GROWTH FACTOR (PDGF) AND PDGF RECEPTORS IN HUMAN MALIGNANT MESOTHELIOMA IN VITRO AND IN VIVO

The expression of platelet-derived growth factor (PDGF) and PDGF receptors was studied in human normal and malignant mesothelial cells in vitro and in vivo. Staining with anti-cytokeratin and ME1 antibodies and ultrastructural analysis confirmed the mesothelial nature of the cell lines used to study PDGF and PDGF receptor expression in vitro. Using antibodies, mesothelioma cell lines were found to express PDGF and both the PDGF α- and the PDGF β-receptor, whereas cultured normal mesothelial cells expressed PDGF and PDGF α-receptor. This PDGF and PDGF receptor staining pattern largely reflects the earlier described mRNA expression in these cell lines. The only exception was the immunocytochemical detection of PDGF α-receptors in the mesothelioma cell lines, which is different from the inability to detect α-receptor transcripts on Northern blots. Expression was also investigated in mesothelial cells in vivo. Expression of PDGF was observed in malignant mesothelioma cells on frozen tissue sections. In pleural effusions, a double immunofluorescence staining procedure for PDGF and epithelial membrane antigen (EMA) revealed PDGF expression by EMA-positive malignant mesothelioma cells. PDGF β-receptors and occasionally PDGF α-receptors were detected in frozen tissue sections of malignant mesotheliomas, whereas mesothelioma cells in effusions showed faint expression of only the PDGF β-receptor. In contrast, in effusions containing non-malignant mesothelial cells, only a very low level of PDGF α-receptor could be detected. Taken together, these results indicate that the pattern of PDGF and PDGF receptor expression in mesothelial cells in vivo largely corresponds to expression of PDGF and its receptors in vitro. Malignant mesothelioma cell lines thus constitute a good model system for studies on the role of PDGF in this malignancy. Furthermore, the data reported in this paper are consistent with the idea that an autocrine growth stimulatory effect of PDGF via PDGF receptors may play a role in the pathogenesis of malignant mesothelioma.

Radioimmunometric demonstration of immunoglobulin G on cancer cells derived from malignant pleural effusions.

Well-preserved viable cancer cells, lymphocytes and mesothelial cells were prepared from various human malignant and non-malignant pleural effusions by a Ficoll-Hypaque discontinuous gradient. Using 125I-labelled protein A from Staphylococcus aureus as tracer, IgG was found on all cancer cells and also on apparently non-malignant mesothelial cells. Loss of cell surface IgG by overnight incubation, and fluctuation of surface IgG levels in the same patient occurred with cancer cells as well as with mesothelial cells. The IgG in vivo coat of cancer cells reflected the IgG levels of the sera rather than those of the pleural effusions. Binding of IgG from autologous and allogeneic sera and from supernatants of acid and mild overnight elution of cancer cells and mesothelial cells did not indicate a cancer-specific uptake of IgG. Various unspecific modes of IgG uptake by cancer cells are discussed including the possibility of a non-immune mechanism. Modification and loss of antigens by metastatic cells have also been considered.