Demethylation of TMS1 gene sensitizes thyroid cancer cells to trail-induced apoptosis

Context:TMS1 is a tumor suppressor gene that encodes for caspase recruitment domain containing regulatory protein and has been shown to be hypermethylated in various cancers. However, its methylation status has not been investigated in thyroid cancer. Therefore, we studied the methylation of TMS1 and its functional consequence in thyroid cancer. Design: The methylation status of the promoter region of the TMS1 gene was determined using methylation-specific PCR in 40 papillary thyroid cancer samples, 10 normal thyroid tissue, and seven thyroid cancer cell lines. RT-PCR and Western blot analysis were used to assess the expression levels. 5-aza-2′-deoxycytidine was used to demethylate the thyroid cancer cell lines. Cell viability and apoptosis was determined by dimethylthiazoldiphenyltetra-zoliumbromide and flow cytometry. Results: Twenty-three percent of the papillary thyroid carcinoma samples were found to be methylated for the TMS1 gene. Two of seven thyroid cell lines were either completely or partially methylated for the TMS1 gene. The treatment of methylated thyroid cancer cell lines with 5-aza-2′-deoxycytidine resulted in the demethylation of the TMS1 gene leading to the restoration of its expression. After demethylation, treatment of cells with TNF-related apoptosis-inducing ligand (TRAIL) led to the induction of apoptosis via activation of caspases-8, caspase-3, and poly(ADP-ribose) polymerase. Interestingly, gene silencing of TMS1 using TMS1-specific small interfering RNA prevented TRAIL-mediated apoptosis. Conclusion: Our results demonstrated that the TMSI gene is methylated in thyroid cancer cells and repression of methylation by 5-aza-2′-deoxycytidine restored expression of the TMS1 gene and sensitized cells to TRAIL-induced apoptosis. These findings suggest that the TMS1 gene can be targeted by combination of demethylating agents with TRAIL to induce efficient apoptosis in thyroid cancer cells

Demethylation of TMS1 gene sensitizes thyroid cancer cells to trail-induced apoptosis

Context:TMS1 is a tumor suppressor gene that encodes for caspase recruitment domain containing regulatory protein and has been shown to be hypermethylated in various cancers. However, its methylation status has not been investigated in thyroid cancer. Therefore, we studied the methylation of TMS1 and its functional consequence in thyroid cancer. Design: The methylation status of the promoter region of the TMS1 gene was determined using methylation-specific PCR in 40 papillary thyroid cancer samples, 10 normal thyroid tissue, and seven thyroid cancer cell lines. RT-PCR and Western blot analysis were used to assess the expression levels. 5-aza-2′-deoxycytidine was used to demethylate the thyroid cancer cell lines. Cell viability and apoptosis was determined by dimethylthiazoldiphenyltetra-zoliumbromide and flow cytometry. Results: Twenty-three percent of the papillary thyroid carcinoma samples were found to be methylated for the TMS1 gene. Two of seven thyroid cell lines were either completely or partially methylated for the TMS1 gene. The treatment of methylated thyroid cancer cell lines with 5-aza-2′-deoxycytidine resulted in the demethylation of the TMS1 gene leading to the restoration of its expression. After demethylation, treatment of cells with TNF-related apoptosis-inducing ligand (TRAIL) led to the induction of apoptosis via activation of caspases-8, caspase-3, and poly(ADP-ribose) polymerase. Interestingly, gene silencing of TMS1 using TMS1-specific small interfering RNA prevented TRAIL-mediated apoptosis. Conclusion: Our results demonstrated that the TMSI gene is methylated in thyroid cancer cells and repression of methylation by 5-aza-2′-deoxycytidine restored expression of the TMS1 gene and sensitized cells to TRAIL-induced apoptosis. These findings suggest that the TMS1 gene can be targeted by combination of demethylating agents with TRAIL to induce efficient apoptosis in thyroid cancer cells

c-Met Inhibitor Synergizes with Tumor Necrosis Factor–Related Apoptosis-Induced Ligand to Induce Papillary Thyroid Carcinoma Cell Death

The Met receptor tyrosine kinase is overexpressed and/or activated in variety of human malignancies. Previously we have shown that c-Met is overexpressed in Middle Eastern papillary thyroid carcinoma (PTC) and significantly associated with an aggressive phenotype, but its role has not been fully elucidated in PTC. The aim of this study was to determine the functional link between the c-Met/AKT signaling pathway and death receptor 5 (DR5) in a large cohort of PTC in a tissue microarray format followed by functional studies using PTC cell lines and nude mice. Our data showed that high expressions of p-Met and DR5 were significantly associated with an aggressive phenotype of PTC and correlated with BRAF mutation. Treatment of PTC cell lines with PHA665752, an inhibitor of c-Met tyrosine kinase, inhibited cell proliferation and induced apoptosis via the mitochondrial pathway in PTC cell lines. PHA665752 treatment or expression of c-Met small interfering (si)RNA resulted in dephosphorylation of c-Met, AKT and its downstream effector molecules. Furthermore, PHA665752 treatment upregulated DR5 expression via generation of reactive oxygen species in PTC cell lines, and synergistically potentiated death receptor–induced apoptosis with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Finally, cotreatment with PHA665752 and TRAIL caused more pronounced effects on PTC xenograft tumor growth in nude mice. Our data suggest that the c-Met/AKT pathway may be a potential target for therapeutic intervention for treatment of PTC refractory to conventionally therapeutic modalities