All samples were histologically confirmed and patient identities had been

Of them, MT1G expression was most significantly induced by these inhibitors in K1 cells. These data buy KU-55933 suggested that epigenetic alterations would be a major mechanism to inactivate MT1G in thy roid cancer cells. MT1G inhibits thyroid cancer cell growth Frequent down regulation or silencing of MT1G medi ated by epigenetic alterations in thyroid cancer cell lines and primary thyroid cancers but not in non malignant thyroid tissues implicated that MT1G may be a tumor suppressor. To test this speculation, we examined the growth inhibitory effect through ectopic expression of MT1G in K1, FTC133, BCPAP and C643 cells, wherein MT1G expression was relatively low and could be dra matically induced by 5 Aza dC and SAHA. MT1G re expression in the transfected cells was confirmed by conventional and real time quantitative RT PCR, respect ively.<br><br> Ectopic expression of MT1G caused a decrease in cell proliferation, par ticularly in K1 and FTC133 cells. The inhibitory effect on thyroid cancer cell growth was further confirmed by colony formation assay. As shown in, was also found in these cell lines, particularly 8305c cells that showed complete methylation. However, down regulation or silencing Linifanib FLT-3 阻害剤 of MT1G was not completely consistent with methylation status of its promoter. For example, methylation level of MT1G was not high in FTC133 cells, although its expression was nearly undetected. Thus, we supposed that other epigen etic mechanisms, such as histone modification, along with DNA methylation, were involved in MT1G inactivation in thyroid cancer cells.<br><br> To explore this, thyroid cancer cell lines were treated with a DNMT inhibitor, 5 Aza dC, and a HDAC inhibitor, SAHA, alone or in combination. MT1G expression was then analyzed LY294002 ic50 using real time quantitative RT PCR. As shown in Figure 1B, 5 Aza dC treatment only caused partial reactivation of MT1G in most of cancer cell lines. Compared with 5 Aza dC treat ment alone, MT1G expression was more significantly re stored in these cancer cells treated with SAHA alone or in the colonies formed in MT1G transfected cells were fewer and smaller than those formed in empty vector transfected cells, particularly in K1 cells. Taken to gether, MT1G exhibits the growth inhibitory ability in thyroid cancer cells and acts as a potential tumor suppressor.<br><br> MT1G induces cell cycle arrest and apoptosis of thyroid cancer cells Suppression of cell growth in cancer cells is usually asso ciated with concomitant cell cycle arrest and activation of cell death pathways. We therefore examined the con tribution of cell cycle arrest and apoptosis to the ob served growth inhibition of MT1G transfected cells. As shown in Figure 2, compared with empty vector, cell cycle was arrested at the G1 phase when cells were transfected with pEGFP N1 MT1G. The percentage of G1 phase was increased from 55. 9% to 62. 1% at 60 h post transfection, and from 59. 1% to 65. 9% at 84 h post transfection in K1 cells, and from 61. 0% to 67. 7% at 48 h post transfection, and from 62. 4% to 68. 0% at 72 h post transfection in FTC133 cells, respectively. In addition, characteristic morphologies of apoptotic nuclei, such as chromatin condensation, margination and nuclear fragmentation, were more frequently observed in cells transfected with pEGFP N1 MT1G compared with empty vector.