And counting cells [47]. Consistent with its proliferative role, pancreatic cancer result, the cells Umbellulone custom synthesis became arrested in the G1 phase and also the proportion of cell cycle progressionphase decreased. These events were anti-TRPM8 siRNA exhibited impairment of cells entering the S [47]. As a result, the cells became CDKN2A and connected withthe G1 phase and of the cyclin-dependent kinases S phase decreased.p27CDKN2B , consistent arrested in accumulation the proportion of cells entering the p21 These events were with related arrestaccumulation of the cyclin-dependent kinases p21CDKN2A and p27CDKN2B, constant cell cycle with in the G1 phase [47]. with cell cycle arrest inside the G1 phase part Consistent with the proliferative[47]. of TRPM8, pancreatic cancer cells with down-regulated Constant using the proliferative role of TRPM8, pancreatic cancer Morphological examination expression of TRPM8 exhibited functions of replicative senescence. cells with down-regulated expression of TRPM8multiple nuclei, suggesting a defect in cell division [49] (Figure two). Utilizing revealed the presence of exhibited attributes of replicative senescence. Morphological examination revealed the presence of multiple nuclei, suggesting a defect in cell division [49] (Figure 2). senescence-associated -galactosidase (SAG) as a marker of cellular senescence, siRNA-mediated Employing senescence-associated -galactosidase (SAG) as a marker of cellular senescence, siRNA-mediated silencing of TRPM8 induced expression of SAG [49]. These findings indicate that TRPM8 is silencing of TRPM8 induced expression of SAG [49]. These findings indicate that TRPM8 is essential expected preserving the uncontrolled proliferation of cancer cells cells by way of regulation ofcyclecycle for for preserving the uncontrolled proliferation of cancer by way of regulation of cell cell progression andand replicative senescence. progression replicative senescence.Cancers 2015, 7, page ageFigure two. Targeted silencing of TRPM8 induces mitotic abnormalities and replicative arrest in pancreatic cancer cells. The BxPC-3 and PANC-1 cells were transfected with anti-TRPM8 siRNA or pancreatic cancer control The BxPC-3 incubated at 37cells until evaluation. Best with anti-TRPM8 siRNA cells. siRNA and and PANC-1 have been transfected panel, phase-contrast non-targeting or non-targeting displaying that TRPM8-deficient cells include various nuclei and cytoplasmic vacuoles. handle siRNA and incubated at 37 C until analysis. Best panel, phase-contrast micrographs micrographspanel, DAPI-stained fluorescent micrographs displaying that nuclei and cytoplasmic vacuoles. Bottom displaying that TRPM8-deficient cells contain several TRPM8-deficient cells contain Bottom panel, DAPI-stained fluorescent micrographs nuclei. For comparison, in each phase-contrast nuclei getting arrested in division constant with many showing that TRPM8-deficient cells contain and fluorescent micrographs, control siRNA-transfected cells include round to comparison, in nuclei becoming arrested in division constant with a number of nuclei. For oval shaped nuclei each having a smooth surface, and no or few cytoplasmic vacuoles. phase-contrast and fluorescent micrographs, manage siRNA-transfected cells include round to oval shaped nuclei having a smooth surface, and no or few cytoplasmic vacuoles. The proliferative function of TRPM8 in cancer cells can also be demonstrated in AR+ prostatic carcinoma (LNCaP), osteosarcoma (MG-63 and U2OS), and colon cancer (Caco-2) cell lines. Within the A.