And counting cells [47]. Constant with its proliferative part, pancreatic cancer outcome, the cells 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 getting into the S [47]. Consequently, the cells became CDKN2A and connected withthe G1 phase and of the cyclin-dependent kinases S phase decreased.p27CDKN2B , constant arrested in accumulation the proportion of cells getting into the p21 These events have been with associated arrestaccumulation on the cyclin-dependent kinases p21CDKN2A and p27CDKN2B, constant cell cycle with within the G1 phase [47]. with cell cycle bis-PEG2-endo-BCN Formula arrest in the G1 phase part Consistent with the proliferative[47]. of TRPM8, pancreatic cancer cells with down-regulated Constant using the proliferative part 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). Using revealed the presence of exhibited characteristics of replicative senescence. Morphological examination revealed the presence of many nuclei, suggesting a defect in cell division [49] (Figure two). senescence-associated -galactosidase (SAG) as a marker of cellular senescence, siRNA-mediated Using senescence-associated -galactosidase (SAG) as a marker of cellular senescence, siRNA-mediated 329059-55-4 manufacturer 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 expected needed keeping the uncontrolled proliferation of cancer cells cells via regulation ofcyclecycle for for maintaining the uncontrolled proliferation of cancer through 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 have been transfected with anti-TRPM8 siRNA or pancreatic cancer control The BxPC-3 incubated at 37cells until analysis. Top rated with anti-TRPM8 siRNA cells. siRNA and and PANC-1 had been transfected panel, phase-contrast non-targeting or non-targeting showing that TRPM8-deficient cells contain many nuclei and cytoplasmic vacuoles. control siRNA and incubated at 37 C until analysis. Leading panel, phase-contrast micrographs micrographspanel, DAPI-stained fluorescent micrographs displaying that nuclei and cytoplasmic vacuoles. Bottom showing that TRPM8-deficient cells contain multiple TRPM8-deficient cells contain Bottom panel, DAPI-stained fluorescent micrographs nuclei. For comparison, in each phase-contrast nuclei getting arrested in division constant with several displaying that TRPM8-deficient cells include and fluorescent micrographs, handle siRNA-transfected cells contain round to comparison, in nuclei being arrested in division constant with several nuclei. For oval shaped nuclei each having a smooth surface, and no or few cytoplasmic vacuoles. phase-contrast and fluorescent micrographs, handle siRNA-transfected cells include round to oval shaped nuclei having a smooth surface, and no or handful of cytoplasmic vacuoles. The proliferative part of TRPM8 in cancer cells is also demonstrated in AR+ prostatic carcinoma (LNCaP), osteosarcoma (MG-63 and U2OS), and colon cancer (Caco-2) cell lines. In the A.