And counting cells [47]. Constant with its proliferative part, 2′-O-Methyladenosine Metabolic Enzyme/Protease pancreatic cancer result, the cells became arrested inside the G1 phase plus the proportion of cell cycle progressionphase decreased. These events have been anti-TRPM8 siRNA exhibited impairment of cells entering the S [47]. Consequently, the cells became CDKN2A and related withthe G1 phase and from the cyclin-dependent kinases S phase decreased.p27CDKN2B , constant arrested in accumulation the proportion of cells entering the p21 These events have been with connected arrestaccumulation from the cyclin-dependent kinases p21CDKN2A and p27CDKN2B, consistent cell cycle with within the G1 phase [47]. with cell cycle arrest in the G1 phase role Constant using the proliferative[47]. of TRPM8, pancreatic cancer cells with down-regulated Consistent using the proliferative part of TRPM8, pancreatic cancer Morphological examination expression of TRPM8 exhibited options of replicative senescence. cells with down-regulated expression of TRPM8multiple nuclei, suggesting a defect in cell division [49] (Figure 2). Employing Clomazone Protocol revealed the presence of exhibited capabilities of replicative senescence. Morphological examination revealed the presence of several nuclei, suggesting a defect in cell division [49] (Figure two). senescence-associated -galactosidase (SAG) as a marker of cellular senescence, siRNA-mediated Making use of 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 expected required preserving the uncontrolled proliferation of cancer cells cells through regulation ofcyclecycle for for sustaining the uncontrolled proliferation of cancer via regulation of cell cell progression andand replicative senescence. progression replicative senescence.Cancers 2015, 7, web page ageFigure 2. 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 manage The BxPC-3 incubated at 37cells till evaluation. Prime with anti-TRPM8 siRNA cells. siRNA and and PANC-1 have been transfected panel, phase-contrast non-targeting or non-targeting showing that TRPM8-deficient cells contain a number of nuclei and cytoplasmic vacuoles. handle siRNA and incubated at 37 C till analysis. Leading panel, phase-contrast micrographs micrographspanel, DAPI-stained fluorescent micrographs displaying that nuclei and cytoplasmic vacuoles. Bottom displaying that TRPM8-deficient cells contain a number of TRPM8-deficient cells include Bottom panel, DAPI-stained fluorescent micrographs nuclei. For comparison, in both phase-contrast nuclei getting arrested in division consistent with several showing that TRPM8-deficient cells contain and fluorescent micrographs, manage siRNA-transfected cells contain round to comparison, in nuclei being arrested in division consistent with many nuclei. For oval shaped nuclei both with a smooth surface, and no or couple of cytoplasmic vacuoles. phase-contrast and fluorescent micrographs, manage siRNA-transfected cells contain round to oval shaped nuclei with a smooth surface, and no or handful of cytoplasmic vacuoles. The proliferative role 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. Inside the A.