Studies On The Proliferation And Apoptosis Effects Of AICAR On Human Cervical Carcinoma Cell Line CaSki And Neonatal Rat Cardiac Fibroblasts And Their Mechanisms

Part IAim:5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) is a widely used activator of AMP activated protein kinase (AMPK), which is an important metabolic sensor of cellular energy status. In this study, we studied the effect of AICAR on the proliferation, cell cycle and apoptosis in human cervical carcinoma cell line, CaSki cells. Methods: Cell count and MTT assay were used to determine the cell proliferation and cell viability. Hoechst 33258 staining was conducted to distinguish the apoptotic cells. Cell cycle and annexin V/PI staining were analyzed by FACS. Western blot assay was used to evaluate the expression of AKT, mammalian target of rapamycin (mTOR), p53 and ERK. Results: AICAR (500μM) significantly inhibited the proliferation of CaSki cells at 24, 48 and 72 hours by cell count. Cells at G1-phase and G2-phase were dramatically decreased while cells at S-phase were increased in respond to AICAR treatment at 24, 48 and 72 hours. Besides, MTT cell viability assay showed less viable cells and Hochest fluorescent staining appeared more apoptotic cells upon AICAR stimulation. Results of Annexin V staining demonstrated a time-dependent increase of apoptosis in cells treated with AICAR for 24, 36 and 48 hours. Furthermore, AICAR activated caspase-3 in a time-dependent manner. It is also found that AICAR inhibited the phosphorylation of AKT and mTOR, which are important kinases regulating cell growth and survival. Moreover, AICAR stimulation obviously increased the expression of the tumor suppressor, p53, and the phosphorylation of ERK. Conclusion: AICAR inhibited proliferation, induced S-phase arrest and promoted apoptosis in CaSki cells, which might be mediated by the downregulation of AKT/mTOR pathway and upregulation of p53/ERK pathway. AICAR may be a useful therapeutic drug against cervical cancer. Part IIAMP-activated protein kinase (AMPK), an energy sensor of cellular metabolism, is also found recently to be involved in myocardial remodeling. In this study, we investigated 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an AMPK activator, on growth and proliferation in neonatal rat cardiac fibroblasts (NRCFs). AICAR significantly inhibited the cell numbers and DNA synthesis under both serum-free and 10% FBS-containing medium, and arrested cell cycle progression by serum stimulation at G1 phase. Furthermore, we demonstrated for the first time that serum inhibited both basal and AICAR-induced AMPK phosphorylation, which further supports the role of negative regulation by AMPK on growth and proliferation.To explore the signaling pathways responsible for the anti-proliferation by AICAR, we focused on the two most important pathways, ERK (extracellular regulated protein kinase) and AKT. Interestingly, AICAR did not inhibit AKT phosphorylation by serum at all time points we examined in 24 hours, we tested three putative downstream substrates of AKT: glycogen synthase kinase 3 (GSK3), mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70S6K). Similar to AKT, AICAR also did not affect GSK3 phosphorylation. However, AICAR rapidly inhibited mTOR and p70S6K at 10 minutes, and the extent of inhibition lessened at 30 minutes but increased at 60 minutes. Unlike mTOR /p70S6K pathway, the second pathway was at about 3 hours starting from inhibition of ERK. Taken together, these results demonstrated that AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts probably by ERK and mTOR /p70S6K.