Experimental Study Of The Antitumous Effect And Mechanism Which Mediated By Panax Notoginseng Saponins In K562 Cells

Objective(1) To detect the effects of Panax notoginseng saponins(PNS) on cell proliferation, apoptosis and cell cycle in K562 cells and investigate the related protein expression in K562 cells.(2) To detect the effects of PNS on m TOR signaling pathway in K562 cells.(3) To detect the effects of PNS combine with anti-cancer drugs Daunorubicin on K562 cells.Methods(1) After K562 cells were treated with PNS(0 ~ 800 μg/m L) for 24 h,48h and72 h, cell proliferation was measured by MTT assay. After K562 cells were treated with PNS(100 μg/m L,200 μg/m L and 400 μg/m L) for 48 h, cellular apoptosis was detected by AO / EB double flumorescent staining and Annexin V-FITC / PI double staining.Cell cycle was observed by flow cytometry. The expression of cleaved caspeas-3,cyclin D1, Fas was assessed by Western Blotting.(2) The expressions of m TOR signaling pathway m RNA was examined by RT-PCR. The expression of m TOR,p-m TOR, p70S6 K, p-p70S6 K, 4E-BPand p-4E-BP1 proteins was assessed by Western Blotting.(3) Daunorubicin(DNR)(0 ~ 1.0 μg/m L) alone and combined treated with PNS and DNR in different periods of time, cell proliferation was measured by MTT assay. Cellular apoptosis was detected by AO / EB double flumorescent staining.Results(1) With the concentration of 100 ~ 800μg/m L, PNS can inhibit proliferation of K562 cells(P<0.05). The IC50 of PNS in K562 cells is(229.07±2.36) μg/m L. With the concentration of 0.0625 ~ 1.0 μg/ml, PNS(100 ~ 400 μg/ml) can promote apoptosis in K562 cells(P<0.05). After treated with PNS(100 ~ 400 μg/ml) for 48 h, the cell cycle was arrested in G0 / G1 phase. Western Blot analysis found that the protein level of cleaved caspase-3 was increased(P<0.05) and the protein level of cyclin D1 was decreased(P <0.05). The protein level of Fas was no significant changes.(2) After treated with PNS(100 ~ 400 μg/m L) for 72 h, the m RNA expression of m TOR was dose-dependently reduced(P<0.05). Western Blot analysis found that the protein level of m TOR was decreased(P<0.05). The protein level of p70S6 K and 4E-BP1 was no significant changes.What’s more, the protein expression of p-m TOR, p-p70S6 K and p-4E-BP1 was also inhibited dose-dependently(P<0.05).(3) DNR can inhibit proliferation of K562 cells(P<0.05). Combination of PNS and DNR in the low concentrations can increase the inhibition rate on K562 cells(P<0.05).Conclusions(1) PNS significantly inhibits the proliferation, promote the apoptosis and arrest the cell cycle in K562 cells.These effect is associated with the increase of cleaved caspase-3 protein and the decrease of cyclin D1 protein.(2) PNS significantly inhibits the activity of the m TOR signaling pathway in K562 cells, reduces the content of m TOR, p-m TOR, p-p70S6 K and p-4E-BP 1 in K562 cells.(3) Combination of PNS and DNR in the low concentrations can increase the inhibition rate on K562 cells induces apoptosis and arrests the cell cycle in K562.