Study On Mechanism Of Mitochondrial In K562/G01Cells Apoptosis Induced By Triptolide

ObjectiveImatinib, a tyrosine kinase inhibitor (TKI) of BCR-ABL, was used as the standard first-line therapy for chronic myeloid leukemia (CML) for almost10years. Imatinib inhibits tyrosine kinase activity by occupying the ATP-binding pocket of BCR-ABL, thus preventing BCR/ABL coding protein from synthesizing and achieving the anti-leukemia effect. However, due to imatinib widely used in clinical, resistance to imatinib has been an emerging problem for therapy of CML. Triptolide, the main biologically active component isolated from the traditional Chinese herbal medicine Tripterygium wilfordii Hook.f., has been shown to have potent antitumor properties. Triptolide could not only inhibit the growth of all kinds of solid tumor, but inhibit the growth and proliferation of leukemia cells.Though during our previous experiment we have proved that triptolide could potently inhibit the growth and proliferation of K562/G01cells, the definitely mechanism of apoptosis in chronic myeloid leukemia cells K562/G01cells induced by triptolide has not been very clear. In this research we intend to investigate the possible mechanism of mitochondrial in chronic myeloid leukemia cells K562/G01cells apoptosis induced by triptolide.Methods1.MTT assay was used to detect the inhibition effect of triptolide, imatinib and two dugs combined on growth of K562/G01cells;2.FCM was used to detect apoptosis rate,cell cycle, mitochondrial membrane potential and of K562/G01cells in triptolide, imatinib and two dugs combined groups;3.The real-time quantitative PCR assay was used to detect the transcription level of caspase-9, cytochrome C and BCR-ABL of K562/G01cells in triptolide,imatinib and two dugs combined groups.4. Western blot assay was used to detect protein level of cytochrome C of K562/G01cells in triptolide, imatinib and two dugs combined groups. Results1.The results of MTT assay showing:with concentration and action time increasing, the effects of growth inhibition induced by triptolide strengthen gradually, and compared with control group, the difference has statistical significance(F=76.742, P<0.001), which suggesting that triptolide inhibits the growth of K562/G01cells in a time-dose-dependent manner;and combined two drugs, this inhibition effects could be strengthened (P<0.01)2.The results of FCM assay(1) The results of cell apoptosis rate showing:the mean apoptosis rate in early stage of20、40nmol/L groups were (3.57±0.15)%,(7.27±0.25)%, which suggesting that triptolide could induce K562/G01cells apoptosising in a dose-dependent manner, and compared with control group, the difference has statistical significance (P<0.01); the mean apoptosis rate in early stage of combined groups were (8.90±0.40)%,(12.00±0.36)%, which suggesting that combined two drugs, this effects could be strengthened (P<0.01)(2) The results of cell cycle showing:with the concentration of triptolide increasing, the cells blocked in Gl phase increase gradually, and the cells blocked in S phase decrease gradually; the block effect in combined groups were more strengthen, but compared with control group, the difference has no statistical significance (P>0.05)(3) The results of mitochondrial membrane potential showing:the cell proportion in green fluorescence region of20、40、80nmol/L groups were (6.70±0.40)%,(10.90±0.30)%,(14.70±0.36)%, compared with control group, the difference has statistical significance (P<0.01), which suggesting that triptolide could make the mitochondrial membrane potential of K562/G01cells fade away, and with the concentration of triptolide increasing, the cell proportion rises in a dose-dependent manner.3. The results of real-time fluorescent quantitative PCRshowing:triptolide could down-regulate the gene expression of BCR-ABL and up-regulate the gene expression of caspase-9and cytochrome C in a dose-dependent manner, compared with control group, the difference has statistical significance (P<0.05), when two drugs combined, these effects could be strengthened (P<0.01).4. The results of Western blot showing:triptolide could up-regulate the protein expression of of cytochrome C, make the amount of cytochrome C released in cytoplasm increase gradually, and with the concentrations of triptolide increasing these effects act in a dose-dependent manner, compared with control group, the difference has statistical significance (P<0.01), when two drugs combined, these effects could be strengthened (P<0.05)Conclusions1. Triptolide could inhibited the growth and proliferation of K562/G01cells in a time-dose-dependent manner, and combined two drugs,this inhibition effect could be strengthened;2. Triptolide could make the mitochondrial membrane potential fade away, enhance the transcriptional level of caspase-9, distinctly increase the amount of cytochrome C in cytoplasm released from the mitochondria in a dose-dependent manner, when two drugs combined, these effects could be strengthened, which suggested that the mitochondria apoptosis pathway maybe the one of the pathway in apoptosis of chronic myeloid leukemia cells induced by triptolide.3.Triptolide could potently reduce the transcriptional level of BCR-ABL...