Autophagy In Adriamycin Induced K562and K562/ADM Cells Apoptosis In The Mechanism Of Action

Objective:To Investigate the mechanism of autophagy in adriamycin (ADM)-indueed K562,K562/ADM cells apoptosis and to examine expression of Beclinl, Survivin,Caspase-3,Caspase-8,Caspase-9mRN A.Method:MTT colorimetry was used to detect the sensitivity of K562, K562/ADM cells to ADM and the effects of ADM on these cell proliferation after the preprocessing of3-MA; Flow cytometry was used to detect cell lines apoptosis rate; Real-time fluorescent quantitative PCR method was used to detect the expression of cell lines Beclinl, Survivin Caspase-3,Caspase-8,Caspase-9mRNA.Results:l.The growth of K562, K562/ADM cells appears different degree of inhibition after they were treated with different concentrations of ADM by24h,48h,72h, According to concentration-inhibition rate curve, the effect of proliferation inhibition of ADM on K562and K562/ADM cells appears concentration and time dependence. This study aimed to investigate the mechanism of the effsets of autophagy on ADM-induced apoptosis, so10μg/ml was choosed as the optimum concentration.2.The effect of Concentration of1,5,10mmol/L of3-MA (ADM joined0.5h before treatment) in combination with10μg/ml ADM on K562, K562/ADM cell proliferation inhibition was more significant than ADM10μg/ml alone (P<0.05), and with the growth of3-MA concentration and the elongation of time, it appeared concentration and time dependence and highest inhibition rate in72h. This study aims to observe the effects of autophagy on the ADM induced K562and K562/ADM cells apoptosis, so the concentration of10mmol/L was choosed to carry out subsequent experiments.3.Flow cytometry shows:using3-MA10mmol/L blocked autophagy before ADM10μg/ml delivery in the first0.5h, K562and K562/ADM cells apoptosis rate was more significant than ADM10mu g/ml single (P<0.05). the apoptosis rates of24h,48h,72h were2.42,2.15,1.31and 2.35,2.26,1.47times larger than ADM group and the apoptosis rate of72h reached the highest.4.RT-PCR shows:compared with the use of ADM10μg/ml monotherapy using3-MA10mmol/L blocked autophagy before ADM10μg/ml delivery in the first0.5h, the relative expression of K562, K562/ADM cells Beclinl, Survivin mRNA was significantly decreased (P <0.05), and they were positively correlated (r=0.827, P<0.01). the expression levelof Caspase-3, Caspase-8. Caspase-9mRNA increased more than ADM drug alone (P<0.05). especially in72h.Conclusion:1.In the early of ADM administion, autophagy has a protective effect on cells. It can be used as a defense mechanism against the cellular damage caused by environmental changes and delayed cell apoptosis.2. Blocking autophagy before ADM10μg/ml delivery,3-MA promotes ADM-induced K562, K.562/ADM apoptosis. It may be related to reduced Beclin1mRNA expression which lead to the inhibition of Survivin expression.3.Compared with ADM alone, Beclin1mRNA expression of the pretreated K562, K562/ADM cells by3-MA was downregulated, and Caspase-3, Caspase-8, Caspase-9mRNA expression was significantly increased (P<0.05). These results showed Beclin1and Caspase-3, Caspase-8, Caspase-9plays an important role in adriamycin-induced K562, K562/ADM cell autophagy and apoptosis. Beclin1may affect apoptosis by regulating Caspase-3, Caspase-8, Caspase-9transcript levels.