Experimental On Bufotaline Induced Senescence-like Phenotype Mechanism Of Chronic Myeloid Leukemia K562 Cells

Objective:This study took Human Chronic Myeloid Leukemia K562 cells as the research object,and explored the proliferation effect of Bufotaline on Human Chronic Myeloid Leukemia,the introduction effect in senescence-like phenotype of K562 cells and its mechanism.The effects of Bufotaline on K562 cells proliferation,aging-related mRNA levels,protein levels and cell morphology were examined to investigate the mechanism of senescence-like phenotype in K562 cells induced by Bufotaline.Methods:1.K562 cells in logarithmic growth phase were divided into control group and experimental group(0.0078125 μg/m L,0.015625 μg/mL,0.03125 μg/mL,0.0625 μg/mL,0.125 μg/m L,0.25 μg/mL,0.5 μg/mL,1 μg/ml Bufotaline)and were cultured for 24 h,48 h,72 h,and 96 h.The effect of Bufotaline on the proliferation of K562 cells was detected by the CCK-8 kit.2.K562 cells in logarithmic growth phase were divided into control group and experimental group(0.01 μg/mL,0.05 μg/mL,0.1 μg/mL Bufotaline)and were cultured for96 h.The changes of proliferation were observed with a microscope every day and take photos.3.K562 cells in logarithmic growth phase were divided into a control group and an experimental group(0.01 μg/mL Bufotaline,0.01 μg/m L Bufotaline + NAC,0.01 μg/mL Bufotaline + PFT-α)and cultured for 96 h.The morphological changes were observed with a microscope every day and take photos.4.K562 cells in logarithmic growth phase were divided into a control group and an experimental group(0.01 μg/mL Bufotaline,0.01 μg/m L Bufotaline + NAC,0.01 μg/mL Bufotaline + PFT-α)and were cultured for 96 h.Real-Time PCR was used to detect the effects of NAC(ROS inhibitor)and PFT-α(p53 inhibitor)on the mRNA levels of aging-related indicators p53 and p21.5.K562 cells in logarithmic growth phase were divided into control group and experimental group(0.01 μg/mL,0.05 μg/mL,0.1 μg/mL Bufotaline)and cultured for 96 h.Western blot was used to detect the effect of aging-related indicators p53,p21,p16 and pRb when treated with different concentrations of Bufotaline.6.K562 cells in logarithmic growth phase were divided into a control group andexperimental group(K562 cells were cultured for 24 h,48 h,72 h,96 h with 0.01 μg / mL Bufotaline).Western blot was used to detect the effect of aging-related indicators p53,p21,p16,pRb and B-myb when treated with different concentrations of Bufotaline.7.K562 cells in the logarithmic growth phase were divided into a control group and experimental group(0.01 μg/mL Bufotaline,0.01 μg/m L Bufotaline + NAC,0.01 μg/mL Bufotaline + PFT-α)and cultured for 96 h.Western blot was used to detect the effects of NAC and PFT-α on the expression of aging-related indicators p53,p21,p16,pRb and B-myb.Results:1.CCK-8 results and the photos of proliferation showed that Bufotaline has a significant inhibitory effect on K562 cells,and its inhibitory effect becomes more pronounced as the increased concentration and time extension.2.According to morphological photos results,Bufotaline can induce senescence-like phenotype of K562 cells,and this result can be weakened by pretreatment of K562 cells with ROS inhibitors or p53 inhibitors.3.Real-Time PCR results showed that the mRNA expression levels of aging-related indicators p53 and p21 were increased.This result can be weakened by pretreatment of K562 cells with ROS inhibitors and p53 inhibitors.4.The results of Western blot showed that the protein expression levels of aging-related indicators p53,p21 and p16 were positively correlated with the concentration of Bufotaline,and the protein expression level of pRb and B-myb was negatively correlated with the concentration of Bufotaline.This result can be weakened by pretreatment of K562 cells with ROS inhibitors and p53 inhibitors.Conclusions:1.Bufotaline has a significant inhibitory effect on K562 cells,and its inhibitory effect becomes more pronounced as the increased concentration and time extension.2.Bufotaline can significantly induce senescence-like phenotype in K562 cells.The mechanism is related to the activation of p53 / p21,p16 / pRb pathways and the expression of B-myb.