Molecular mechanism of mitochondrial autophagy induced by small molecule targeted drug SKLB-BH128 against colorectal cancer
Objective To investigate the molecular mechanism of mitochondrial autophagy induced by SKLB-BH128 in colorectal cancer.Methods Colorectal cancer cell line SW620 cells were randomly divided into control group,50 ng/ml SKLB-BH128 group and 100 ng/ml SKLB-BH128 group.The cells in control group,50 ng/ml SKLB-BH128 group and 100 ng/ml SKLB-BH128 group were treated with 0 ng/ml,50 ng/ml and 100 ng/ml SKLB-BH128 for 24 h,respectively,and the viability and number of clones were analyzed by cell counting kit-8(CCK-8)assay.Protein immune stress analysis was used to si-lence the expression of regulatory protein 3 sirtuin 3(SIRT3),translocase of outer mitochondrial membrane 20(TOM20),forkhead box O3 A(FOXO3A),phosphatase and tensin homologue deleted on chromosome ten-induced putative kinase(PINK1),Parkin,microtubule-associated protein 1 light chain 3(LC3)pro-teins.The LC3-Ⅱ/LC3-Ⅰ ratio was calculated.The effects of SKLB-BH128 on the expression of mito-chondrial autophagy protein were analyzed in SIRT3 overexpressed colon cancer cells.The cell apoptosis was analyzed by flow cytometry.The measurement data of SKLB-BH128 were compared by single factor a-nalysis.Results The absorbance value and clonogenic rate of cells in the control group[1.75±0.07,(83.50±8.88)％]were higher than those in the 50 ng/ml SKLB-BH128 group[1.34±0.16,(72.25±6.09)％],and the difference was statistically significant(t=5.681,4.974,P＜0.05).The absorbance value and clonogenic rate of cells in the 50 ng/ml SKLB-BH128 group[1.34±0.16,(72.25±6.09)％]were significantly higher than those in the 100 ng/ml SKLB-BH128 group[0.85±0.05,(61.88±5.22)％,t=4.998,5.441,P＜0.05].The relative activity of SIRT3 in the control group(1.08±0.19)was significantly higher than that in the 50 ng/ml SKLB-BH128 group(1.48±0.15,t=6.158,P＜0.05).The relative activity of SIRT3 in the 50 ng/ml SKLB-BH128 group(1.48±0.15)was significantly higher than that in the 100 ng/ml group(1.85±0.14,t=4.612,P＜0.05).The acetylation levels of FOXO3A and Beclin1 in the control group(1.10±0.07,1.30±0.06)were significantly higher than those in the 50 ng/ml SKLB-BH128 group(0.81±0.06,0.98±0.08,t=6.879,7.562,P＜0.05).The acetylation levels of FOXO3A and Beclin1 in the 50 ng/ml SKLB-BH128 group(0.81±0.06,0.98±0.08)were significantly higher than those in the 100 ng/ml SKLB-BH128 group(0.54±0.07,0.76±0.10,t=8.224,6.325,P＜0.05).The expression level of TOM20 in the control group(1.22±0.07)was significantly higher than that in the 50 ng/ml SKLB-BH128 group(0.92±0.06,t=3.561,P＜0.05).The expression level of TOM20 in the 50 ng/ml SKLB-BH128 group(0.92±0.06)was signifi-cantly higher than that in the 100 ng/ml SKLB-BH128 group(0.64±0.10,t=3.120,P＜0.05).The PINK1 expression level and LC3-Ⅱ/LC3-Ⅰ ratio in the control group(0.81±0.07,0.71±0.08)were significantly lower than those in the 50 ng/ml SKLB-BH128 group(1.17±0.11,1.07±0.07,t=6.312,4.589,P＜0.05).The PINK1 expression level and LC3-Ⅱ/LC3-Ⅰ ratio in the 50 ng/ml SKLB-BH128 group(1.17±0.11,1.07±0.07)were lower than those in the 100 ng/ml group(1.41±0.10,1.39±0.08,t=5.140,3.441,P＜0.05).The apoptosis rate of the control group[(2.55±0.27)％]was sig-nificantly lower than that of the 50 ng/ml SKLB-BH128 group[(7.16±1.24)％,t=5.123,P＜0.05].The apoptosis rate of the 50 ng/ml SKLB-BH128 group[(7.16±1.24)％]was significantly lower than that of the 100 ng/ml SKLB-BH128 group[(19.07±2.41)％,t=6.210,P＜0.05].Conclusion SKLB-BH128 activates SIRT3,induces mitochondrial autophagy,inhibits proliferation of colon cancer cells,and induces apoptosis of tumor cells.
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