Overexpression of NAT10 induced platinum drugs resistance in breast cancer cell / 中华肿瘤杂志

Objective: To observe the platinum drugs resistance effect of N-acetyltransferase 10 (NAT10) overexpression in breast cancer cell line and elucidate the underlining mechanisms. Methods: The experiment was divided into wild-type (MCF-7 wild-type cells without any treatment) group, NAT10 overexpression group (H-NAT10 plasmid transfected into MCF-7 cells) and NAT10 knockdown group (SH-NAT10 plasmid transfected into MCF-7 cells). The invasion was detected by Transwell array, the interaction between NAT10 and PARP1 was detected by co-immunoprecipitation. The impact of NAT10 overexpression or knockdown on the acetylation level of PARP1 and its half-life was also determined. Immunostaining and IP array were used to detect the recruitment of DNA damage repair protein by acetylated PARP1. Flow cytometry was used to detect the cell apoptosis. Results: Transwell invasion assay showed that the number of cell invasion was 483.00±46.90 in the NAT10 overexpression group, 469.00±40.50 in the NAT10 knockdown group, and 445.00±35.50 in the MCF-7 wild-type cells, and the differences were not statistically significant (P>0.05). In the presence of 10 μmol/L oxaliplatin, the number of cell invasion was 502.00±45.60 in the NAT10 overexpression group and 105.00±20.50 in the NAT10 knockdown group, both statistically significant (P<0.05) compared with 219.00±31.50 in wild-type cells. In the presence of 10 μmol/L oxaliplatin, NAT10 overexpression enhanced the binding of PARP1 to NAT10 compared with wild-type cells, whereas the use of the NAT10 inhibitor Remodelin inhibited the mutual binding of the two. Overexpression of NAT10 induced PARP1 acetylation followed by increased PARP1 binding to XRCC1, and knockdown of NAT10 expression reduced PARP1 binding to XRCC1. Overexpression of NAT10 enhanced PARP1 binding to LIG3, while knockdown of NAT10 expression decreased PARP1 binding to LIG3. In 10 μmol/L oxaliplatin-treated cells, the γH2AX expression level was 0.38±0.02 in NAT10 overexpressing cells and 1.36±0.15 in NAT10 knockdown cells, both statistically significant (P<0.05) compared with 1.00±0.00 in wild-type cells. In 10 μmol/L oxaliplatin treated cells, the apoptosis rate was (6.54±0.68)% in the NAT10 overexpression group and (12.98±2.54)% in the NAT10 knockdown group, both of which were statistically significant (P<0.05) compared with (9.67±0.37)% in wild-type cells. Conclusion: NAT10 overexpression enhances the binding of NAT10 to PARP1 and promotes the acetylation of PARP1, which in turn prolongs the half-life of PARP1, thus enhancing PARP1 recruitment of DNA damage repair related proteins to the damage sites, promoting DNA damage repair and ultimately the survival of breast cancer cells.

Effect of atorvastatin on autophagy and cholesterol level in foam cells by activating transcription factor EB / 医学研究生学报

ObjectiveRecent studies revealed that the transcription factor EB (TFEB) plays an important role in regulating autophagy, reducing intracellular lipids, and inhibiting atherosclerosis. This study aims to explore the effects of atorvastatin (ATV) on autophagy and cholesterol levels of foam cells by activating TFEB.MethodsHuman mononuclear cell line THP-1 was cultured in vitro and induced to differentiate into macrophages using phorbol ester. Oxidized low-density lipoprotein (oxLDL) was added to macrophages, which were induced for 48 hours to establish a foam cell model. The experiment was divided into four groups: blank group, model group (oxLDL group), oxLDL+ Chloroquine (CQ) group, oxLDL+ ATV group, and oxLDL+CQ+ ATV group. Cells in each group were treated with drugs for 48 h. The toxicity of ATV and chloroquine on the cells was detected by the CCK8 method. Oil red O staining was used to test the level of lipid droplets. Oxidase method was used to detect levels of intracellular free cholesterol (FC), total cholesterol (TC) and others related to. Cholesterol efflux fluorescence analysis was used to determine the cholesterol efflux rate of the cells. Expression of I, P62, TFEB, LAMP 1 protein was determined by Western blot.ResultsThe results of the CCK8 method showed that the cell survival rate decreased significantly with the increase of ATV and CQ concentrations (P<0.01). Compared to the blank group, the levels of lipid droplets, FC, TC, and CE/TC in the model group significantly increased (P<0.05), and the cholesterol outflow rate significantly decreased (P<0.05). Compared to the model group, the intracellular lipid droplets, FC, TC, and CE/TC levels in the oxLDL+CQ group elevated significantly (P<0.05), while the cholesterol outflow rate decreased significantly (P<0.05). The intracellular lipid droplets, FC, TC, and CE/TC contents in the oxLDL+ATV group decreased significantly (P<0.05), and the cholesterol outflow rate increased significantly (P<0.05). Compared to the oxLDL+CQ group, the intracellular lipid droplets, FC, TC, and CE/TC content in the oxLDL+CQ+ATV group decreased significantly (P<0.05), while the cholesterol outflow rate increased significantly (P<0.05). Western blotting results showed that protein expression levels of LC3II/I, P62 and TFEB were decreased in the model histone in comparison to the blank group (P<0.05). Compared to the expression levels of LC3II/I, P62, TFEB and LAMP1 ((1.006±0.052), (0.183±0.013), (0.333±0.020), and (0.957±0.026)) in the model group, the expression levels of oxLDL+CQ histamine ((1.594±0.017), (0.257±0.006), (0.477±0.024), and (0.957±0.026)) were significantly higher (P<0.05).The protein expression levels of LC3II/I, TFEB and LAMP1 in oxLDL+ATV group ((1.146±0.060), (0.540±0.031), and (1.027±0.054)) were increased, while the protein expression levels of P62 (0.115±0.009) were decreased (P<0.05). Compared to LC3II/I and P62 in oxLDL+CQ group, the expression level of oxLDL+CQ+ATV histone was decreased ((1.419±0.036) and (0.165±0.006)), and the difference was statistically significant (P< 0.05).ConclusionATV can increase the cholesterol outflow rate of macrophages, reduce the level of cholesterol and lipid droplets in macrophages, and reduce the formation of foam cells. The mechanisms behind this are still unknown, which may be related to the activation of TFEB by ATV and influencing the process of autophagy.