| Objective: The effect of down-regulation of acyl-Co A cholesterol acyltransferase-1(ACAT-1)expression on proliferation,migration and apoptosis of LLC cells was observed at the cell level in vitro.The effects of ACAT-1 inhibitor avasimibe on tumor growth and metastasis in Lewis lung carcinoma(LLC)mice were observed at the animal level in vivo,and the expression of ACAT-1 in Lewis lung carcinoma tissues was detected to add new content for lung cancer research and provide new strategies for clinical lung cancer treatment.Methods: 1.The effect of avasimibe on the expression of ACAT-1 protein in LLC cells was detected by flow cytometry,western blot and immunofluorescence.2.The effects of different concentrations of avasimibe on the proliferation and migration of LLC cells were studied by Cell Counting Kit-8(CCK-8)method and cell scratch test.3.The effect of ACAT-1 protein level on apoptosis of LLC cells was detected by Annexin V-FITC/PI double staining flow cytometry.4.In addition,we observed the inhibitory effect of avasimibe on tumor growth in Lewis lung carcinoma mice.5.Tissues from Lewis lung carcinoma mice and wild mice were collected and the expression of ACAT-1 was detected by Western blot.Results: 1.Avasimibe could down-regulate the expression of ACAT-1 in LLC cells effectively.LLC cells were treated with avasimibe for 72 hours.Flow cytometry,western blotting and immunofluorescence assay showed that avasimibe could effectively inhibit the expression of ACAT-1(P < 0.05).2.The decreased expression of ACAT-1 inhibited the proliferation ability of LLC cells.LLC cells were treated with avasimibe to inhibit the expression of ACAT-1.CCK-8 assay showed that avasimibe could attenuate the proliferation of LLC cells in a concentration-and time-dependent manner.The cell viability of the control group and the avasimibe group(2.5,5,10,and 20 ?M)were 100 ± 0%,63.57 ± 4.88%,45.47 ± 5.35%,37.66 ± 3.72%,and 30.59 ± 1.24%,respectively.The cell viability of control group and different time groups(1,2,3,and 4 days)was 100 ± 0%,72.21 ± 4.50%,58.60 ± 5.25%,46.11 ± 3.9%,and 39.02 ± 3.04%,respectively.3.The decreased expression of ACAT-1 inhibited the migration ability of LLC cells.LLC cells were treated with avasimibe to inhibit the expression of ACAT-1.Cell scratch assay showed that avasimibe could reduce the migration ability of LLC cells(P < 0.05).The wound healing rates of avasimibe treatment group(5,10 ?M)and control group were 18.90±2.37%,11.07±1.27% and 40.63±4.98%,respectively.4.The decreased expression of ACAT-1 promoted LLC cells apoptosis.LLC cells were treated with avasimibe and flow cytometry revealed that avasimibe promoted apoptosis in LLC cells(P < 0.05).5.Avasimibe inhibits LLC growth and metastasis of Lewis lung carcinoma mice..We established a LLC mouse model to verify the anti-tumor effect of avasimibe,an ACAT-1 inhibitor,in vivo.Our results showed that avasimibe inhibited tumor growth,reduced tumor volume(P < 0.01),and increased spleen weight and spleen index(P < 0.01)compared with the control group.Compared with the CTX and avasimibe monotherapy groups,CTX + avasimibe combination therapy inhibited tumor growth more effectively than monotherapy(P < 0.01).In addition,Avasimibe did not cause changes in body weight over time.Metastatic lesions of distant organs(liver and lung)were also assessed at the end of the study,at least one metastatic lesion was observed in the liver and lung of each control mouse,and there was no evidence of metastatic disease in the other groups.These results further confirmed that avasimibe inhibited the growth and metastasis of Lewis lung carcinoma mice and enhanced the immune response of mice.In addition,avasimibe enhances the anti-tumor effect of CTX.6.The expression of ACAT-1 in Lewis lung carcinoma-bearing mice was higher than that in control mice.The expression of ACAT-1 in lung tissue was higher in Lewis lung carcinoma mice than in control mice(P < 0.05).Conclusion: Avasimibe can inhibit the growth and metastasis of Lewis lung carcinoma by down-regulating the expression of ACAT-1,which may be a potential therapeutic target for lung cancer. |
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