Triptolide Inhibits The Expression Of Matrix Metalloproteinase 9(MMP-9) Via Elevated DNA Methylation In Human Fibrosarcoma HT-1080 Cells

Objective1.To investigate the effects of triptolide on the expression and activities of matrix metalloproteinase-9(MMP-9)in human fibrosarcoma HT-1080 cells in vitro.2.To investigate the effects of triptolide on HT-1080 cells migration in Matrigel Matrix-coated Cell Culture Inserts in vitro.3.To identify the mechanism by which triptolide regulates the expression of MMP-9 in HT-1080 cells.Methods1.The antiproliferative effect of triptolide on HT-1080 cells was performed by MTr assay.2.Flow cytometry(FCM)was used to analyse triptolide-induced apoptosis in HT-1080 cells after 72 hours of treatment.3.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the expression of 9 genes at mRNA level in HT-1080 cells after 72 hours of treatment with triptolide.The 9 genes were MMP-9/-2, TIMP-1/-2/-3/-4,and DNMT-1/-3A/-3B.4.Western blotting was used to identify the expression of DNMT-1, -3A and-3B at protein level in HT-1080 cells after 72 hours of treatment with triptolide. 5.Gelatin zymography was employed to determine the expression and activities of MMP-9 and MMP-2 in triptolide treated HT-1080 cells.6.In vitro migration assays were performed in Matrigel Matrix-coated Cell Culture Inserts to examine the ability of triptolide-treated HT-1080 cells to migrate through Matrigel Matrix(artificial extracellular matrix).7.Methylation-specific PCR(MSP)was applied to assess the methylation status of MMP-9 promoter in HT-1080 cells after 72 hours of treatment with triptolide.Results1.The proliferation of HT-1080 cells was inhibited in a dose-dependent manner after 72 hours of treatment with triptolide.And the triptolide IC₅₀for HT-1080 cells was 25nM.2.The percentages of apoptotic HT-1080 cells were 16.0%,17.3% and 18.8%after 72 hours of treatment with 6nM,12nM or 18nM triptolide,respectively.And the countpart of control group was 17.7%.3.The expression of MMP-9 at mRNA level was significantly down-regulated in HT-1080 cells after 72 hours of treatment with 18nM triptolide(P＜0.05)However,the counterparts of MMP-2 and TIMP-1/-2/-3/-4 were not significantly changed(P＞0.05)And no significant change was identified in the expression of the above genes at mRNA level after 72 hours of treatment with 6nM or 12nM triptolide (P＞0.05). 4.Following 72 hours of treatment with 12nM or 18 nM triptolide, gelatinolytic activity of MMP-9 in serum-free media conditioned by HT-1080 cells was statistically reduced,P＜0.05,and P＜0.01,respectively. However,triptolide did not influence the gelatinolytic activity of MMP-2 in HT-1080 cells(P＞0.05).5.The number of HT-1080 cells migrating through the artificial extracellular matrix was significantly reduced to 65%or 50%after 72 hours of incubation with 18nM triptolide or 3μg/ ml anti-MMP-9 antibody, respectively,relative to the control group(P＜0.05).6.The methylation level of the MMP-9 promoter was elevated in HT-1080 cells after 72 hours of treatment with 18 nM triptolide(P＜0.05).7.The expression of DNMT-1 and -3A at both mRNA and protein level was significantly up-regulated in HT-1080 cells after 72 hours of treatment with 18nM triptolide(P＜0.05).However,18nM triptolide did not modulate DNMT-3B Expression in HT-1080 cells(P＞0.05).And no significant change was detected in the expression of the above genes in HT-1080 cells after 72 hours of treatment with 6nM or 12nM triptolide(P＞0.05)Conclusion1.Triptolide decreases the proliferation of HT-1080 cells.2.We detected triptolide significantly inhibits HT-1080 cell invasion in vitro,which may be related to inhibition of the expression and activities of MMP-9 by triptolide.3.For the first time,we demonstrated that triptolide statistically modulates gene expression via elevated DNA methylation.4.For the first time,our research suggested that triptolide significantly elevates the expression of DNMT-1 and -3A.