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OBJECTIVE The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo,as well as the potential mechanisms.METHODS First,the model of C6 glioma in Wistar rats was established and verified by hemotoxylin and eosin staining,immunohistochemical staining and magnetic resonance imaging(MRI).Then different doses of lapachol were gavaged and tumor volumes of the C6 glioma were detected by MRI.The effects of lapachol on C6 cell proliferation,apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)/phen-azinemethosulfate(PMS)assay,Hoechst33358 staining,AnnexinⅤ-FITC/PI staining,and comet assay.Effects of lapachol on topoisomeraseⅠ(TOPⅠ)and topoisomeraseⅡ(TOPⅡ)activities were detected by TOPⅠand TOPⅡmediated supercoiled p BR322 DNA relaxation assay.Molecular docking was used to predict the interaction of lapachol-TOPⅠand lapachol-TOPⅡ.TOP I and TOPⅡexpression levels in C6 cells were determined by Enzymelinked immunosorbent assay kits and real-time polymerase chain reaction(RT-PCR).RESULTS The rat C6 glioma model was successfully established.High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats(P<0.05).MTS/PMS assay,Hoechst 33258 staining,AnnexinⅤ-FITC/PI staining,and comet assay showed that lapachol could inhibit proliferation,induce apoptosis and DNA damage of C6 cells in dose dependent manners.Lapachol could inhibit the activities of both TOPⅠandⅡ.Molecular docking showed that lapachol-TOPⅠshowed relatively stronger interaction than that of lapachol-TOPⅡ.Enzyme-linked immunosorbent assay and RT-PCR showed that lapachol could inhibit TOPⅡexpression levels,but not TOPⅠexpression levels.CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro,which might be related with inhibiting TOPⅠand TOPⅡactivities,as wel as TOPⅡexpression.
OBJECTIVE The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo, as well as the potential mechanisms. METHODS First, the model of C6 glioma in Wistar rats was established and verified by hemotoxylin and eosin staining, immunohistochemical staining and magnetic resonance imaging (MRI). Different doses of lapachol were gavaged and tumor volumes of the C6 glioma were detected by MRI. The effects of lapachol on C6 cell proliferation, apoptosis and DNA damage were detected by 3- (4-sulfophenyl) -2H-tetrazolium (MTS) / phen-azinemethosulfate (PMS) assay, Hoechst33358 staining, AnnexinⅤ- FITC / PI staining, and comet assay. Effects of lapachol on topoisomerase I (TOP I) and topoisomerase II (TOP II) activities were detected by TOP I and TOP II mediated supercoiled p BR322 DNA relaxation assay. Molecular docking was used to predict the interaction of lapachol-TOP I and lapachol-TOP II.TOP I and TOPⅡexpression levels in C6 cells were determined by Enzymelinked immunosorbent assay kits and real-time polymerase chain reaction (RT-PCR) .RESULTS The rat C6 glioma model was successfully established. High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats ( P <0.05) .MTS / PMS assay, Hoechst 33258 staining, Annexin V-FITC / PI staining, and comet assay showed that lapachol could inhibit proliferation, induce apoptosis and DNA damage of C6 cells in dose dependent manners. Both TOP I and II. Molecular docking showed that lapachol-TOP Ishowed relatively stronger interaction than that of lapachol-TOP II. Enzyme-linked immunosorbent assay and RT-PCR showed that lapachol could inhibit TOP II expression levels, but not TOP I expression levels. CONCLUSION These results showed that lapachol could be significantly inhibit C6 glioma both in vivo and in vitro, which might be related with inhibiting TOP I and TOP II activations, as wel as TOP IIe xpression.