| Objective:This study was aimed to investigate the effect and its mechanisms of betulinic acid on the proliferation of human cervical cancer HeLa cells and nude mice with its subcutaneously transplanted tumor. Method:MTT assay was used to determine the inhibitory rate of betulinic acid on the proliferation of HeLa cells. Apoptosis induced by betulinic acid was detected by Hoechst33258staining. Apoptotic rate was quantified by flow cytometry (FCM) using Annexin-V/PI dual staining method. Western blot was used to evaluate the changes of caspase-3and CytoC proteins in HeLa cells. Nude mice model with transplanted tumor of human cervical cancer HeLa cells were established successfully by injecting HeLa cells(4X106) under the right axillary subcutaneously. Twenty-four nude mice were divided into4groups randomly, solvent group(control group), BA high dosage group(80mg/kg), BA medium dosage group(40mg/kg) and BA low dosage group(20mg/kg). Each group was6. All the mice medicated by intraperitoneal injection once every two days for21days. Then all mice were sacrificed24h after the final medication, and the transplanted tumor mass volume and weight were measured to calculate the tumor inhibitory rate; the antitumor effect of BA was evaluated. Morphology changes of transplanted tumor tissues were observed under light microscope after HE staining. Apoptosis index was tested by TUNEL assay. The protein expressions of caspase-3and CytoC were detected by immunohistochemical method. Result:Betulinic acid induced HeLa cells apoptosis in a time-and dose-dependent manner. The IC50of a24h,48h and72h time course for HeLa cells was20.36μg/mL,15.45μg/mL and12.02μg/mL, respectively. Typical morphological changes of apoptosis were observed in HeLa cells with Hoechst staining after induced by betulinic acid. FCM showed the early apoptotic cells by Annexin-V/PI staining, early apoptotic rates were (3.27±0.43)%,(7.51±0.84)%,(18.39±2.17)%,(28.94±3.88)%, late apoptotic rates were (2.14±0.38)%,(16.35±2.56)%,(23.52±3.13)%,(40.38±4.27)%, Early apoptosis and late apoptosis rate compared with control group,or compared with each group, there were significant differences (P<0.01). The protein expressions of caspase-3and CytoC in HeLa cells were both up-regulated after treatment with betulinic acid. Compared with control group, BA significantly inhibited the growth of transplanted tumors, the tumor inhibitory rate in BA high, medium, and low dosage groups were,56.2%,40.4%,24.6%(P<0.01). The necrosis and apoptosis cells of the transparent tumors were found under light microscope in the BA treated group by HE staining assay. The results of TUNEL demonstrated that BA obviously induced apoptosis of HeLa cells in vivo, the apoptosis index was8.36±2.78in control group, and were20.98±3.01,28.74±4.77,39.34±6.15in low, medium, and high dosage group respectively, the apoptosis index increased as the BA dosage multiplied(P<0.01). Immunochemical method showed that as compared with the control group, expressions of caspase-3and CytoC in transparented tumors were upregulated in all the three treated groups to different extent. Conclusion:Betulinic acid could significantly inhibit the proliferation of both HeLa cells and transparented tumors of HeLa cells in nude mice, and induce apoptosis through caspase-3pathway by up-regulating caspase-3and CytoC proteins. The substance of this thesis is looking for new medicine which have effect of antitumor from Chinese herbal medicine. Betulinic acid is proved out a good promising medicine by this experiment.
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