| Objective:1. To study the effects of betulinic acid on biological behaviors of colorectal cancer SW480 cells and detect the states of the related proliferation genes.2. To construct colorectal cancer model by injecting colorectal cell SW480 into BALB/C nude mice.3. To detect the effects of betulinic acid on colorectal cancer and explore a new treatment method.Methods:1. The mRNA and protein levels of transcription factor Spl and vascular endothelial growth factor was detected by real time PCR and Western blot respectively after betulinic acid treatment.2. The effects of betulinic acidon cell proliferation, cell apoptosis, migration were assayed by the MTT, flow cytometry (FCM) and scratch method respectively.3. The xenografts derived from colorectal cell SW480 were established in BALB/C nude mice. Inoculated mice were randomly divided into negative control (corn oil), low dose betulinic acid group (20 mg/kg/d) and high dose group (40 mg/kg/d). After 22 days, the animals were sacrificed; tumor volume and weights were measured.4. After betulinic acid treatment, the mRNA level of vascular endothelial growth factor was analyzed by quantitative real-time polymerase chain reaction. The expression and localization of vascular endothelial growth factor protein was detected by immunohistochemistry.Results:1. The expression of vascular endothelial growth factor and transcription factor Sp1 in the colorectal cells after betulinic acid treatment was significantly down-regulated at both mRNA and protein levels, the difference was significant (P<0.05). The relative expression of vascular endothelial growth factor mRNA were 0.97±0.01,0.82±0.03,0.39±0.03,0.28±0.01 in SW480 different dose groups(5,10,15,20μg/ml), the ratio of inhibition against the expression of vascular endothelial growth factor protein was 17%,34%,63.8%,78.8% in 48th hour; the relative expression of transcription factor SplmRNA were 0.98±0.01,0.88±0.02,0.46±0.03,0.31±0.01 in SW480 different dose groups(5,10,15,20μg/ml),the ratio of inhibition against the expression of protein was 14.8%,35.2%,63%,78.7% in 48th hour; Betulinic acid can effectively inhibit the expression of vascular endothelial growth factor and itranscription factor Spl in colorectal cell line.2. Cell growths were obviously repressed and the characteristic morphology of treatment cells changed greatly against negative controls. The proliferation and migration ability of cells was inhibited, which was detected by MTT analysis, flow cytometry (FCM) and scratch method respectively. FCM showed betulinic acid could induce cell apoptosis and have dose-dependence. The apoptosis rates of SW480 cells was 1.2%,3.6%,9.8%,13.2% and 0.5% in experimental group(5,10,15,20μg/ml) and controlled group (DMSO) respectively, the difference was significant (P<0.05).3. The tumor weight was significantly lower in low and high dose groups than in corn oil group(1.12±0.04,0.43±0.02vs2.08±0.07, P<0.05).4. The mRNA levels of vascular endothelial growth factor was also significantly lower in betulinic acid treated groups (0.72±0.02,0.38±0.01, P< 0.05) than in control group(1.08±0.04). H&E staining showed tumor tissue necrosis was observed in treatment groups. Immunohistochemistry showed vascular endothelial growth factor was expressed in the nuclear of cancer cells, the positive expression of vascular endothelial growth factor was lower in low and high dose groups than in corn oil group. Gray scale increased in low dose group and high dose group (121.1±2.8,156.2±3.3, P<0.05).Conclusion:1. Betulinic acid had significant inhibitory effect on the expressions of vascular endothelial growth factor and transcription factor Spl and suppressed the proliferation and migration of colon cancer cells, possibly through down-regulating transcription factor Sp1 gene expression.2. Colorectal cancer model by injecting colorectal cell SW480 into nude mice was successfully constructed. Betulinic Acid could vascular endothelial growth factor expression of tussie and tumors growth of human colorectal cancer xenografts in vivo, It may provide a novel therapeutic approach for color cancer. |
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