Effect Of Flavonoids Of Astragalus Complanatus On Anti-Hepatocarcinoma And Its Mechanism

Objective: To study the anti-hepatocarcinoma effects of flavonoids of Astragalus complanatus (ACF) in vitro. and in vivo. and to explore its molecular mechanism.Methods: The inhibitions of ACF on proliferations of SMMC-7721 cell line and mice Hepal-6 cell line were observed in vitro. by colorimetric MTT assay and ³H-TdR assay. The inhibitions of ACF on transplantation tumor H₂₂ in Kunmming species mice and transplantation tumor induced by SMMC-7721 in BALB/cA nude mice were assayed. The Hoechst 33258 staining, transmision electron microscope (TEM), agarose gel electrophoresis, and flow cytometry were used to detect the ACF-induced apoptosis and the cell cycle of SMMC-7721 cells by characteristic morphological changes and bichemical features. The mechanism of apoptosis was explored by series apoptosis and cell cycle gene array and western bolts annlysis. The proliferation of endothelial cells ECV-304 of human umbilical vein was evaluated in normal ECV304 cells and in ECV304 cells treaded with tumour-secreted factors. The chick chorioallantoic membrane (CAM) test and microvascular density (MVD) assay in the tumor tissues were used to investigate the inhibitory effect of ACF on tumor angiogenesis. The tumor tissues were stained by immunohistochemical methods to detect the protein expression of vascular endothelial growth factor(VEGF), VEGFR1/Flt-1, VEGFR2/KDR/Flk-1, VEGFR3/Flt-4 and endostatin (ES). Proliferation assay and cytotoxicity analysis of the human natural killercell line——NK-92 cells were performed as MTT assay. Surface molecules of NK-92cells were detected by flow cytometric analysis and gene expression of NK-92cell-activating receptors——KLRK1,NCR1,NCR2 and NCR3 were assayed byReal Time PCR.Results: The proliferations of SMMC-7721 cells and mice Hepal-6 cells were dramatically inhibited by ACF in concentration-dependent manners. After the cells exposed to ACF for 48h, the IC₅₀ values were 26.6 mg/L and 199.4 mg/L, respectively. ACF had significantly inhibitory effect on the growth of H₂₂ transplantaion solid tumor and transplantation tumor induced by SMMC-7721 in BALB/cA nude mice. The pathological alterations were observed under light-microscopy and TEM. In ACF groups, degrees of necrosis in tumor tissues were markedly increased respectively, and cell apoptosis can be observed. Morphological features of apoptotic nuclei were observed by Hoechst 33258 staining. A ladder molecule strap of the DNA in the SMMC-7721 treated with ACF of 400 mg/L was presented in agarose gel electrophoresis and a hypodiplid peak in front of the G1 peak (Sub-G1 peak) was displayed by flow cytometry. The analysis of cells apoptosis and cell cycle gene array indicated that there were 76 genes levels changed, 35 gene were up-regulated and 41 genes were down-regulated. The result of protein expressions of cyclinB1, cyclinD1, CDK1, CDK4, caspase3, caspase8, Bax, bcl-2, P21 and P27 were consistent with that of genes array. Moreover, it was observed that ACF induced the enhancement of toxicity against ECV304 in the presence of tumour-secreted factors, strongly inhibited the angiogenesis in the chick chorioallantoic membrane, significantly reduced the protein expressions of VEGF, Fit-1and Flk-1 in the tumor tissues and significantly enhanced the protein expression of ES. Experimental data also showed that ACF enhanced the activations of NK-92 cells, up-regulated the surface molecules expression levels of CD25 and CD69, and the transcription levels of KLRK1, NCR1 and NCR2.Conclusion: ACF showed dramatically inhibibory effects on the growth of H₂₂ and SMMC-7721 in vitro. and in vivio. The anti-tumor mechanisms of ACF were maybe associated with the cell apoptosis induction related to the up and down regulated expression of cells apoptosis and cell cycle gene. Moreover, the inhibition on angiogenesis in tumor tissue and activation of NK cell might be involved in the anti-tumor mechanism. Therefore, ACF could be a new prospective, high effective and low toxic anticancer candidates. It is worthy of more investigation and exploiture.