Study On Antitumor Effect Of Huoxia Capsule In Vivo And In Vitro

Objective:To explore the anti-tumor activity of Huoxia capsule in vivo and in vitro.Methods:1.The inhibitory effects of serum containing Huoxia capsule on NCI-H460,Hep G₂ and SKOV-3 cells were detected by MTT assay in vitro.Effects of drugs on morphology in hunman cancer cells were obeserved by AO-EB.2.Subcutaneous transplantation tumor model of xenografts in nude mice was established.The nude mouse model were randomly divided into five groups（n=6）:model group,positive control group,low,middle and high does group.Another 6 normal nude mice were used as normal control group.The model and normal group were orally administered with equal volume of saline.The positive control group was injected cyclophosphamide at a dosage of 0.025g/kg,three times a week.The mice in high,middle and low does group were orally administered with Huoxia capsule at a dosage of 2.4g/kg,1.2g/kg,0.6g/kg respectively.Duration of drugs groups were given drougs for two weeks.At the end of the treatment,weighed and measured the volume of the transplanted tumor,then,the nude mice were executed,and the blood and the tumor was colleted.3.The contents of AST,ALT,ALP,UA,BUN,CREA,GLB,ALB and TP were counted by extract serum.IL-6 and TNF-αlevels in serum were measured by ELISA.The content of SOD was determined by hydroxylamine method,and the content of MDA was determined by thiobarbituric acid method.4.The pathological changes of tumor tissue by HE staining were observed with light microscope.The protein expression of Bcl-2,Bax,P53 and Caspase-3in tumor tissues were determined by immunhistochemistry after treatment of different doeses of Huoxia capsules.Results:1.MTT assay revealed that the Huoxia capsule could inhibit the proliferation of NCI-H460,Hep G₂ and SKOV-3 cells.The inhibitory effect on the NCI-H460 cells was more significantly than the Hep G₂,SKOV-3 cells.The AO-EB double staining results showed that the normal control group developed green fluorescence,and the cell structure was normal,and the living cells grew densely;in the CTX group,the amount of cells were significantly reduced,the number of early apoptotic cells were large,the fragments were numerous,the nucleus were contracted,and apoptotic cells were stained.Dyeing bright orange;different concentrations of the amount of viable cells in the administration group decreased with the increase of drug concentration,some nuclei were shrinking,there was debris agglutination,apoptotic nuclei were stained orange red.2.The nude mice were all observed tumor appearancing.Compared with the normal group,the body weight of the model group and the treatment group were significantly reduced（P<0.01）;compared with the model group,the weight of the nude mice was significantly reduced in the CTX group and the high dose group of Huoxia extract（P<0.01）;The inhibitory rates of the model group,expermental-L,expermental-M,expermental-H and CTX group were0.00%,13.0%,21.5%,43.9%,71.7%;the tumor volume of nude mice in the 5groups were（3271.19±1777.70）,（2169.87±1678.50）,（1817.21±1191.77）,（1672.39±707.06）,（135.26±128.40）mm³,respectively;The tumor weight in the5 groups were（2.23±0.07）,（1.94±0.36）,（1.76±0.48）,（1.25±0.61）,（0.63±0.39）g,respectively;The tumor volume and tumor weight of CTX group and Huoxia capsule high dose group were smaller than those of the model group（P<0.01 or P<0.05）.3.There were no significant difference was found among the each treatment group compared with the model group in the contents of ALP,ALT,AST,UA,BUN,GLB,ALB and TP（P>0.05）;compared with that of the model group,the CRE of the Huoxia capsule cancer group was no significant difference（P>0.05）;CTX group had statistical significance compared with the model group（P<0.05）.The content of TNF-αin serum and tumor tissues of nude mice in each administration group was significantly higher than that of the model group（P<0.01 or P<0.05）.The IL-6 levels in serum of nude mice in high dose group and CTX group were lower than those of the model group（P<0.05）.The IL-6 levels in tumor tissues in middle and high dose group and CTX group were lower than those of the model group（P<0.05）.The content of MDA in the tumor tissues of each treatment group was significantly lower than that of the model group（P<0.01 or P<0.05）.The SOD activity in the tumor tissues of each treatment group was significantly higher than that of the model group（P<0.01）.4.The HE staining results showed that the cancer cells of the model group arranged closely,with strong growth and large nuclei;necrotic cells were more in each administration group,and the nucleus was small and the cells were loose.The results of immunohistochemistry showed that the Bax positive expression rate in the tumor tissues of each administration group was statistically significant compared with the model group（P<0.05 or P<0.01）;the Caspase-3positive expression rate in the tumor tissues of the middle and high dose group of Huoxia capsule and the CTX group were statistically significant compared with the model group（P<0.01）.Conclusion:It was initially verified that Huoxia capsule had inhibitory effects on NCI-H460,Hep G₂ and SKOV-3 cancer cells,and the inhibitory effect on NCI-H460 cells was more significant than Hep G₂ and SKOV-3 cells.Huoxia capsule inhibited the transplanted tumor of NCI-H460 in nude mice in vitro and could induce apoptosis.The mechanism may be related to scavenging free radicals,increasing TNF-αcontent,inhibiting IL-6 secretion,and increasing the expression of Bax and Caspase-3 genes.