Initial Exploration On The Effects Of Improved Aitongxiao Prescription On Hepatocellular Carcinoma Cells And Their Exosomes

Objective: To observe the effects of improved Aitongxiao prescription(I-ATXP)on proliferation,apoptosis and cell cycles of different hepatocellular carcinoma(HCC)cells,and also to investigate the effects of I-ATXP on secretion of exosomes derived from these HCC cells,which would provide the basis for further understand functions of I-ATXP on proliferation of hepatocellular carcinoma cells via regulation of HCC-derived exosomes on HCCs.Methods:(1)Study on effects of I-ATXP on different HCC cell proliferation:different HCC cell lines(HepG2,SMMC7721 and HKCI-C3)and one immortalized hepatocyte line(MIHA)were treated with various concentrations of I-ATXP(0.125-1 mg/ml)for 24,48 and 72 h,cytotoxicity and cell proliferation were determined by the CCK-8 assay.(2)Study on I-ATXP effects on apoptosis and cell cycle of different HCC cell lines:HepG2,SMMC7721 and HKCI-C3 cells were treated with various concentrations of I-ATXP(0.125-1 mg/ml)for 24 h,apoptosis and cell cycles were determined by flow cytometry using Annexin V-FITC/PI and PI staining.(3)Study on I-ATXP effects on exosomes derived from different HCC cell lines:HepG2SMMC7721and HKCI-C3 were treated with various concentrations of I-ATXP(0.125-1mg/ml)for 24 h,exosomes derived from different HCC cell lines were extracted by Exosome Isolation Resgeat,and then acetylcholinesterase(AchE)were measured for exosome specific enzyme activity and protein expression of CD63 and Alix were measured by Western blot.Results:(1)After treatment for 24,48 and 72 h,the IC50 values of HepG2,SMMC7721 and HKCI-C3 were significantly less than that of MIHA cells,especially at 24 h,the IC50 value in MIHA was 5.052±0.229 mg/ml,which was twice larger than that of three HCC cell lines(P<0.001).These data indicate that the cytotoxicity of I-ATXP on three HCC cell lines were more powerful than MIHA.(2)The results showed that I-ATXP readily inhibited the HCC cell proliferation in a time-and dose-dependent manner.Moreover,the inhibitory effect on HCC cells were significantly stronger than immortalized hepatocyte line MIHA.(3)With I-ATXP concentration increased,the apoptosis rate of SMMC7721 and HKCI-C3 were increased in a dose-dependent manner.Compared with the untreated group,the apoptosis rate of HepG2 cell was significantly higher(P<0.001).These data indicate that I-ATXP can induce apoptosis in HCC cell line cells.(4)After various concentrations of I-ATXP(0.125-1mg/ml)treatmeant for 24 h,the ratios of G0/G1 phase in the HCC cells(HepG2,SMMC7721 and HKCI-C3)were significantly higher than that in the untreated group(P<0.0001).These data indicate that I-ATXP can induce cell cycle arrest in G0/G1 phase in HCC cell lines.(5)After various concentrations of I-ATXP(0.125-1mg/ml)treatment for 24 h,the AchE value of the three HCC cell lines(HepG2,SMMC7721,and HKCI-C)were significantly lower than that in the untreated group(P<0.01).Compared with 5-Fu,under the existing concentration conditions,the I-ATXP exhibits the weaker inhibition effects on HCC cell lines.(6)After various concentrations of I-ATXP(0.125-1mg/ml)treatment for 24 h,exosomes derived from three different HCC cells expressed exosome specific proteins Alix and CD63.Compared with the untreated group,with the increasement of the concentration of I-ATXP,the expression of exosome specific proteins Alix and CD63 were reduced.These results suggest that I-ATXP can inhibit the expression of specific proteins Alix and CD63 in exosomes from the HCC cells.Conclusions:(1)I-ATXP can effectively inhibit cell proliferation of different HCC cells in a time-and dose-dependent manner.Compared with 5-Fu,I-ATXP exhibits more selective on proliferation inhibition in HCC cells,which display the advantages of traditional Chinese medicine on tumor therapy,and also provide experimental basis for I-ATXP clinical application.(2)I-ATXP can induce apoptosis and cell cycle arrest in HCC cells.Correlated with the results of CCK-8 assay,these data suggest that I-ATXP could inhibit HCC cell proliferation mediated by apoptosis and cell cycle arrest.(3)I-ATXP can inhibit both the exosome releases and expression of CD63 and Alix derived from HCC cells.These suggest that I-ATXP may function on HCC cells via regulation of exosomes of HCC cells.