Anti-tumor Effect And Its Mechanism Of Paeonol Combined With Fluorouralcil On Different Human Hepatic Cellular Cancer Lines

Objective:On the basis of previous study of the anti-oncogene PTEN,to investigate the expression of PTEN and AKT of human hepatocellular cell line and 7 common human hepatocellular carcinoma cell lines.According to the Traditional Chinese Medicine theory,to investigate the anti-tumor effect of the drugs(Paeonol,5-Fluorouracil and combined of them)on the hepatocellular cell lines BEL-7404, SMMC-7721,MHCC97-H and its molecular mechanisms.Methods:Partâ… (1)The hepatocellular cell line and 7 common human hepatocellular carcinoma cell lines were cultured in the medium of its' respectively.Doubling time of each cell lines were computed and growth curve were drew.(2)The expression of PTENmRNA and Akt1, Akt2mRNA were examined by reverse transcription-polymerase chain raction(RT-PCR),and the protein of PTEN and p-AKT were detected by Western Blot in 8 cell lines.Partâ…¡(1)Hepatocellular carcinoma lines (BEL-7404,SMMC-7721,MHCC97-H)were treated by Paeonol,5-Fluorouracil and combined of them in various concentrations and different time points respectively;and then the cell proliferation was assayed by light microscope,MTT method.Meanwhile,the IC₅₀were computed of each drugs at every time point.(2)The concentrations of Paeonol and 5- Fluorouracil in the combined of each cell lines were calculated by middle-effect principle,and the effects of the different sequence of administration were observed en masse.The combination index of each effect of every cell lines were calculated,in the same time, the synergia or antagonism of the two drugs were computed.(3)DNA ladder agarose gel electrophoresis and TUNEL were used to detect the apoptosis which induced by the drugs.(4)The effect of the expression of PTENmRNA and Akt1,Akt2mRNA of the drugs were examined by reverse transcription-polymerase chain raction,and the changes of expression of the protein of PTEN and p-AKT were detected by immunocytochemical ABC and Western Blot.Results:Partâ… (1)There have been expressed PTENmRNA,Akt1,Akt2mRNA in the 8 cell lines,and the PTENmRNA was expressed highly in L-02 cell lines,and Akt1,Akt2mRNA was little.In contrast, expression of PTENmRNA of human hepatocellular cell line was significantly higher than other HCCs',and Akt1,Akt2mRNA were significantly lower.(2)Compared with the human hepatocellular cell line,the protein of PTNE of HCC were significantly lower,and p-AKT were significantly higher(except the HepG2 cell line,for there have no detected the protein PTEN's expression).Partâ…¡(1)Compared with the control groups,the drugs(individual or combined)obviously increased the inhibitory rate of HCC,and had dose-dependent effect and timedependent effect.And the IC₅₀(72h)of the HCC(BEL-7404, SMMC-7721,MHCC97-H)respectively were Pae(20.77,19.19,19.47 mg·L^-1);5-FU(12.60,12.93,13.57 mg·L^-1).(2)Used meddle-effect principle,got the concentration of the combined of IC₅₀(72h) respectively were 6.91 mg·L^-1(Pae:6.45;5-FU:2.26),9.12 mg·L^-1 (Pae:6.49,5-FU2.63),11.46mg·L^-1(Pae:6.44,5-FU 5.02).The two drugs was antagonistic at higher concentration and synergistic at lower concentration,and the sequence and time of administration ware factors influencing the killing effect.(3)After the treated by the drugs,it showed a typical apoptotic morphology and DNA depicted a ladder pattern characteristic of the apoptosis,indicating the presence of DNA fragmentation,and apoptosis rate were increased along with the time longer of drug treated.(4)Compared with the control group,the PTENmRNA were up-regulated increased significantly and Akt1,Akt2 mRNA were down-regulated significantly in all the treated groups(all P＜0.01).This effect were surpassed in two drugs combined than individual(P＜0.05 or P＜0.01).The expression of PTENmRNA of all groups were negative correlation with the Akt1,Akt1mRNA(P＜0.001). (5)Compared with the control group,the protein PTEN were upregulated significantly and p-AKT were down-regulated significantly in all the treated groups(all P＜0.01).This effect were surpassed in two drugs combined than individual(P＜0.05 or P＜0.01).And along with the treated time,all the groups' protein PTEN were expressed higher and p-AKT expressed lower.The expression of PTEN protein of all groups were negative correlation with the P-AKT(P＜0.001).Conclusions:1.There had the expression of PTENmRNA and protein in human hepatocellular cell line and 7 common human hepatocellular carcinoma cell lines(PTEN protein except HepG2 cell line) and the expression of PTEN and AKT had the negative correlation relationship.2.The PI3K/AKT signal conduction pathway maybe be part in the apoptosis of HCC.3.The two drugs of Paeonol and 5-Fluorouracil either individual or combined could increase the inhibitory proliferation rate of 3 HCC cell lines and induced them apoptosis.The two drugs were antagonistic at higher concentration and synergistic at lower concentration,and the sequence and time of administration ware factors influencing the killing effect.4.Paeonol individual or combined 5-Fluorouracil could upregulated the expression of PTEN and down-regulagted the expression of AKT.This maybe was the mechanism of Paeonol inhibit HCC proliferation and induce it apoptosis.