The Effects And Mechanism Of Shenmai Injection On The Apoptosis Of Hepatocellular Carcinoma Cells

Liver cancer is the most common malignancy cancer and the incidence is 6th among the malignancy cancer worldwide.There are 626000 new cases every year,about half of which happened in China where liver cancer is the second leading cause of cancer death.The malignant of liver cancer is high and most patients lost the opportunity for operation because of diagnosis delaying.Radiotherapy,chemotherapy and molecular targeted therapy are the main treatment of advanced hepatocellular carcinoma.But due to the treatment related adverse effects and drug resistance,the 5 years survival rate was not improved greatly.Traditional Chinese medicine(TCM)has been widely used for a long history.Developing novel drugs from TMC with fewer side effects is of significance for treatment of patients with liver cancer.Shenmai Injection originated from the ancient prescription "Shengmaisan" is a traditional Chinese herbal compound injection extracted and refined by Red Ginseng and Radix.The drug has various effects on the patients,including invigorating Qi for relieving desertion,nourishing Yin for retaining the body fluid and other effects.SM Injection is widely used to treat clinical patients of Qi-Yin Deficiency type of shock,coronary heart disease,viral myocarditis and tumors etc.Some clinical researches have proven that the drug can significantly improve cancer patients' immune function,and play an important role in anti-tumor effects alone or combined with chemotherapy while reducing the toxicity caused by chemotherapy.Previous studies showed that Shenmai injection significantly inhibited the proliferation of human hepatocellular carcinoma SMMC-7721 cells and it also reduced the adverse reactions and improved the life quality of patients after interventional therapy.Although the Shenmai injection has been widely used in clinic,its molecular mechanism in inhibition of tumor is not fully understood.The function of P53 gene plays an important role in carcinogenesis.About 50%of cancer patients harbor P53 gene mutation,which is the most common genetic mutation in cancer.Cells which express P53 mutations are defective in DNA repair and cell division.In hepatocellular carcinoma,P53 gene will loss the tumor suppressor gene function or even active oncogene and cause drug resistant because of the mutation of P53.In this study,we establish Shenmai injection treatment model in two p53 proficient cells L-02 and HepG2,and p53 deficiency Hep3B.Using the molecular biology techniques flowcytometry,immunofluorescence and Western Blotting,we elucided the role of Shenmai injection in proliferation and apoptosis in different cell lines,especially explored the P53 independent cell signaling pathway in DNA damage response induced by shenmai injection.Taken together these data point to possible mechanism of Shenmai injection to inhibit tumor growth,and provide theoretical basis for clinical treatment of liver cancer.Part I:Effects of Shenmai injection on Proliferation and apoptosis in hepatocellular carcinoma cellsObjective:To explore the possible mechanism of anti-tumor effect of Shenmai injection,its impact on the proliferation and apoptosis of L-02,HepG2,Hep3B(p53 deficiency)were investigated.Methods:The viability of L-02,HepG2 and Hep3B cells were determined by MTT after treated with 4?8?16?32mg/mL of Shenmai injection for 12h and 24h.After treated with Shenmai injection,cell apoptosis were detected by flow cytometry after staining with Annexin V/PI.Results:1.cell viabilityNo significant inhibition on L-02 treated for 12h and 24h were observed,except that the cell viability increased at 16 mg/mL for 24h.We observed that Shenmai injection inhibited proliferation of liver cancer cells HepG2 and Hep3B in a dose-dependent manner.Treated with Shenmai injection at 32mg/mL for 24h,the cell viability of HepG2 and Hep3B was reduced to about 70%and 50%of the control,respectively.2.cell apoptosisWe also observed that Shenmai injection induced apoptosis on HepG2 and Hep3B cells in a dose-dependent manner.Early-apoptosis dominated when treated for 12h,while the percentage of later-apoptosis cells increased when treated for 24h.Conclusion:Shenmai injection has no inhibition effect on proliferation of L-02 cells,while could inhibit the proliferation of HepG2 and Hep3B,and induce apoptosis in time-and dose-dependent manner.Part ?.Mechanisms of Shenmai injection inducing oxidative response on hepatocellular carcinoma cellsObjectives:To futher explore the inhibitory effect of Shenmai injection on hepatocellular carcinoma cells,the mechanisms of Shenmai injection promoting the apoptosis of HepG2 and Hep3B cells(p53 deficiency)were investigated.Methods:The intracellular ROS level were detected by DCFH-DA probe after HepG2 and Hep3B were treated with 4?8?16?32mg/mL Shenmai injection for 24h.The content of SOD,GSH-Px and MDA were analyzed by kits.The DNA damage was detected by immunofluorescence.The proliferation and apoptosis of HepG2 and Hep3B treated with Shenmai injection were examined using MTT combined with flow cytometry.Results:1.Intracellular ROSTreated with 4?8?16?32mg/mL for 24h on HepG2 and Hep3B,ROS positive cells and intracellular ROS fluorescence intensity increased as the concentration of drug increased,showed a dose dependent trend and significant difference compared with control.2.Intracellular SOD activityTreated with 4?8?16?32mg/mL for 24h on HepG2 and Hep3B,we found that both cellular SOD activity decreased compared with control.The SOD activity of HepG2 decreased significantly treated with drug concentration between 8?32mg/mL.The SOD activity of Hep3B decreased significantly treated with concentration of 16mg/mL and 32 mg/mL Shenmai injection.3.Intracellular GSH-Px activityTreated with 4?8?16?32mg/mL for 24h on HepG2 and Hep3B,we found that cellular GSH-Px activity decreased significantly in HepG2 at concentration of 16 mg/mL and 32mg/mL Shenmai injection.The GSH-Px activity of Hep3B decreased significantly treated with drug concentration between 8?32mg/mLShenmai injection.The SOD activity of Hep3B decreased significantly treated with concentration of 16mg/mL and 32 mg/mL Shenmai injection.4.Intracellular MDA levelTreated with 4?8?16?32 mg/mL for 24h on HepG2 and Hep3B,the MDA level increased significantly compared with control.The MDA level of HepG2 rised with increase of drug concentration with an exception of 32 mg/mL.The MDA level of Hep3B increased in a dose-dependent manner.5.Intracellular DNA damage(yH2AX foci)Treated with 4,8,16,32 mg/mL Shenmai injection for 24h on HepG2,number of cells without ?H2AX foci decreased from 56%to 9%,while number of cells with 10-20 foci more than 20 foci increased by 32%,34%respectively in 32 mg/mL treated group.Hep3B showed a similar trend after treated with a series of concentrations.Number of cells with 10-20 foci and above increased by 21%,28%respectively in 32 mg/mL treated group.6.Effect of Shenmai injection combined with NAC on cell viabilityTreated with different concentrations of Shenmai injection for 24h on HepG2 and Hep3B,the cell viability decreased to 70%and 50%respectively of control.Combing with NAC,the inhibitive effect of Shenmai injection on cell viability HepG2 and Hep3B was attenuated.There is no significant difference of cell viability between treatment and control,although the viability of combination group was obviously higher than that of control after 32 mg/mL drug treatment.No overturn of inhibitory effect of NAC on viability of HepG2 and Hep3B induced by Shenmai injection was observed.7.Effect of Shenmai injection combined with NAC on cell apoptosisTreated with different concentrations of Shenmai injection on HepG2 and Hep3B,the rate of apoptosis increased with increase of concentration.After treated with 32mg/mL Shenmai injection for 24 h,the apoptosis ratio rose to 25%and 42%respectively for HepG2 and Hep3B.Combing Shenmai injection with NAC,the apoptosis of HepG2 and Hep3B were significantly undermined.No significant differences were observed between treatment and control,although the apoptosis of combination group decreased slightly than that of control after 32 mg/mL drug treatment.It is concluded that no overturn of NAC on apoptosis of HepG2 and Hep3B induced by Shenmai injection.Conclusion:The experimental model of Shenmai injection treated HepG2 and Hep3B was established.Shenmai injection could cause reactive oxygen species and DNA damage,further induce apoptosis in HepG2 and Hep3B under servere treatment condition.No connection between Shenmai injection induced oxidative response and p53 status was established.Part ? Effect on apoptotic pathway of liver cancer cells triggered by Shenmai injectionObjective:To investigate the molecular mechanism of apoptosis induced by Shenmai injection using HepG2(p53 wide type)and Hep3B(p53 deficiency)cell lines.Methods:Changes of mitochondrial membrane potential of HepG2 and Hep3B treated by Shenmai injection was determined by flow cytometry.Expression of apoptosis related proteins,p53,Caspase 3,Caspase 9,PARP and Cytochrome C were detected by western blot.Results:1.Mitochondrial membrane potentialChange of mitochondrial membrane potential was evaluated by JC-1 intensity using flow cytometry.It was found that,after treated by 8?32 mg/mL Shenmai injectioin for 24 h,the mitochondrial membrane potential of HepG2 and Hep3B was significantly decreased in a dose dependent manner.2.Expression of apoptosis related proteinsExpression of Pro-caspase3,Pro-caspase9 and Pro-PARP proteins decreased with increase of concentration,accordingly,expression of Cleaved Caspase3,Cleaved-caspase 9 and Cleaved-PARP proteins increased.The same situation also occurred for Cytochrome C.Conclusion:Shenmai injection could exert an anti-tumor effect on HepG2 and Hep3B cells through mitochondrial-mediated apoptotic pathway,and the activation of ROS might be involved in.Shenmai injection can induce the apoptosis of hepatocellular carcinoma cells by p53 dependent or independent matter.