Experimental Study On Hepatocellular Carcinoma Treated By Vitamin K2 In Vitro And Vivo

Primary hepatocellular carcinoma(HCC) is a common malignant tumor with poor prognosis.It was reported that the incidence rate of HCC was ranked at the 5^th, and the death rate was at the 3^rd of tumor-related death rate in the world and at the 2^nd in China,respectively.There are many patients with type B hepatitis in China and type B hepatitis is one of the main reasons leading to HCC.According to statistics,in China,85%-90%patients suffering from HCC are complicated with liver cirrhosis, most of which are developed from viral hepatitis,especially from type B hepatitis. And 50%patients suffer from liver cirrhosis concurrent hepatocellular carcinoma.The research of HCC in China started from clinic in 1950s and experienced many kinds of treatments such as exairesis,TACE(Transcatheter arterial chemoembolization),PEI (Percutaneous ethanol injection),RFA(Radio frequency ablation),liver transplantation,biotherapy,molecular targeted therapy and so on.Notwithstanding there has been much progression both in clinical and experimental research of HCC in the past fifty years,and five-year survival rate has been elevated strikingly.Patients with HCC are still faced with poor prognosis,most of which often loss therapeutic chances of operation or intervention because of poor liver function caused by concurrent hepatic cirrhosis.So it is emergent to find therapeutic tools or medicine of more effective but fewer side effects.Vitamin K2 is such a remedy with effects of anti-tumor found recently.Vitamin K2 acts as a cofactor in the enzymatic carboxylation by gamma-glutamylcarboxylase of glutamic acid residues forming gamma-carboxyglutamic acid in plasma proteins,such as thrombinogen and other blood coagulation factors, participating in the process of blood clotting.It has been reported recently that vitamin K2 has effects on inhibiting human tumor cells growth.And it was confirmed in clinic that vitamin K2 had inhibition on hepotocellular carcinoma.For example, when Japanese researcher Habu reviewed curative effects on osteoporosis of female patients treated by vitamin K2 who suffered from liver cirrhosis,he found unexpectedly that HCC incidence rate in female patients with liver cirrhosis taking vitamin K2 orally was much lower(2/21) than control group(9/19),which showed that vitamin K2 could prevent female patients with liver cirrhosis from developing into HCC.However,the mechanisms are not be elucidated.Some studies showed that vitamin K2 induced a variety of solid neoplasitc cells apoptosis,such as pulmonary carcinoma,ovarian cancer,gastric cancer and so on,as well as malignant tumor cells of hematological system.The research team in which I am found that vitamin K2 could inhibit hepatocellular carcinoma cell line MHCC97-H with a highly metastatic potential growth,adhesion and invasion in vitro.This experiment continued with research of prophase and used MHCC97-H as targeted cells as well to approach apoptosis induction of vitamin K2 and its mechanisms.Further,we used animal model of nude mice bearing human hepatocellular carcinoma with high metastasis which was established by using the same cell line,in order to observe inhibitory effects of vitamin K2 on pulmonary metastasis and life span of nude mice,so as to provide theoretical and experimental evidence for applying vitamin K2 to treat or prevent beforehand hepatocellular carcinoma in clinic.PartⅠ.Experiment in vitro——apoptosis induction of vitamin K2 on hepatocellular carcinoma cells and its mechanismsSome studies showed that vitamin K2 induced many kinds of cells apoptosis.But it was uncertain whether it had the same effects on human hepatocellular carcinoma cells with a highly metastatic potential or not.So we used MHCC97-H as targeted cells to detect apoptosis induction of vitamin K2 and studied its mechanisms further by detecting mRNA expression of kinds of apoptosis-ralated genes.1.Apoptosis induction of vitamin K2 on human hepatocellular carcinoma cell line MHCC97-HHuman hepatocellular carcinoma cell line MHCC97-H were co-incubated with vitamin K2 of final concentration 100μM for 3h or 6h.Control group was set up. Cells were digested by pankrin and resuspensed by Binding buffer,picked up 100μl, added 5μl FITC and 5μl PI,incubated in 25℃protected from light,then added with 400μl Annexin V Binding buffer,at last detected apoptosis rate by flow cytometer. The cells in right up and right low quadrantic area were recognized as apoptotic cells. The result showed that vitamin K2 had apoptosis induction on MHCC97-H.When cells were treated with vitamin K2 6 hours later,the apoptosis rate was(28.5±1.6)%. There was an obvious difference compared with control group(2.8±4.8)%.But when treated 3 hours later,the difference was not significant compared with control group.2.mRNA expression of easpase-3,Bcl-2 and Bax was detected by RT-PCRHuman hepatocellular carcinoma cell line MHCC97-H were treated with vitamin K2 of different final concentration(20μmol/L,50μmol/L and 100μmol/L). Expression of caspase-3,Bcl-2 and Bax mRNA was detected by RT-PCR after 24 hours,48 hours and 72 hours respectively.The results showed that the expression of caspase-3 mRNA has concentration-dependence and time-dependence.When treated with 100μl vitamin K2 72 hours,expression of caspase-3 mRNA was 0.603±0.098. There was a significant difference compared with control group(0.270±0.132), P＜0.05.And when action time was prolonged,the expression of caspase-3 mRNA was also augmented.Expression of Bcl-2 mRNA could not be detected in MHCC97-H whether treated with vitamin K2 or not.And there was no significant difference of Bax mRNA expression whether treated with vitamin K2 or not.PartⅡ:Experiment in vivo——Influence of vitamin K2 on pulmonary metastasis and life span of nude mice bearing human hepatocellular carcinomaSmall scale clinical trials showed that vitamin K2 lowered HCC incidence rate of female patients with liver cirrhosis and postoperative recurrence rate of patients suffering from HCC,prolonged their life span as well.In order to identify the role which vitamin K2 plays in resisting hepatocellular carcinoma in vivo,we used nude mice bearing human hepatocellular carcinoma animal model established by liver cancer institute of Fudan university as objects to observe influence of vitamin K2 on their pulmonary metastasis and life span.1.Establishment of nude mice bearing human HCC ModelHighly metastatic human hepaotcellular carcinoma orthotopic transplantation model was established by Liver Cancer Insititute of Zhongshan Hospital affiliated Fudan University.Human hepatocellular carcinoma cell strain MHCC97-H with a highly metastatic potential was injected into nude mice subcutaneously.Six weeks later,tumor shaped.Then the tumor was removed and cut into small pieces about 2mm in diameter and implanted orthotopically into the liver of 24 BALB/c-nu/nu nude mice,which were randomly divided into treatment group and control group each with 12 mice.2.Influence of vitamin K2 on pulmonary metastasis and life span of nude miceThe treatment group received vitamin K2 daily by intragastric administration at the dose of 30mg·kg^-1·d^-1 beginning from the second day of postoperativeday.The control group received only vector(edible oil) also by intragastric administration daily. Six weeks later,six mice were extracted randomly from each group and sacrificed. The lungs were taken out,fixed with formalin,embeded with paraffin,sliced and stained by H-E.Pulmonary metastases of each mouse were recorded under microscope and mean values were calculated.The rest kept to be bred until died a natural death and life span was recorded.The results showed that mean value of pulmonary metastases in treatment group was 4.6±1.95.There was a significant difference compared with control group(12±6.6),which showed that vitamin K2 decreased nude mice bearing human hepatocellular carcinoma pulmonary metastases. Life span was analyzed by Kapalan-Meier and Log-rank test showed P＜0.05,so the difference was significant.The results showed that vitamin K2 could prolong life span of nude mice bearing human hepatocellular carcinoma.Main conclusions1.Vitamin K2 can induce human hepatocellular carcinoma cell line MHCC97-H with a highly metastatic potential apoptosis in vitro.2.The expression of caspase-3 mRNA was increased when cells were treated with vitamin K2,which suggested that caspase-3 may be involved in the regulation of apoptotic process.3.Vitamin K2 can decrease pulmonary metastases of nude mice bearing human hepatocellular carcinoma,prolong their life span.The results may be caused synergistically by the inhibitory effects of vitamin K2 on adhesion,invasion, growth of hepatocellular carcinoma cells and its apoptosis induction.