A Study On The Use Of Anti-Cancer Herbal Immune Booster, Vitamin C And Their Combinations On Hepatocarcinoma Treatment

Objective:Human primary hepatocellular carcinoma,with its notoriously difficult early detection,high degree of malignancy,aggressive development,and high mortality rate,is one of the most common cancers in China.Presently,there is no effective clinical cure for this disease.Therefore, in the field of clinical oncology,investigation into effective therapies that promise minimal side effects,increased rates of survival,and improved quality of life against this disease has become the subject of popular research.Anti-Cancer Herbal Immune Booster(ACHIB) is an effective Chinese herbal remedy developed by this researcher for the treatment of hepatocarcinoma.In recent years,concurrent studies in theoretical oncology and clinical applications of intravenous delivery of Vitamin C(VC) have also proved worthwhile for exploration.Based on previous clinical trials and related research studies overseas,this study used Walker256 implanted rat hepatoma model,DEN (diethylnitrosamine)-induced rat hepatoma model,CBRH7919(in vitro rat hepatoma cells),and BRL(in vitro normal rat liver cells).These were used to observe what possible interventional roles were made by ACHIB,VC,and their combinations on the onset and development of hepatocarcinogenesis in rats and to further explore their possible mechanisms.Methods:1.The carcinosarcoma Walker 256 was implanted into the rats' livers and the rats were divided into several groups:a normal group,a sham-operated group,a model group,a low-dose Chinese herbal medicine group(ACHIB delivered by gastric gavage,12.1g/kg),a medium-dose Chinese herbal medicine group(ACHIB delivered by gastric gavage,24.2g/kg), and a high-dose Chinese herbal medicine group(ACHIB delivered by gastric gavage, 48.4g/kg).The general conditions,such as weight,eating/drinking data,were recorded at regular intervals.In week 1,week 2,and week 3 of the experiment,the subjects' serum total protein(STP),albumin(ALB),globulin(GLB),ratio of albumin to globulin(A/G),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and the enzyme tumor markers alkaline phosphatase(ALP),γ-glutamyl transferase(GGT) and fucosidase(AFU) were tested; the tumor's weights and sizes by its long and short diameters were measured;the pathological development of tumor tissues and their surrounding tissues were observed;tumor metastasis was recorded;and the survival time of the remaining rats in each group was observed;the inhibiting roles of different doses of ACHIB on implanted liver hepatoma in rats and the most effective dose-result relationship were also investigated.2.The carcinosarcoma Walker 256 was implanted into the rats' livers and the rats were divided into a normal group,a sham-operated group,a model group,a low-dose VC group(VC intravenous,2.83g/kg),and a high-dose VC group(VC intravenous,5.65g/kg).The general condition of the rats was observed;their liver functions(STP,ALB,GLB,A/G,ALT,AST,ALP, GGT,and AFU) were tested;each subject's tumor weight and size of long and short diameters were measured;and the pathological development of tumor tissue was observed.The inhibiting roles of different doses of VC on implanted liver hepatoma in rats and the most effective dose-result relationship were also investigated.3.Rats were fed with DEN to create test groups with liver cancer.These groups were given, respectively,extracts of ACHIB 48.4g/kg,VC injection 2.83g/kg,combination of ACHIB 48.4g/kg with VC injection 2.83g/kg,and UFT 0.09g/kg.In weeks 8,14,and 20,rats in each group were randomly selected,their body weight and quality of life were observed;their STP, ALB,GLB,A/G,ALT,AST,ALP,GGT,AFU were tested,the changes in liver function were observed;peripheral blood CD⁴⁺/CD⁸⁺ and Interleukin-2(IL-2) expression level were detected to understand the immune function of rats in each group;liver tissue homogenate superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),malondialdehyde(MDA) expression were investigated to understand the oxidative damage in the rat liver and anti-free radical capacity;HE staining was used to observe pathological picture changes in the rat liver; immunohistochemistry was used to detect liver marker alpha-fetoprotein(AFP) expression and hepatocellular carcinoma vascular endothelial growth factor(VEGF) expression;PCR and real-time quantitative PCR technology were used to detect p53 gene mutations and c-myc, cyclin D1 expression;and the survival time of rats in each group was recorded.4.Serum pharmacology was used to prepare ACHIB,VC,ACHIB+VC,and UFT containing serum for the rats,with concentrations of 2.5%,5%,and 10%respectively.CCK8 assay was used to observe these serums,cultured at different times(24h,48h,72h),and its effects on normal rat liver cells(BRL) and rat hepatoma cells(CBRH7919) proliferation;the best combination of drug containing serum and incubation time for selective killing of CBRH7919 cells was determined;flow cytometry was used to further observe the groups' CBRH7919 cell cycle and apoptosis rate of change;and chemical colorimetric was used to detect apoptosis CBRH7919 cells in each group and changes of expression of apoptosis-related protein, Caspase-3.Results:1.ACHIB's Inhibiting Effect on Implanted Walker256 Liver Cancer in SD Rats1.1 The protection of liver function by different doses of ACHIB at different times.Liver function results demonstrated that,in comparison with the model group,during week 1 of the experiment,the high-dose ACHIB group's AST decreased(P＜0.05).In week 2,the low-dose group's ALB and A/G increased(P＜0.05),GGT decreased(P＜0.05),and ALT, ALP significantly decreased(P＜0.01);the middle-dose group's ALB and A/G increased(P＜0.05),while ALT,ALP,and GGT significantly decreased(P＜0.01);the high-dose group's ALB increased(P＜0.05),A/G also significantly increased(P＜0.01),while ALT,ALP and GGT were significantly lower than the model group(P＜0.01).In week 3,the low-dose ACHIB group's ALT,AST,GGT decreased(P＜0.05),while ALP significantly decreased(P＜0.01);the middle-dose group's ALB was higher than the model group(P＜0.05),while ALT,AST,ALP,and GGT decreased significantly(P＜0.01);the high-dose group's A/G increased(P＜0.05),ALB was also significantly higher than that in the model group(P＜0.01),while ALT,AST,ALP,GGT,and AFU were significantly reduced(P＜0.01).1.2 The results of tumor inhibition by different doses of ACHIB at different times.During weeks 1 to 3 of the experiment,different doses of ACHIB groups all demonstrated inhibition of tumor growth.Tumor weight inhibition rate in week 3 of the experiment was higher in the high-dose group than that in the low-dose group(P＜0.05).Pathology results showed that a high-dose ACHIB in the early- and medium-term(week 1,week 2) can inhibit the infiltration of cancer cells to surrounding tissues.In the advanced stage(week 3),ACHIB promoted cancer cell necrosis.The scores of tumor metastasis in week 3 of the experiment for low-,middle-,and high-doses of ACHIB were all lower than that of the model group(P＜0.05).1.3 The effects of different doses of ACHIB on the survival time of experimental rats.The high-dose ACHIB group's survival time had statistical differences when compared with the model group(P＜0.05). 2.Inhibiting Effects of VC on Implanted Liver Cancer in Rats2.1 The protective effects of VC on rat liver.Serum A/G in the low-dose VC group was higher than the model group(P＜0.05),while ALT was significantly lower than the model group(P＜0.01),GGT was also lower than the model group(P＜0.05).Serum ALT in the high-dose VC group was lower than that in the model group(P＜0.05).2.2 The tumor inhibition role of VC on liver cancer.The inhibition effects of VC on tumor volume and weight were not obvious(P＞0.05),but pathology results showed that VC could promote tumor cell necrosis,in which the extent of liver necrosis in the low-dose VC group was higher than that in the model group(P＜0.05).3.Protective Effect of ACHIB,VC and ACHIB+VC in DEN-Induced Rat Liver Cancer3.1 Each group's changes in liver function were observed at different times.In week 8 of the experiment,ALP levels were lower in the ACHIB and VC groups than in the model group(P＜0.05).ACHIB+VC not only had a significant reduction in ALP(P＜0.01),but also inhibited AST release from the liver,and showed statistical differences when compared with the model group(P＜0.05).By week 14 of the experiment,ACHIB,VC,and ACHIB+VC showed obvious effects on liver function protection,in which the ACHIB group's ALT and AST were significantly lower than the model group(P＜0.01).ACHIB also inhibited the synthesis of liver GGT,with lower content than the model group(P＜0.05);the VC group's and ACHIB+VC group's ALT decreased(P＜0.05) in comparison with that of model group,while AST decreased significantly(P＜0.01),they also demonstrated improvement of protein synthesis,with STP and GLB recovery in the VC group(P＜0.05). The improvement in the ACHIB+VC group's protein synthesis was even more significant, with STP and GLB significantly higher than the model group's(P＜0.01).By week 20 of the experiment,ACHIB still inhibited the release of AST compared to the model group(P＜0.05);AST was significantly lower in the ACHIB+VC group than in the model group(P＜0.01),and ALT was also lower than the model group(P＜0.05).The treatment results of the chemotherapy group compared with the model group showed no difference(P＞0.05).3.2 Comparison of the progression of liver cancer in rats at different times.Pathology results showed that ACHIB,VC,and ACHIB+VC all delayed the progress of liver cancer including the induction of cancer cell necrosis in its terminal stage.UFT also promoted cancer cell necrosis.In addition,AFP levels in the ACHIB+VC group were lower than the model group in weeks 8 and 14(P＜0.05).By week 20,ACHIB+VC group showed further reduction of the AFP level compared with the model group(P＜0.01),making it lower than that of the chemotherapy group(P＜0.05);the body weight/liver weight of rats in the ACHIB+VC group recovered in comparison with the model group's(P＜0.05).3.3 Immune function changes in different medication groups at different times.In rats with liver cancer,there are some barriers to cell-mediated immunity,reflected in CD⁴⁺/CD⁸⁺ reduction,and reduction of immune-enhancing factor IL-2 expression.By week 8,the VC treated rats' CD⁴⁺/CD⁸⁺ had increased(P＜0.05).ACHIB and ACHIB+VC showed more obvious effects,with CD⁴⁺/CD⁸⁺ significantly higher than the model group's(P＜0.01);by week 14,ACHIB+VC group's CD⁴⁺/CD⁸⁺ were also significantly higher than the model group's(P＜0.01);by week 20,compared with the model group,both the ACHIB group and the ACHIB+VC group not only had significant increases in CD⁴⁺/CD⁸⁺(P＜0.01),but also had an increase in the serum IL-2 expression(P＜0.05).3.4 The extent of oxidative damage of rats in each group at different times.Rats,after drinking DEN,showed increases in their liver MDA expression.After medical treatment, MDA expression decreased in the VC group,and the ACHIB+VC group by week 8(P＜0.05).By week 14,the SOD levels increased in the ACHIB group(P＜0.05).SOD levels significantly increased in the VC group and the ACHIB+VC group(P＜0.01).By week 20, the MDA levels in the ACHIB group,the VC group,the ACHIB+VC group,and the chemotherapy group were significantly lower than the model group(P＜0.01).There were also increased SOD and GSH-PX expressions in the ACHIB group(P＜0.05).The VC group,the ACHIB group,and the ACHIB+VC group recorded significant increases in the SOD and GSH-PX expressions,which were significantly higher than those of the model group(P＜0.01).3.5 Changes in VEGF expression in rats at different times.The immunohistochemical method was used to detect VEGF expression in the livers of each group.Results demonstrated that by week 8,VEGF expression showed an obvious decrease in the VC group and the ACHIB+VC group,and was significantly lower when compared with the model group(P＜0.01);the expression of VEGF in the ACHIB group also decreased(P＜0.05).By week 14,VEGF expressions were significantly inhibited(P＜0.01) in the ACHIB group,the VC group,and the ACHIB+VC group;in the terminal stage of cancer by week 20, the VEGF expression in the ACHIB+VC group was not only lower than the model group(P＜0.05),but also lower than the chemotherapy group(P＜0.05). 3.6 The rate of p53 mutation and c-myc,cyclin D1 expression at different times in each group.DEN-induced liver cancer in rats had a high rate of p53 mutation,with the existence of multiple point mutations.By week 14,the mutation points of the ACHIB,VC, and ACHIB+VC groups were significantly reduced(P＜0.01).As the rat's liver cancer progressed,the liver cancer gene expression of c-myc,cyclin D1 gradually increased.But c-myc,cyclin D1 expressions were significantly inhibited by ACHIB,VC,ACHIB+VC,and UFT,with the best effect produced by ACHIB+VC;by week 20,c-myc,cyclin D1 expressions in the ACHIB+VC group were only about 1/18 and 1/16 of the model group.3.7 Comparison of survival time of rats in each group.The survival times of the ACHIB group and ACHIB+VC group were longer compared with the model group's survival time (P＜0.05).The ACHIB+VC group's survival times were significantly longer than the chemotherapy group's(P＜0.01),while ACHIB group's was also longer than the chemotherapy group's(P＜0.05).4.The Effect of ACHIB,VC and ACHIB+VC on the Proliferation,the Cell Cycle and the Apoptosis Rat of Rate Hepatoma CellsDrug Containing Serum 5%respectively of ACHIB,VC,ACHIB+VC and UFT for 48h can Selectively Inhibit the Proliferation in vitro of CBRH7919 rat hepatoma cell line.The result of the ACHIB+VC group was better than that of ACHIB and UFT groups(P＜0.01).The ACHIB serum made the G₁ period:CBRH7919 cell quantity ratio higher than that of the serum control group(P＜0.01) and kept cell proliferation in the G₁ phase;the S period:CBRH7919 cell quantity ratio increased significantly in VC and ACHIB+VC groups (P＜0.01),the S period:CBRH7919 cell quantity ratio also increased in the UFT group(P＜0.05);hence,proliferation of cancer cells was blocked in its S phase.The expression of Caspase-3 increased in the ACHIB,VC,ACHIB+VC and the UFT groups(P＜0.05) resulting in an escalation of CBRH7919 apoptosis.The apoptosis rates of ACHIB+VC group and UFT group increased significantly compared to serum control group(P＜0.01),and better than that of the ACHIB group(P＜0.05) and VC group(P＜0.01).Conclusion:1.ACHIB,VC and ACHIB+VC inhibit Walker256 rat implanted liver carcinosarcoma.Their main roles are:liver protection,inhibition of cancer cell proliferation resulting in retardation of liver cancer development,inhibition of AFP expression,improvement in the quality of life and prolongation of the survival times of rats with hepatocarcinoma.2.ACHIB+VC inhibits the increase of hepatocarcinoma cells and induces their apoptosis better than the mono-therapies of ACHIB and VC.ACHIB and ACHIB+VC significantly prolong the survival times of rats with hepatocarcinoma much better than the chemotherapeutic UFT. Furthermore,ACHIB+VC inhibits the expressions of AFP and VEGF much better than chemotherapeutic UFT.3.The anti-liver cancer mechanisms of ACHIB,VC and ACHIB+VC are related to the following:3.1 Increasing the ratio of serum CD⁴⁺/CD⁸⁺,and increasing the expression of IL-2,thereby strengthening the cell's immune capability.3.2 Increasing the activity of liver's SOD and GSH-PX,and decreasing the expression of MDA, thereby increasing the anti-free radical capability of the liver and reducing the oxidative damage due to free radicals.3.3 Inhibiting the liver's expression of VEGF thereby inhibiting the cancer tumor's blood vessel growth.3.4 Lowering the c-myc,cyclin D1 expressions thereby disrupting the cancer cell cycle and inhibiting the increase of cancer cells.3.5 Inhibiting the mutation of cancer gene p53,and increasing the expression of Caspase-3 thereby speeding up the apoptosis rate of cancer cell.