| It is widely believed that Hepatocellular carcinoma(HCC)is a kind of the most common malignant tumors,which has the characteristics of high morbidity and high mortality and is a serious threat to human health.Case fatality rate result from HCC ranks in the third place on a global scale,but particularly ranks in the second place in china.Fast spread,high transfer capability and high postoperative recurrence rate of HCC have verified its high malignancy and poor prognosis.So,it is a big problem faced in the clinical treatment to overcome poor treatment effect and relapse metastasis.Although,it has made great progress in diagnosis and treatment of HCC,as for solid tumor with high invasive HCC,there is no effective way to change or improve the prognosis of patients.Depending on relevant data shows that the 5-year survival rate for patients with HCC is 35%to 45%after surgical removal,and is 47%to 61%after liver transplantation.The prognosis is always poor.Therefore,it is of paramount importance to search for developing new therapeutic agents that can effectively act on multiple oncogenes pathways to inhibit tumor metastasis and improve both the quality of life and survival of cancer patients with postoperative recurrence.It has been one of the current difficult task in the field of medical research.At present,chemical drug development is facing more and more problems such as excessive development difficulties,time-consuming and large amount of early investment.What's more important is that chemical drug has the characteristics of poor efficacy,side effects and small application scope.Fortunately,natural compounds are valuable source of chemotherapeutic agent.According to relevant statistics,many of approved anti-cancer drugs are resulted from natural compounds for various advantages of herbal medicine.There is a recent trend that the use of herbal medicines as alternative medicines for cancer throughout the world.So,a pristine natural source of anticancer agents would be a valuable tool in prevention and treatment of cancer.Isodon lophanthoides var gerardianus(Benth)Hara is a perennial member of the mint,or Lamiaceae,a kind of Xihuangcao in Guangdong province."Xihuangcao" are common folk herbal medicine for many years.As early as 2000 years ago,ancient folk doctors apply "xihuangcao" for patients who suffer from diseases of liver and gall as removing jaundice medicine in china.Now,it is a common medicine for hepatobilitary diseases.There are many preparations varieties listed."Xihuangcao" in this paper derive from Isodon lophanthoides var.gerardianus(Benth.)H.Hara.With the development of medical technology,a large number of studies report extract from Isodon lophanthoides var.gerardianus(Benth.)H.Hara could restrain activity of HepG2 cells in vitro.Other studies demonstrate that it could significantly improve the quality of survival in clinical treatment for patients with hepatocellular carcinoma.Our research team preliminary findings show the water-soluble total flavonoids from Isodon lophanthoides var.gerardianus(Benth.)H.Hara(WSTF)has an inhibitory effect on proliferation of HepG2 cell.The experimental conclusion showed has induced apoptosis,likely to be antitumor drug candidates.However,few studies have investigated activity and relevant mechanisms of extract from WSTF on cancer cells in vitro and in vivo.Therefore,because of widely used in the treatment of liver diseases,there is a very necessary to research clearly active and relevant mechanisms of WSTF on cancer cells in vitro and in vivo.ObjectiveBased on being used for many years in Chinese medicine and previous research achievements about "xihuangcao",this paper is to improve the purification process WSTF,to study investigation of mechanisms underlying WSTF induces growth inhibition and apoptosis in human hepatocellular carcinoma HepG2 cells,to observe the effect of WSTF on tumor growth inhibition of human hepatoma cells-induced nude mice model,and investigate the molecular mechanisms of WSTF inhibiting tumor.Methods1.Investigation of underlying WSTF induces growth inhibition in human hepatocellular carcinoma HepG2 cells.First,we adopt MTT assay to test growth inhibition ratio of underlying WSTF induces growth inhibition in human hepatocellular carcinoma HepG2 cells as well as concentration and time dependence.After statistical analysis,the 50%inhibitory concentration(IC50)was obtained for expressing the cytotoxic effects.Growth inhibition was also assessed by cosmogenic assay.Cells that underwent exponential growth were seeded in 6-well plates at a density of 5*105 per well.After synchronized,cells were treated with WSTF at the different concentrations for 24 h.Then 20,000 cells from each group were undergone for cell cycle analysis by a FACS cantonTM II flow cytometer according to the manufacturer's instructions2.The effects of the WSTF on apoptosis of human hepatocellular carcinoma HepG2 cells.We applied Rhodamine 123 staining to measure mitochondrial energization.The effects of WSTF on mitochondrial membrane potential were identified using a Rhodamine 123 assay kit according to the manufacturer,s instructions.Quantitative measurement of Rhodamine 123 fluorescence was examined by fluorescence plate reader at wavelengths of 505nm(excitation)/530nm(emission)pairs.The intracellular ROS was measured using Reactive oxygen species assay kit,which is use of the characteristics of the fluorescent probe(2,7-dichlorodihydrofluorescein diacetate,DCFH-DA).It is a non-fluorescentdye which can freely through the cell membrane.After entering the cell,the dye is hydrolyzed by intracellular esterases to non-fluorescent 2,7-dichlorohydrofluorescein(DCFH,not through the cell membrane).Then DCFH is oxidized to the fluorescent substance 2,7-dichlorofluorescein(DCF,be detected)by the intracellular ROS.Cells were treated with WSTF and then cells from each group were undergone according to the manufacturer's instructions.Finally,the fluorescence intensity was detected at wavelengths of 488nm(excitation)/525nm(emission)pairs using a fluorescence microplate reader.The values were expressed as the relative intensity of DCF fluorescence,compared with the control.To determine the effects of the WSTF on the percentage of apoptosis cells and necrosis cells,the double-staining[AnnexinV-FITC and propidium iodide(PI)]and flow cytometry(FCM)according to the manufacturer's instructions were applied.In different stages of the cell apoptosis and necrotic cells were determined by fluorescence-activated cell sorting(FACS)analysis performed with a FACS cantoTM II flow cytometer.3.Investigation of the mechanisms underlying the WSTF induces apoptosis of hepatocellular carcinoma HepG2 cellsWe detect autophagy in hepatocellular carcinoma HepG2 cells by transmission electron microscopy.RT-PCR for genetic expression of main effectors(Bcl-2 mRNA,Bax mRNA,Survivin mRNA).The cells were interpreted with WSTF for the indicated time.After that,cells were washed once with cold PBS and harvested by pipeline.Total RNA from the cells was separated using the Trizol kit according to the manufacturer' s instructions.The Advantage RT-PCR kit was used for reverse transcription according to the manufacturer' s protocol.All reactions were repeated independently at least 3 times to make sure the reproducibility of the results.The specificity of the amplified PCR products was assessed using a melting curve analysis.The relative expression of target genes in comparison with the&-actin gene was assessed by the comparative CT threshold method using the Bio-Rad software tool,Genex-Gene Expression MacroTM(Bio-Rad).Western blot analysis for activation of main effectors(protein:cytochrome c,cytologic AIF,Bcl-2,Bax,Survivin,Caspase-3)involved in the potential apoptosis signaling pathways.In the study,the antibodies and dilutions were used for Western blots as follows:cytochrome c(1:800),AIF(1:800),bcl-2(1:800),mcl--(1:800),bax(1:800),surviving(1:800)and beta-actin(1:2000).4.Effect of WSTF on tumor growth inhibition of human hepatoma cells-induced nude mice model.Nude mice model of tumor was established by transplantation of hepatocellular carcinoma HepG2 cells.Nude mice were allocated randomly to seven groups."There were model group(blank control group,BC),high dose group of WSTF(WSTF-H),middle dose group of WSTF(WSTF-M),low dose group of WSTF(WSTF-L),5-Fu group,cinobufagin group,and high dose group of WSTF+5-Fu group,6 nude mice per group.Intervention was administered in each group when the tumor was size of 100 mm3.WSTF groups were administered WSTF by lavage according 200mg/kg ·d,100mg/kg ·d,50mg/kg ·d respectively,once daily.5-Fu group was administered 5-Pluorouracil(5-Fu)via intraperitoneal injection,twice a week,while NS was administered saline via lavage,at some other time.Cinobufagin group was administered cinobufagin by lavage according 0.624g/kg · d,once daily.High dose group of WSTF+5-Fu group was administered WSTF by lavage according 200mg/kg · d,and at the same time,also was administered 5-Pluorouracil(5-Fu)via intraperitoneal injection,twice a week.Another 6 nude mice served as a normal control group.After the treatment,researchers observed nude mice every day,weight every two days and the parameters of tumor growth every three days.Based on these data,volume was calculated and the tumor growth-curve of tumor volume was drawn according to time in every group.After intervention of 21 days,nude mice were sacrificed and researchers severed carefully the tumor and weighed up for calculating the inhibition rate,compared with model gruop.The tumor was examined by histopathological method and changes of AFP in the peripheral blood were detected.We employed tunnel dyeing to evaluate tumor cells apoptosis.5.Investigation the molecular mechanisms of WSTF inhibiting tumor.We employed a transmission electron microscope(TEM)to assess formation of autophagosome and cell ultrastructure.RT-PCR for genetic expression of main effectors(Beclinl,Atg5,CyclinDl,Caspase-3,bax,bcl-2,Survivin,nm23-Hl and MMP-9 mRNA)and western blot analysis for activation of main effectors(protein:Beclinl,Atg5,CyclinDl,Caspase-3,bax,bcl-2,Survivin,nm23-H1 and MMP-9)involved in the potential apoptosis and autophagy signaling pathways.Results1.Investigation of underlying WSTF induces growth inhibition in human hepatocellular carcinoma HepG2 cells.After treatment HCC cells with different concentrations of WSTF(0.0625,0.125,0.25,0.5,1.0,2.0 mg/mL),the result shows that WSTF inhibited concentration dependent manner.When treatment HCC cells with different times of WSTF(24?48?72 h),the result further shows that WSTF inhibited the time-dependent manner.Morphology of cell apoptosis observed by Hoechst 33342 fluorescence staining.We examined the effects of WSTF on colony formation of the HCC cell line HepG2 on plates and showed that WSTF inhibited anchorage-dependent growth in a dose dependent manner.FCM analysis shows that after the treatment with different concentrations of WSTF resulted in an accumulation of cells in the G0/G1 phase and a decrease in the number of S-phase cells in a dose dependent manner.This suggests WSTF inhibited cell proliferation and growth by interfering with the cell cycle.2.The effects of the WSTF on apoptosis of human hepatocellular carcinoma HepG2 cells.WSTF could significantly increase early-and late-stage apoptotic/necrotic cells and the findings showed a dose-and time-dependent manner in Ann+/PI-and Ann+/PI+cells,respectively.Collectively,the results from experiment suggest that growth inhibitory of WSTF may be associated with induction of cell apoptosis in HepG2 cells.These results show a red fluorescence was observed in WSTF-treated cells in a dose-dependent manner,and MFI value decrease significantly compared with control group.The findings indicate that WSTF probably induce HepG2 cell apoptosis through enhancing mitochondrial membrane permeability.Compared to the control,the relative amounts of intracellular ROS in HepG2 cells increased in a dose-dependent manner.The findings suggest that increase in amount of intracellular ROS in HepG2 cells probably induces cell apoptosis.3.Investigation of the mechanisms underlying the WSTF induces apoptosis of hepatocellular carcinoma HepG2 cells.Transmission electron microscopic show that WSTF could reduce numbers of autobiographies found in these cells.RT-PCR results show the expression levels of Bcl-2,Survivin mRNA were both significantly downregulated by WSTF treatment(P<0.01).In contrast,the expression of Bax mRNA and Caspase-3 mRNA was both significantly upregulated after WSTF treatment for 48 h(P<0.05).These results collectively testify that WSTF may probably induce cell apoptosis through decreasing the levels of anti-apoptotic factors and increasing the levels of promoting-apoptotic factors.It was found that,exposure to WSTF for 24 h,the level of the anti-apoptotic proteins(bcl-2 and mcl-1)and the level of survivin diminished in a concentration-dependent manner.In contrast,the level of the apoptosis-promoting protein Bax increased in a concentration-dependent manner.Moreover,exposure to 1.0 mg/ml of WSTF for 12-48 h,the changes of protein levels in bcl-2,mcl-1,and Bax in cells was also time-dependent.Collectively,these findings from experiment show that WSTF may promote apoptosis,through regulating the levels of apoptosis-promoting factors and anti-apoptotic factors.Western blot analysis also showed,exposure to different concentrations of WSTF for 24 h,we observed the relative band densities of cytochrome c in mitochondria fraction were weakened respectively,while it was shown reverse trend in the cytosolic fraction.In other words,the activity of cytochrome c was significantly decreased in mitochondria fraction and presented the concentration dependent manner.In contrast,it was increased in cytosolic fraction,compared to the control.Moreover,the activity of cytosolic AIF was gradually increased,compared to the control.These eventually lead to cell apoptosis.4.Effect of WSTF on tumor growth inhibition of human hepatoma cells-induced nude mice model.After intervention of 21 days,the tumor growth-curve of tumor volume was drawn.Compared to the model group,ratio to inhibitory tumor was 53.5%,27.7%,5.7%and 61.6%respectively in WSTF high,middle,low dose groups and 5-Fu group.WSTF high,middle dose and 5-Fu groups have an advantage over the model group(P<0.05)and presented the concentration dependent manner,while WSTF low dose group has little effect on growth of inhibitory tumor(P>0.05).Compared to the model group,each dosage group could significantly decrease the AFP,which is the diagnosis sign material of liver cancer.Experimental studies have found that nude mice are more thin,lazy to move,eat less,diarrhea in 5-Fu group because of the side effects of 5-Fu.The most attention is WSTF high dose +5-Fu group has an advantage over 5-Fu group not only in reduction of the tumors weight but also in improvement the tumor weight/body weight ratio.These findings imply WSTF combined with 5-Fu in clinical practice could both inhibit tumor cell growth and overcome the side effects of 5-Fu.The result of tumor-pathohistologic examination showed,compared to the model group,WSTF high,middle,low dose groups and 5-Fu group have obvious change as well as tumor cell apoptosis and organization with heavy/medium necrosis.These findings imply WSTF could promote tumor cell apoptosis and inhibit cellular cleavage and proliferation as well as presented the concentration dependent manner.Pathohistologic examination also showed there have no effusion in chest,abdominal cavity of tumor-burdened nude mice,and there have no cancer metastasis nodule and other abnormalities in the important visceras such as heart,liver,kidney,spleen,lungs and brain.These findings imply WSTF have no harmful effect on these visceras.Tunnel dyeing found that a lot of tumor cells apoptosis were perceived in WSTF high,middle dose groups and 5-Fu group compared with the model group.5.Investigation the molecular mechanisms of WSTF inhibiting tumor.Transmission electron microscopic showed that WSTF could reduce numbers of autophagosomes found in these cells.These findings are consistent with the conclusion from the previous in vitro cell experiments.We found that there are marked changes in morphology of cell such as the nucleus fragmentation,vacuoles in cells and the large number of apoptotic bodies by TEM examination.RT-PCR results show the expression levels of Bcl-2,Survivin mRNA were both significantly downregulated by WSTF treatment(P<0.01).In contrast,the expression of Bax mRNA and Caspase-3 mRNA were both significantly strengthened after WSTF treatment(P<0.05).These results collectively testify that WSTF may probably induce cell apoptosis through decreasing the levels of anti-apoptotic factors and increasing the levels of promoting-apoptotic factors.Moreover,RT-PCR results also show the expression levels of Belin1 mRNA?Atg5 mRNA were both significantly downregulated by WSTF treatment.These findings imply WSTF could decrease autophagic activity,which may validate the conclusion from TEM examination.We also found that the expression levels of CyclinDl mRNA?MMP-9 mRNA were both markedly downregulated by WSTF treatment by RT-PCR.In contrast,the expression of nm23-H1 mRNA was obviously strengthened after WSTF treatment(P<0.05).Western blot analysis showed,after treatment of WSTF,we observed the relative band densities of bcl-2 and Survivin were weakened respectively,while it was shown reverse trend in relative band densities of Bax and Caspase-3.Collectively,these findings from experiment show that WSTF may induce apoptosis cells,by regulating the levels of anti-apoptotic protein and apoptosis-promoting protein.Moreover,Western blot analysis also showed WSTF could suppress the protein expression of Beclinl?Atg5?CyclinD1?MMP-9,but strengthen the protein expression of nm23-H1.These findings further validate experiment conclusion from RT-PCR using protein levels.ConclusionTo sum up,in vitro cellular level,WSTF could inhibit significantly the proliferation of HepG2 cells,block effectively HepG2 cells in the G0/G1 phase,enhance mitochondrial membrane permeability,increase amount of intracellular ROS in HepG2 cells,and ultimately induce cell apoptosis.The most relevant mechanisms could be WSTF may probably induce cell apoptosis through decreasing the levels of anti-apoptotic protein(bcl-2,Survivin and mcl-1)and increasing the levels of promoting-apoptotic protein(Bax).Moreover,WSTF could increase MOMP,release cytochrome c and cytosolic AIF from mitochondria to cytoplasma.These eventually lead to cell apoptosis.In vivo,WSTF could inhibit tumors growth in nude mice,overcome the side effects of pharmaceutical chemical,improve living quality.The most relevant mechanisms could be WSTF could on the one hand,prevent HepG2 cells from energy and nutrients from the surrounding stromal cells through inhibiting autophagy,and on the other hand,WSTF may probably induce cell apoptosis to inhibit tumors growth through decreasing the levels of anti-apoptotic protein(bcl-2 and Survivin)and increasing the levels of promoting-apoptotic protein(Bax and Caspase-3).The study also found that WSTF could effectively suppress the ability invasion and metastasis of tumor cells in vivo.Our study provided scientific basis for effectiveness of xihuangcao in clinical medication and promoted its development and application of health care products. |
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