| Objective: Through theoretical research,we summarized the understanding and treatment methods of traditional Chinese and western medicine of liver cancer,and summarized the research status of traditional Chinese medicine in radiotherapy sensitizer.To investigate the effect of Compound Taxus on proliferation,migration,invasion,angiogenesis,apoptosis,cell cycle,enhanced radiosensitivity,and DNA repair after radiotherapy of hepatocellular carcinoma and the possible mechanisms.Methods: Theoretical research:by reading ancient books,we summarized the literature description,etiology,syndrome differentiation and treatment of hepatocellular carcinoma;through literature in Chinese and foreign languages,we summarized the etiology and pathogenesis,pathological classification,clinical manifestations,examination,diagnosis and treatment,and summarized the research status of Chinese medicine in radiotherapy sensitizers.Experimental research: twenty SD rats were randomly divided into two groups;one group was intragastrically given Compound Taxus solution and the other group was given equal volume saline solution.After fourteen days of intragastric administration,blood was collected from abdominal aorta to prepare the drug-containing serum and the control serum.Two hepatoma cell lines cultured in vitro,Smmc7721 and Bel7402,were treated with 0%,2.5%,5%,10%,20% and 40% Compound-Taxus-containing serum for 24 hours.cck-8 experiments were performed to observe survival rate of the cells,and the half inhibition concentration(IC50)was calculated.The IC50 obtained was used for the following experiments.Smmc7721 and Bel7402 cells were treated with 0%,5% and 10% CompoundTaxus-containing serum for 24 hours.The effects of Compound Taxus on cell migration and invasion were observed using scratch experiment and invasion experiment.HUVEC,Smmc7721 and Bel7402 cells were treated with 0%,5% and 10%Compound-Taxus-containing serum for 24 h to observe the effects of Compound Taxus on angiogenesis of HUVEC and vasculogenic mimicry of hepatocellular carcinoma cells.Smmc7721 and Bel7402 cells were treated with 0%,5% and 10% CompoundTaxus-containing serum for 12 hours(or 24 hours),and cell cycle status and apoptosis were detected by flow cytometry.Smmc7721 and Bel7402 cells were treated with 0%,5% and 10% CompoundTaxus-containing serum for 24 hours,and the expression levels of Ncadherin,Vimentin,E-cadherin,MMP-9,NF-?B p65,p-NF-?B p65,Bax and Bcl-2 were detected by western blot.Smmc7721 and Bel7402 cells were treated with 0% or 5% Compound-Taxuscontaining serum for 12 hours,and then exposed to X-ray irradiation(0-10 Gy).Radiation survival curves of Smmc7721 and Bel7402 were obtained from the colony formation results.Sensitizing enhancement ratio(SER)was obtained according to the one-hit,multi-target model.Smmc7721 and Bel7402 cells were treated with 0% or 5% Compound-Taxuscontaining serum for 12 hours,and then exposed to X-ray irradiation(4Gy).At 12-hour post-irradiation,Rad51 was detected by cell immunofluorescence assay.We established the Bel7402 hepatocellular carcinoma model in nude mice.Then all the model mice were randomly separated into the following groups(n=5): control(Con),Compound Taxus(CT),X-ray irradiation(RT)and combination(Comb).159mg/kg/day Compound Taxus and saline solution were given intragastrically to the CT and Con group,respectively.A single dose of 8Gy X-ray irradiation was given to the RT group.The Comb group received one dose of Compound Taxus,was exposed to X-ray irradiation two hours later on the first day,and then received other daily doses of Compound Taxus from the next day.Tumor volumes were monitored and recorded once every two days to evaluate the treatment responses.On day 14 after the continuous intervention,all nude mice were executed.The weight of nude mice and tumors were recorded,and the tumor inhibition rate,tumor growth delay time and sensitization coefficient(EF)were calculated.Immunohistochemical staining was performed to detect the expression levels of CD31,VEGFA,NF-?B p65,Bax and Bcl-2.Results: Theoretical research: The pathogenesis of hepatocellular carcinoma is complicated.Ininvasion of dampness and heat is the main factor of hepatocellular carcinoma.It is said the strong antipathogenic qi keeps pathogen away.Deficiency of antipathogenic qi leads to cancer toxin.The cancer toxin itself need senergy for its growth,which must consume qi and blood in human body.The current therapeutic methods of hepatocellular carcinoma consume qi and damage yin.Experimental research: The survival rates of Smmc7721 and Bel7402 were significantly reduced by Compound-Taxus-containing serum(p<0.0001),and IC50 was(8.938±0.444)% and(7.948±0.190)%,respectively.The drug-containing serum of Compound Taxus significantly inhibited the migration(p<0.0001)and invasion(p<0.05)of hepatocellular carcinoma cells and increased the apoptosis rate(p<0.01).The drug-containing serum of Compound Taxus significantly inhibited the angiogenesis of HUVEC and the vasculogenic mimicry of hepatocellular carcinoma cells in vitro(p<0.0001).Significant cell cycle arrest in G2/M phase was found in Smmc7721 and Bel7402 cells treated with the drug-containing serum of Compound Taxus(p<0.05).The drug-containing serum of Compound Taxus significantly decreased the expression levels of N-cadherin,Vimentin,MMP-9,NF-?B p65,p-NF-?B p65 and Bcl-2,while increased E-cadherin and Bax expression in Smmc7721 and Bel7402 cells.Those colonies with more than 50 cells were counted to establish the radiation survival curves.The survival fractions of smmc7721 and bel7402 cells were decreased by Compound-Taxus-containing serum treatment.Furthermore,the drug-containing serum of Compound Taxus enhanced the radiosensitivity of Smmc7721 and Bel7402 with radiation enhancement ratios of 1.347 and 1.360,respectively.Compared with the group of radiotherapy alone,pretreatment with 5%Compound-Taxus-containing serum followed by 4Gy X-ray irradiation inhibited the formation of Rad51 foci in both Smmc7721 and Bel7402 cells.In the Bel7402 hepatocellular carcinoma model of nude mice,the tumors grew the fastest in the control group,while grew the slowest in the combination group.There was significant statistical difference between every two groups(p<0.05).EF value of the combination group was greater than 1,suggesting that Compound Taxus might have the effect of enhanced radiosensitivity.Immunohistochemical staining results showed that expression levels of CD31,VEGFA,NF-?B p65 and Bcl-2 were decreased,while Bax level was increased in the Compound Taxus group,compared with the control group.Conclusions: Internal invasion of dampness and heat and antipathogenic qi deficiency are important reasons for the formation of hepatocellular carcinoma.and it is appropriate to dispel damp,clear heat,detoxification and replenish qi and nourish blood.Compound Taxus significantly inhibited cell migration,invasion,angiogenesis,vascular mimicry and other malignant behaviors of hepatocellular carcinoma cells.In addition,it inhibited the homologous recombination DNA repair response after radiotherapy,and improved the radiosensitivity of hepatocellular carcinoma cells,indicating that Compound Taxus had the effect of enhanced radiosensitivity.Compound Taxus affected NF-?B activity and its downstream protein expression.Its anti-tumor and enhanced radiosensitivity effect might be achieved,at least in part,through inhibiting the NF-?B signaling pathway. |
PDF Full Text Request