The Improved Efficacy And Reduced Toxicity Of Adriamycin In Targeted Therapy Of Hepatocellular Carcinoma By Carrier PEPT1

Background Hepatocellular carcinoma(HCC)accounts for the majority of Primary carcinoma of the liver.China is a high incidence of hepatitis B,so the incidence of HCC is particularly high.HCC results in 250,000 to 1,000,000 deaths per year,while China accounts for 45% of the total,and HCC has become the fifth and seventh most common malignant tumor in men and women respectively.The preferred treatment for HCC is liver resection,but this treatment is restricted to the early stages of HCC.Due to the difficulty associated with early diagnosis,the rapid progression of the disease and the fact that the majority of patients have liver cirrhosis,few patients are able to undergo the operation.Thus,non-operative therapy including chemotherapy is an important treatment strategy for advanced HCC.However,chemotherapy drugs is not specific to liver cancer cells,it may also affect normal cells and may result in serious adverse effects.Therefore,the currently available therapies offer limited benefits to patients.A targeted drug delivery system that can be used as an effective specific treatment may have good prospects.In particular,this system has many advantages.For example,the drug can be specifically transported to the specific location of a tumor and may achieve directional and focal inhibition of tumor cells as well as also causing less injury to normal cells,which may promote the highest treatment effects with minimal toxic effects on normal cells,resulting in increases in overall efficacy and safety as well as in patient compliance with therapy.The search for a safe and effective targeted drug carrier is the focus of ongoing research.Proton-independent oligopeptide transporter 1(PEPT1)plays an important role in the absorption and distribution of many drugs.The results of the present study demonstrated that PEPT1 was relatively limited expressed in normal tissues but highly expressed in various types of tumor cells,which suggest that PEPT1 may be specifically expressed in liver cancer cells and tissues to a large extent.The aim of this study was thus to verify the expression and function of PEPT1 in HCC.At the same time,Adriamycin was designed and modified to a certain structure,so that it can be identified and transported into HCC cells by a carrier of PEPT1 in a targetedmanner.This approach may lead to the development of a chemotherapy drug with increased efficacy and reduced toxic effects.This study was divided into three parts as below:Part One: The expression profile and drug targeted-transport function of PEPT1 in HCCObjective To verify the expression profile of PEPT1 in HCC and to explore the performance of its drug targeted-delivery function.Method We explored the expression profile and functional activity of PEPT1 in the HCC by Western blot and Immunohistochemistry and Real-time RT-PCR respectively,while we verified the drug transport activity of PEPT1.Result The results suggested that PEPT1 and PEPT1 m RNA were expressed in HCC cells respectively.The positive expression rate of PEPT1 in HCC tissues(86.00%)was higher than that observed in adjacent tissues(53.85%)and normal liver tissues(52.63%)(P<0.05).Significant differences in the expression of PEPT1 were observed between three different pathological grades of liver cancer,its expression positive rates were 92.31%,90.00% and 25.00% respectively in pathological grade 1,2 and 3 of HCC(P<0.05).What’s more,the uptake of PEPT1 substrate was time-dependent and concentration-dependent,and with a competitive inhibition.Conclusion PEPT1 was overexpressed in HCC,and its expression was correlated with its pathological grade.The uptake of PEPT-1 substrate was time-dependent,concentration-dependent and with a competitive inhibition phenomena.Part Two: The vitro studies of Adriamycin in targeted anti-HCC by carrier PEPT1 with improved efficacy and reduced toxicityObjective To synthesize a tumor-targeted drug Adriamycin peptide conjugate,and to verify the transport efficiency of PEPT1 to this drug through vitro cell experiments.Method To synthesize and identify the Adriamycin peptide conjugate.the distribution of this drug in the liver cancer cells was observed by fluorescencemicroscope,and the concentration of it in the liver cancer cells was detected by HPLC method.To verify the recognition between Adriamycin peptide conjugate and PEPT1,dynamic experiment,uptake experiment and substrate competition inhibition test were carried out.Result The Adriamycin peptide conjugate was successfully synthesized.compared with Adriamycin,Adriamycin peptide conjugate was more likely to enter the liver cancer cells with a strong fluorescence intensity by fluorescence microscopy and a high concentration by HPLC(P<0.05).The uptake of Adriamycin peptide conjugate was also time-dependent and concentration-dependent and significantly reduced by inhibitors.Conclusion Adriamycin peptide conjugate based on PEPT-1 carrier can be successfully synthesized.The Adriamycin peptide conjugate can be identified and targeted to the liver cell by PEPT1 with a stable drug concentration.Part Three: The vivo studies of Adriamycin in targeted anti-HCC by carrier PEPT1 with improved efficacy and reduced toxicityObjective To verify the transport efficiency of PEPT1 to Adriamycin peptide conjugate,and to study the efficacy and security of this tumor-targeted drug.Method The HCC mice models were established by the HCC cell lines Hep G2 and Bel-7402,which then were randomly divided into three groups: the control group without any treatment,the Adriamycin peptide conjugate group and the Adriamycin group.Meanwhile administration was divided into low dose group(Hep G2)and high dose group(Bel-7402)for comparative analysis.Result In the low dose group(HepG2): Compared with control group and Adriamycin group,the general condition in Adriamycin peptide conjugate group was better with a unobvious weight loss.The survival time of Adriamycin peptide group was 8.57%(3.0 d)longer than that of the control group and 24.28%(8.5 d)longer than that of the Adriamycin group.The Adriamycin peptide conjugate and Adriamycin can decrease the growth of tumor volume and weight.Their tumor inhibition rates were 42.13% and 25.280% respectively.The difference between themhad statistical significance(P<0.05),and there was statistically significant compared with the control group respectively(P<0.05).It was suggested that the drug Adriamycin peptide conjugate was less damage to the heart and bone marrow(P<0.05).In the high dose group(Bel-7402): Adriamycin peptide conjugate was mainly distributed in subcutaneous tumor tissue and liver metastatic tumor tissue compared with Adriamycin group by small animal imaging technique.When the drug dosage was increased,the Adriamycin peptide conjugate had more effective in cancer treatment with less side effects,While the tumor inhibition rate of the Adriamycin was improved but associated with drug toxicity increased.Conclusion This study provided a experimental evidence that PEPT1 can be used as a carrier of Adriamycin in targeted therapy for HCC.Furthermore,it was proved that the administration of antitumor drugs through PEPT1 may represent a potential cancer-targeted treatment with greater efficacy and lower toxicity.