Matrine Induces Apoptosis Of Multidrug Resistant CRBH-7919/m Drl Cells

Liver cancer incidence rate showed an increasing trend, and hepatocellular carcinomahas become the second largest malignancy, patients die each year from liver cancer rankedfirst in the world[1].The incidence and prognosis stagnant increased year by year more andmore serious challenges for the clinical treatment of hepatocellular carcinoma. There is noeffective means of treatment of hepatocellular carcinoma, surgery is only expected to getcure, but in fact in clinical patients suitable for radical treatment, less than half, sochemotherapy with advanced liver cancer and surgery recurrent hepatocellular carcinomaafter treatment. Generation of multi-drug resistance greatly affects the efficacy ofchemotherapy drugs, and even chemotherapy-sensitive hepatoma cells after chemotherapyare often drug resistance, eventually leading to failure of chemotherapy. Multi-drug resistant(multidrug resistance, MDR) refers to tumors in contact with a chemotherapy drug, not onlyfor this drug resistance and cross-tolerance phenomenon, and other structures andmechanisms of different chemotherapy drugs. MDR's main features include:â‘ the nature ofthe lipophilic cell poisons and anticancer drug tolerance;â‘¡through the ATP-dependentpump to intracellular drug pump thereby reducing its effective concentration;â‘¢mainlythrough multi-drug resistance overexpression of the gene and its encoded protein and lead todrug resistance. Mdrl overexpression of MDR in the main mechanism[2,3].Researchers found that[4,5]will contain the full-length cDNA of the human mdrlrecombinant vector was transfected into cells, the reorganization of mdrl cDNA can producefunctional P-gp protein, high expression of these genes or proteins causing cells to theformation of the MDR phenotype. Therefore, inhibition of mdr1mRNA encoding P-gpprotein expression is the focus of the research reversal of MDR.MDR is a pressing problemin the process of chemotherapy. In recent years, screening of natural anti-cancer drugs hasbecome a hot research.Matrine alkaloids present in the active ingredient in the herb of the leguminous plantSophora, Sophora alopecuroides. And found that matrine has anti-tumor effect in a variety oftumor cells including cervical cancer, ovarian cancer, stomach cancer, leukemia, coloncancer, liver cancer, malignant glioma and other matrine in vitro and in vivo on a varietyof tumor cell function, inducing tumor cell apoptosis and no significant toxic sideeffects. However, the the matrine Antitumor activity and apoptosis induction mechanism is not clear. Vigorously develop the traditional Chinese herbal medicine, the use of molecularbiology by means of its pharmacological effect, and is committed to the development of newanticancer drugs, has become the development trend of Chinese medicine research. In thiscontext, the depth of matrine on tumor and the molecular mechanism is undoubtedly aprospect is extensive and the subject of great research value. In this experiment, the rathepatoma cell line CRBH-7919cells and their MDR cell lines CRBH-7919/mdr1matrine invitro studies of rat hepatoma and its drug-resistant cell proliferation and resistance clearwhether matrine can be induced in vitro in rat hepatoma cell line CRBH-7919cells and theirMDR cell lines CRBH-7919/mdr1apoptosis; matrine CRBH-7919and CRBH-7919/mdr1apoptosis induced bymolecular mechanism of action; clear matrine apoptosis regulatorymolecules p53, Fas and Bcl-2protein expression levels of impact; study was designed toconfirm the anti-tumor activity of matrine, and looking for potential targets of possible andmechanisms.First, build the multidrug resistance gene (of mdrl) overexpression cell modelSo far, the MDR cell lines induced by in vitro research has led to the molecular basis oftumor drug resistance. In this study, gene cloning, of mdrl cDNA and the eukaryoticexpression vector connected to the PCI-neo, and the Import rat hepatoma cell lineCRBH-7919cells, G418screening stable MDR cell lines CRBH-7919/mdr1cells. Andreal-time quantitative PCR and Western blot detection CRBH-7919/mdr1cells mdrl geneand its encoded protein P-gp expression than cell CRBH-7919, to determine the model wasconstructed successfully.Second,matrine on rat hepatoma cells the strains CRBH-7919and its drug-resistantcells CRBH-7919/mdr1growth inhibition and resistanceMatrine of different drugs CRBH-7919, and CRBH-7919/mdr1cell MTT assay wasused to measure the cell,Inhibition of proliferation, the results show that differentconcentrations of matrine for48h, both cell proliferation to varying degrees by inhibition,and this phenomenon was dose and time dependent; real-time quantitative PCR and Westernblot results: After concentrations were0,0.25,0.50,1.00mg/ml matrine CRBH-7919/mdr1cells at24h and48h after dosing control cells P-gp was mRNA and protein expressionflavescens alkali concentration in a dose-dependent decline. Prompted the matrine not onlycan inhibit the CRBH-7919and CRBH-7919/mdr1cell proliferation, but also to some extent,reversed the resistance of MDR cells. Third,of matrine on the induction of apoptosis in rat hepatoma cell line CRBH-7919andresistant cells CRBH-7919/mdr1Many methods can detect cell apoptosis, such as electron microscopy, DNA gelelectrophoresis, flow cytometry with PI staining and TUNEL[2]. In this study, opticalmicroscopy, DNA gel electrophoresis and flow cytometry were used to detect apoptosis.These results demonstrated that matrine can not only induce the apoptosis of drug-sensitiveCRBH-7919cells but also markedly induce the apoptosis of its MDR cells. This finding maysuggest that matrine can overcome MDR of cancer cells and induce apoptosis to kill tumorcells.Matrine may be one kind of ideal natural anti-tumor agents, especially for the treatmentof recurrence or metastasis in patients with chemotherapy-resistance[4-6]. In this study, flowcytometric analysis showed that matrine results in the increase in S-phase fraction in a certaindegree and the decrease in G2/M fraction, indicating that matrine-induced apoptosis of cancercells may be associated with G1/S phase arrest. We can assume that matrine arrest cancer cellsin G1/S phase and combine with S-phase specific drugs, the combined chemotherapy shallincrease the sensitivity to chemotherapy. After the action of matrine, the fraction of apoptosiscells is not significant difference between24h and48h, indicating that the apoptosis canoccur within24h.Initiation of apoptosis is a complex process involving multiple genes, such as wild-type p53, c-myc, Fas, bcl-2/Bax in mobilized to participate in the regulation of apopt-osis. Our experiments found that matrine processing CRBH-7919and CRBH-7919/mdr1cells at different times with different drug concentrations, the proapoptotic gene P53and Fasgene mRAN of and protein expression was increased, while the inhibition of apop-tosis Bcl-2mRAN decrease and protein table. And these two phenomena are related to the role ofmatrine in a concentration and time depen-dent. Matrine induced tumor cell apoptosisprocess regulation of gene activation and transcription, with or without the MDR-relatedgene expression changes, we will further study.