Study On Reversing HCC Multidrug Resistance Combination Therapy With Tumor Necrosis Factor α And Bromocriptine In Vitro And In Vivo

Human hepatocellular carcinoma (HCC) is one of the most common malignancy tumors. General given by individual treatment but giving priority to operation had been become the standard therapy project for HCC. Chemotherapy, as a critical adjuvant treatment method, still hold the important status in clinical therapy, but the MDR phenomena was one of the major factors which are restrict the chemotherapy effect.In recent years, to find an effective gene therapy method and high effective but low virulence drugs for HCC treatment was still the hot topic for the scholars. Any single method for reversing HCC multidrug resistance couldnot obtain the ideal treatment effect because of the complicated mechanism to MDR, and combining two effective means were more likely to win enhancing the susceptibility of HCC cells to cytotoxic drugs. However, cytokines administered by the conventional intravenous method can cause severe side effects. Transduction of cytokine genes into tumor cells constitutes an alternative approach for production and release of the cytokine proteins in the local tumor microenvironment, which may reduce problems of toxicity associated with systemic administration but enhance the susceptibility of the tumor cells to the cytotoxic drugs. Human tumor necrosis factor-a was one of the most powerful anti-cancer cytokines up to now. Some scholars also found that BCT, described as a dopaminergic receptor agonist, could inhibit the P-gp ATPase activity and modulates P-glycoprotein function. BCT is used in clinics to treat hyperprolactinemia and Parkinson's disease and accepted by patients because of its little side-effect. Therefore systemic study on reversing HCC multidrug resistance combining with human tumor necrosis factor-a and bromocriptine would be carried through in this experiment which concludes four parts: Part 1 Construction of a recombinant eukaryotic vectorexpressing human tumor necrosis factor alpha andexpressing stably in cell line HepG₂/ADM[Objective] To construct a eukaryotic expression vector expressing human tumor necrosis factor alpha(hTNF-α) and expressing stably in cell line HepG₂/ADM.[Methods) The full length gene of hTNFα cDNA was identified and cloned(cDNA) from human monocyte stimulated by LPS using reverse transcription polymerase chain reaction(RT-PCR). The cDNA was incorporated into the pMD18-T plasmid and then inserted into a dual expression vector pBK-CMV and called pBK-hTNF-α. Furthermore, pBK-hTNF-α vector was used to infect E.coli DH5α. The expression of the recombinant hTNFα protein(rhTNFα) by E.coli DH5α was analyzed using SDS-PAGE and westernblot test. After gene sequence of hTNFα was confirmed by cleavage of restriction enzymes and DNA sequence analyzed, expression of hTNFα mRNA and protein of hTNF-α transfected into drug resistant cell line HepG₂/ADM were detected by RT-PCR and ELISA.[Results] The sequence of hTNFα cDNA cloned from human monocyte was completely correct,compared with the sequence in Genbank. Digestion with BamH I and Hind III confirmed that hTNFα cDNA was inserted correctly into pBK-CMV eukaryotic expression vector. The genetically engineered E.coli DH5α did express hTNF-α confirmed by westernblot and the drug resistant cell line of HepG₂/ADM was transfected with an eukaryotic expression plasmid containing hTNFα gene by Lipofectamine2000, and then expressed hTNFα cDNA steadily. [ Conclusion ] The pBK-hTNFα, a eukaryotic expression plasmid for the full length gene of hTNFα cDNA was constructed successfully by DNA recombinant technique and expressed steadily in vitro . Part 2 Tumor necrosis factor-α and bromocription induceapoptosis and sensitize resistant hepatic cancercells to chemotherapy[Objective] To investigate the effect of bromocriptine(BCT) combining with human tumor necrosis factor a(hTNFα) inducing apoptosis in resistant hepatic cancer cells.[Methods] Firstly, to isolate and identification of hTNFα expressing cells, and to use liposome carrying hTNFα gene to transfect HepG₂ /ADM cell line and establish a cell model expressing the hTNFα protein stably. The hTNFα secreting cell clone HepG₂ /ADM/ TNF-α was obtained by G418 selection, and the integrating and secreting of hTNFα were analyzed by RT-PCR and ELISA method. All experiments were divided into four groups and named blank control group(A), drug resistant group HepG₂/adriamycin(ADM)(B) , hepatocarcinoma cell line transfected with hTNFα gene HepG₂/ADM/TNF (C ) and group BCT(D) respectively. Among these groups, group BCT was that group C treated with bromocriptine simultaneously. MTT assay was tested to detect the sensitivity to ADM for every groups' cells, and Rhodaminel23(Rhl23) applied to test the function of P-gp by Flow Cytometric Analysis(FCM). MDR associated genes and proteins(MDRmRNA, P-gp) and PKC-α protein were detected by immunohistochemistry (IHC), Westernblot and reverse transcriptase polymerase chain reaction (RT-PCR) methods respectively, and Bcl-2 protein expression and apoptosis rate of hepatocarcinoma cells was detected by FCM.[Results] The cell line transfected with hTNFα gene were established successfully by RT-PCR. The levels of hTNFα secreted by HepG₂ /ADM/ TNF-α and HepG₂ /ADM cell line were (789.68±34.43) pg/ml, (34.48±13.62) pg/ml respectively, there was significant difference between them. At the same time, there were significant difference between group C and group D in aspect of the rate of reversing resistance and the intracellular Rho 123 accumulation (P<0.01) . MDR1mRNA and P-gp protein expression in group C and D were low similar to that in group A, but no difference could be found among them(P<0.05). As we found that PKC-a protein expression was downregulated in group D but Bcl-2 protein expression was downregulated in group C, and there were significant difference compared to other groups. The apoptosis rate of hepatocarcinoma cells was much higher in group D than that in group C(P<0.05) with FCM, but similar to that in group A(P>0.05).[Conclusion] Synergistic effect of between bromocription and tumor necrosis factor-α on reversing hepatocellular carcinoma multidrug resistance could be obtained and enhancing the susceptibility of HepG₂/ADM cells to cytotoxic drugs Part 3 Establishing multidrug resistant model in nude micevia orthotopic implantation of human multidrug resistanthepatocellular carcinoma cells directed by B ultrosound[Objective] To establish multidrug resistant model in nude mice via orthotopic implantation of human multidrug resistant hepatocellular carcinoma cells directed by B ultrosound.[Methods] Human hepatocellular carcinoma cells HepG₂ and multidrug resistant human hepatocellular carcinoma cells HepG₂/ADM were maintained in 1640 containing 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in a humid atmosphere of 5% CO2 and an orthotopic mdr1 hepatoma was obtained by injecting the cell lines HepG₂ and HepG₂/ADM subserosally into the mice liver directed by B ultrasound(control group 15 cases, multidrug resistant groups 15 cases). Ultrasonography and laparotomy were used to detect the tumor growth, and integrity mdrl gene from multidrug resistant human hepatocellular carcinoma cells HepG₂/ADM and corresponding implantated tumor tissues were amplificated by long PCR technique and sequenced. Furthermore, the MDR1mRNA and P-gp protein expression were evaluated by means of reverse transcription and the polymerase chain reaction(RT-PCR), Westernblot , Immunohistochemistry (IHC) methods respectively.[Results] The successful rate of tumor implantation and multidrug resistant induced is 100%(30/30), 100% (15/15)respectively. MDR1 integrity gene 3.8kbp band can be detected from both HepG₂/ADM cells line and corresponding implantated tumor tissues by long RT-PCR technique, and the MDR1gene sequence is in coincidence to that reported in genebank. The expression of MDRlmRNA and P-gp protein in multidrug resistant groups were significantly higher than that in control groups by RT-PCR and Westernblot methods respectively. There were notable difference between multidrug resistant groups and controls which P-gp protein expression were(42.6±1.7)%and(2.6±0.1)%respectively by immunohistochemistry. Furthermore, P-gp protein expression from implanted tumors after HepG₂/ADM cells implanted 2 and 8 weeks were detected respectively and no difference were found.[Conclusion] Multidrug resistant model in nude mice via orthotopic implantation of multidrug resistant human hepatocellular carcinoma cells could be successfully established directed by B ultrasound, and provided good plateau for studying the reversal strategy of PHC multidrug resistant phenomenon. Part 4 Study on reversing HCC multidrug resistance by combination therapy with tumor necrosis factor a and bromocriptme in nude mice mdr model of liver neoplasm.[Objective] To investigate the reverse effect on HCC multidrug resistance(MDR) by combination with tumor necrosis factor a and bromocriptine in nude mice mdr model of liver neoplasm.[Methods] Human hepatocarcinoma cell line HepG₂, drug resistant hepatocarcinoma cell HepG₂/ADM and hepatocarcinoma transfected into human tumor necrosis factor a gene cell HepG₂/ADM/TNF were injeceted into the liver of nude mice via orthotopic implantation respectively and MDR model of liver neoplasm in vivo was established[named group HepG₂(A), ADM(B), TNF(C), BCT(D) respectively]. BCT group was group TNF which treated together with bromocriptine using gastric canal.Each group was divided into control and chemotherapy group and size , weight of the tumors were measured ,tumor histological characters by HE and growth of the nude mice were observed and its chemosensitivity were tested.MDR associated genes and proteins(MRP, LRP) of implantated tumors were detected by immunohistochemistry (IHC) and RT-PCR technique, and the apoptosis rates of hepatocarcinoma cell were measured with TUNEL assay.[Results] The nude mice model of every cell line were all inoculated successfully. The tumor growth rates and weights were different significantly among every groups(P<0.05), with the lowest group by hTNF α transfected cell line compared to the other two groups. After the same chemotherapy though abdominal cavity tumor growth inhibitory rate was highest in D group (67%) compared with B and C group respectively (P<0.01),and similar to HepG₂ group (54%) .MDR1 and LRPmRNA could all be detected in all groups, but TNFmRNA be detected only in C and D groups. Furthermore, MDR1 protein expression of tumors in C and D groups were lower than that in group B and were similar to group A, but difference could be detected between the group C and D. Additionally the apoptosis rates of hepatocarcinoma cell were much highest in group D than that of other groups(P<0.05) with TUNEL assay.[Conclusion] TNF-α gene can down-regulate the MDR associated genes andproteins expression for example MDR1 , LRP, and lower its tumorgenesis. Moreover, enhancing the susceptibility of HepG₂/ADM cells to cytotoxic drugs combination therapy with bromocriptine,...