【Abstract】ObjectiveTo construct a mrp1 expression vector and investigate its biological characteristics in HepG2 cells in vitro. MethodsThe 6.5 kb multidrug resistanceassociated protein （MRP) cDNA obtained from plasmid pGEM-mrp1 was cloned into the pCI-neo mammalian expression vector, which was later transferred into human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and proliferated, and the mrp1 mRNA and MRP in these HepG2 cells were detected by means of RT-PCR and FCM respectively. ResultsThe mrp1 expression vector was established successfully, and the stable MDR hepatocarcinoma cell line (HepG2/mrp1) was developed as well. The content of the specific fragment of mrp1 mRNA was (56.8±6.37)% and MRP was 7.89 in the HepG2/mrp1 cells, the corresponding value in HepG2 cells was (9.67±3.26)% and 0.79 respectively. The difference was statistically significant (P＜0.05). ConclusionIt is practicable to establish MDR hepatocarcinoma cell line by transferring mrp1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
Objective To review the application advancements of ATP-binding cassette (ABC) transporter in medical research.Methods Relevant literatures about the applications of ABC families in medical research were reviewed. Results ABC families mainly took roles in transporting substances across cell membrane. Some of them were useful for the prediction of drug resistance and the prognosis of malignant tumors. Others were target s for molecular researches. Their expressions or mutations might be related with the occurrence of diseases. Conclusion ABC families are very important in the diagnosis and therapy for diseases. Thus they are very promising tools for future medical research.
Objective To dynamically study the formation of multidrug resistance(MDR) of human hepatocellular carcinoma cell SMMC-7721 induced by Adriamycin (ADM) and the role of multidrug resistance-associated protein(MRP) in its mechanisms.Methods Hepatocellular carcinoma cell SMMC-7721 was cultured in RPMI-1640 medium containing ADM with progressively increased concentration or directly cultured in medium containing different concentrations of ADM. Resistant index of drug-resistant variants of SMMC-7721 cell was determined by drawing cell dosage-reaction curves.Levels of MRP mRNA expression were detected by reverse transcription-polymerase chain reaction(RTPCR). Intracellular rubidomycin(DNR) concentration was examined by flow cytometry(FCM).Results With progressive increasing of ADM concentration in medium resistant index and levels of MRP mRNA expression were correspondingly increased but intracellular DNR concentration was markly reduced. When parental cells were directly cultured in medium containing different concentrations of ADM, the higher the ADM concentration, the higher the level of MRP mRNA expression, but intracellular DNR concentration was kept at the similar high level and most cells died. Conclusion ADM may progressively induce SMMC-7721 cell resistant to multiple chemotherapeutic drugs with reduced intracellular DNR accumulation associated with the overexpression of MRP gene.
Multidrug resistance (MDR) remains the major obstacle to the success of clinical cancer chemotherapy. P-glycoprotein (P-gp), encoded by the MDR1, is an important part with complex mechanisms associated with the MDR. In order to overcome the MDR of tumors, we in the present experimental design incorporated small interfering RNA (siRNA) targeting MDR1 gene and anticancer drug paclitaxel (PTX) into the solid lipid nanoparticles (SLNs) to achieve the combinational therapeutic effects of genetherapy and chemotherapy. In this study, siRNA-PTX-SLNs were successfully prepared. The cytotoxicity of blank SLNs and siRNA-PTX-SLNs in MCF-7 cells and MCF-7/ADR cells were detected by MTT; and the uptake efficiency of PTX in MCF-7/ADR cells were detected via HPLC method; quantitative real-time PCR and flow cytometry were performed to investigate the silencing effect of siRNA-PTX-SLNs on MDR1 gene in MCF-7/ADR cells. The results showed that PTX loaded SLNs could significantly inhibit the growth of tumor cells, and more importantly, the MDR tumor cells treated with siRNA-PTX-SLNs showed the lowest viability. HPLC study showed that SLNs could enhance the cellular uptake for PTX. Meanwhile, siRNA delivered by SLNs significantly decreased the P-gp expression in MDR tumor cells, thus increased the cellular accumulation of rhodamine123 as a P-gp substrate. In conclusion, the MDR1 gene could be silenced by siRNA-PTX-SLNs, which could promote the growth inhibition efficiency of PTX on tumor cells, leading to synergetic effect on MDR tumor therapy.
Objective To investigate the reversal effect of antisense phosphorothioate oligonucleotide （ASOND) on human hepatoma resistant cells. Methods Human hepatoma resistant cells SMMC-7721 was transfected with synthetic antisense phosphorothioate oligonucleotide complementary to the 5′ region flanking the AUG initiation codon mediated by lipofectamine. In vitro drug sensitivity was measured by MTT assay. The expression of P-170 was determined by flow cytometry and mRNA was assessed by RT-PCR. Results ASOND inhibited the expression of mRNA and p-170 in SMMC-7721, enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug. The best inhibitory effect was achived by the dose of 0.5μmol/L. Conclusion ASOND enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug and reversed the multidrug resistance of SMMC-7721 partially.
ObjectiveTo explore the relationship between mdr1 gene expression of hepatocellular carcinoma (HCC) and pathological characteristics,chemotherapy and prognosis. MethodsThe mdr1 gene expression of HCC in 56 patients with the methods of immunohistochemistry was studied. The results were analysed with the pathological data by statistic methods. ResultsThe positive expression of mdr1 gene in cancer tissues and pericancerous tissues of HCC were 30/56(53.6%) and 19/56 (33.9%) respectively. The difference was statistically significant (χ2=4.39,P＜0.05). The positive expression of mdr1 gene in cancer tissues of untreated patients and in recurrent patients were 22/48(45.8%) and 8/8(100%) respectively.The expression of mdr1 gene was not associated with tumor size, number, tumor thrombus, differentiation, HBsAg and liver cirrhosis. The patients with positive mdr1 expression had a shorter survival time than that of negative ones. But the difference was not statistically significant. Conclusion The positive expression of mdr1 in HCC is 53.6%. It is not associated with tumor size, number, tumor thrombus, tumor differentiation, HBsAg and liver cirrhosis. There are innate multidrug resistance in HCC.
ObjectiveTo construct the recombinant adenovirus vector carrying antisense multidrug resistanceassociated protein (MRP) and transfect the human drugresistant hepatocellular carcinoma cell line(SMMC7721/ADM). MethodsThe fragment of MRP gene encoding 5′region was cloned reversely into the shuttle plasmid pAdTrackCMV, with the resultant plasmid and the backbone plasmid pAdEasy1,the homologous recombination took place in the bacteria and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in 293 cells. Then the cell line of SMMC7721/ADM was transfected with the resultant adenoviruses.ResultsThe recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5×109 efu/ml, and more than 90% SMMC7721/ADM cells could be transfected when the multiplicity of infection（MOI） was 100. ConclusionThe recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drugresistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.
Objective To study the effects of survivin antisense RNA on SGC7901 cell’s apoptosis and chemosensitivity to taxotere, and to investigate its effect on the expression of multi-drug resistance gene-1 (MDR-1). Methods Survivin antisense eukaryotic vector anti-pcDNA3-svv was transfected into SGC7901 cell lines by lipofectamine and positive clones were screened out then. Survivin protein and MDR-1 mRNA were measured by western blot and RT-PCR, respectively. Apoptosis that was induced by anti-pcDNA3-svv was observed by electronic microscope, and the sensitivity of SGC7901 cell to taxotere was examined by MTT. Results The expressions of survivin protein and MDR-1 mRNA in transfected SGC7901 cells both decreased more significantly than that of non-transfected cells (P＜0.05， P＜0.01), and the indices of MDR of transfection group and non-transfection group were 0.196±0.013 and 3.126±0.019, respectively, at the late phase of apoptosis, which had a significant difference between each other (P＜0.01), IC50 of the transfected cells to taxotere was （16.7±1.98） ng/ml and that of the non-transfected cells was (55.7±1.89) ng/ml, which also had a significant difference (P＜0.01). Conclusion Surivivin antisense RNA could induce the apoptosis of SGC7901 cancer cell line and could increase the cells’ sensitivity to taxotere, which may help to reverse drug resistance.