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.
Objective To review the recent studies on the multidrug resistance of breast cancer. Methods The literatures of recent years on the studies of multidrug resistance, multidrug resistance protein and breast cancer resistance protein were reviewed. Results Multidrug resistance resulted from multiple factors. How to identify the sensibility of chemotherapy drugs and select individual therapeutic regime early were important to improve the survival rate and life quality of breast cancer patients. Conclusion These studies on multidrug resistance of breast cancer are helpful to predicting the effect and outcome of chemotherapy and overcoming the barrier of drug resistance.
ObjectiveTo establish multidrugresistance cell substrain of human hepatocellular carcinoma and to investigate its characteristics.MethodsSMMC7721 cell strain was cultured in Adriamycin(ADM). The multidrugresistance cell substrain SMMC7721/ADM was harvested after a long period of culture by gradually increasing the concentration of ADM and its characteristics were investigated. Results①The drug resistance of SMMC7721/ADM to ADM increased by 33.3 times, to Vincristine 16.8 times, to Diamminedichloroplatinum 2.8 times. ②The drug resistance cell substrain had almost the same growth velocity as its parental generation. The doubling time was 32.0 hours and 30.5 hours respectively. They had the analogous growth curves. ③The obvious difference between the drug resistance cell substrain and its parental generation was that the former’s microvilli became thick, short and scattered by scanning and transmitting electron microscopy. ④The multidrug resistance cell substrain kept the characteristics of hepatocellular carcinoma, it could be transplanted into the subcutaneous tissue of nude mice. ⑤The drug resistance of the cell substrain reduced to 28.0% and 9.2%after removal of the drug for 1 month and 2 months respectively, its drug resistance could remain stable (35.4 times) after 2 months of culture in ADM (0.04 μg/ml).ConclusionThe SMMC7721/ADM cell substrain has the stable fundamental characteristics of a drug resistance cell strain.
Objective To investigate the effect of phosphorothioate antisense oligonucleotides(AS-ODN) on suppressing multidrug resistance-associated protein gene(MRP) in human drug-resistant hepatocellular carcinoma cell line (SMMC-7721/ADM). Methods Cell line was transfected with a synthetic S-ODN complementary to the coding region of MRP mRNA, Lipofectamine acting as carrier. The drug sensitivity was measured by MTT assay. The expression of MRP mRNA was detected by RT-PCR and the expression of P190 was detected by flow cytometry. Results AS-ODN inhibited expression of MRP mRNA and P190 and promoted sensitivity to daunorubicinum and adriamycinum. Conclusion AS-ODN can reduce the expression of MRP gene. MDR caused by MRP is an important cause of multidrug resistance of SMMC-7721/ADM.
【Abstract】ObjectiveTo construct an mdr1 expression vector and detect its expression in HepG2 cells in vitro. MethodsThe 4.5-kb mdr1 cDNA was obtained from the plasmid pHaMDR1 cloned into the PCIneo 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 specific fragment of mdr1 cDNA, mRNA and the Pgp in these HepG2 cells were detected by means of PCR, RT-PCR and FCM respectively. ResultsThe mdr1 expression vector was constructed successfully，and the stable multidrug resistance(MDR) hepatocarcinoma cell line （HepG2/mdr1） was developed as well. The outcome of PCR analysis showed that the specific fragment of mdr1 cDNA could be found in HepG2/mdr1 cells, but not in the nontransfection HepG2 cells. Furthermore,the content of the specific fragment of mdr1 mRNA and the expression of P-gp in HepG2/mdr1 cells were (59.7±7.9)% and (12.5±5.45)% respectively, the corresponding value in HepG2 cells were (16.9±3.2)% and (4.63±2.59)% respectively. The difference was statistically significant (P＜0.05). ConclusionIt is praticable to develop MDR hepatocarcinoma cell line by transferring mdr1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
Objective To detect the characteristic of multidrug resistance gene products expressions in gastric cancer tissues, including glutathione-s-transferase π (GST-π), P-glycoprotein (P-gp), topoisomerase Ⅱ (Topo-Ⅱ), thymidylate synthase (TS), and multidrug resistance related protein (MRP), and analyze their clinical significance for the therapy of gastric cancer. Methods SP immunohistochemical stain was used to detect GST-π, P-gp, Topo-Ⅱ, TS and MRP expressions in sample of 48 gastric cancer tissues and 10 normal gastric mucosa. And their corresponding clinical data were comprehensive analyzed. Results The expressions of GST-π, P-gp, Topo-Ⅱ, TS and MRP had notable differences between the gastric cancer tissues and normal gastric mucosa (GST-π： P＜0.01； P-gp： P＜0.01； Topo-Ⅱ： P＜0.01； TS： P＜0.05； MRP： P＜0.05). Positive expression rates of GST-π, P-gp, Topo-Ⅱ, TS and MRP in gastric cancer tissues were 72.9% (35/48), 56.3% (27/48), 83.3% (40/48), 41.7% (20/48) and 39.6% (19/48), and positive expression rates of them in normal gastric mucosa were 10.0% (1/10), 0 (0/10), 0 (0/10), 0 (0/10) and 0 (0/10) corresponding. Their positive expression rates were closely relevant to the degree of differentiation (P＜0.01), but not to the patients’ sex, age, tumour site, size of tumour, invasive depth and lymph node metastasis (Pgt;0.05). Conclusions The expressions of GST-π, P-gp, Topo-Ⅱ, TS and MRP in gastric cancer tissues exist obvious heterogeneity. Their overexpression underlie the multidrug resistance of gastric cancer. The joint detection of GST-π, P-gp, Topo-Ⅱ, TS and MRP can be looked as an important symbol for guiding its chemotherapy.
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.
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 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.