ObjectiveTo evaluate the effect of neoadjuvant chemotherapy and find the mechanism of multidrug resistance. MethodsTwenty patients with gastric cancer and 31 patients with colorectal cancer underwent neoadjuvant chemotherapy and then operations. The preoperative specimens were stained by immunohistochemical techniques for testing p53,multidrug resistanceassociated protein (MRP), glutathione S transferase（GST）, telomerase. Resection specimens were evaluated for chemotherapy effect by routine histology; at the same time, the postoperative morbidity and mortality were observed. ResultsIn 51 patients, the response rate of neoadjuvant chemotherapy was 27.45%(14/51),so multidrug resistance was a kind of common phenomena in gastrointestinal carcinomas. The postoperative morbidity was 15.69%(8/15), the main operation complication was infection,the mortality was 1.96%(1/51),only one person died from severe infection.The expression rate of p53, MRP, GST, telomerase was 58.0%,51.0%,66.7%,74.0%respectively, the location of p53 was at cell nucleus,location of MRP,GST was at cell memberane and cytoplasm,location of telomerase was at cytoplasm.The response rate had nothing to do with age, sex and metastasis. But it was related with p53 and telomerase expression. ConclusionNeoadjuvant chemotherapy is an effective, safe therapy. But the rate of drug resistance is high in gastrointestinal carcinomas, and the response rate is related to p53, telomerase expression.
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.
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.
Objective To study the expression and significance of multidrug resistance-associated protein (MRP) gene in hepatocellular carcinoma (HCC). Methods Reverse transcription polymerase chain reaction (RT-PCR) assay was used to detect the expression of MRP mRNA in 25 fresh specimens of the primary HCC and its surrounding liver tissues. Immunohistochemistry LSAB technique was adopted to test MRP in 60 HCC specimens. The drug sensitivity was also tested by flow cytometry.Results The positive expression rates of MRP mRNA and MRP protein in primary HCC were 44.00%(11/25) and 45.00%(27/60) respectively. All the intensity of expression was low, but significant higer than its surrouding liver tissues (P＜0.05). The intensity and expression rate of MRP protein in 5 recurrent HCC had a tendency to increase. There was a correlation between the expression of MRP mRNA and MRP protein in 25 patients using RT-PCR and immunohistochemistry assay (Plt;0.05). Detected by flow cytometry, the average sensitivity of drugs in vitro of 60 HCC sp-cimens were 5-FU （15.80±7.63）%，DDP（18.45±9.59）%，ADM（17.95±7.99）%，MMC（16.60±8.69）% and CTX（17.40±10.14）%. Only 5FU and ADM were significantly affected by the expression of MRP protein (Plt;0.05).Conclusion The expression of MRP in primary HCC may be one of the important mechanisms of the intrinsic and acquired drug resistance in HCC. To study the expression of MRP could give a predictive value in HCC chemotherapy.
【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.
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 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.
【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.
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 reversal of the multidrug resistant gene mdr1 in vivo by antisense oligodeoxynucleotide （ASODN） on the basis of study in vitro. Methods The cultured drug-resistant human hepatocellular carcinoma cells were injected under the skin of axilla to establish the tumor model of nude mice. mdr1 ASODN accompanied by Lipofectamine were injected locally and ADM was injected intraperitoneally. Control 1 and control 2 were locally injected by Lipofectamine and normal saline separately, and ADM was also injected intraperitoneally. Results As time went on the tumor size increased and from the 5th day on alterations were marked, tumor size in different time phase showed marked difference to the prior time phase with significant difference (P＜0.05）. Tumor size in group ASODN was marked smaller than that of other 3 groups after the 5th day (P＜0.05)，while tumor size of group control 1,2 and group SODN in different phase showed no significant difference (Pgt;0.05). The results suggested that SODN and Lipofectamine showed no marked effect on tumor growth of nude mice and ASODN had marked inhibition effect on tumor growth. Conclusion mdr1 ASODN can also reverse multidrug resistance of drug-resistant human hepatocellular carcinoma cells in vivo. After the treatment the tumor’s growth in nude mice will slow down in a range of time.