Objective: To investigate the relationship between the expression of multidrug resistance-associated protein (MRP) and clinicopathological factors and prognosis. Methods: The expression of MRP in 62 cases with non-sma...Objective: To investigate the relationship between the expression of multidrug resistance-associated protein (MRP) and clinicopathological factors and prognosis. Methods: The expression of MRP in 62 cases with non-small cell lung cancer (NSCLC) was detected using immunohistochemistry method. The expression of MRP in 30 cases of NSCLC and corresponding normal lung tissues were detected using immunohistochemistry and Western Blot. Results: this study of tumor tissues confirmed the plasma membrane and/or cytoplasm locations of MRP. There was apparent difference between normal lung tissues and NSCLC in MRP. The survival analysis of 62 NSCLC showed that the mean survival time of the patients with negative MRP expression was 69.8117.41 months and that of patients with positive MRP expression, 25.384.46 months. Log-rank test suggested that the difference between them was significant (P=0.0156). It was also found that in squamous cell lung cancer the statistically significant difference between the mean survival time of patients with positive MRP expression and those with negative MRP expression (P=0.0153). Multivariate Cox model analysis suggested that the survival time was significantly related to expression of MRP (P=0.035) and lymphatic metastasis (P=0.038). Conclusion: MRP expression in NSCLC is significantly higher compared with normal lung tissues. The mean survival time of patients with negative MRP was relative longer and expression of MRP was an independent factor for prognosis.展开更多
To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hamme...To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted alternatively regularly and the plasmid of pGEM-MDR1/MRP1 used to transcribe the MDR1/MRPl(196/210) substrate containing double target sites was also constructed by DNA recombination. Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct. The clea- vage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system. The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively, and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively. The multi- ribozyme expression system of the [Coat'A196Rz/Coat'B210Rz]5 is more significantly superior to that of the [Coat'A 196Rz/Coat'B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat'A 196Rz/Coat'B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg^2+ on cleavage efficiency indicate that cleavage reaction is dependent on Mg^2+ ions concentration. The plot of lg(kobs) vs. lgc(Mg^2+) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg^2+. It suggests that Mg^2+ ions play a crucial role in multi-ribozyme cleavage on the substrate.展开更多
Objective: To investigate the effects of neoadjuvant chemotherapy on the expression of drug resistance genes, multidrug resistance-1 (MDR1) and multidrug resistance-associated protein (MRP), in patients with primary b...Objective: To investigate the effects of neoadjuvant chemotherapy on the expression of drug resistance genes, multidrug resistance-1 (MDR1) and multidrug resistance-associated protein (MRP), in patients with primary breast cancer. Methods: MDR1 and MRP expression were detected by semi-quantitative RT-PCR in 20 patients with primary breast cancer, before and after chemotherapy. Results: Before chemotherapy, MDR1 and MRP expression can be detected in 15 cases (75%) and 18 cases (90%) respectively. After chemotherapy, expression of MDR1 is not significantly different from that before chemotherapy, but expression of MRP is significantly different from that before chemotherapy. Conclusion: Expression of drug resistance gene MRP, but not MDR1, is enhanced in patients with primary breast cancer submitted to neoadjuvant chemotherapy.展开更多
To study the effects of hypoxia on the expression of P-gp and mutltidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. The expre...To study the effects of hypoxia on the expression of P-gp and mutltidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. The expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein was immunohistochemically detected by culturing human lung adenocarcinoma A549 cell under hypoxia (2 % O_2) for 24 h. After interaction with adriamycin or cisplatin under hypoxia (2 % O_2) for 24 h, the cell survival rate was detected by MTT. Our results showed that the expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein under hypoxia were higher than the expression under normoxia, and correlations between the expression of HIF-1α and P-gp or multidrug resistance-associated protein was observed (P<0.05). The resistance of adriamycin of A549 cell was enhanced under hypoxia. It is concluded that the resistance of tumor chemotherapy is enhanced in hypoxia. The expression of HIF-1α is obviously correlated with the expression of P-gp and mutltidrug resistance protein.展开更多
OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these differen...OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.展开更多
Introduction: Multidrug resistance (MDR) is one of the major problems of chemotherapy. Overexpression of efflux pumps, such as P-glycoprotein (Pgp), multiple resistance-related protein 1 (MRP-1) and lung resistance pr...Introduction: Multidrug resistance (MDR) is one of the major problems of chemotherapy. Overexpression of efflux pumps, such as P-glycoprotein (Pgp), multiple resistance-related protein 1 (MRP-1) and lung resistance protein (LRP) can lead to MDR. Verapamil and L-buthionine-sulfoximine (BSO) are two modulators of these proteins. This study aims to compare 99mTc-Sestamibi transport kinetics in human colorectal adenocarcinoma cell lines, in the presence and absence of the MDR modulators verapamil and BSO. Material and Methods: MDR proteins expression was evaluated in sensitive (WiDr) and resistant (LS1034) human colorectal adenocarcinoma cell lines. Intracellular and plasma membrane Pgp and MRP1, and LRP expression was analyzed by flow-cytometry and western blot. Cellular transport kinetics was assessed using 99mTc-Sestamibi. MDR modulation was evaluated though retention studies in resistant cells after incubation with the modulators. Results: Pgp expression was significantly higher (p≤0.001) in resistant cells. These results were confirmed by western blot analysis. 99mTc-Sestamibi uptake and retention percentage were significantly higher (p99mTc-Sestamibi, there were differences among the MDR modulators used (p99mTc-Sestamibi could indicate MDR phenotype in colorectal adenocarcinoma cells. As the modulators used showed a reversion of the retention profile only for the first minutes, their administration should occur immediately before the administration of cytotoxic drugs.展开更多
OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI)...OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.展开更多
文摘Objective: To investigate the relationship between the expression of multidrug resistance-associated protein (MRP) and clinicopathological factors and prognosis. Methods: The expression of MRP in 62 cases with non-small cell lung cancer (NSCLC) was detected using immunohistochemistry method. The expression of MRP in 30 cases of NSCLC and corresponding normal lung tissues were detected using immunohistochemistry and Western Blot. Results: this study of tumor tissues confirmed the plasma membrane and/or cytoplasm locations of MRP. There was apparent difference between normal lung tissues and NSCLC in MRP. The survival analysis of 62 NSCLC showed that the mean survival time of the patients with negative MRP expression was 69.8117.41 months and that of patients with positive MRP expression, 25.384.46 months. Log-rank test suggested that the difference between them was significant (P=0.0156). It was also found that in squamous cell lung cancer the statistically significant difference between the mean survival time of patients with positive MRP expression and those with negative MRP expression (P=0.0153). Multivariate Cox model analysis suggested that the survival time was significantly related to expression of MRP (P=0.035) and lymphatic metastasis (P=0.038). Conclusion: MRP expression in NSCLC is significantly higher compared with normal lung tissues. The mean survival time of patients with negative MRP was relative longer and expression of MRP was an independent factor for prognosis.
基金Supported by Fund of Shenzhen Bureau of Science and Technology, China(No.20008).
文摘To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted alternatively regularly and the plasmid of pGEM-MDR1/MRP1 used to transcribe the MDR1/MRPl(196/210) substrate containing double target sites was also constructed by DNA recombination. Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct. The clea- vage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system. The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively, and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively. The multi- ribozyme expression system of the [Coat'A196Rz/Coat'B210Rz]5 is more significantly superior to that of the [Coat'A 196Rz/Coat'B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat'A 196Rz/Coat'B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg^2+ on cleavage efficiency indicate that cleavage reaction is dependent on Mg^2+ ions concentration. The plot of lg(kobs) vs. lgc(Mg^2+) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg^2+. It suggests that Mg^2+ ions play a crucial role in multi-ribozyme cleavage on the substrate.
文摘Objective: To investigate the effects of neoadjuvant chemotherapy on the expression of drug resistance genes, multidrug resistance-1 (MDR1) and multidrug resistance-associated protein (MRP), in patients with primary breast cancer. Methods: MDR1 and MRP expression were detected by semi-quantitative RT-PCR in 20 patients with primary breast cancer, before and after chemotherapy. Results: Before chemotherapy, MDR1 and MRP expression can be detected in 15 cases (75%) and 18 cases (90%) respectively. After chemotherapy, expression of MDR1 is not significantly different from that before chemotherapy, but expression of MRP is significantly different from that before chemotherapy. Conclusion: Expression of drug resistance gene MRP, but not MDR1, is enhanced in patients with primary breast cancer submitted to neoadjuvant chemotherapy.
文摘To study the effects of hypoxia on the expression of P-gp and mutltidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. The expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein was immunohistochemically detected by culturing human lung adenocarcinoma A549 cell under hypoxia (2 % O_2) for 24 h. After interaction with adriamycin or cisplatin under hypoxia (2 % O_2) for 24 h, the cell survival rate was detected by MTT. Our results showed that the expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein under hypoxia were higher than the expression under normoxia, and correlations between the expression of HIF-1α and P-gp or multidrug resistance-associated protein was observed (P<0.05). The resistance of adriamycin of A549 cell was enhanced under hypoxia. It is concluded that the resistance of tumor chemotherapy is enhanced in hypoxia. The expression of HIF-1α is obviously correlated with the expression of P-gp and mutltidrug resistance protein.
基金The project supported by National Natural Science Foundation of China(81373439,81473234 and U1303221)
文摘OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.
文摘Introduction: Multidrug resistance (MDR) is one of the major problems of chemotherapy. Overexpression of efflux pumps, such as P-glycoprotein (Pgp), multiple resistance-related protein 1 (MRP-1) and lung resistance protein (LRP) can lead to MDR. Verapamil and L-buthionine-sulfoximine (BSO) are two modulators of these proteins. This study aims to compare 99mTc-Sestamibi transport kinetics in human colorectal adenocarcinoma cell lines, in the presence and absence of the MDR modulators verapamil and BSO. Material and Methods: MDR proteins expression was evaluated in sensitive (WiDr) and resistant (LS1034) human colorectal adenocarcinoma cell lines. Intracellular and plasma membrane Pgp and MRP1, and LRP expression was analyzed by flow-cytometry and western blot. Cellular transport kinetics was assessed using 99mTc-Sestamibi. MDR modulation was evaluated though retention studies in resistant cells after incubation with the modulators. Results: Pgp expression was significantly higher (p≤0.001) in resistant cells. These results were confirmed by western blot analysis. 99mTc-Sestamibi uptake and retention percentage were significantly higher (p99mTc-Sestamibi, there were differences among the MDR modulators used (p99mTc-Sestamibi could indicate MDR phenotype in colorectal adenocarcinoma cells. As the modulators used showed a reversion of the retention profile only for the first minutes, their administration should occur immediately before the administration of cytotoxic drugs.
基金Shanghai Municipal Health Bureau:Traditional Chinese Medicine in Treating with Advanced Hepatocellular Carcinoma(No.ZYSNXD-CC-ZDYJ032)
文摘OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.