1.Effect of intracellular acidification on P-glycoprotein in drug-resistant K562/A02 cells.
Ying LU ; Qing-Hua LI ; Li MA ; Bin LI ; Wen-Su YUAN ; Yong-Xin RU ; Jian-Xiang WANG ; Tian-Xiang PANG
Journal of Experimental Hematology 2009;17(3):568-573
The aim of this study was to investigate the effect of intracellular acidification on the P-gp in K562/A02 cells. Confocal laser microscope was used to determine the intracellular acidification. MTT assay was used to detect the cytotoxicity of intracellular acidification on K562 and K562/A02 cells. Flow cytometry was applied to measure the influence of intracellular acidification on the activity of P-gp. The P-gp expression at protein and mRNA levels were determined by Western blot and real-time RT-PCR respectively. The results indicated that intracellular acidification had no obvious cytotoxicity on K562 and K562/A02 cells. The function of P-gp in K562/A02 cells weakened along with decrease of intracellular acidification, the intracellular acidification significantly increased the accumulation of Rhodamine 123 (Rh 123) and suppressed the efflux of Rh 123 mediated by P-gp. The intracellular acidification also inhibited the expression of P-gp in K562/A02 cells at protein and mRNA levels which showed intracellular acidification with time-dependence. It is concluded that the intracellular acidification can inhibit the expression and function of P-gp in K562/A02 cells.
ATP Binding Cassette Transporter, Sub-Family B
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ATP-Binding Cassette, Sub-Family B, Member 1
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metabolism
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Drug Resistance, Multiple
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Drug Resistance, Neoplasm
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Humans
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Hydrogen-Ion Concentration
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K562 Cells
4.Correlation of MDR1 and ABCG2 genetic polymorphisms with the efficacy and adverse events of irinotecan chemotherapy in patients with colorectal cancer.
Zhi-wei SUN ; Xi-cheng WANG ; Jing GAO ; Jie LI ; Yan-yan LI ; Yun-zhi DANG ; Lin SHEN
Chinese Journal of Gastrointestinal Surgery 2013;16(6):524-528
<b>OBJECTIVEb>To investigate the correlation of MDR1 and ABCG2 genetic polymorphisms with the efficacy and adverse events of irinotecan chemotherapy in patients with colorectal cancer (CRC).
<b>METHODSb>Clinical data of CRC patients treated with irinotecan-based chemotherapy in the Peking University Cancer Hospital between January 1996 and December 2011 were collected, and their blood samples were collected accordingly. Genomic DNA was extracted from blood samples. The following SNP detection of MDR1 and ABCG2 genes was conducted by direct sequencing method. The correlation of genetic SNPs with efficacy and toxicity of irinotecan treatment was further analyzed.
<b>RESULTSb>Allele frequencies of MDR1 2677 G>T/A, ABCG2 421 C>A, 34 G>A, 376 C>T were comparable with previous studies. Genetic SNPs results from peripheral blood samples and tumor tissues were highly consistent. Patients carrying MDR1 2677 wild type had higher clinical benefit than those carrying mutant genotype, while the differences were not significant. The progression-free survival (PFS) was longer in wild-type patients as compared to mutant-type patients in second-line chemotherapy (P=0.012). There were no significant correlations between ABCG2 421 C>A, 34 G>A, 376 C>T and chemotherapy efficacy. No significant correlations were observed between MDR1 2677 G>T/A, ABCG2 421 C>A, ABCG2 34 G>A, ABCG2 376 C>T and irinotecan-related grade 3 and 4 neutropenia or diarrhea.
<b>CONCLUSIONb>MDR1 2677 G>T/A may be served as a biomarker in predicting the efficacy of irinotecan chemotherapy in patients with colorectal cancer.
ATP Binding Cassette Transporter, Sub-Family B ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; Adult ; Aged ; Camptothecin ; analogs & derivatives ; therapeutic use ; Colorectal Neoplasms ; drug therapy ; genetics ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; Polymorphism, Single Nucleotide ; Retrospective Studies ; Treatment Outcome ; Young Adult
5.Expression of breast cancer resistance protein and p-glycoprotein in residual breast cancer tissue after chemotherapy and its correlation with cancer stem cells.
Hong-bo QU ; Yuan-ming FAN ; Ming-li HAN ; Hao-jun LUO ; Jia XIE ; Hong LIU ; Hao LIU ; Cheng-yi WU ; Wei-xue TANG
Acta Academiae Medicinae Sinicae 2013;35(2):171-176
<b>OBJECTIVEb>To compare the expression differences of breast cancer resistance protein(BCRP/ABCG2) and P-glycoprotein(P-gp) in breast cancer tissue before chemotherapy and in residual breast cancer tissue, and to explore its correlation with breast cancer stem cells.
<b>METHODSb>Immunohistochemistry was used to detect the expression of ABCG2, P-gp, and breast cancer stem cells(BCSCs) markers(CD44 and CD24) in breast cancer tissue before chemotherapy and residual breast cancer tissue after chemotherapy. Immunofluorescence was applied for determination of the CD44 and CD24 protein expressions of BCSCs microspheres cells. The monoclone-forming ability of BCSCs microspheres cells was detected by limited dilution assay. The expressions of ABCG2, P-gp, CD44, and CD24 proteins were detected by Western blot.
<b>RESULTSb>Compared with those in breast cancer tissue before chemotherapy, the expression levels of ABCG2 and P-gp were positively correlated with the expression level of CD44 protein(Χ(2)=41.34, r=0.83;Χ(2)=22.81, r=0.61) in residual breast cancer tissue after chemotherapy;meanwhile, they were negatively correlated with the expression of CD24 protein(Χ(2)=-21.25, r=0.72;Χ(2)=-17.26, r=0.65) (all P<0.05) .The diameter of BCSCs microspheres were increased significantly after chemotherapy.The content of BCSCs increased by about 2.5 times after chemotherapy.The expressions of ABCG2, P-gp and CD44 proteins significantly increased and that of CD24 protein significantly declined(P<0.05) .
<b>CONCLUSIONb>Chemotherapy endows residual breast cancer tissue with cancer stem cells-like features, leading to multidrug resistance of breast cancer.
ATP Binding Cassette Transporter, Sub-Family B ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adult ; Aged ; Breast Neoplasms ; drug therapy ; metabolism ; CD24 Antigen ; metabolism ; Cell Culture Techniques ; Drug Resistance, Neoplasm ; Female ; Humans ; Hyaluronan Receptors ; metabolism ; Middle Aged ; Neoplasm Proteins ; metabolism ; Neoplasm, Residual ; Neoplastic Stem Cells ; cytology ; metabolism
6.Effect of tetrandrine on the doxorubicin-induced expression of mdr1 gene in K562 cells.
Xu-jing LU ; Wen-lin XU ; Wen-juan LUO ; Fa-chun WANG ; Qiao-yun CHEN
Chinese Journal of Hematology 2008;29(7):468-471
<b>OBJECTIVEb>To investigate the effect of tetrandrine (TTD) on doxorubicin-induced mdr1 gene expression and its mechanism.
<b>METHODSb>MTT assay was used to detect the cytotoxicity of TTD to K562 cells. K562 cells were treated with doxorubicin alone or 0.6 microg/ml doxorubicin combined with various concentrations of TTD. RT-PCR was used to detect the mRNA expression of mdr1 and NF-kappa B. Flow cytometry was used to assay the expression of P-glycoprotein (P-gp). Intracellular rhodamine 123 (Rho123) retention assay was applied to test the P-gp function.
<b>RESULTSb>After treatment with 0.6 microg/ml doxorubicin for 24 hours, the expressions of mdr1 mRNA, NF-kappa B mRNA and P-gp in K562 cells were increased from 0.171 +/- 0.012, 0.783 +/- 0.090, 7.85 +/- 0.15 to 0.428 +/- 0.012, 1.075 +/- 0.047 and 73.68 +/- 1.84, respectively. The intracellular Rho123 retention was decreased from 711.9 +/- 63.6 to 347.8 +/- 60.6, indicating up-regulation of P-gp function (P<0.05). Pretreatment of K562 cells with 2.0 microg/ml TTD for 24 hours and then incubated for another 24 h with doxorubicin, the expressions of mdr1 mRNA, NF-kappa B mRNA, P-gp and up-regulation of P-gp function induced by doxorubicin were prevented in K562 cells (0.148 +/- 0.006, 0.627 +/- 0.098, 7.18 +/- 0.38 and 799.7 +/- 45.8, respectively P<0.05). But 0.5 microg/ml and 1.0 microg/ml TTD had little effect.
<b>CONCLUSIONSb>TTD inhibits the expression of mdr1 mRNA, P-gp and up-regulated P-gp function induced by doxorubicin in a dose dependent manner. The mechanism of this effect may be down-regulation of NF-kappa B by TTD.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Benzylisoquinolines ; pharmacology ; Doxorubicin ; pharmacology ; Humans ; K562 Cells ; NF-kappa B ; metabolism ; RNA, Messenger ; genetics ; Up-Regulation ; drug effects
7.Clinical significance of NF-kappaB continual activity and expression of WT1 and MDR1 in acute nonlymphocytic leukemia.
Journal of Experimental Hematology 2007;15(2):253-257
The study was aimed to explore the NF-kappaB continual activity and the expression of WT1 and MDR1 in acute non-lymphocytic leukemia (ANLL) patients, and to investigate if the three factors affect the curative effect of ANLL together as to provide some theoretical basis for finding new measures to improve the curative effect of refractory ANLL. The bone marrow samples of 45 ANLL patients was collected. 45 patients including 20 primary ANLL patients (A group) and 25 refractory ANLL patients. Refractory ANLL patients were divided into 2 sub-groups (B, C groups). The primary patients who was no effect after more than two courses of treatment were taken as group B, and the patients with more than two relapses were taken as group C. At the same time, 15 patients with simple iron deficiency anemia were collected as negative control. The NF-kappaB continual activity was measured by using electrophoretic mobility shift assay (EMSA) and the expressions of WT1, MDR1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that the activity of NF-kappaB and the expressions of WT1, MDR1 were not detected in 15 samples of simply iron deficiency anemia subjects. The NF-kappaB continual activity, the expression levels of WT1 and MDR1 in the refractory group were significantly higher than that in primary group (P<0.001). But the NF-kappaB continual activity, the expression of WT1 gene and MDR1 gene were not significantly different between group B and group C (P>0.05). By assaying the relativity between the them the NF-kappaB continual activity and the expression of WT1 or MDR1 had positive correlation in ANLL patients. It is concluded that the NF-kappaB continual activity, the overexpression of WT1 and MDR1 may be one of the reasons causing poor curative effect in acute non-lymphocytic leukemia. The NF-kappaB continual activity and the expression of WT1, MDR1, all show positive correlation in ANLL patients.
ATP Binding Cassette Transporter, Sub-Family B
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ATP-Binding Cassette, Sub-Family B, Member 1
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biosynthesis
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genetics
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Female
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Humans
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Leukemia, Myeloid, Acute
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genetics
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metabolism
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Male
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NF-kappa B
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metabolism
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WT1 Proteins
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biosynthesis
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genetics
8.A review on regulation of drug transporters during inflammation.
Hang ZENG ; Hui-Chang BI ; Min HUANG
Acta Pharmaceutica Sinica 2011;46(7):773-779
Drug metabolism will change significantly during inflammation, including the reduction of expression and activity of many drug metabolizing enzymes and transporters. Body would release a series of inflammatory cytokines which can regulate drug metabolizing enzymes. Recent studies have revealed that drug transporters are also regulated by the cytokines with obvious species difference. Mechanism studies show that several transcription factors play important roles during the signal pathways of regulation. This review focuses on the progress in the regulation of drug transporters during inflammation.
ATP Binding Cassette Subfamily B Member 11
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ATP Binding Cassette Transporter, Sub-Family B
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metabolism
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ATP Binding Cassette Transporter, Sub-Family G, Member 2
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ATP-Binding Cassette Transporters
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metabolism
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Animals
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Biological Transport
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Humans
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Inflammation
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metabolism
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Membrane Transport Proteins
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metabolism
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Multidrug Resistance-Associated Proteins
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metabolism
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Neoplasm Proteins
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metabolism
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Organic Anion Transporters
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metabolism
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Organic Cation Transport Proteins
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metabolism
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Signal Transduction
9.Study on reversing mechanism of multidrug resistance of K562/A02 cell line by carnosic acid.
Xiao-Ning YU ; Hao LI ; Xue-Liang CHEN ; Xiang-Xin LI ; Ran WANG ; Fang GAO
Chinese Journal of Hematology 2010;31(6):381-384
<b>OBJECTIVEb>To investigate the effects of carnosic acid (CA) on reversal of the multidrug resistance (MDR) of human leukemia cell line K562/A02 and its mechanism.
<b>METHODSb>MTT assay was used to determine the sensitivity of K562/A02 cells to adriamycin (ADM) pre-and post-treated with CA. Flow cytometry (FCM) and laser scanning confocal microscopy (LSCM) were used to measure intracellular fluorescence intensity and concentration of ADM respectively. The expression level of mdr1 was detected by semi-quantitative RT-PCR. P-glycoprotein (P-gp) expression was detected by FCM and Western blot.
<b>RESULTSb>CA decreased IC(50) of ADM in K562/A02 cells from 16.31 µg/mL to 1.35 µg/mL, being a 12.08-fold decrease. The intracellular ADM fluorescence intensity of K562/A02 was increased from 17.05 to 60.53 after treated with CA (P < 0.01). In living K562/A02 cells, after treated with CA, the diffuse distribution of intracellular ADM was recovered in both nuclear and cytoplasm, and the concentration of intracellular ADM increased from 4.93µg/mL to 15.43µg/mL. RT-PCR assay showed that CA inhibited the expressions of mdr1 mRNA in K562/A02 cells (P < 0.01). Mean fluorescence intensity of P-gp detected by FCM in CA-treated K562/A02 was decreased to 22.80 as compared with that in untreated K562/A02 cells (44.40, P < 0.05).
<b>CONCLUSIONb>CA can reverse the MDR of K562/A02 cells in vitro. The mechanism may be associated with down-regulation of mdr1 and inhibition of P-gp function.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; K562 Cells
10.Expression of MDR1 gene in cancer stem cells in breast cancer tissues of different molecular subtypes.
Guohua YANG ; Fangqin XUE ; Xiaogeng CHEN
Journal of Southern Medical University 2012;32(11):1636-1638
<b>OBJECTIVEb>To investigate the association between MDR1 gene expression in breast cancer stem cells and the molecular subtypes of breast cancer tissue.
<b>METHODSb>According to ER, PR, Her-2 and CK5/14 expression profiles, 153 breast cancer specimens were divided into 5 molecular molecular subtypes, in which the expression of MDR1 was detected to analyze the relationship between MDR1 gene expression and the subtypes of breast cancer stem cells.
<b>RESULTSb>The expression of MDR1 in Luminal A subtype breast cancer was 0.26∓0.04, which showed no significant difference from that of Luminal B subtype (0.31∓0.03, P>0.05). Compared with these two subtypes, HER-2 (+) subtype breast cancer tissues showed a significantly higher MDR1 expression(0.56∓0.05, P<0.05). MDR1 expression in Basal-like subtype and Normal-like subtype breast cancers was comparable (0.98∓0.01 vs 0.90∓0.15, P<0.05), but both significantly higher than that in Luminal A and B subtypes and HER-2 (+) subtype (P<0.05).
<b>CONCLUSIONb>The expression of MDR1 gene in cancer stem cells is related with the molecular subtypes of breast cancer tissue.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Adult ; Breast Neoplasms ; genetics ; metabolism ; Female ; Humans ; Middle Aged ; Neoplastic Stem Cells ; metabolism