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
;
ATP-Binding Cassette, Sub-Family B, Member 1
;
metabolism
;
Drug Resistance, Multiple
;
Drug Resistance, Neoplasm
;
Humans
;
Hydrogen-Ion Concentration
;
K562 Cells
2.Expression of leucocyte cell-surface antigens on colon cancer cell line HR8348.
Shi-long YANG ; Yon-min TANG ; Hong-qiang SHEN ; Bo-qin QIAN ; Hua SONG ; Shu-wen SHI ; Bo-tao NING ; Ying-hu CHEN ; Wei-qun XU
Journal of Zhejiang University. Medical sciences 2004;33(2):118-120
<p><b>OBJECTIVEb>To define the immune phenotype of colon cancer cells.p><p><b>METHODSb>Using a panel of 40 anti-human monoclonal antibodies (MoAbs), the cells of colon cancer HR8348 were analyzed with three-color flow cytometry after direct immunofluorescent staining.p><p><b>RESULTSb>HR8348 cell line did not express CD2, CD3, CD4, CD5, CD7, CD8, TCR, CD10, CD11b, CD14, CD16, CD19, CD22, CD25, CD28, SmIg, CD33, CD35, CD36, CD41a, CD45, CD45RA, CD45RO, CD56, CD61, CD64, CD66b, CD69, CD71, CD117, CD122 and P-glycoprotein but expressed CD13, CD15, CD20, CD38, CD95 and HLA-DR.p><p><b>CONCLUSIONb>The results demonstrate that colon cancer cell line HR8348 shares some antigenic determinants with leucocyte lineage.p>
ATP-Binding Cassette, Sub-Family B, Member 1
;
analysis
;
Antigens, CD
;
analysis
;
Cell Line, Tumor
;
Colonic Neoplasms
;
chemistry
;
Humans
3.Celecoxib enhances the chemotherapy sensitivity of KB/VCR cell lines to vincristine.
West China Journal of Stomatology 2013;31(4):403-407
<p><b>OBJECTIVEb>To investigate the influence of celecoxib, cycloxygenase-2 (COX-2) selective inhibitor, upon the proliferation of KB/VCR cells, and analyze the effect of celecoxib on the expression of P-glycoprotein (P-gp).p><p><b>METHODSb>MTT method was employed to study the inhibitory effect of celecoxib on KB/VCR cells, which were divided into vincristine (VCR) group, Celecoxib group, Celecoxib + VCR group, Celecoxib + VCR + prostaglandin E2 (PGE2) group. Western blot was employed to detect the expression of P-gp, Bcl-2 and Bcl-X(L). Flow cytometry was used to evaluate the apoptosis of KB/VCR cells. All of the data were statistically analyzed using SPSS 13.0 software package.p><p><b>RESULTSb>The growth inhibition rate of KB/VCR cells in Celecoxib+VCR group was significantly higher than that in Celecoxib group, VCR group and Celecoxib + VCR + PGE2 group (P < 0.01). The expression of P-gp in Celecoxib + VCR group and Celecoxib group were markedly lower, compared with those in VCR group and Celecoxib + VCR + PGE2 group (P < 0.01). The expression of Bcl-2, Bcl-XL in Celecoxib+VCR group, Celecoxib+VCR+PGE2 group and Celecoxib group were significantly lower than those in VCR group (P < 0.01). The apoptosis rate of Celecoxib + VCR group, Celecoxib + VCR + PGE2 group were significantly higher than those in VCR group and Celecoxib group (P < 0.01). The apoptosis rate of Celecoxib+VCR+PGE2 group were significantly lower than those in Celecoxib+VCR group (P < 0.01).p><p><b>CONCLUSIONb>Celecoxib could enhance the toxicity of VCR against KB/VCR cells. The mechanism probably correlates with the downregulation of celecoxib on the expression of P-gp and the increase of apoptosis.p>
ATP-Binding Cassette, Sub-Family B, Member 1
;
Apoptosis
;
Celecoxib
;
Cell Line, Tumor
;
Drug Resistance, Neoplasm
;
Humans
;
Pyrazoles
;
Sulfonamides
;
Vincristine
4.Role of ABCB1 and ABCG2 in the multidrug resistance of hypopharyngeal carcinoma FaDu cell line.
Ju-ke MA ; Su-mei LU ; Liang YU ; Jia-jun TIAN ; Jian-feng LI ; Hai-bo WANG ; Wei XU
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2012;47(4):305-310
<p><b>OBJECTIVEb>To investigate the expression of multidrug resistance gene ABCB1 and ABCG2 in FaDu cells (human hypopharyngeal carcinoma cell line) and the multidrug resistance (MDR) cell lines FaDu/T transformed from FaDu cells by taxol and underlying mechanisms of MDR.p><p><b>METHODSb>The multidrug resistance sensitivities of FaDu and FaDu/T to cisplatin (DDP), 5-fluorouracil (5-FU), doxorubicin (Dox), and vincristine (VCR) were examined by methyl-thiazolyl-tetrazolium (MTT) assay. The mRNA and protein expressions of multidrug resistance genes ABCB1 and ABCG2 were analysed with RT-PCR, Western blot and laser confocal microscopy. JNK signal proteins were detected through Western blot.p><p><b>RESULTSb>The multidrug resistance of FaDu/T cells to Taxol, DDP, 5-FU, ADM and VCR was more than that of FaDu cells. The expression of ABCB1 in FaDu/T cells was significantly higher than that in FaDu cells (t = 22.42, P < 0.05), but the expression of ABCG2 in FaDu/T cells was significantly lower than that in FaDu cells (t = 10.06, P < 0.05). JNK signal was inhibited in FaDu or FaDu/T cells and the inhibited JNK was reactivated by taxol or anisomycin (an activator for MAPK signal transduction pathways). Anisomycin down-regulated the expression of ABCB1 (F = 33.72, P < 0.05) and up-regulated the expression of ABCG2 (F = 220.16, P < 0.05) in FaDu/T cells, but not in FaDu/T cells pretreated by JNK inhibitor SP600125 (P > 0.05).p><p><b>CONCLUSIONb>The overexpression of ABCB1 and the down-regulation of ABCG2 in FaDu/T cells were the main features of MDR in hypopharyngeal carcinomas, in which JNK signal transduction pathways could play an important role.p>
ATP Binding Cassette Transporter, Sub-Family B
;
ATP-Binding Cassette Sub-Family B Member 2
;
ATP-Binding Cassette Transporters
;
genetics
;
ATP-Binding Cassette, Sub-Family B, Member 1
;
genetics
;
Cell Line, Tumor
;
Drug Resistance, Multiple
;
genetics
;
Drug Resistance, Neoplasm
;
genetics
;
Humans
;
Hypopharyngeal Neoplasms
;
genetics
5.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
<p><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).p><p><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.p><p><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.p><p><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.p>
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
6.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
<p><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.p><p><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.p><p><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) .p><p><b>CONCLUSIONb>Chemotherapy endows residual breast cancer tissue with cancer stem cells-like features, leading to multidrug resistance of breast cancer.p>
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
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
;
ATP-Binding Cassette, Sub-Family B, Member 1
;
biosynthesis
;
genetics
;
Female
;
Humans
;
Leukemia, Myeloid, Acute
;
genetics
;
metabolism
;
Male
;
NF-kappa B
;
metabolism
;
WT1 Proteins
;
biosynthesis
;
genetics
8.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
<p><b>OBJECTIVEb>To investigate the effect of tetrandrine (TTD) on doxorubicin-induced mdr1 gene expression and its mechanism.p><p><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.p><p><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.p><p><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.p>
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
9.Analysis of the impact of extracellular acidity on the expression and activity of P-glycoprotein and on the P-glycoprotein-mediated cytotoxicity of daunorubicin in cancer cell by microfluidic chip technology.
Yuan LI ; Jiao XIANG ; Sha-sha ZHANG ; Bei-zhong LIU ; Fang GONG ; Ming-qing PENG
Acta Academiae Medicinae Sinicae 2015;37(1):75-81
<p><b>OBJECTIVEb>To explore the impact of extracellular acidic environment on the expression and activity of P-glycoprotein (P-gp) and on the P-gp-mediated cytotoxicity of daunomycin in cancer cells by using microfluidic chip technology.p><p><b>METHODSb>The A549 cells cultured on a microfluidic chip were divided into experiment group and control group. The experiment group was exposed to an acidic cell culture medium (pH 6.6), while the control group was treated with a neutral cell culture medium (pH 7.4). The expression of P-gp was detected by cell immunofluorescense analysis and the activity of P-gp was evaluated by Rhodamine 123 efflux experiment. Meanwhile, the cytotoxicity of daunomycin was analyzed by cell live/dead fluorescence staining method.p><p><b>RESULTSb>Microfluidic chip designed in this study could provide a suitable microenvironment for the growth of A549 cells and the A549 cells reached the confluence of 90% after inoculation for 72 h. Treatment of the acidic cell culture media on A549 cells did not make a significant difference on the expression level of P-gp. However, the activity of P-gp was significantly enhancement and peaked at 6 h after treatment with acidic cell culture media. Meanwhile, the cytotoxicity of daunomycin reduced significantly after treatment with acidic cell culture medium for 6 h,and a reversal effect was obtained when synergy with verapamil.p><p><b>CONCLUSIONSb>Microfluidic chip technology can shorten the analysis time and reduce the reagent consumption. It can be used as a new technology platform for understanding the mechanisms of multi-drug resistance and for screening highly efficient multi-drug resistance reversal agents.p>
ATP Binding Cassette Transporter, Sub-Family B
;
ATP-Binding Cassette, Sub-Family B, Member 1
;
Cell Culture Techniques
;
Cell Line, Tumor
;
Culture Media
;
Daunorubicin
;
Extracellular Space
;
Humans
;
Hydrogen-Ion Concentration
;
Microfluidics
10.Orphanin FQ combined with adriamycin reverses multi-drug resistance of K562/ADM and its molecular mechanism.
Xiao-Xia WANG ; Xiao-Qin LIU ; Wei ZHANG ; Xuan CHEN ; Li ZHAO
Journal of Experimental Hematology 2012;20(3):574-578
Our study have confirmed that orphanin FQ (OFQ) alone can reverse the multi-drug resistance of K562/ADM at the cellular level. Thus, this study was purposed to investigate the molecular mechanism of OFQ combined with ADM that reverses multi-drug resistance of K562/ADM, as well as its correlation with the expression of MDR1 mRNA and P-glycoprotein (P-gp). MTT method was used to detect the proliferation ability of K562/ADM treated with OFQ and ADM alone and their combination; flow cytometry was performed to measure the cell apoptosis rate; real time-PCR was applied to detect the MDR1 mRAN expression; Western blot was used to determine the P-gp expression. The results showed that OFQ (0.1 µmol/L) combined with ADM (15 mg/L) significantly inhibited the cell proliferation of K562/ADM, compared with ADM group; the date gained at 48 h was statistically significant (P < 0.05), and cell apoptosis rate was significantly raised (P < 0.01); MDR1 mRNA and P-gp expression levels of OFR combined with ADM were significantly lower than that of ADM alone, and were time-dependent within 48 h. It is concluded that OFQ combined with ADM can reverse the multi-drug resistance of K562/ADM in time-dependent manner, and the 48 h after treatment with these 2 drugs is the best reverse time, which may be related with down regulating the expression of MDR1 mRNA and P-gp.
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
;
Opioid Peptides
;
pharmacology