OBJECTIVES: Hypoxia can alter the oral bioavailability of drugs, including various substrates (drugs) of P-glycoprotein (P-gp), suggesting that hypoxia may affect the function of P-gp in intestinal epithelial cells. Currently, Caco-2 monolayer model is the classic model for studying the function of intestinal epithelial P-gp. This study combines the Caco-2 monolayer model with hypoxia to investigate the effects of hypoxia on the expression and function of P-gp in Caco-2 cells, which helps to elucidate the mechanism of changes in drug transport on intestinal epithelial cells in high-altitude hypoxia environment. METHODS: Normally cultured Caco-2 cells were cultured in 1% oxygen concentration for 24, 48, and 72 h, respectively. After the extraction of the membrane proteins, the levels of P-gp were measured by Western blotting. The hypoxia time, with the most significant change of P-gp expression, was selected as the subsequent study condition. After culturing Caco-2 cells in transwell cells for 21 days and establishing a Caco-2 monolayer model, they were divided into a normoxic control group and a hypoxic group. The normoxic control group was continuously cultured in normal condition for 72 h, while the hypoxic group was incubated for 72 h in 1% oxygen concentration. The integrity and polarability of Caco-2 cells monolayer were evaluated by transepithelial electrical resistance (TEER), apparent permeability (Papp) of lucifer yellow, the activity of alkaline phosphatase (AKP), and microvilli morphology and tight junction structure under transmission electron microscope. Then, the Papp of rhodamine 123 (Rh123), a kind of P-gp specific substrate, was detected and the efflux rate was calculated. The Caco-2 cell monolayer, culturing at plastic flasks, was incubated for 72 h in 1% oxygen concentration, the expression level of P-gp was detected. RESULTS: P-gp was decreased in Caco-2 cells with 1% oxygen concentration, especially the duration of 72 h (P<0.01). In hypoxic group, the TEER of monolayer was more than 400 Ω·cm², the Papp of lucifer yellow was less than 5×10^-7 cm/s, and the ratio of AKP activity between apical side and basal side was greater than 3. The establishment of Caco-2 monolayer model was successful, and hypoxia treatment did not affect the integrity and polarization state of the model. Compared with the normoxic control group, the efflux rate of Rh123 was significantly reduced in Caco-2 cell monolayer of the hypoxic group (P<0.01). Hypoxia reduced the expression of P-gp in Caco-2 cell monolayer (P<0.01). CONCLUSIONS Hypoxia inhibits P-gp function in Caco-2 cells, which may be related to the decreased P-gp level.
Humans ; ATP Binding Cassette Transporter, Subfamily B, Member 1 ; Caco-2 Cells ; ATP Binding Cassette Transporter, Subfamily B ; Hypoxia ; Oxygen

OBJECTIVE: To investigate the effect of ursane triterpenoids 3β,19α-dihydroxyursu-12-ene-23,28-dicarboxylic acid (Rotundioic acid, RA) on the sensitivity of adriamycin-resistant K562 cells (K562/ADM Cell) anti-tumor drug, and to explore the effect and mechanism of RA on the multidrug resistance of K562/ADM cells. METHODS: CCK-8 method was used to detect the effect of RA on the sensitivity of K562 cells and K562/ADM cells to anti-tumor drug. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression level of mRNA and the protein in K562 and K562/ADM cells, and the effect of RA on the expression of MDR1 mRNA and P-gp in K562/ADM cells was also detected; Western blot was used to detect the expression of p-JNK, p-p38 and p-ERK1/2 in K562/ADM cells. RESULTS: RA could increased the sensitivity of K562/ADM cells to adriamycin(the reversal factor was 1.61 times), the difference showed statistically significantly (P<0.05); the resistance factor of K562/ADM to ADM was 41.76 times. The expression of MDR1 mRNA in K562 cells was extremely low, and the protein product P-glycoprotein (P-gp) was almost not expressed; MDR1 mRNA and P-gp in K562/ADM cells were highly expressed; RA could down-regulate the expression levels of MDR1 and P-gp in K562/ADM cells. In addition, RA could upregulate the phosphorylation levels of p38 and ERK1/2 in K562/ADM cells, but it has no effect on the expression of p-JNK. CONCLUSION RA may participate in the regulation of MAPK signaling pathway by upregulating the expression levels of p-p38 and p-ERK1/2 in K562/ADM cells, and thus inhibit the transcription and translation levels of MDR1, and finally reverse the multidrug resistance of leukemia cells.
ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1 ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; K562 Cells

Drug transport is an important source of inter-individual variations in drug responses and is also a common site where drug-drug interactions happen. In recent years, more and more novel identified transporters have been added into the transporter super family, and this trend will continue in the future. Among the transporter members of this family, ATP-dependent efflux transporter P-glycoprotein (MDR1) and organic anion transporters (OATP) are the most important proteins involved in drug transport. MDR1 is the most well known transporter. Widely distributed in tissues such as the gastrointestinal tract, liver, kidney and so on, MDR1 plays an important role in drug absorption, distribution and excretion. Its functional genetic polymorphisms have significantly changed the pharmacokinetics of its substrate drugs, which has important clinical implications. OATP expressed in multiple tissues, and it mediated the drug excretion through the bile acid and kidney. Some genetic polymorphism of OATP genes is the cause of some abnormal drug responses.
ATP Binding Cassette Transporter, Subfamily B, Member 1 ; genetics ; Drug Interactions ; genetics ; Humans ; Membrane Transport Proteins ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; Pharmaceutical Preparations ; metabolism ; Polymorphism, Genetic

OBJECTIVES: To investigate the effect of adriamycin (ADM), idelalisib or ADM and their combination on cell proliferation and intracellular concentration of ADM, and to explore the reversal effect of idelalisib on drug resistance to ADM. METHODS: The K562 and K562/ADM cells were respectively treated with ADM and idelalisib at different concentrations. The 50% inhibitory concentration (IC RESULTS: The cell survival rates were significantly decreased in a dose-dependent manner when the cells were treated with different doses of ADM (0.001-10.000 mg/L ). The IC CONCLUSIONS Idelalisib exerts effect on inhibition of the proliferation in myeloid leukemia K562 and K562/ADM cells, which may partially reverse the drug resistance of K562/ADM cells to ADM. The mechanisms for the effect of idelalisib may be related to increasing the accumulation of ADM and inducing the cell apoptosis in the K562 and K562/ADM cells.
ATP Binding Cassette Transporter, Subfamily B, Member 1 ; Cell Proliferation ; Doxorubicin/pharmacology* ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; K562 Cells ; Leukemia, Myeloid ; Purines ; Quinazolinones

<b>OBJECTIVEb>To establish the adriamycin(ADR)-resistant ALL cell lines and to investigate their drug-resistan mechanisms.

<b>METHODSb>The drug-resistant cell lines SUP-B15/ADR and RS4;11/ADR were derived by exposing the parental cells [SUP-B15(Ph) and RS4;11(Ph)] to the ascending concentrations of ADR. The cell viability was detected by CCK-8 method. The expression of P-gp was examined by Western blot, and RT-qPCR was performed to detect the expression of MDR1.

<b>RESULTSb>The drug-resistant cell lines SUP-B15/ADR and RS4;11/ADR were successfully established, their resistance indexes were 14.088±0.763 and 10.473±1.024, respectively. After the cryopreserved SUP-B15/ADR and RS4;11/ADR cells were resuscitated, their survival rates were 88.4±1.2% and 89.3±1.6% respectively, while their resistance indexes were 13.976±0.967 and 10.342±0.846 respectively (P>0.05). When the drug-resistant cells were cultured in the medium without ADR for 1 month, their drug-resistance indexes dropped down to 12.893±1.255 and 9.327±0.321 respectively(P<0.05). Drug-resistant cell lines had the cross-resistance to cytarabine and etoposide. The expression of P-gp and MDR1 in drug-resistant cells was significantly higher than that in wild-type cells.

<b>CONCLUSIONb>Two drug-resistant ALL cell lines have been successfully established by exposing to the ascending concentration of ADR. The over-expression of MDR1 and P-gp in drug-resistant cells may be one of the mechanisms underlying the drug resistance.

OBJECTIVES: Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer. But the problem of drug resistance severely limits their clinical application. This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil (5-FU) and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition. METHODS: Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment, and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group. The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression level of hypoxia inducible factor-1α (HIF-1α), multidrug resistance (MDR1) gene, P-glycoprotein (P-gp), and vascular endothelial growth factor (VEGF) which were related to 5-FU drug-resistance. We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU. RESULTS: Compared with the Normoxic+5-FU group, the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced (P<0.05), and the expression of apoptosis promoter protein caspase 8 was also decreased. Compared with the the Normoxic+5-FU group, HIF-1α mRNA expression in the Hypoxia+5-FU group was significantly increased (P<0.05), and the mRNA and protein expression levels of MDR1, P-gp and VEGF were also significantly increased (all P<0.05). The increased expression of MDR1, P-gp and VEGF had the same trend with the expression of HIF-1α. CONCLUSIONS Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy. Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.
Humans ; Fluorouracil/therapeutic use* ; Vascular Endothelial Growth Factor A/metabolism* ; Stomach Neoplasms/drug therapy* ; Drug Resistance, Multiple ; Vascular Endothelial Growth Factors/metabolism* ; Hypoxia ; ATP Binding Cassette Transporter, Subfamily B/genetics* ; ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics* ; Cell Line, Tumor ; Cell Hypoxia ; RNA, Messenger/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Tumor Microenvironment

OBJECTIVE: To investigate the suppression of MDR1 and P-glycoprotein induced by small interfering RNA and the restoration of sensitivity to chemotherapeutic drugs in multidrug-resistant hepatocellular carcinoma cell line Bel7402/5-Fu. METHODS: MDR1j targeted small interfering RNA duplexes were introduced into multidrug-resistant hepatocellular carcinoma cell line Bel7402/5-Fu. The suppression of MDR1 and its gene product P-glycoprotein was examined by RT-PCR and Western blot. MTT assay was performed to measure the reverse effect of small interfering RNA based on the results of IC50. Cell apoptosis was assessed by flow cytometry after various cell lines were treated with chemotherapeutic drugs. RESULTS: The overexpression of MDR1 and P-glycoprotein was suppressed efficiently by the introduction of small interfering RNA, which caused sequence-specific gene silence. The level of MDR1 in the transfected Bel7402/5-Fu cells reduced to 22.55% and P-glycoprotein to 25.49% compared with those of the controls. The apoptosis rate of Bel7402/5-Fu cells increased significantly in the siRNA group during the chemotherapy (P<0.01). Their resistance to 5-Fu was reversed by 14.88 folds, which indicated the restoration of sensitivity to drugs. CONCLUSION Small interfering RNA can inhibit MDR1 expression effective and reverse the multidrug resistance mediated by P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1 ; genetics ; metabolism ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVE: To explore the multidrug resistance (MDR) mechanism of ABC superfamily transporters in the tumor stem cells(TSC) from human brain glioma tissues. METHODS: Samples of glioma were obtained from 30 patients undergoing microsurgical tumor resection. The CD133(+) cells and CD133(-) cells from these tumor specimens were isolated by magnetic activated cell sorting(MACS). These cells were cultured, proliferated and passaged. The protein and activity expression of multidrug-resistance protein 1(MDR1) and multidrug-resistance associated protein 1(MRP1) were analyzed between CD133(+) and CD133(-) cells by immunocytochemistry and RT-PCR respectively. RESULTS: CD133(+) cells generated free floating neurosphere like brain tumor spheres(BTS) and abnormal proliferating capacity in the serum-free medium(SFM) in vitro. Three cases from glioblastoma stem cells could form BTS in the complete medium, and could be cultured for 1-3 passages. The range of positive cell proportion for MDR1 and MRP1 expression in CD133(+) cells was 18%-67% and 23%-73% respectively. The expression levels of MDR1 and MRP1 mRNA were higher in CD133(+) glioma stem cells than those in the differentiated tumor cells(TC), the protein activity was increased to 16.1 and 19.6 times respectively compared with that of TC. The protein and activity expression were positively related to the pathological grades of tumors. MDR1 or MRP1 drug resistance was not expressed in all the tumors and there was obvious correlation between MDR1 and MRP1. CONCLUSION Only a small proportion of cells in the heterogeneous glioma is CD133(+) brain tumor stem cells which display the strong capacity of self-renewing, abnormal proliferation and intrinsic multidrug resistance to traditional chemotherapy. The high expression of MDR1 and MRP1 by the CD133(+) brain tumor stem cells is one of the main mechanisms in the chemoresistance of tumors. CD133(+) brain tumor stem cells can be served as the root of multidrug resistance and key therapeutic target for glioma chemotherapy.
AC133 Antigen ; ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1 ; genetics ; metabolism ; Antigens, CD ; immunology ; metabolism ; Brain Neoplasms ; metabolism ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Glioma ; metabolism ; Glycoproteins ; metabolism ; Humans ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Neoplastic Stem Cells ; drug effects ; metabolism ; Peptides ; metabolism ; Spheroids, Cellular ; drug effects ; Tumor Cells, Cultured

OBJECTIVE: To determine the effect of neferine (Nef) combined with mdr-1shRNA on the expression of mdr/P-gp in K562/A02 cell line. METHODS: MTT assay was used to observe the cell proliferation. The expression level of P-gp was determined by Western blot and the transcription of mdr-1 gene was detected by semi-quantitative RT-PCR. RESULTS: After K562/A02 cells were treated by Nef or mdr-1shRNA alone or both for 24 h, the proliferation of K562/A02 cells was significantly higher in the Nef combined with mdr-1shRNA treatment group than that of Nef or mdr-1shRNA alone group (P<0.01).The expression of mdr-1/P-gp in the Nef with mdr-1 shRNA group was significantly lower than that of Nef or mdr-1shRNA alone group. CONCLUSION Nef enhances the inhibition of mdr-1shRNA expression vector on K562/A02 cell proliferation and on P-gp protein to effectively reverse multidrug resistance induced by mdr-1 gene encoding P-gp.
ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1 ; genetics ; metabolism ; Benzylisoquinolines ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drug Synergism ; Drugs, Chinese Herbal ; pharmacology ; Humans ; K562 Cells ; RNA, Small Interfering ; genetics ; pharmacology

OBJECTIVE: To determine the effect of different heating Methods combined with neferine(Nef) on the proliferation and expressions of γH2AX and mdr-1/P-gp in MCF-7/Adr breast cancer cells. METHODS: MTT assay was used to determine block heating, water submerged heating, medium heating, and oven heating combined with 10 μg/mL Nef on adriamycin cultured MCF-7/Adr cell proliferation. The mdr-1mRNA expression was detected by real-time quantitative PCR. γH2AX and P-gp expressions were detected by Western blot. RESULTS: The absorbance values of MCF-7/Adr cells in different heating groups at 42 degree and 45 degree were significantly decreased, the mdr-1/P-gp expression was decreased, and γH2AX expression was upregulated compared with those of the 37 degree control group (all P<0.01). The absorbance values (P<0.01) and mdr-1/P-gp expression(P<0.05) were significantly lower and γH2AX expression(P<0.05) was significantly higher in the hyperthermia combined with 10 μg/mL Nef group than those of 10 μg/mL Nef group and hyperthermia group in MCF-7/Adr cells. The water submerged heating group had the lowest P-gp expression and the highest γH2AX expression among different heating groups at 42 degree and 45 degree in MCF-7/Adr cells (P<0.05). CONCLUSION Hyperthermia can increase the cell toxicity of adriamycin to multidrug resistant breast cancer cells. Hyperthermia significantly damages DNA of MCF-7/Adr cells and the higher temperature, the worse effect. Multidrug resistant breast cancer cells may respond differently to the different heating methods. Combined treatment of hyperthermia with Nef can increase the sensitivity in adriamycin chemotherapy.
ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1 ; genetics ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Benzylisoquinolines ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Histones ; genetics ; metabolism ; Hot Temperature ; Humans ; RNA, Messenger ; genetics ; metabolism