Reversal of P-glycoprotein-mediated Multidrug Resistance in SGC7901/VCR Cells by PPARγ Activation by Troglitazone

Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma(PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Western blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR) alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

Multidrug Resistance P-glycoprotein Function of Bone Marrow Hematopoietic Cells and the ReversalAgent Effect

The multidrug resistance P-glycoprotein (P-gp) expression and func-tion in hematopoietic stem/progenitor cells were studied to investigate whether the inhibition of hematopoietic cell P-gp function by multidrug resistance reversal agent increases the cytotoxicity of chemotherapy drugs on the hematopoietic cells.The expression of P-gp on the surface of CD cells from healthy human marrow was examined by flow cytometry. The multidrug resistance reversal agent MS-209 was used to measure the effects of MS-209 on the Rhodamin-123 uptaking o fCD hematopoietic cells. By using methylcellulose semi-solid culture, normal human granulocyte-macrophage clonal formation unit (CFU-GM) was cultured. The changes in CFU-GM inhibitory rate caused by daunorubicin were determined in the presence or absence of MS-2O9. The results showed that the P-gp expression rate of bone marrow CDL cells was 13. 3 %. MS-209 obviously increased the Rhodamin-123 uptake of CD positive cells. The mean inhibitory rate of daunorubicin for CFU-GM was 29. 6 %, but it was increased to 43. 3 % in the presence of MS-209 with the difference being significant (P< 0. 05). It was concluded that hematopoietic cells expressed P-gp protein and possessed active function- MS-209could inhibit the membrane efflux pump and increase the cytotoxicity of chemotherapy drugs to the clonal growth of hematopoeitic stem cells, suggesting the side effects of these drugs on the hematopoietic system should be taken into consideration in the clinical use.

REVERSION OF MULTIDRUG RESISTANCE IN THE P-GLYCOPROTEIN POSITIVE BREAST CANCER CELL LINE(MCF-7/ADR) BY INTRODUCTION OF HAMMERHEAD RIBOZYME

A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

JNK1,JNK2,and JNK3 are involved in P-glycoprotein-mediated multidrug resistance of hepatocellular carcinoma cells

BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK)activation could be a new method to reverse MDR.However,the relationship between JNK activity and MDR in HCC cells is unknown.This study aimed to explore the relationship between MDR and JNK in HCC cell lines with different degrees of MDR.METHODS:A MDR human HCC cell line,SMMC-7721/ ADM,was developed by exposing parental cells to gradually increasing concentrations of adriamycin.The MTT assay was used to determine drug sensitivity.Flow cytometry was used to analyze the cell cycle distribution and to measure the expression levels of P-glycoprotein(P-gp)and MDR-related protein(MRP)-1 in these cells.JNK1,JNK2 and JNK3 mRNA expression levels were quantified by real-time PCR.Expression and phosphorylation of JNK1,JNK2,and JNK3 were analyzed by Western blotting.RESULTS:The MDR of SMMC-7721/ADM cells resistant to 0.05 mg/L adriamycin was mainly attributed to the overexpression of P-gp but not MRP1.In addition,these cells had a significant increase in percentage in the S phase,accompanied by a decrease in percentage in the G0/G1 phase,which is likely associated with a reduced ability for cell proliferation and MDR generation.We found that JNK1,JNK2,and JNK3 activities were negatively correlated with the degree of MDR in HCC cells.CONCLUSION:This study suggests that JNK1,JNK2,and JNK3 activities are negatively correlated with the degree of MDR in HCC cells.

Apatinib reduces liver cancer cell multidrug resistance by modulating NF-κB signaling pathway

Objectives:This investigation aimed to elucidate the inhibitory impact of apatinib on the multidrug resistance of liver cancer both in vivo and in vitro.Methods:To establish a Hep3B/5-Fu resistant cell line,5-Fu concentrations were gradually increased in the culture media.Hep3B/5-Fu cells drug resistance and its alleviation by apatinib were confirmed via flow cytometry and Cell Counting Kit 8(CCK8)test.Further,Nuclear factor kappa B(NF-κB)siRNA was transfected into Hep3B/5-Fu cells to assess alterations in the expression of multidrug resistance(MDR)-related genes and proteins.Nude mice were injected with Hep3B/5-Fu cells to establish subcutaneous xenograft tumors and then categorized into 8 treatment groups.The treatments included oxaliplatin,5-Fu,and apatinib.In the tumor tissues,the expression of MDRrelated genes was elucidated via qRT-PCR,immunohistochemistry,and Western blot analyses.Results:The apatinibtreated mice indicated slower tumor growth with smaller size compared to the control group.Both the in vivo and in vitro investigations revealed that the apatinib-treated groups had reduced expression of MDR genes GST-pi,LRP,MDR1,and p-p65.Conclusions:Apatinib effectively suppresses MDR in human hepatic cancer cells by modulating the expression of genes related to MDR,potentially by suppressing the NF-κB signaling pathway.

Identification of FDA-Approved Drugs as Modulators of Multidrug Resistance Protein 2 (MRP2/ABCC2) Expression Levels in MRP2-Overexpressing Cells: Preliminary Data

Multidrug Resistance Protein 2 (MRP2) is an ATP-dependent transmembrane protein that plays a pivotal role in the efflux of a wide variety of physiological substrates across the plasma membrane. Several studies have shown that MRP2 can significantly affect the absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles of many therapeutic drugs and chemicals found in the environment and diet. This transporter can also efflux newly developed anticancer agents that target specific signaling pathways and are major clinical markers associated with multidrug resistance (MDR) of several types of cancers. MDR remains a major limitation to the advancement of the combinatorial chemotherapy regimen in cancer treatment. In addition to anticancer agents, MRP2 reduces the efficacy of various drug classes such as antivirals, antimalarials, and antibiotics. The unique role of MRP2 and its contribution to MDR makes it essential to profile drug-transporter interactions for all new and promising drugs. Thus, this current research seeks to identify modulators of MRP2 protein expression levels using cell-based assays. A unique recently approved FDA library (372 drugs) was screened using a high-throughput In-Cell ELISA assay to determine the effect of these therapeutic agents on protein expression levels of MRP2. A total of 49 FDA drugs altered MRP2 protein expression levels by more than 50% representing 13.17% of the compounds screened. Among the identified hits, thirty-nine (39) drugs increased protein expression levels whereas 10 drugs lowered protein expression levels of MRP2 after drug treatment. Our findings from this initial drug screening showed that modulators of MRP2 peregrinate multiple drug families and signify the importance of profiling drug interactions with this transporter. Data from this study provides essential information to improve combinatorial drug therapy and precision medicine as well as reduce the drug toxicity of various cancer chemotherapies.

Genotyping Characteristics of Human Fecal Escherichia coli and Their Association with Multidrug Resistance in Miyun District, Beijing

Objective To explore the genotyping characteristics of human fecal Escherichia coli(E. coli) and the relationships between antibiotic resistance genes(ARGs) and multidrug resistance(MDR) of E. coli in Miyun District, Beijing, an area with high incidence of infectious diarrheal cases but no related data.Methods Over a period of 3 years, 94 E. coli strains were isolated from fecal samples collected from Miyun District Hospital, a surveillance hospital of the National Pathogen Identification Network. The antibiotic susceptibility of the isolates was determined by the broth microdilution method. ARGs,multilocus sequence typing(MLST), and polymorphism trees were analyzed using whole-genome sequencing data(WGS).Results This study revealed that 68.09% of the isolates had MDR, prevalent and distributed in different clades, with a relatively high rate and low pathogenicity. There was no difference in MDR between the diarrheal(49/70) and healthy groups(15/24).Conclusion We developed a random forest(RF) prediction model of TEM.1 + baeR + mphA + mphB +QnrS1 + AAC.3-IId to identify MDR status, highlighting its potential for early resistance identification. The causes of MDR are likely mobile units transmitting the ARGs. In the future, we will continue to strengthen the monitoring of ARGs and MDR, and increase the number of strains to further verify the accuracy of the MDR markers.

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,

Survey of Multidrug Resistance and Class I Integron Prevalence in Chicken-derived Salmonella

[Objective] This study aimed to investigate the multidrug resistance and prevalence of class I integrons in Salmonel a. [Method] Salmonel a strains were isolated from chicken farms in Shandong Province. Kauffmann-White classification method was employed to analyze the serotypes of Salmonel a strains. Minimum in-hibition concentration （MIC） of Salmonel a strains against 19 common antimicrobial drugs was analyzed determined with microdilution method. The class I integrons and carried drug resistance gene cassettes were detected by PCR. [Result] A total of 311 Salmonel a strains were isolated and classified into two serotypes, including 133 Salmonel a Indiana strains and 178 Salmonel a Enteritidis strains. Drug sensitivity test showed that the isolated Salmonel a strains were general y resistant to sulfadiazine, sulfamethoxazole, nalidixic acid, ampicil in, tetracycline, doxycycline and trimethoprim, with a multidrug resistance rate of 91.0% （283/311）; 99% strains were sensitive to amikacin and colistin. PCR assay indicated that the detection rate of class I integrons was 65.0% （202/311）; the positive rate of class I integrons in Salmonel a strains with multidrug resistance was 92.6%; among 202 positive strains, six strains carried gene cassette dfr17-aadA5. [Conclusion] According to the above results, class I integrons exist general y in Salmonel a and are closely associated with the multidrug resistance of Salmonel a strains.

Effects of arsenic trioxide on drug transporting molecules in multidrug resistance malignant neoplasma MR2 cell line

Objective： To study the effect of arsenic trioxide （As203） on the expression of drug transporting molecules in multidrug resistance malignant neoplasma acute promyelocytic leukemia （APL） MR2 cell line. Methods： MR2 resistant to alltrans retinoic acid （ATRA） and non-ATRA resistant APL cell line NB4 were used. Expressions of P-glycoprotein （Pgp）, multidrug resistance protein （MRP） and lung resistance-related protein （LRP） were detected by immunocytochemical assay. Results： The expression of Pgp was significantly higher in MR2（30%-40%） than that in NB4（10%-20%） （P 〈 0.001）, and the expression of MRP was also higher in MR2 （56.9 ± 3.4 - 21.2 ± 1.1） than that in NB4 （20.6 ± 5.3 - 16.7 ± 1.2） （P 〈 0.001）. As2O3 ranging from 0.5-2.0 μmol/L, could significantly decrease the expressions of Pgp and MRP. The expression of Pgp and MRP in MR.2 cell line were negatively correlated with the dose and duration of action of As2O3. Conclusion： Pgp and MRP may be the sensitive targets of As2O3 to overcome drug-resistance. ATRA might be the substrates of Pgp and MRP.

Using ^(99m)Tc-MIBI to Evaluate the Effects of Chemosensitizer on P-glycoprotein in Multidrug-resistant Carcinoma Cells

To establish a method to evaluate the effects of chemosensitizer onP-glycoprotein using ^(99m)Tc-MIBI, and observe the changes of ^(99m)Tc-MIBI uptake kinetics andP-glycoprotein levels after using verapamil in MDR human breast cells MCF-7/Adr. Methods: MDR breastcarcinoma cells, MCF-7/Adr, were incubated and different protocols were performed. Protocol Ⅰ: achemosensitizer, verapamil (10 μmol/L), was added into cell culture medium, while in control group,the same volume of DMEM was given. Cells were harvested after 2 h incubation with ^(99m)Tc-MIBI.Protocol Ⅱ: Verapamil (10 μmol/L) was added into cell culture medium and incubated for 20 min, 40min, 60 min, 80 min, 8 h, 24 h, 48 h and 72 h respectively. Cells were harvested after 2 hincubation with ^(99m)Tc-MIBI. The radioactivity of the cells was measured and P-glycoproteinexpression levels were determined with immunohistochemical stain. Results: Protocol Ⅰ: After 2hincubation with verapamil the cellular uptake of ^(99m)Tc-MIBI was remarkably higher than controlgroup (t=2.33, P 【 0.05), but there was no difference in P-glycoprotein expression levels betweentwo groups (P 】 0.05). Protocol Ⅱ: In verapamil group, ^(99m)Tc-MIBI uptake was increased withincubation time prolonging (F=58.2, P 【 0.05). When verapamil incubation time surpassed 8 h the^(99m)Tc-MIBI uptake negatively correlated to the P-glycoprotein expression levels (r=-0.73, P 【0.01). However, when incubation time was less than 80 min, there was no correlation between^(99m)Tc-MIBI accumulation and P-glycoprotein levels (r=0.16, P 】 0.05). Conclusion: ^(99m)Tc-MIBImay be used to evaluate the qualitative as well as quantitative change of P-glycoprotein expressionlevels induced by the chemosensitizer, verapamil.

Structure-activity Relationship of Phenothiazines for Inhibition of Protein Kinase C and Reversal of Multidrug Resistance

Studies on structure-activity relationship of phenothiazines (PTZs) forinhibition of protein kinase C (PKC) and reversal of multidrug resistance (MDR) has been made invitro. The results showed that the order of potency of reversal effect of PTZs on MDR is as follows:2-COC_3 H_7 > 2-CF_3 > 2-COCH_3 > H. The type of piperazinyl substitution also significantlyaffected potency against MDR. The results show the order: CH_3 > COOC_2 H_5 > C_2 H_4 OH. Inaddition, PKC plays a marked role in diverse cellular process including MDR. Some derivatives of PTZwas tested for inhibition of PKC, of which PTZ11 showed the highest inhibitory effect of MDR andPKC, implying a potential reversal agent of MDR for tumor therapy in the future. We also tried toexplore the possible binding model of PTZs to PKC. Our molecular-modeling study preliminarilysuggests how these PTZs bind to PKC and provides a structural basis for the design of high affinityPKC-modulator. The infor-mation may be used in the rational design of more effective drugs.

Reversal of Multidrug Resistance by Neferine in Adriamycin Resistant Human Breast Cancer Cell Line MCF-7/ADM

Objective: To study the reversal effect of neferine on adriamycin (ADM) resistant human breast cancer cell line MCF-7/ADM. Methods: The cytotoxic effect of Nef or ADM was determined by 3-[4, 5-dimethylthiazol-2.-yl], 5-diphenyl tetraxolium bromid (MTT) assay. Apoptosis and the expression of P-glycoprotein (P-gp) were detected by flow cytometry (FCM). The intracellular ADM concentration was measured by HPLC. Results: Nef at 1, 5, 10 mol/L decreased the IC50 of ADM to MCF-7/ADM from 11.63 g/mL to 4.59, 2.44, 0.27 g/mL respectively. MCF-7/ADM could resist the apoptosis induced by ADM while Nef (1-10 mol/L) could augment ADR-mediated apoptosis. Nef (10 mol/L) increased the accumulation of ADM up to 2.88 fold in MCF-7/ADM but not in sensitive cells MCF-7/S and reduced the expression of P-gp in MCF-7/ADM cells. Conclusion: Nef can circumvent multidrug resistance (MDR) of MCF-7/ADM cells and the mechanism was associated with the increase of intracellular accumulation of ADM and the reduced expression of P-gp in MCF-7/ADM cells.

EXPRESSION AND CLINICAL SIGNIFICANCE OF MULTIDRUG RESISTANCE GENE AND MULTIDRUG RESISTANCE-ASSOCIATEDPROTEIN GENE IN ACUTE LEUKEMIA

Objective: To evaluate the expression and clinical significance of multidrug resistance gene (mdr1) and multidrug resistance-associated protein (MRP) gene in acute leukemia. Methods: The expression of mdr1 and MRP assay in 55 patients with acute leukemia (AL) by reverse transcription polymerase chain reaction (RT-PCR). Results: The mdr1 and MRP gene expression levels in the relapsed AL and the blastic plastic phases of CML were significantly higher than those in the newly diagnostic AL and controls. The mdr1 and MRP gene expression levels in the clinical drug-resistant group were significantly higher than those in the non-drug-resistant group. The complete remission (CR) rate in patients with high mdr1 expression (14.3%) was significantly lower than that with low mdr1 expression (57.5%); similarly the CR rate in patients with high MRP level was also lower than that with low MRP level. Using both high expression of mdr1 and MRP gene as the indicator for evaluating multidrug resistance (MDR), the positive predictive value and accuracy increased in comparison with single gene high expression. Conclusion: Elevated level of mdr1 or MRP gene expression might be unfavorable prognostic factors for AL patient and may be used as an important index for predicting drug-resistance and relapse in AL patient. Measuring both mdr1 and MRP gene expression would increase accuracy and sensibility of evaluating MDR in acute leukemia.

Influence of efflux pump inhibitors on the multidrug resistance of Helicobacter pylori

AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine and pantoprazole), and the minimum inhibitory concentrations (MICs) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H. pylori infection, on MICs of antibiotics were evaluated.RESULTS: Four strains of MDR H. pylori were induced successfully, and the antibiotic susceptibilities of MDR strains were partly restored by CCCP and pantoprazole, but there was little effect of reserpine. Rabeprazole was the most effective of the 5 PPIs which could decrease the MICs of antibiotics for MDR H. pylori significantly.CONCLUSION: In vitro, some EPIs can strengthen the activities of different antibiotics which are the putative substrates of the efflux pump system in H. pylori.

Synergistic Effect of Hyperthermia and Neferine on Reverse Multidrug Resistance in Adriamycin-resistant SGC7901/ADM Gastric Cancer Cells

Multidrug resistance（MDR） plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine（Nef） in adriamycin（ADM） resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein（P-gp）,γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

The multidrug resistance transporter P-glycoprotein confers resistance to ferroptosis inducers

Aim:Ferroptosis is a non-apoptotic form of cell death caused by lethal lipid peroxidation.Several small molecule ferroptosis inducers(FINs)have been reported,yet little information is available regarding their interaction with the ATP-binding cassette(ABC)transporters P-glycoprotein(P-gp,ABCB1)and ABCG2.We thus sought to characterize the interactions of FINs with P-gp and ABCG2,which may provide information regarding oral bioavailability and brain penetration and predict drug-drug interactions.Methods:Cytotoxicity assays with ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2 were used to determine the ability of the transporters to confer resistance to FINs;confirmatory studies were performed in OVCAR8 and NCI/ADR-RES cells.The ability of FINs to inhibit P-gp or ABCG2 was determined using the fluorescent substrates rhodamine 123 or purpuin-18,respectively.Results:P-gp overexpression conferred resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin.P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1.The FINs ML-162,GPX inhibitor 26a,and PACMA31 at 10µM were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells.GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells.Conclusion:Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain.The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.

Reversing multidrug resistance by RNA interference through the suppression of MDR1 gene in human hepatoma cells

AIM： To reverse the multidrug resistance （MDR） by RNA interference （RNAi）-mediated MDRI suppression in heparoma cells.METHODS： For reversing MDR by RNAi technology, two different short hairpin RNAs （shRNAs） were designed and constructed into pGenSil-1 plasmid, respectively. They were then transfected into a highly adriarnycin-resistant HepG2 hepatorna cell line （HepG2/ADM）. The RNAi effect on MDR was evaluated by real-time PCR, cell cytotoxicity assay and rhodarnine 123 （Rh123） efflux assy. RESULTS： The stably-transfected clones showed various degrees of reversal of MDR phenotype. Surprisingly, the MDR phenotype was completely reversed in two transfected clones. CONCLUSION： MDR can be reversed by the shRNAmediated MDRI suppression in HepG2/ADM cells, which provides a valuable clue to make multidrug-resistant hepatoma cells sensitive to anti-cancer drugs.

Multidrug resistance 1 gene in inflammatory bowel disease: A meta-analysis

The MDR1 gene is an attractive candidate gene for the pathogenesis of inflammatory bowel disease （IBD） and perhaps response to therapy, with evidences at both functional and genetic levels. Its product, the P-glycoprotein （P-gp） functions as a transmembrane efflux pump thus influencing disposition and response of many drugs, some of whom （i.e. glucocorticoids） central to IBD therapy. In addition P-gp is highly expressed in many epithelial surfaces, included gastrointestinal tract （G-I） with a putative role in decreasing the absorption of endogenous or exogenous toxins, and perhaps host-bacteria interaction. Many genetic variations of MDR1 gene has been described and in some instances evidences for different P-gp expression as well drugs metabolism have been provided. However data are often conflicting due to genetic heterogeneity and different methodologies employed. Perhaps the greatest piece of evidence of the physiological importance of P-gp in the G-I tract has come from the description of the mdrl knock-out mice model, which develops a spontaneous colitis in a specific pathogen-free environment. Studies investigating MDR1 gene polymorphism and predisposition to IBD have also shown conflicting results, owing to the known difficulties in complex diseases, especially when the supposed genetic contribution is weak. In this study we have undertaken a metaanalysis of the available findings obtained with two SNPs polymorphism （C3435T and G2677T/A） in IBD; a significant association of 3435T allele and 3435TT genotype has been found with UC （OR = 1.17, P = 0.003 and OR = 1.36, P = 0.017, respectively）. In contrast no association with CD and the G2677T/A polymorphism could be demonstrated.

Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.