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.

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.

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.

Correlative Expression of Glutathion S-Transferase-π and Multidrug Resistance Associated Protein in Bladder Transitional Cell Carcinoma

In order to elucidate the mechanisms of multidrug resistance (MDR) in bladder cancer, the expression of glutathione S-transferase-π (GST-π) and multidrug resistance associated protein (MRP) in tissue samples resected from 44 patients and 6 normal bladder mucosa as control was de- tected by using immunohistochemical method, and the results were analyzed by computer-assisted im- age analyzing system (IAS) to achieve semi-quantitative data. In addition, correlation between the expression of both factors was studied. The results showed that the positive expression rate of GST- π and MRP in bladder cancer was 72. 7 % (32/44) and 68. 2 % (30/44) respectively, significantly higher than those in normal bladder mucosa, being 16. 7% and 33. 3% respectively. The rate of GST-πpositive staining was increased correspondingly with tumor grade and stage elevated, being higher in recurrent tumors treated by chemotherapy, but not significantly (P>0. 05). There was no significant differences between the expression of MRP and tumors' behaviors and clinical characters. However, the results demonstrated that the correlation between the expression of both resistant fac- tors was very evident (r=0. 695, P<0. 0025). It was suggested that the activation of GST-π and MRP might occur during malignant transformation of normal mucosa, but tumors' differentiation and progression could not be the unique factors that influenced both overexpression. Chemotherapy might be another important reason. The correlation of both indicated that there was a common mech- anism regulating their expression probably, which made them play a pivotal role in chemotherapy drug resistance of bladder cancers.

Construction of the recombinant adenovirus vector carrying antisense multidrug resistance gene

BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.

Association of single nucleotide polymorphisms of brain-derived neurotrophic factor gene and multidrug resistance 1 gene to refractory epilepsy in Chinese Han children

BACKGROUND： There are two hypotheses for the underlying cause of refractory epilepsy： ＂target＂ and ＂transport＂. Studies have shown that brain-derived neurotrophic factor （BDNF） is over-expressed in refractory epilepsy. Multidrug resistance 1 （MDR1） gene encodes for P-glycoprotein, the primary ATP-binding cassette transporter in the human body. Some single nucleotide polymorphisms of the MDR1 gene have been associated with refractory epilepsy. OBJECTIVE： To investigate the association between BDNF gene C270T polymorphism and MDR1 T-129C polymorphism with refractory epilepsy in Chinese Han children through the use of polymerase chain reaction （PCR）-restriction fragment length polymorphism analysis. DESIGN, TIME AND SETTING： A case-control, genetic association study was performed at the Central Laboratory, Third Xiangya Hospital of Central South University from June 2005 to November 2007. PARTICIPANTS： A total of 84 cases of unrelated children with epilepsy, including 41 cases of refractory epilepsy and 43 cases of drug-responsive epilepsy, were enrolled. An additional 30 healthy, Chinese Han children, whose ages and gender matched the refractory epilepsy patients, were selected as normal controls. METHODS： Venous blood was collected and genomic DNA was extracted from the blood specimens. C270T polymorphism in BDNF gene and T-129C polymorphism in MDR1 gene were genotyped using PCR-restriction fragment length polymorphism analysis. Association analysis using the Ftest and Chi-square test was statistically performed between C270T polymorphism in BDNF gene and T-129C polymorphism in MDR1 gene and refractory epilepsy. MAIN OUTCOME MEASURES： The distribution of genotypes and allele frequencies of C270T polymorphism in BDNF gene and T-129C polymorphism in MDR1 gene. RESULTS： The distribution of CC, CT, and TT genotypes, as well as C and T allele frequencies, in the BDNF gene was not significantly different between the refractory epilepsy group, drug-responsive epilepsy group, or the normal control group （P 〉 0.05）. The distribution of TT genotype and T allele frequencies of the MDR1 gene was significantly different in the refractory epilepsy group compared with the drug-responsive epilepsy and normal control groups （P 〈 0.05）. Comparison of haplotype combinations demonstrated that there were no significant differences in combinations of TT＋CC, -FI-＋CT, TC＋CC, and TC＋CT among the three groups （P 〉 0.05）. CONCLUSION： C270T polymorphism of the BDNF gene was not associated with refractory epilepsy in Chinese Han children, but T-129C polymorphism in the MDR1 gene was associated with refractory epilepsy in Chinese Han children. The TT genotype and T allele frequencies could serve as susceptibility loci for refractory epilepsy. Interactions between C270T in BDNF gene and T-129C in MDR1 gene were not observed in refractory epilepsy in Chinese Han children.

Expression of multidrug resistance 1 gene and C3435T genetic polymorphism in peripheral blood of patients with intractable epilepsy

BACKGROUND： Increased expression of multidrug resistance 1 （MDR1） mRNA in peripheral blood of patients with intractable epilepsy is not due to epilepsy drugs, but epilepsy behavior. Monitoring MDR1 expression in peripheral blood is a target for MDR1 gene evaluation. OBJECTIVE： To investigate the influence of antiepileptic drugs and seizures on MDR expression in intractable epilepsy, and to analyze the genetic polymorphisms of C3435T in the MDRl gene. DESIGN, TIME AND SETTING： Factorial designs and comparative observations at the experimental center of the Affiliated Hospital of Qingdao Medical College, Qingdao University between October 2003 and October 2004. PARTICIPANTS： A total of 120 subjects were recruited from the epilepsy clinical department of the Affiliated Hospital of Qingdao Medical College. Four groups （n = 30） were classified according to statistical factorial design： intractable epilepsy, treatment response, no treatment, and normal control groups. METHODS： One-step semi-quantitative reverse-transcription polymerase chain reaction technology was used to test expressions of the MDR1 gene in 120 subjects. C3435T polymorphisms in intractable epilepsy group and normal control groups were analyzed by polymerase chain reaction-restriction fragment length polymorphism. MAIN OUTCOME MEASURES： Expression of MDR1 mRNA in the four groups, and C3435T genetic polymorphisms in intractable epilepsy and normal control groups. RESULTS： MDRl gene expression was increased in the intractable epilepsy group, due to the factor seizures, but not the antiepileptic drugs. However, the interaction between the two factors was not statistically significant. Of the 30 subjects in the intractable epilepsy group, the following genotypes were exhibited： 3 （10%） C/C genotype, 9 （30%） C/T genotype, and 18 （60%） T/T genotype at the site of C3435T, while 4 （13%）, 10 （33%）, and 16 （53%） subjects were determined to express these genotypes in the normal control group, respectively. C and T allele frequency were 25% and 75% in the intractable epilepsy group, and 30% and 70% in the normal control group, respectively. However, there was no statistical difference between the groups. CONCLUSION： Results demonstrated that seizures, not antiepileptic drugs, induced MDR1 gene expression in intractable epilepsy. Genetic polymorphisms of C3435T in the MDR1 gene did not contribute to the development of multidrug resistance in patients with intractable epilepsy.

THE CORRELATION BETWEEN THE EXPRESSION OF MULTIDRUG RESISTANCE RELATED GENE AND CELL APOPTOSIS AND CLINICAL SIGNIFICANCE IN NON-SMALL CELL LUNG CANCER

Objective: To explore the correlation and clinical significance between expression of MDR (multidrug resistance) related gene MRP, MDR1, C-erbB-2 and cell apoptosis in non-small cell lung cancer (NSCLC). Methods: RT-PCR, Immunohistochemistry were used to examine the expression of mRNA and protein in the MDR and apoptosis related gene. Apoptosis cells were assayed by Terminal deoxynucleotidyl transferase (TdT)- mediated biotin dUTP nick end-labeling (TUNEL). Results: The positive rates of MRP, MDR1, C-erbB-2, bc1-2, C-myc mRNA in 63 cases NSCLC were 81.0% (51/63), 38.1%(24/63), 47.6%(30/63), 65.1%(41/63), 76.2%(48/63) respectively. Their levels were higher than those of corresponding proteins (74.6%, 34.9%, 46.0%, 61.9%, 71.4%, respectively). The significant association was found between the mRNA level and the protein expression (r =+0.764, P<0.02). The C-myc expression in 2 cases adjacent and benign lung tissue were light positive, and another 3 cases were negative. The positive correlation were demonstrated between C-myc and C-erbB-2 (r=+0.547, p=0.001) as well as bcl-2 and C-erbB-2 (r =+0.486, p=0.023) in NSCLC. There is no any correlation among bcl-2, C-myc and MRP or MDR1. There exists inverse correlation between apoptotic index and bcl-2 (r = -0.587, p = 0.017), and no any correlation among apoptotic index and MRP or MDR1 or C-erbB-2 or C-myc. The average apoptotic index were higher in the effective chemotherapy group (27.2( 2.1, 30.5(1.8) than that in the non-effective chemotherapy group (9.4( 1.3, 12.6( 2.4) with adenocarcinoma and squamous cell carcinoma (p =0.01, p=0.004). The positive rates of bcl-2, MRP, C-erbB-2 expression in the effective chemotherapy group (31.8%, 40.9%, 22.7%, respectively) were lower than those in the non-effective chemotherapy group (77.4%, 90.3%, 67.7%, respectively) (p=0.036, p=0.012, p=0.01), but MDR1 and C-myc expression have no any significant difference (p=0.067, p=0.282). The median survival time in the patients with coexpression of more than three MDR and/or apoptosis related genes are shorter (8.6 months) than that in those patients with coexpression of less than three MDR and/or apoptosis related genes (15.5 months)(p=0.01). Conclusion: The multidrug resistance in NSCLC is not only related to many drug resistance genes, but also involved in cell apoptosis and apoptosis related gene expression. The coexpression of MDR and apoptosis related gene is related to the survival time.

Differential expression of breast cancer-resistance protein,lung resistance protein,and multidrug resistance protein 1 in retinas of streptozotocin-induced diabetic mice

AIM：To investigate the altering expression profiles of efflux transporters such as breast cancer-resistance protein（BCRP）,lung resistance protein（LRP）,and multidrug resistance protein 1（MDR1） at the inner blood-retinal barrier（BRB） during the development of early diabetic retinopathy（DR） and/or aging in mice.METHODS：Relative m RNA and protein expression profiles of these three efflux transporters in the retina during the development of early DR and/or aging in mice were examined.The differing expression profiles of Zonula occludens 1（ ZO-1） and vascular endothelial growth factor-A（ VEGFA） in the retina as well as the perfusion characterization of fluorescein isothiocyanate（FITC）-dextran and Evans blue were examined to evaluate the integrity of the inner BRB.RESULTS：There were significant alterations in these three efflux transporters＇ expression profiles in the m RNA and protein levels of the retina during the development of diabetes mellitus and/or aging.The development of early DR was confirmed by the expression profiles of ZO-1 and VEGFA in the retina as well as the compromised integrity of the inner BRB.CONCLUSION：The expression profiles of some efflux transporters such as BCRP,LRP,and MDR1 in mice retina during diabetic and/or aging conditions are tested,and the attenuated expression of BCRP,LRP,and MDR1 along with the breakdown of the inner BRB is found,which may be linked to the pathogenesis of early DR.

Roles of sulfonylurea receptor 1 and multidrug resistance protein 1 in modulating insulin secretion in human insulinoma

BACKGROUND:Sulfonylurea receptor 1(SUR1)and multidrug resistance protein 1(MRP1)are two prominent members of multidrug resistance proteins associated with insulin secretion. The aims of this study were to investigate their expression in insulinomas and their sole and synergistic effects in modulating abnormal insulin secretion. METHODS:Fasting glucose,insulin and C-peptide were measured in 11 insulinoma patients and 11 healthy controls. Prolonged oral glucose tolerance tests were performed in 6 insulinoma patients.Insulin content,SUR1 and MRP1 were detected in 11 insulinoma patients by immunohistochemistry. SUR1 and MRP1 were also detected in 6 insulinoma patients by immunofluorescence. RESULTS:Insulinoma patients presented the typical demons-trations of Whipple’s triad.Fasting glucose of each insulinoma patient was lower than 2.8 mmol/L,and simultaneous insulin and C-peptide were increased in insulinoma patients. Prolonged oral glucose tolerance tests showed that insulin secretion in insulinoma patients were also stimulated by high glucose.Immunohistochemistry and immunofluorescence staining showed that SUR1 increased,but MRP1 decreased in insulinoma compared with the adjacent islets. CONCLUSIONS:The hypersecretion of insulin in insulinomas might be,at least partially,due to the enrichment of SUR1. In contrast,MRP1,which is down-regulated in insulinomas, might reflect a negative feedback in insulin secretion.

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.

REVERSAL EFFECTS OF MIFEPRISTONE ON MULTIDRUG RESISTANCE(MDR) IN DRUG-RESISTANT BREAST CANCER CELL LINE MCF7/ADR IN VITRO AND IN VIVO

To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human breast cancer cell line MCF7/ADR and its mechanisms. Methods: Expression of MDR1 and MDR-associated protein(MRP) mRNA in MCF7/ADR cells was detected using reverse transcription- polymerase chain reaction(RT-PCR). Western blotting was used to assay the protein levels of P-glycoprotein (P-gp) and MRP. Intracellular rhodamine 123 retention and [3H]vincristine (VCR) accumulation were measured by flow cytometry and liquid scintillation counter, respectively. MTT reduction assay was used to determine the sensitivity of cells to the anticancer agent, adriamycin (ADR). Additionally, a MCF7/ADR cell xenograft model was established to assess the reversal effect of mifeprisone on MDR in MCF7/ADR cells in vivo. Results: Miferpristone dose-dependently down- regulated the expression of MDR1 and MRP mRNA in MCF7/ADR cells, accompanied by a significant decrease in the protein levels of P-gp and MRP. After exposure to 5, 10, and 20 mmol/L mifepristone, MCF7/ADR cells showed a 3.87-, 5.81-, and 7.40-fold increase in the accumulation of intracellular VCR(a known substrate of MRP), and a 2.14-, 4.39-, and 5.53-fold increase in the retention of intracellular rhodamine 123(an indicator of P-gp function), respectively. MTT analysis showed that the sensitivity of MCF7/ADR cells to ADR was enhanced by 7.23-, 13.62-, and 20.96-fold after incubation with mifepristone as above-mentioned doses for 96 h. In vivo, mifepristone effectively restored the chemosensitivity of MCF7/ADR cells to ADR. After 8 weeks of administration with ADR(2 mg穔g-1穌-1) alone or in combination with mifepristone(50 mg穔g-1穌-1), the growth inhibitory rate of xenografted tumors in nude mice was 8.08% and 37.25%, respectively. Conclusion: Mifepristone exerts potent reversal effects on MDR in MCF7/ADR cells in vitro and in vivo through down- regulation of MDR1/P-gp and MRP expression and inhibition of P-gp- and MRP-dependent drug efflux, thus increasing the sensitivity to anticancer drug.

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.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective： Multidrug resistance（MDR） is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia（ALL） which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction（FQ-RT-PCR）, and combine minimal residual desease（MRD） detection by flow cytometry（FCM） and to study their relationship with treatment response and prognosis of ALL. Methods：The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission（CR）, 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results：Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group（P 〉 0.05）, but significantly higher in the recurrence group than that in newly diagnosed disease group and control group（0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05）. 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration（0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01）. For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group（P〉 0.05）. In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group（0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05）. Conclusion：The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration（within 3 years）. Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

Photodynamic therapy of cancer-Challenges of multidrug resistance

Photodynamic therapy(PDT)of cancer is a two-step drug device combination modality,which involves the topical or systemie administration of a photosensitizer followed by light ilumnination of cancer site.In the presence of oxygen molecules,the light illmnination of photosensitizer(PS)can lead to the generation of cytotoxic reactive oxygen species(ROS)and consequently destroy cancer.Similar to many other anticancer therapies,PDT is also subject to intrinsic cancer resistance mediated by mutidrug resistance(MDR)mechanisms.This paper will review the recent progress in understanding the interaction between MDR transporters and PS uptalke.The strategies that can be used in a clinical setting to owercome or bypass MDR will also be discussed.

Reversal Effect of BM-cyclin 1 on Multidrug Resistance by Down-regulating MRP2 in BALB/C Nude Mice Bearing C-A120 Cells

Our previous study demonstrated that BM-cyclin 1, a traditional anti-mycoplasma drug, could effectively reverse the multidrug resistance （MDR） of C-A120 cells. The present study aims to explore the reversal effect of BM-cyclin 1 on MDR and its mechanisms in BALB/C nude mice bearing C-A120 cells. Irnmunoblotting analysis and reverse transcription-polymerase chain reaction （RT-PCR） were used to study the change in multidrug resistance-associated protein 2 （MRP2） induced by BM-cyclin 1. We found that the expression levels of MRP2 protein and mRNA in C-A120 cells treated with BM-cyclin 1 were reduced significantly. Chemical colorimetry revealed no significant change in the level of glutathione （GSH）. In the xenografl model, the inhibitory rate of C-A120 cells growth in BM-cyclin 1 plus adriamycin （ADM） group was 52%, which was significantly higher than in control group （P〈0.01）. The immunoblotting and RT-PCR results conclusively demonstrated that BM-cycin 1 could significantly reduce the expression of MRP2 in transplanted tumor. In conclusion, BM-cyclin 1 could effectively reverse the MDR of C-A 120 cells in vivo by suppressing the expression of MRP2.

Reversal Effects of Ivermectin and Moxidectin on Multidrug Resistance in C6/adr Cells in vitro

Multidrug resistance(MDR)is a serious obstacle encountered in cancer treatment.This study was performed to explore the reversal MDR activity of ivermectin(IVM)from avermectin family and moxidectin(MOX)belonging to milbemycin family.The two compounds(5μmol•L-1)showed strong potency to increase adriamycin cytotoxicity toward adriamycin-resistant rat glioma cells C6/adr with fold reversal(FR)of 31.02 and 13.40,respectively.In addition,the mechanisms of them on p-glycoprotein(P-gp)-mediated MDR demonstrated that the two compounds significantly increased the intracellular accumulations of adriamycin and Rh123 via inhibiting P-gp efflux.Based on the analysis of P-gp,MDR1 and MRP1 gene expressions by using immunofluorescence flow cytometry and RT-PCR,the results revealed that the two compounds could down regulate the expression of P-gp,and that MDR1 and MRP1 gene expressions were down regulated.These findings suggested that ivermectin and moxidectin probably represented potent agents for reversing MDR in cancer therapy,and especially ivermectin was a better modulator.

Inhibition of apoptosis-regulatory protein Siva-1 reverses multidrug resistance in gastric cancer by targeting PCBP1

Introduction:Siva-1,as a pro-apoptotic protein,has been shown to induce extensive apoptosis in a number of different cell lines.In our previous study,we showed that overexpressed Siva-1 decreased the apoptosis of gastric cancer cells.So,we believe that it can also work as an anti-apoptotic protein.The present study aimed to determine the specific role of Siva-1 in anticancer drug resistance in gastric cancer in vivo and in vitro and preliminarily reveal the mechanism.Materials and Methods:A vincristine-resistant MKN-28/VCR gastric cancer cell line with stably downregulated Siva-1 was established.The effect of Siva-1 downregulation on chemotherapeutic drug resistance was assessed by measuring the IC50 and pump rate of doxorubicin.Proliferation,apoptosis of cells,and cell cycle were detected via colony formation assay andflow cytometry,respectively.Additionally,migration and invasion of cells was detected via wound healing and transwell assays.Moreover,we determined in vivo effects of LV-Siva-1-RNAi on tumor size,and apoptotic cells in tumor tissues were detected using TUNEL and hematoxylin and eosin staining.Results:Siva-1 downregulation reduced the pump rate of doxorubicin and enhanced the response to drug treatment.Siva-1 negatively regulated proliferation and promoted apoptosis of cells by potentiality G2-M phase arresting.Inhibition of Siva-1 expression in MKN-28/VCR cells significantly weakened wound healing ability and decreased invasion ability.Poly(C)-binding protein 1(PCBP1)was identified as a Siva-1-interacting protein in yeast two-hybrid screening.Semiquantitative RT-PCR and western blotting revealed that Siva-1 downregulation could inhibit expression of PCBP1,Akt,and NF-κB and eventually decrease the expression of MDR1 and MRP1.Conclusion:The current study demonstrated that the downregulation of Siva-1,which functions as a regulator of MDR1 and MRP1 gene expression in gastric cancer cells by inhibiting PCBP1/Akt/NF-κB signaling pathway expression,enhanced the sensitivity of gastric cancer cells to certain chemotherapies.