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.

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.

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.

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.

Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance

AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of eryth-romycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (ΔH pylori LZ1026) for (puta-tive) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. RESULTS: The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased signifi cantly (≥ 4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P = 0.033). A H pylori LZ1026 mutant was successfully constructed and the ΔH pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that con-fi rmed the in vitro expression of these genes.CONCLUSION: The efflux pump gene hefA plays an important role in multidrug resistance of H pylori.

Tamoxifen can reverse multidrug resistance of colorectal carcinoma in vivo

AIM: To investigate the effect of tamoxifen (TAM) on multidrug resistance (MDR) of colorectal carcinoma in vivo and its relationship with estrogen receptor (ER). METHODS: Multidrug resistance was determined by means of semi-quantitative retro-transcription polymerase chain reaction (RT-PCR) to test mdr1 gene mRNA and ER expression was studied by immunohistochemistry. Tumor tissues from three cases of human colon carcinoma, which had mdr1(+)/ER(+),mdr1(+)/ER(-), mdr1(-) expressions, were planted subcutaneously in the neck of nude mice to establish three xenograft models. These models were subdivided into four subgroups randomly: Doxorubicin (DOX)-treated group, TAM-treated group, DOX and TAM group and control group. The dimensions of these xenografts were measured after each course of treatment and the xenografts were removed at the end of the experiments for measurements of weight and the variation of mdr1 mRNA level with RT-PCR. In each course, TAM [15 mg/(kg/d)] was administrated orally per day in the first seven days and DOX (3.6 mg/kg) was injected peritoneally on the first day. Data was evaluated by q and t tests. RESULTS: In the animal models with mdr1(-) tumor, the weights and volumes of the planted tumor in DOX group [(39.1±2.29) mg, (31.44±1.61) mm3] and TAM and DOX group [(38.72±2.56) mg, (31.31v1.74) mm3], which were lesser than that of control group [(45.48±3.92) mg, (36.42±2.77) mm3, P= 0.037, P= 0.016 respectively] significantly. In the animal models with mdr1(+)/ER(+) tumor, the weights and volumes of planted tumor were not affected by DOX or TAM treatment; however, in TAM and DOX group [(425.5±28.58) mg, (340.35±22.28) mm3], they were significantly less than that of control group [(634.23±119.41) mg, (507.45±93.34) mm3, P= 0.022, P = 0.045 respectively], which are similar to that in the models with mdr1(+)/ER(-) tumor. No significant changes were found in the expressive level of mdr1 mRNA following these treatments. CONCLUSION: The expression of mdr1 gene corresponds to the sensitivity of colon cancer to anti-tumor drugs in vivo. TAM can reverse the MDR of colorectal carcinoma in nude mice, which is independent of the expression of ER; however, no change was observed in the expressive level of mdr1 mRNA.

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.

Multidrug resistance protein 3 R652G may reduce susceptibility to idiopathic infant cholestasis

AIM:To evaluate the role of genetic factors in the pathogenesis of idiopathic infant cholestasis.METHODS:We performed a case-control study,in-cluding 78 infants with idiopathic infant cholestasis and 113 healthy infants as controls.Genomic DNA was extracted from peripheral venous blood leukocytes us-ing phenol chloroform methodology.Polymerase chain reaction was used to amplify the multidrug resistance protein 3(MDR3)R652G fragment,and products were sequenced using the ABI 3100 Sequencer.RESULTS:The R652G single nucleotide polymorphism(SNP)was significantly more frequent in healthy infants(allele frequency 8.0%)than in patients(allele frequency 2.60%)(P < 0.05),odds ratio,0.29;95% confidence interval,0.12-0.84.The conjugated bilirubin in patients with the AG genotype was significantly lower than in those with the AA genotype(44.70 ± 6.15 μmol/L vs 95.52 ± 5.93 μmol/L,P < 0.05).CONCLUSION:MDR3 R652G is negatively correlated with idiopathic infant cholestasis.Children with the R652G SNP in Guangxi of China may have reduced susceptibility to infant intrahepatic cholestasis.

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.