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 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.

Therapeutic efficacy and bone marrow protection of the mdr1 gene and over-dose chemotherapy with doxorubicin for rabbits with VX2 hepatocarcinoma

BACKGROUND : Malignant tumors are common diseases threatening to the health and life of human being. Clinically, the multidrug resistance of tumor cells and bone marrow depression caused by chemotherapeutic agents are the main obstacles to the treatment of tumors, and both are related to the mdr1 gene. The over expression of the mdr1 gene in tumor cells contributes to the multidrug resistance of malignant tumor cells. With little expression of the mdr1 gene, bone marrow cells particularly susceptible to multidrug resistance-sensitive agents, which cause serious toxicity in bone marrow. This study was undertaken to assess therapeutic efficacy of transplantation of bone marrow mononuclear cells transferred with the mdr1 gene and over-dose chemotherapy with doxorubicin for VX2 hepatocarcinoma of rabbits. METHODS: The mdr1 gene was transferred into the bone marrow mononuclear cells of rabbits, which was co- cultured with retroviral vector-containing supernatant, and the cells were autotransplanted into a rabbit model with VX2 hepatocarcinoma. After chemotherapy with doxorubicin, the protective effects of the mdr1 gene and therapeutic efficacy of over-dose chemotherapy were observed. RESULTS: The mdr1 gene was transferred successfully into the bone marrow mononuclear cells, with a transduction efficiency of 35%. After autotransplantation, the mdr1 gene was expressed functionally in bone marrow with a positive rate of 8%, indicating that the gene played animportant role in bone marrow protection. The rabbits with VX2 hepatocarcinoma, which had received the mdr1 gene-transduced cells, survived after chemotherapy with a 3-fold dose of adriamycin, and their white blood cell counts were (4.26±1.03)×104/L. Since hepatocarcinoma cells were eradicated, the survival time (97.00±46.75 d) of the rabbits was extended (P<0.05) and the healing rate of the tumor was increased (P<0.05). CONCLUSIONS: The transferring of the mdr1 gene into bone marrow mononuclear cells could confer chemoprotection to bone marrow, and over-dose chemotherapy could be prescribed for the treatment of malignant tumors.

Quantitative detection of peripheral MDR genes in breast cancer before chemotherapy and their clinical significances

Objective： To explore expressions of multidrug resistance gene （MDR） product P170 and peripheral blood MDR1 mRNA in breast cancer tissue and their clinical significances. Methods： Fluorescent quantitative RT-PCR and immunohistochemistry were used to detect peripheral blood MDR1 mRNA before chemotherapy in breast cancer tissue samples from 32 cases with P170 positive （trial group） and 11 cases with P170 negative （control group）. Results： There were 14 cases （43.75%） peripheral blood MDR1 mRNA positive and 18 cases （56.25%） negative in 32 cases P170 positive breast cancer patients, while there were 6 cases （54.55%） peripheral blood MDR1 mRNA positive and 5 cases （45.45%） negative in 11 cases P170 negative breast cancer patients; there wasn＇t a significance difference with peripheral blood MDR1 mRNA express rate in P170 positive （43.75%） group and P170 negative （54.55%） group （x2 = 0.383, P 〉 0.05）. There were 12 cases （37.50%） Her-2 positive and 20 cases （62.50%） negative in P170 positive group; there were 2 cases （18.18%） Her-2 positive and 9 cases （81.82%） negative in P170 negative group; there wasn＇t a significance difference with Her-2 positive rate in P170 positive group （37.50%） and P170 negative （18.18%） group （χ^2= 1.391, P 〉 0.05）. There were 13 cases （40.63%） ER positive and 19 cases （59.37%） negative in P170 positive group; there were 7 cases （63.64%） ER positive and 4 cases （36.36%） negative in P170 negative group; there wasn＇t a significance difference with ER positive rate in P170 positive group （40.63%） and P170 negative （63.64） group （χ^2 = 1.742, P 〉 0.05）. There were 20 cases （62.50%） PR positive and 12 cases （37.50%） negative in P170 positive group; there were 4 cases （36.36%） PR positive and 7 cases （63.64%） negative in P170 negative group; there wasn＇t a significance difference with PR positive rate in P170 positive group （62.50%） and P170 negative （36.36%） group （χ^2 = 2.267, P 〉 0.05）. There were 9 cases （28.12%） with lymphaden metastasis and 23 cases （71.88%） without lymphaden metastasis in P170 positive group; there were 5 cases （45.45%） with lymphaden metastasis and 6 cases （54.55%） without lymphaden metastasis in P170 negative group; there wasn＇t a significance difference with lymphaden metastasis rate in P170 positive group （28.12%） and P170 negative （45.45%） group （χ^2 = 1.120, P 〉 0.05）. Conclusion： 1） There were possibly MDR expressing infrequently in carcinoma tissue and peripheral blood before chemotherapy. 2） FQ-RT-PCR for detecting of peripheral blood MDR1 mRNA is special, sensitive and reliable. It can be used as new molecular biology diagnostic maker dynamic detecting peripheral blood MDR1 mRNA for regulating chemotherapy proposal and elevating chemotherapy effect.

IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1(MDR1) mRNA. Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5’ and 3’ untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdr1 mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry. Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

Expression of the human multidrug resistance gene mdr1 in leukemic cells and its application in studying P-glycoprotein antagonists

Objective To investigate the retrovirus mediated transfer and expression of multidrug resistance gene (mdr1) in hematopoietic cells and to develop a model for studying the possible reversal of the MDR mediated phenotype Methods A retroviral vector HaMDR expressing the human mdr1 gene was packaged by PA317 cells with a titer of up to 8 5×10 5CFU/ml K562 leukemia cells were infected with MDR retrovirus, and transfectant K562/MDR cells were generated The integration and expression of the exogenous mdr1 gene in K562/MDR cells were determined by polymerase chain reaction and flow cytometry The reversal ability of P glycoprotein (P gp) antagonists was analyzed by in vitro drug sensitivity, accumulation and efflux of rhodamine 123 (Rh123) in this model Results Transduction with amphotropic MDR retrovirus resulted in integration and expression of the mdr1 gene in the resistant cells, where an aberrant splicing transcript of the mdr1 gene was found The K562/MDR cells displayed a classic MDR phenotype with a 41-78 fold resistance to vincristine and colchicine in comparison with parental K562 cells The drug sensitivity of K562/MDR cells to vincristine can be completely restored by cyclosporin A (CsA, 2?mg/L) and Cremophor EL (CRE 132?mg/L), either individually or in combination ( P <0 05) CsA (3 ?mg/L) can block the efflux pump function of P gp shown by the significantly increased accumulation and efflux reduction of Rh123 in K562/MDR cells Conclusions Retroviral vector HaMDR allows transfection with high level expression of the mdr1 gene in human myeloid progenitor cells K562 The transfected K562/MDR provides a simple, sensitive model for developing antagonists of P gp and studying their mechanism of action

Chemoprotection of transfer of multidrug resistance gene into human hematopoietic progenitor cell

Objective To observe the effect of the transfer of multidrug resistance gene (mdr1) into human hematopoietic progenitor cells (HPC) on the chemoprotection Methods Human CD34 + cells served as a target of mdr1 gene transfer Retroviral vector SF mdr containing human total length mdr1cDNA was introduced into packing cells GP envAM12 by liposome mediated transfection The mdr1 gene was transduced into human CD34 + cells by retroviral supernatants of packing cells The integration and expression of the mdr1 gene and its protein (P170) in transduced cells were determined by PCR, RT PCR, and flow cytometry The drug resistance of chemotherapy in transduced HPC was determined by culturing colonies Results The mdr1 gene was integrated and expressed in transduced CD34 + cells The efficiency of mdr1 gene transfer was 10%-14% Compared with untransduced controls, within a certain range of drug concentration, the number of drug resistant colony in transduced HPC for taxol, doxorubicin,VCR and VP16 were increased by 3 6±2 1 fold, 2 9±0 3 fold, 1 9±0 4 fold, and 3 5±0 5 fold, respectively Conclusion The transfer of the mdr1 gene into human HPC can increase the drug resistance of the transduced cells to corresponding chemotherapeutic drugs that may provide some degree of chemoprotection for HPC

Effects of Jianpi Jiedu Recipe (健脾解毒方)on Reversion of P-Glycoprotein-Mediated Multidrug Resistance through COX-2 Pathway in Colorectal Cancer

Objective： To evaluate the underlying mechanism of Jianpi Jiedu Recipe （健脾解毒方, JJR） in the reversion of multidrug resistance concerning colorectal cancer in vitro and in vivo. Methods： Mice were treated orally with JJR at a daily 4.25 g/（kg.day） or injected with vinblastine （VCR） 2.5 mg/（kg day） for 3 weeks after having been inoculated with HCT8N cells; tumor tissues were assayed by hematoxylin and eosin staining. Firstly, the effects of JJR on the expression of cyclooxygenase-2 （COX-2） were tested by real-time polymerase chain reaction （PCR） technique and COX-2 gene silenced by siRNA. Secondly, the variation of intracellular concentration of oxaliplatin （L-OHP） was evaluated by the inductively coupled plasma mass spectroscopy （ICP- MS） in HCT8N and its COX-2 siRNA cells; the concentration of J JR combined with chemotherapeutic drugs and the reverse effect of multidrug resistance （MDR） in HCT8N cells was evaluated by the MTT assay. Thirdly, real-time quantitative PCR and Western blot analysis were used to detect the multidrug resistance gene 1 （MDR1） mRNA and P-gp expression. Results： JJR had an inhibitory effect on the growth of tumors in vivo, and it, in combination with chemotherapeutic drugs, could reverse the drug-resistance of HCT8N cells and increase the sensitivity of HCT8N cells to VCR, DDP, 5-Fu, and THP. ICP-MS results showed that JJR could increase the concentration of drugs in HCT8/V cells （P〈0.01）. Furthermore, it was shown that JJR could reverse drug resistance of colorectal cancer cells by decreasing MDR1 expression and P-gp level via downregulation of COX-2, which has been represented as one of the major mechanisms that contributes to the MDR phenotype （P〈0.01）. Conclusion： JJR reversed multidrug resistance and enhanced the sensitivity to chemotherapy, which could be attributed to the down-regulation of COX-2 in MDRl/P-gp-mediated MDR colorectal cancer after chemotherapy.

Ursolic acid sensitized colon cancer cells to chemotherapy under hypoxia by inhibiting MDR1 through HIF-1α

Objective： To explore the efficacy of ursolic acid in sensitizing colon cancer cells to chemotherapy under hypoxia and its underlying mechanisms. Methods： Three colon cancer cell lines （RKO, LoVo, and SW480） were used as in vitro models. 5-Fluorouracil （5-FU） and oxaliplatin were used as chemotherapeutic drugs. Cell viability and apoptosis were tested to evaluate the sensitivity of colon cancer cells to chemotherapy. The transcription and ex- pression levels of hypoxia-inducible factor-1α （HIF-1α）, multidrug resistance gene 1 （MDR1）, and vascular endothelial growth factors （VEGF） were assessed by quantitative real-time polymerase chain reaction （qRT-PCR） and im- munoblotting. Cycloheximide and MG132 were used to inhibit protein synthesis and degradation, respectively. In vitro tube formation assay was used to evaluate angiogenesis. Results： We demonstrated the chemosensitizing effects of ursolic acid with 5-FU and oxaliplatin in three colon cancer cell lines under hypoxia. This effect was correlated to its inhibition of MDR1 through HIF-la. Moreover, ursolic acid was capable of inhibiting HIF-1α accumulation with little effects on its constitutional expression in normoxia. In addition, ursolic acid also down-regulated VEGF and inhibited tumor angiogenesis. Conclusions： Ursolic acid exerted chemosensitizing effects in colon cancer cells under hypoxia by inhibiting HIF-la accumulation and the subsequent expression of the MDR1 and VEGF.