MDR1 Gene Expression Research Articles

Reversal Effect of A Novel N-sugar Substituted Thalidomide Analogue on Multidrug Resistant Human Nasopharyngeal Carcinoma Cells*

One major problem to successful treatment of cancer is the development of resistance by tumor cells to multiple chemotherapeutic drugs, a phenomenon named multidrug resistance (MDR). Searching for the novel chemotherapeutical agents is one of the important strategies for overcoming MDR. By using a cytotoxicity assay, flow cytometry analysis, Western-blotting and RT-PCR, a drug (Taxol, TAX) resistant human nasopharyngeal carcinoma KB cell line (KB/TAX) was established by addition of the drug to the cell cultures gradually, then a novel N-sugar substituted thalidomide analogue (STA-35) was investigated for its reversal effect on MDR of KB/TAX cells and possible mechanism. The results showed that KB/TAX cells were resistant to several chemotherapeutical agents, and the relative resistance to TAX was 73.1. Compared with parental KB cells, the function and protein expression of P-glycoprotein (P-gp), as well as mdr1 gene in the KB/TAX cells were remarkable reduced. Moreover, both KB and KB/TAX cells were sensitive to STA-35, the relative resistance to TAX on KB/TAX cells was decreased by the addition of STA-35. Furthermore, STA-35 (5 ～20 μmol/L)was capable to reduced the activity of P-gp by increasing the accumulation of rhodamine 123, decreasing P-gp expression in KB/TAX cells in a dose dependent manner , but had no effect on the mdr1 gene expression. These results suggest a potential action of STA-35 as MDR reversing agent, and one of the possible mechanisms could be the suppression of P-gp function and protein expression.

Antitumor effect of BPR-DC-2, a novel synthetic cyclic cyanoguanidine derivative, involving the inhibition of MDR-1 expression and down-regulation of p-AKT and PARP-1 in lung cancer

In our previous study, a series of novel cyclic cyanoguanidine compounds, eg. 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4- pyrimidinone derivatives have been successfully synthesized and showed remarkable cytotoxicity in several cancer cell lines. In this present study, it is our aim to screen more potential candidates among the cyclic pyridyl cyanoguanidine compounds (BPR-DC-1, 2, 3) by in vitro and in vivo studies for the therapy of lung cancer, alternatively. Our results showed that BPR-DC-2 significantly inhibited proliferation of tumor cells with an IC50 of 3.60 ± 1.27 and 14.81 ± 4.23 μM in human lung carcinoma cells, H69 and A549, respectively by the MTT assay at 48 hr; BPR-DC-2 also obviously suppressed the tumor proliferation and MDR-1 gene expression, even induced cell apoptosis in the ex vivo histocultured lung tumor. We further demonstrated that, in the nude mouse model of metastatic lung cancer, BPR-DC-2 could diminish the tumor mass, retard the progression of metastasis, and prolong the survival time. In addition, it was found that BPR-DC-2 exerted its anti-tumor effects through the inhibition of MDR-1 gene expression and down-regulation of tumor anti-apoptosis signals (activated p-AKT and over-expression of PARP-1) by western blotting analysis. In conclusion, in this present study we have demonstrated that BPR-DC-2, derived from a series of novel synthetic cyclic cyanoguanidine compounds, has proved its potential as an anti-tumor drug candidate in treating lung cancer.

Development of radioresistance in drug resistant human MCF-7 breast cancer cells

AbstractBackground and purpose: Radiotherapy is used for the treatment of malignant tumours, and may be used as the primary therapy. It is also common to combine radiotherapy with surgery, chemotherapy, hormone therapy or some combination of them. Even if the tumour is treated intensively, women diagnosed with breast cancer may develop a recurrence. Most recurrences may be in the form of distant metastases, development of multi-drug resistance phenotype or both together. This study demonstrated that some of the multi-drug resistant cancer cells may also become radioresistant.Materials and Methods: Chemoresistance in paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), vincristine (MCF-7/Vinc), doxorubicin (MCF-7/Dox) and zoledronic acid (MCF-7/Zol) resistant MCF-7 cells were demonstrated by XTT assay. MDR1 gene expression was detected by real-time PCR in human MCF-7 breast cancer cells. Drug resistant and sensitive cells were exposed to γ-radiation and development of radioresistance was investigated.Results: Results have indicated that paclitaxel, docetaxel, vincristine, doxorubicin and zoledronic acid–selected cells gained varying degrees of resistance to their selective drugs when compared with original MCF-7/S. MCF-7/Pac, MCF-7/Doc, MCF-7/Vinc and MCF-7/Dox cells have all acquired MDR1 expression. Among the resistant sub-lines, MCF-7/Pac and MCF-7/Doc cells were significantly cross-resistant to irradiation compared to the sensitive cells.Conclusion: MCF-7/Pac and MCF-7/Doc cell lines were found radioresistant to γ-radiation. On the contrary, doxorubicin, vincristine and zoledronic acid resistant cancer cells were still sensitive to radiation.

Small interfering RNA targeting MDR1 inhibits ovarian cancer growth and increases efficacy of chemotherapy in vivo

To further validate a knockdown approach for circumventing the multidrug resistance gene (MDR1), we used small interfering RNA(siRNA) targeting MDR1 gene to inhibit the expression of MDR1 gene and P-glycoprotein(P-gp) in vivo. Ascite tumor xenografts were established by implanting human ovarian carcinoma cells SKOV3/AR intraperitoneally into the nude mice. The mice were randomized into the following three treatment groups with each group six mice respectively: Taxol, Taxol with lipofectamine and Taxol with siRNA/MDR1-lipofectamine intraperitoneal injection. The tumor growth rate and the ascite growth rate of mice were investigated. The expressions of MDR1 gene and P-gp in mice were determined by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry respctively. The growth of tumors and ascites in mice treated with Taxol and siRNA/MDR1-lipofectamine was significantly inhibited compared with those in mice of other groups. After 28 days’ treatment, the average tumor weight and ascite volume decreased by 43.6% and 29.7% in the group treated with Taxol and siRNA/MDR1-lipofectamine compared with these treated with Taxol alone (P<0.001). The expressions of MDR1 gene and P-gp in the group treated with Taxol and siRNA/MDR1-lipofectamine were also decreased compared with those in the group treated with Taxol alone (P<0.001). Small interfering RNA targeting-MDR1 can effectively and specifically suppress the expression of MDR1(P-glycoprotein) and inhibit ovarian cancer growth in vivo.

Expression of FLT3 Internal Tandem Duplication in Pediatric Patients with Acute Myeloid Leukemia and Its Correlation with Multidrug Resistance.

Abstract 4427FLT3 Internal Tandem Duplication is a kinase mutation frequently observed in cases of adult acute myeloid leukemia (AML). This study was aimed to investigate the expression of FLT3 internal tandem duplication (FLT3-ITD) in pediatric patients with acute myeloid leukemia (AML) and to analyse the clinical features of patients with mutations and the relation of FLT3-ITD with multidrug resistance gene 1 (mdr1). Samples were analysised from eighty-one new diagnosed pediatric patients with AML. The diagnosis was based on French-American-British (FAB) criteria. Immunophenotyping was done by flow cytometry. For each patient, several clinical and biological characteristics were analyzed (age, WHO performance status, WBC count at diagnosis, bone marrow blasts, FAB subtypes, immunologic phenotype, and cytogenetics). The expressions of FIT3-ITD and mdr1 gene in bone marrow samples was determined by RT-PCR. patients were treated with uniform chemotherapy. Statistical analysis were carried by SAS. The results indicated that the FLT3-ITDs were detected in 8 out of 81 pediatric patients (9.88%) and all mutations detected were hybrid, while less frequently this mutation was detected in adult patients. Although they were irrelevant with sex and immunophenotypes, the mutations seemed predominant in older pediatric patients. The average age is 14-year-old in the patients with mutations, but 9-year-old in those without mutation (P=0.039). The leukocyte counts and bone marrow blast cell counts in pediatric patients with FLT3-ITD at diagnosis were higher than those in pediatric patients without FLT3-ITD (median of 58.4×10∧9/L versus 19.49 ×10∧9/L, p = 0.001 and median of 78.5% versus 54.4%,p = 0.041 respectively). The normal chromosomes were found in most pediatric patients with FLT3-ITD, although no significane in the stastistics. The patients with FLT3-ITD had lower induction remission rate (only 25%), but the patients without FLT3-ITD had higher remission rate (76.1%). According results detected by RT-PCR, the mdr1 gene was found in 27 pediatric patients, but only 3 out of 8 pediatric patients with FLT3-ITD were detected to express both FLT3-ITD and mdr1. It is concluded that the FLT3-ITD is a frequent mutation in pediatric patients with AML, especially in the elder pediatric patients. It is related with higher leukocyte counts and bone marrow blast cell counts, which may due to the continual activation of Flt3 tyrosine kinase. The prognosis of these patients with mutations is worse and the induction remission rate is lowe. But the FLT3-ITD not relates with the mdr1, which suggests that the low induction rate is not resulted from the multidrug resistance, at least not from the P-glycoprotein (Pgp) activity. So the common MDR modulators may be uneffective for therapy of the patients with FLT3-ITD. Disclosures:No relevant conflicts of interest to declare.

Reversal of Multidrug Resistance by Magnetic Fe3O4 Nanoparticle Copolymerizating Daunorubicin and mdr1-shRNA Expression Vetor in Leukemia Cells.

Abstract 4829 ObjectIn many instances, Multidrug resistance (MDR) is mediated by increased expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe3O4 (MNP(Fe3O4)) and mdr1-shRNA Expression vetor.in K562/A02 leukemic cells. MethodsTo synthesis short hairpin RNA (shRNA)aiming divectly at the target sequence,we choice the 3491-3509,1539-1557and 3103-3121 nucleotide of mdr-1 mRNA as targets. Cloning in the plasmid vetor PGCSilencer-U6-neo-GFP, The recombinant plasmid vetors were called for PGY1-1,PGY1-2 and PGY1-3.The recombinant plasmid vetors were transfected into the cell 1ines K562/A02 by lipofection. After transfected 48 hours,the inhibition of mdr-1mRNA expression and the expression of P-gp was detected by realtime–PCR and Weston-blot, screening the recombinant plasmid vetor which has the most greatest mdr-1 gene inhibition ratio is PGY1-2.Analysis of the reveral ratio of multidrug resistance, the concentration of DNR and the content of mdr-1 gene and P-gp in K562/A02 cell line. ResultsThe combination of daunorubicin (DNR) with either MNP(Fe3O4) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while MNP(Fe3O4) and PGY1-2 cotreatment can synergistically down regulation the expression of mdr-1 gene and the expression of P-gp(P<0.05). The transfection efficiency was 20%; the concentration of DNR in K562/A02 cell line was obviously elevated (P<0.05);the multidrug resistance index of K562/A02 cell line was obviously decreased (P<0.05). ConclusionMNP(Fe3O4) and PGY1-2 cotreatment can synergistically reveral multidrug resistance. Thus our in vitro data strongly suggests a potential clinical application of MNP(Fe3O4) and PGY1-2 combination on CML. DisclosuresNo relevant conflicts of interest to declare.

A novel podophyllotoxin derivative (YB-1EPN) induces apoptosis and down-regulates express of P-glycoprotein in multidrug resistance cell line KBV200

Our group synthesized a new potent anti-tumor podophyllotoxin derivative, YB-1EPN. In our study, we found that it was more potent than etoposide (VP-16). Interestingly, we found that the KBV200 cell line and K562/A02 cell line were rendered resistant towards VP-16 but not towards YB-1EPN. In vitro, both the cytotoxicity of YB-1EPN and its ability to inhibit KBV200 and K562/A02 cells were determined by MTT assay and growth curve. The IC(50) value of YB-1EPN on KBV200 cell was (2.52+/-0.28)microM in contrast to VP-16 (10.1+/-0.220)microM. And YB-1EPN showed a dose-dependent and broad-spectrum antiproliferative activity. Inducing apoptosis by YB-1EPN in KBV200 was assessed by various morphological and biochemical characteristics, including cell shrinkage, chromatin condensation, membrane blebbing, formation of apoptotic bodies, and DNA ladder formation. Rates of apoptosis and cell cycle were also checked through flow cytometry. Reverse transcription-polymerase chain reaction(RT-PCR) was used to detect mdr-1,p53,bcl-2,and bax gene expression. Western-blot assay was used to assess P-glycoprotein (P-gp) expression. We found that YB-1EPN could down-regulate expression level of the mdr-1, bcl-2, and up-regulate expression level of p53, and bax mRNA, as compared to the control. These results suggest that YB-1EPN has the potentiality to overcome P-glycoprotein-mediated multidrug resistance in the KBV200 cell line.

Expression and distribution of CYP3A genes, CYP2B22, and MDR1, MRP1, MRP2, LRP efflux transporters in brain of control and rifampicin-treated pigs

The in vivo effect of rifampicin, a potent ligand of PXR, on gene expression of CYP2B22, 3A22, 3A29, 3A46, CAR, PXR and MDR1, MRP1, MRP2, LRP transporters in liver and cortex, cerebellum, midbrain, hippocampus, meninges and brain capillaries of pig was investigated. Animals were treated i.p. with four daily doses of rifampicin (40 mg/kg). The basal mRNA expressions of the individual CYP3As, CYP2B22, CAR, and PXR in various brain regions, except meninges, were about or below 10% of the corresponding hepatic mRNA values, whereas the mRNAs of brain transporters were closer or comparable to those in liver. After pig treatment with rifampicin, the mRNA expression of CYPs and transporters from brain regions did not appear to change, except CYP3A22 and 3A29 in cortex and hippocampus, CYP2B22 in meninges. An enzymatic analysis for CYP3As and CYP2B, in microsomes and mitochondria from liver and brain tissues using the marker activities 7-benzyloxyquinoline O-debenzylase and the anthraldehyde oxidase, showed the lack of rifampicin induction in all the brain regions, unlike liver. Taken together, our results demonstrate that CYP2B22, CYP3As, and MDR1, MRP1, MRP2, and LRP transporters are all expressed, although at different extent, in the brain regions but, despite the presence of PXR and CAR, are resistant to induction indicating that the regulation of these proteins is more complex in brain than in liver. These data obtained in vivo in the brain regions and liver of pig may be of interest to human metabolism in CNS.

Enhanced MDR1 Expression and Chemoresistance of Cancer Stem Cells Derived from Glioblastoma

ABSTRACTWe established a cancer stem (CS) cell line, U87CS, by means of spheroid culture of U87MG cells derived from glioblastoma (GBM) in neuronal stem cell medium. U87CS cells presented positive immunohistochemical staining for multidrug resistance (MDR)1 and CD133, a marker for a subset of leukemia and GBM CS cells. The gene expression of MDR1 and CD133 on U87CS cells increased by an average of 8.51 and 47.18 times, respectively, compared to the levels on U87MG cells by real-time quantitative RT-PCR. U87CS cells possessed stronger drug-resistance to conventional anti-cancer drugs, such as doxorubicin (Dox), etoposide (VP-16), carboplastin, and BCNU than U87MG cells. Double immunofluoresence staining showed co-expression of MDR1 and CD133 on U87CS cells transplanted into nude mice brains. In addition, we identified the crossreactivity of CD133 and MDR1 in a surgical specimen of GBM. Our results suggest that CS cells may be resistant to current chemotherapy and represent a novel target for GBM therapeutics.

Synergism of hydroxyapatite nanoparticles and recombinant mutant human tumour necrosis factor-α in chemotherapy of multidrug-resistant hepatocellular carcinoma

Locoregional chemotherapy continues to be the mainstay for the treatment of unresectable hepatocellular carcinoma (HCC). One of the principal obstacles implicated in its unsuccessful therapy is multidrug resistance (MDR). Former studies have identified the multidrug-resistant nature and possible mechanisms of hepatoma cells both in vitro and in vivo. This work aimed to develop an effective strategy for the treatment of HCC with MDR. The treatment was exploited to inhibit the MDR cells by co-administration of the recombinant mutant human tumour necrosis factor-alpha (rmhTNF-alpha), a sublethal dose of chemicals [adriamycin (ADM), mitomycin and 5-FU] and hydroxyapatite nanoparticles (nHAPs). Real-time quantitative reverse transcriptase-polymerase chain reaction and electrochemiluminescence Western blot were used to detect the expression of several related genes. The chemicals acted synergistically with rmhTNF-alpha and nHAP in suppressing the growth of hepatoma cells and inducing apoptosis of the cells, with the MDR phenotype reversed, as measured by intracellular ADM retention. Analysis of mRNA and protein revealed that rmhTNF-alpha inhibited the gene expression of XIAP, survivin, Ki67, PCNA, MDR1 and BCRP to some extent. Moreover, the inhibitory effects mentioned above could be as good or better than when nHAP is incorporated into the regimens. rmhTNF-alpha was not only able to restore the chemotherapeutic sensitivity to HepG2/ADM, its xenograft model and clinical samples but also further inhibited the growth of these tumours by a combination of nHAP. These results strongly suggested that chemicals in combination with rmhTNF-alpha and nHAP may be beneficial for the local treatment of advanced HCC.

Upregulation of antioxidants and chemoresistance genes in human granulosa cells following Pregnane X receptor activation: a potential role for fertility preservation

OBJECTIVE: The nuclear Pregnane X receptor (PXR; NR1I2) is an important component of the body's adaptive defense mechanism against toxic substances including foreign chemicals (xenobiotics). PXR stimulate CYP3A4 and MDR1 gene expression in the mouse liver. Here we investigate the expression of chemoresistance (PXR and MDR1) and antioxidants (CYP3A4 and NOQ1) genes in mural and granulosa cells from patients undergoing IVF as well as in human ovarian tissues.

High Gene Expression of MDR1 (ABCB1) is Associated with Medical Treatment Response and Long-Term Remission in Patients with Ulcerative Colitis

High Gene Expression of MDR1 (ABCB1) is Associated with Medical Treatment Response and Long-Term Remission in Patients with Ulcerative Colitis

Effects of Compound Zhebei Granule plus doxorubicin on mdr1 gene expression in nude mice with K562/A02 tumor xenografts

To investigate the effects of Compound Zhebei Granule (CZBG), a compound traditional Chinese herbal medicine, combined with adriamycin (ADM) on mdr 1 gene expression in tumor xenografts in nude mice. A tumor xenografts model was established by subcutaneously injecting multidrug resistance cell line K562/A02 into the axillary flank of BALB/c-nu mice. Sixty tumor-bearing nude mice were divided into untreated group, ADM group and high-, medium-, and low-dose CZBG plus ADM groups. The nude mice in the CZBG plus ADM groups were intragastrically administered with CZBG once per day and intraperitoneally injected with ADM once every other day. The nude mice in the untreated group and the ADM group were administered with normal saline or ADM respectively. The course of treatment was continuous for 14 days. The weight of tumor xenografts was measured after treatment and the inhibition rate of tumor xenografts was calculated. The mdr 1 gene expression in tumor xenografts was tested by quantitative polymerase chain reaction method. Compared with the untreated group, CZBG (high-, medium- and low-dose) plus ADM could significantly decrease the weight of tumor xenografts in nude mice (P<0.05). Compared with ADM, high- and medium-dose CZBG plus ADM could decrease the weight of tumor xenografts in nude mice with statistical significance (P<0.05). The high- and medium-dose CZBG plus ADM could also significantly decrease the mdr 1 gene expression in tumor xenografts as compared with ADM alone (P<0.05). CZBG at high- and medium-dose plus ADM can inhibit the growth of tumor xenografts and decrease the mdr 1 gene expression in tumor xenografts in nude mice.

Moderate Increase in Mdr1a/1b Expression Causes In vivo Resistance to Doxorubicin in a Mouse Model for Hereditary Breast Cancer

We have found previously that acquired doxorubicin resistance in a genetically engineered mouse model for BRCA1-related breast cancer was associated with increased expression of the mouse multidrug resistance (Mdr1) genes, which encode the drug efflux transporter ATP-binding cassette B1/P-glycoprotein (P-gp). Here, we show that even moderate increases of Mdr1 expression (as low as 5-fold) are sufficient to cause doxorubicin resistance. These moderately elevated tumor P-gp levels are below those found in some normal tissues, such as the gut. The resistant phenotype could be completely reversed by the third-generation P-gp inhibitor tariquidar, which provides a useful strategy to circumvent this type of acquired doxorubicin resistance. The presence of MDR1A in drug-resistant tumors with a moderate increase in Mdr1a transcripts could be shown with a newly generated chicken antibody against a mouse P-gp peptide. Our data show the usefulness of realistic preclinical models to characterize levels of Mdr1 gene expression that are sufficient to cause resistance.

Synergic effect of Na+-K+ ATPaseB1 and adriamycin on inhibition of cell proliferation and reversal of drug resistance in breast cancer MCF-7 cells

Na(+)-K(+)ATPase (Na(+)-K(+) pump) is an important cell energy conversion system which is probably associated with tumor metastasis. Expression of its B1 subunit gene-ATP1B1 is high in well differentiated and low in poorly differentiated tumor cells. This study was to investigate the synergic effect of Na(+)-K(+) ATPaseB1 and adriamycin (ADM) on inhibition of cell proliferation and reversal of drug resistance in MCF-7 and MCF-7/ADM cells. Growth of MCF-7 and MCF-7/ADM cells transfected with PEGFP-ATP1B1 and shATP1B1 were measured by MTT. Intracellular fluorescence intensity of ADM was analyzed by inverted fluorescence microscopy and flow cytometry. ATP enzyme activity was measured by ultramicro-ATP enzyme, and mRNA expression of multi-drug resistance gene MDR1 was detected by RT-PCR and real-time PCR. The expression of P-glycoprotein (P-gp) was analyzed by western blot. The sensitivity of MCF-7 and MCF-7/ADM cells transfected with pEGFP-ATP1B1 to ADM was higher in comparing to the negative control ADM-C3 (transfected with vector pEGFP-C3) and the control ADM-RPMI-1640 (cultured with RPMI-1640), and the differences between ADM-ATP1B1 and ADM-RPMI-1640 groups were statistically significant at different concentrations of adriamycin (P<0.05). After the B1 subunit was silenced, the sensitivity of cells to ADM in the ADM-shNC group was higher than that in the shATP and ADM-RPMI-1640 groups. The mean fluorescence intensity of ADM in the ADM-ATP1B1 group was higher than that in the ADM-C3 and ADM-RPMI-1640 groups (P<0.05). ATP enzyme activity was significantly higher in ADM-ATP1B1 group comparing to the ADM-RPMI-1640 group (P<0.05). mRNA expression of MDR1 gene and protein expression of P-gp were not significantly different among the ADM-ATP1B1 group and two control groups (P>0.05). Na(+)-K(+) ATPase B1 can synergize with ADM and reverse drug resistance to ADM in the MCF-7/ADM cell line. This may be related to ATP enzyme activity, but not to influencing the expression of MDR1 gene.

Chemosensitivity of radioresistant cells in the multicellular spheroids of A549 lung adenocarcinoma

BackgroundThe relapse of cancer after radiotherapy is a clinical knotty problem. Previous studies have demonstrated that the elevation of several factors is likely in some way to lead to the development of treatment tolerance, so it is necessary to further explore the problem of re-proliferated radioresistant cells to chemotherapeutic agents. In the present study, we aimed to investigate the chemosensitivity of radioresistant cells originated from the multicellular spheroids of A549 lung adenocarcinoma.MethodsAfter irradiated with 25 Gy of 6 MV X-ray to A549 multicellular spheroids, whose 10th re-proliferated generations were employed as radioresistant cells, and the control groups were A549 parental cells and MCF7/VCR resistant cells. The chemo-sensitivity test was made by six kinds of chemotherapeutic drugs which were DDP, VDS, 5-Fu, HCP, MMC and ADM respectively, while verapamil (VPL) was used as the reversal agent. Then the treatment effect was evaluated by MTT assay, and the multidrug resistant gene expressions of mdr1 and MRP were measured by RT-PCR.ResultsBoth A549 parental cells and A549 derived radioresistant cells were resistant to DDP, but sensitive to VDS, 5-Fu, HCP, MMC and ADM. The inhibitory rates of VPL to these two types of cell were 98% and 25% respectively (P < 0.001). In addition, without drugs added, the absorbance value (A value) of A549 parental cells was 2-folds higher than that of their radioresistant cells (P < 0.001). As to the MCF7/VCR cells, they were resistant to DDP and VDS, but slight sensitive to MMC, ADM, 5-Fu, and HCP with 80% of inhibitory rate to VPL. The subsequent RT-PCR demonstrated that the Mdr1/β2-MG and MRP/β2-MG of all A549 cells were about 0 and 0.7 respectively, and those of MCF7/VCR cells were 35 and 4.36.ConclusionThe chemosensitivity of A549 radioresistant cells had not changed markedly, and the decreased sensitivity to VPL could not be explained by the gene expression of mdr1 and MRP. It is possible that the changes in the cell membrane and decreased proliferate ability might be attributed to the resistance. Unlike multidrug resistance induced by chemotherapy, VPL may be not an ideal reverser to radioresistant cells. Therefore, the new biological strategy needs to be developed to treat recurring radioresistant tumor in combination with chemotherapy.

Correlation of multidrug-resistance gene (MDR1) expression levels and outcome to adjuvant cisplatin/MTX based chemotherapy in patients enrolled in the AUO-AB 05/95 phase III trial

5026 Background: The human MDR1 gene encodes an integral membrane protein, P glycoprotein (Pgp), whose function is the energy dependent export of substances from the inside of cells, and from membranes, to the outside. MDR1 mRNA levels have been shown to influence metabolism of cancer drugs. We tested whether MDR1 gene expression is associated with outcome in patients with locally advanced bladder cancer. Methods: 221 formalin fixed paraffin embedded (FFPE) tumor samples from patients with locally advanced and/or lymph node-positive bladder cancer enrolled for adjuvant therapy in the AUO-AB 05/95 phase III trial (Lehmann et al, J Clin Oncol. 2005;23:4963–4974) were analyzed (110 pts treated with cisplatin/MTX, 101 pts treated with MTX, vinblastine, epirubicin, and cisplatin, M-VEC).FFPE tissues were dissected using laser-captured microdissection and analyzed for MDR1 and ERCC1 mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference gene (beta-actin). Results: MDR1 was significantly correlated to progression free and overall survival. We included tumor stage (pT), lymph node status (pN) and the blood vessel invasion along with the measured gene expression in a stepwise multivariate Cox proportional hazards regression model. The overall model fit had a significance level of p = 0.0001 (&lt;0.05). The relative risk for shorter survival in patients with High MDR1 expression was 2.8. Regarding survival MDR1 was strongly associated to ERCC1 expression in patients with M-VEC treatment. Conclusions: These results suggest that MDR1 gene expression levels predict response and overall survival in patients with locally advanced urothelial cancer enrolled in AUO-AB 05/95. MDR1 gene expression levels may allow the selection of patients who benefit likely from adjuvant cisplatin/MTX based chemotherapy. Further studies are warranted to study this association. [Table: see text]

Use of multidrug-resistance gene (MDR1) expression levels to predict outcome to adjuvant cisplatin/gemcitabine based chemotherapy in patients with with locally advanced urothelial cancer

e22081 Background: The human MDR1 gene encodes an integral membrane protein, P glycoprotein (Pgp), whose function is the energy dependent export of substances from the inside of cells, and from membranes, to the outside. MDR1 mRNA levels have been shown to influence metabolism of cancer drugs. We tested whether MDR1 gene expression is associated with outcome in patients with locally advanced bladder cancer. Methods: 43 formalin fixed paraffin embedded (FFPE) tumor samples from patients with locally advanced and/or lymph node-positive bladder cancer undergoing treatment with cisplatin/gemcitabine were analyzed.FFPE tissues were dissected using laser-captured microdissection and analyzed for MDR1 expression using a quantitative real- time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference gene (beta-actin). Results: MDR1 was significantly correlated to overall survival. We included tumor stage (pT), lymph node status (pN) and the blood vessel invasion along with the measured gene expression in a stepwise multivariate Cox proportional hazards regression model. The overall model fit had a significance level of p=0.04 (&lt;0.05). The relative risk for shorter survival in patients with High MDR1 expression was 1.9. Conclusions: These results suggest that MDR1 gene expression levels predict response and overall survival in patients with locally advanced urothelial cancer. MDR1 gene expression levels may allow the selection of patients who benefit likely from adjuvant cisplatin/gemcitabine based chemotherapy. Further studies are warranted to study this association. [Table: see text]

Gene therapy for pretreated ovarian cancer via a novel fiber modified mdr1 targeting conditionally replicating adenovirus

e16513 Background: Ovarian cancer (OC) is a leading cause of death from gynecologic malignancies. Oncolytic adenoviruses (CRAd) are a new approach for cancer treatment. In this study, we constructed a fiber-modified CRAd containing the multi drug resistance gene (mdr) 1 promoter to control viral replication (Ad5/3MDR1). Antineoplastic agents induce mdr1-gene expression triggering chemoresistance. Methods: RT-PCR was performed for expression of mdr1 in cell lines and primary patient samples. We constructed Ad5/3MDR1luc1 to determine relative activity in a variety of cell lines, patient samples, and normal control cells. Replication and oncolysis of Ad5/3MDR1 were assessed in naïve and pretreated OC cell lines. The specificity of viral replication was analyzed in co-culture of pretreated OC cell lines and human fibroid cells. A mouse model of peritoneal carcinomatosis was used to evaluate the efficacy of a combined chemo- and gene therapy for OC. Results: RT-PCR reveals a significantly (p &lt; 0.05) increased expression of the mdr1 gene in OC cell lines and patient samples. Compared to the ubiquitous cmv promoter, mdr1 showed a high level of activity in chemoresistant OC cell lines (7.3%-11.5 %) and OC patient samples (8.8%-12.4%), whereas activity in normal fibroblasts (&lt;1%) was low. Ad5/3MDR1 shows highly selective replication in chemoresistant OC cells in a co-culture with fibroblast cells. Cell killing of Ad5/3MDR1 was comparable to Ad5/3Δ24, a CRAD that replicates in cancer cells inactive in the Rb/p16 pathway, in chemotherapy naïve OC cells but displayed significantly (1.5 log; p &lt; 0.05) higher oncolytic potency in pretreated OC cell lines and primary cells from pretreated OC patients. Moreover, in a therapeutic orthotopic mouse model of peritoneal carcinomatosis, dramatically enhanced survival was noted with Ad5/3MDR1. Conclusions: Ad5/3MDR1 is a promising candidate for gene therapy of metastatic chemoresistant OC with great potential for clinical testing. No significant financial relationships to disclose.

The proliferative effect on silencing of endogenetic mdr1 gene expression by RNA interference in ovarian cancer resistant strain SKOV3/TAXOL

Objective To construct the short hairpin RNA recombinant plasmids targeting mdr1 gene which expresses highly in ovarian cancer resistance strain SKOV3/TAXOL to silence endogenefic mdr1 gene expression and investigate the role of mdr1 gene in the development of resistant ovarian cancer. Methods The pGPU6/GFP/Neo-mdr1 were constructed by gene clone technology. The influence on proliferation and apoptosis were investigated by CCK-8 in SKOV3/TAXOL after transfected pGPU6/GFP/Neo-mdr1. Results The expression against mdr1 proteins were inhibited by pGPU6/GFP/Neo-mdr1. The cell proliferation were inhibited after transfected pGPU6/GFP/Neo-mdr1 by CCK-8. The apoptosis were observed in DAB experiments and the apoptosis rate increased. Conclusion mdr1 plays an important role in proliferation of resistant ovarian cancer and the short hairpin RNA of mdr1 can efficiently suppress mdr1 expression and enhance the apoptosis in SKOV3/ATAXOL. Key words: Ovarian neoplasms; RNA interfering; Cell line, tumor; Genes, mdr1; Hyperplasia