Effect Of Altered Expression Of RLIP6on Biological Behavior And Temozolomide Sensitivity Of Glioma

Malignant astrocytomas (glioblastoma multiforme and anaplastic astrocytoma) remainthe most frequent primary neoplasms of the central nervous system in adults, with an annualincidence of approximately0.005%[1]. Although relatively infrequent, malignant gliomasare associated with significant morbidity and mortality [2,3]. Even with aggressiveintervention, including surgery, chemotherapy, and radiotherapy, the median survival periodis only14-15months after diagnosis in the last5years [4]. However, a considerate part ofglioma is to chemotherapeutic drug resistance.,the overall efficacy of chemotherapy is stillnot good enough. Now it is resported that some genes (such as MGMT, Survivn and P53)with abnormal expression may influence glioma chemotherapy resistance. But a single genecan not accurately predict the sensitivity to chemotherapeutic drugs. The response is oftendetermined by multiple genes. Therefore, to find new resistance genes and clarify themolecular mechanism of drug resistance is one focus in neurosurgery.Ral binding protein1(RLIP76) is a multi-functional protein that transportsglutathione-electrophile conjugates (GS-Es) as well as chemotherapy drugs across theplasmalemma [5-12]. It is also required for such diverse cellular functions as mitosis [13,14],apoptosis [12,15,16], and endocytosis [17], and is overexpressed in a variety of malignancies[18-20]. Previous studies have shown the importance of the transport function of RLIP76,especially for chemotherapeutic agents and anti-epileptic drugs [21] and electrophilicconjugates [5]. Its substrates range from weakly cationic compounds including: doxorubicin(DOX), vinblastine (VBL), vincristine (VCR), vinorelbine (VRL)[9,11,22], colchicine,sunitinib and sorafenib [7,23], to anionic metabolites such as glutathione conjugates ofelectrophiles [5]. It is also required for diverse cellular signaling pathways including mitosis,proliferation, differentiation, apoptosis and endocytosis [12,17,24]. Its wide substratespecificity and ubiquitous presence in different tissues, especially its overexpression incancer tissues, make RLIP76an important regulator of chemotherapy drugs [25].Thedramatic increases in cancer cell radio-and chemosensitivity induced by RNAi-mediatedRLIP76knockdown and RLIP76antibody inhibition are due mainly to suppression ofRLIP76ATPase-dependent transporter activity, allowing accumulation of endogenouslyformed GS-Es and drugs that induce apoptosis in cancer cells [26]. In addition to membranetransport, RLIP76is a Rho-selective GTPase activating protein (RhoGAP) and negativeregulator of Cdc42and Rac1[18,27,28]. However, a previous study found that suppression of Rac1activity induced apoptosis and decreased proliferation of human glioma cells bystimulating the JNK signaling pathway [29], a result seemingly at odds with the inhibitoryeffects of RLIP76on Rac1activity and cancer cell apoptosis. While it is clear that RLIP76interacts with the Rho pathway, the regulation of Rac1by RLIP76may be more complexthan that of a simple negative regulator of activated Rac1-GTP.Here, we tested the hypothesis that RLIP76plays a requisite role in gliomatumorigenesis. We demonstrate that human malignant gliomas abundantly overexpressRLIP76and that RLIP76overexpression is associated with higher tumor grade and shortersurvival. Knockdown of RLIP76expression reduces the survival of glioma cancer cells invitro and inhibits tumorigenesis in mice. Furthermore, we demonstrate that RLIP76promotes proliferation and suppresses glioma cell apoptosis through a RhoGAP-independentmechanism. Instead, RLIP76ATPase activity controls Rac1activity by regulating Rac1ubiquitylation and degradation. These results strongly suggest that the ATPase-dependentactivity of RLIP76contributes to glioma cell survival and morbidity through both GS-Etransport and Rac1suppression. Our findings also provide important novel insights intoglioma Temozolomide (TMZ) resistance and indicate that the drug efflux function of RLIP76may be involved. The potential contribution of RLIP76to gliomal TMZ resistance isemphasized by the observation that expression of the ABC transporters MDR1and MRP1,which also efflux alkylating agents, is not influenced by decreased RLIP76expression inglioma cells. Our findings highlight RLIP76as a novel molecular target that can bespecifically inhibited to sensitize glioma cells to TMZ, thereby facilitating antitumor therapy.Part I Expression of RLIP76in astrocytic tumorsObjective: To investigate the expression level of RLIP76gene in astrocytomas of differentgrades and associated with decreased patient survival.Methods: By using immunohistochemistry, expression levels of RLIP76protein wereassessed in99astrocytic tumors of different pathological grades. By using Western blot,expression levels of RLIP76protein were assessed in34astrocytic tumors of differentpathological grades. The expression levels of RLIP76mRNA were evaluated by real-timequantitative PCR. The correlation between RLIP76and Ki-67immunoreactivity wasexamined by Spearman’s correlation coefficient. Univariate survival analysis was performedusing the Kaplan–Meier method and analyzed by the log-rank test to assess survivaldifferences between groups. Five possible prognostic factors were analyzed by univariate analysis for potential association with survival. Factors with a result of p<0.2in theunivariate analysis were included in multivariate analysis for potential association withsurvival.Results: Glioblastoma (WHO grade IV) exhibited greater immunoreactivity for RLIP76,Rac1, JNK and Ki-67proteins compared to diffuse astrocytoma (WHO grade II). Statisticalanalyses revealed that RLIP76immunostaining positively correlated with immunostainingfor Ki-67, a nuclear protein highly expressed by proliferating cells (r=0.581, p<0.001bySpearman’s correlation coefficient). Consistent with these immunohistochemical results,glioblastoma exhibited dramatically higher RLIP76and Rac1mRNA expression than diffuseastrocytoma, but JNK mRNA expression did not differ significantly between glioma grades(Figure1D). Western blotting confirmed that RLIP76, Rac1, and JNK protein levels werehigher in glioblastoma than diffuse grade II astrocytoma. Analysis of immunohistochemicalstaining intensity revealed high RLIP76expression in32patients (32/63,50.8%) and lowRLIP76expression in31patients (31/63,49.2%). Univariate survival analysis revealedsignificant relationships between expression of RLIP76, expression of Rac1, age andprognosis, while no significant associations were found between prognosis and sex, tumorsize. Survival probability was higher in glioma patients exhibiting lower RLIP76and Rac1expression. Multivariate analysis using the Cox proportional hazards model for all variablesincluded in univariate analysis revealed that high RLIP76expression at diagnosis (stainingscore≥4) and age were independent prognostic factors for patients with glioblastoma. Theseresults suggest that RLIP76expression may be a key prognostic index for glioblastomapatient survival.Conclusion: In summary, we found that RLIP76gene expression was higher in gliomas ofmore advance pathological stage and that expression reached a maximum in high-gradeastrocytomas. To determine whether RLIP76expression in glioma is associated withenhanced proliferative activity, we measured RLIP76immunostaining against that of theproliferation marker Ki-67in99glioma tumors and found a strong positive correlation,suggesting that RLIP76overexpression leads to a highly proliferative phenotype.Furthermore, statistical analysis also revealed a significant correlation between RLIP76andRac1expression. Multivariate analysis revealed low RLIP76expression as an independentprognostic indicator of prolonged survival time. Thus, this is first study to show that RLIP76protein expression is positively correlated with the pathological stages and tumorigenicity ofgliomas. Part II Knockdown of RLIP76inhibits proliferation, inducesapoptosis in human malignant gliomas cellsObjective: To investigate the effects of siRNA targeting human RLIP76on tumor apoptosisof U87, U251and SW1088glioma cells.Methods: Human malignant glioma U87, U251and SW1088cells were stably transfectedwith antisense RLIP76and ectogenic RLIP76using Lentivirus-mediated transfection,followed by in-vitro growth assays, cell cycle analyses, and invasion assays. Theproliferation assa were determined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide measured mRNA and protein expression levels of Bcl-2,Caspase-3, and P53were measured by RT-PCR and Western blot analysis.Results: Western blot analysis showed decreased levels of RLIP76protein expression inboth the U251and U87siRNA-transfected cell lines, whereas a line stably expressing GFPdid not significantly reduce RLIP76protein expression. Consistent with an important role ofRLIP76in cell survival, stably siRNA-transfected U251and U87cells showed significantlower survival rates compared to untransfected cell lines and GFP-expressing cell lines asmeasured by daily MTT cell viability assays. In contrast to glioma cells underexpressingRLIP76, U251and SW1088lines stably overexpressing RLIP76showed enhanced survival.Transfection of U251and U87cells with Lenti-RLIP76resulted in an approximate3.6-foldincrease in RLIP76mRNA expression in U251cells and a4.2-fold increase in SW1088cellscompared to the parent cell lines or lines stably expressing GFP. Western blot analysisshowed increased RLIP76protein expression in both the RLIP76-transfected U251andSW1088cell lines compared to the parental lines and lines stably expressing GFP.Overexpression of RLIP76induced a time-dependent increase in cell survival compared tocontrol U251and SW1088cells. Defective apoptotic signaling is thought to be a commonfeature of transformed cells, including glioma cells. In light of the effects of RLIP76expression on cell number, we speculated that RLIP76underexpression would increase theapoptosis rate while overexpression would decrease the number of apoptotic cells. Indeed,flow cytometry of Annexin V-stained glioma cell lines indicated that the rate of apoptosiswas significantly higher in U87and U251cells stably transfected with the RLIP76-targetedsiRNA compared to U87cells transfected with the GFP siRNA. Conversely, SW1088stablyoverexpressing RLIP76demonstrated a significantly lower rate of apoptosis relative to control SW1088cells or SW1088cells stably expressing the Lenti-GFP vector. Similarresults were also found in U251cells overexpressing RLIP76. These results suggest thatdownregulation of RLIP76expression promotes cell apoptosis, whereas RLIP76overexpression suppresses apoptosis and thus enhances glioma cell survival. We used TEManalysis in the present study to confirm in evaluation that Annexin V stain showed theapoptotic changes in U87and U251cells stably transfected with the RLIP76-targeted siRNA.The RLIP76-siRNA transfected U87and U251cell showed significant typical apoptosisfeatures, such as nuclear fragmentation, chromatin condensation and membrane blebbingcompared with control U87and U251cells.Conclusion: Our results suggest that overexpression of RLIP76increases the survival andanchorage-independent growth of human glioma cells in addition to suppressing apoptosis.We also suggest that downregulation of RLIP76expression promotes cell apoptosis, whereasRLIP76overexpression suppresses apoptosis and thus enhances glioma cell survival. Allthese findings strongly suggest that RLIP76plays a critical in mediating glioblastoma cellapoptosis in vitro. Part Ⅲ Therapeutic effect of RLIP76shRNA on xenografts in nudemice.Objective: To observed the therapeutic effect of T RLIP76shRNAon U251cell linexenografts in nude mice.Methods: Approximately1×106human glioma cells of the U251line were injected into thecaudate-putamen of nude mice as described previously [30]. In brief,6-to8-week-old malerats (200–250g) were anesthetized and immobilized on a rodent stereotaxic device. A1-cmincision was made retro-orbitally along the sagittal plane to exposed the skull and a burr holewas drilled2mm lateral to bregma and1mm anterior to the coronal suture. Glioma cellswere then injected slowly at a depth of5mm below the skull surface through a syringe heldon the stereotaxic frame and fitted with a27-gauge needle (needle tip toward the midline).After5minutes, the needle was slowly withdrawn and the skin incision sutured closed [31].Two independent repetitions with12mice per group were performed for each experiment.One hour before sacrifice, mice were injected i.p. with100μg/l BrdUrd. All animalexperiments conformed to the U.S. Public Health Service Policy on Humane Care and Useof Laboratory Animals. The brain of each mouse was harvested, fixed in4%formaldehyde, and embedded in paraffin. Formalin-fixed brain tumors were analyzed for proliferation usingBrdUrd incorporation as described previously [32]. Orthotopic brain tumors were evaluatedfor apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL) of fragmented DNA[33].Results: Mice injected with control U251cells, U251cells transfected with GFP siRNA, andcells transfected with Lenti-GFP exhibited median survivals of49,51, and49daysrespectively, whereas the median survival for mice injected with U251cells expressingRLIP76siRNA was134days. Medium survival was only26days for mice injected withU251cells transfected with Lenti-RLIP76. We then examined in vivo apoptosis of xenograftgliomas expressing Lenti-RLIP76or RLIP76siRNA using TUNEL staining. Tumorsresulting from injection of RLIP76siRNA-transfected cells displayed a significantly higherpercentage of TUNEL-stained (apoptotic) cells compared gliomas derived from cellstransfected with GFP-siRNA. Furthermore, we found that tumor cell proliferation wassignificantly inhibited in gliomas expressing the RLIP76siRNA compared to tumors derivedfrom U251cells transfected with GFP-siRNA as evidenced by differences in BrdUrdincorporation. Conversely, U251stably overexpressing RLIP76demonstrated a significantlylower rate of apoptosis and higher proliferation relative to U251cells stably expressing theLenti-GFP vector.Conclusion: Knockdown of RLIP76increases median survival in an orthotopic xenograftmouse model, and decreased RLIP76expression reduces tumor size and increases apoptosisin vivo. Furthermore, RLIP76and Rac1expression are positively correlated in gliomaxenografts. Part IV RLIP76regulates glioma cell apoptosis and growthby modulating the Rac1–JNK pathwayObjective: To investigate the RLIP76effect on Rac1-JNK pathway on glioam cell apoptosisand growth after RLIP76inhibition by RNAinterference.Methods: RLIP76siRNA was transfected into brain glioma cell line U251, the expression ofRLIP76was determined by western blot. The Rac1ubiquitylation assay was describedpreviously [34]. In brief, cells were transfected with6His-myc-tagged ubiquitin24hoursafter lentivirus transfection. Following24h incubation, cells were washed with PBS at roomtemperature and lysed in urea buffer (20mM Tris-HCl, pH7.5,200mM NaCl,10mM imidazole,0.1%Triton X-100in8M urea) for5minutes. Cells were scraped, collected,incubated for5minutes at37℃, and then centrifuged for5minutes at11,000g, Thesupernatant was retrieved, incubated with25μl TALON beads (Clontech) that wereprewashed and blocked (1hour at room temperature) with200μg/ml BSA. The suspensionwas incubated under slow rotation at room temperature for1h. The beads were recovered bybrief centrifugation, washed five times with urea buffer and the urea buffer resuspended inSDS sample buffer. Ubiquitylated Rac1was detected by the gel shift on Western blots.Results: Expression of the GAP-deficient RLIP76(R208L/K244R) reversed the impairedproliferation and enhanced apoptosis in cells expressing the RLIP76-specific siRNA(underexpressing RLIP76). Furthermore, similar effects were observed after transfection ofwild type RLIP76. We found that downregulation of RLIP76decreased Rac1expressionindependently of catalytic GAP activity, while RLIP76containing the ATP-binding siteK425M mutation (alone or in combination with K74M) failed to increase the expression ofRac1and Rac1-GTP in cells expressing the RLIP76-targeted siRNA. The level ofmonoubiquitylated Rac1, which migrates at a molecular weight of30kDa on SDS-PAGE,was reduced to approximately28%of that of GFP-siRNA controls in glioma cellsunderexpressing RLIP76. Furthermore, overexpression of WT RLIP76but not RLIP76ATPase-deficient mutants increased monoubiquitylated endogenous Rac1in cells expressingRLIP76siRNA. In contrast to the ATPase deficient mutants, a GAP-deficient RLIP76(R208L/K244R) still successfully increased monoubiquitylated endogenous Rac1.Conclusion: RLIP76regulates glioma cell apoptosis and growth by modulating theRac1–JNK pathway. RLIP76GAP-deficient mutants decrease apoptosis and increaseproliferation in glioma cells expressing RLIP76siRNA. RLIP76ATP-binding mutants fail toreduce apoptosis or promote proliferation in cells expressing RLIP76siRNA Part V Suppression of Brain Glioma Cells Expression of RLIP76by siRNA and itsEffect on TMZ ChemosensitivityObjective: To investigate the TMZ chemosensitivity change in U87and U251glioma cellafter RLIP76inhibition by RNAinterference.Methods: RLIP76siRNA was transfected into brain glioma cell line U87and U251, theexpression of RLIP76was determined by western blot. The effects of drugs on proliferationof puser-scambled, siRNA, siRNA+TMZ, TMZ negative and control cells were assessed by MTT assay.Results: Incubation with TMZ resulted in inhibition of cell proliferation, as indicated bydaily MTT assays, and shRNA-transfected cells exhibited enhanced TMZ sensitivitycompared to control and GRP-transfected U87cells. Combined RLIP76shRNA transfectionand TMZ treatment resulted in a significant increase in apoptotic cell death compared withcontrol cells. In addition, combined transfection and TMZ treatment decreased Bcl-2expression and increased Caspase3expression compared with control and lenti-GFP cellstreated with TMZ alone, indicating that RLIP76knockdown contributes to the increasedchemoresistance. Similar increases in chemosensitivity to TMZ were observed forshRNA-transfected U251cells revealed. Compared with TMZ alone (7.77±1.78and46.68±1.99g/mL in U87and U251cells, respectively) or combined shGFP and TMZ(7.37±1.07and48.20±1.77g/mL in U87and U251cells, respectively), combined shRNAand TMZ therapy resulted in a significant decrease in IC50value (4.08±1.99and23.68±2.59g/mL in U87and U251cells, respectively). Decreased expression of RLIP76inshRNA-transfected U87or U251cells did not affect MRP1or MDR mRNA or proteinlevels.Conclusion: RLIP76downregulation increased the sensitivity of glioma cells to TMZ bytriggering apoptosis and the enhanced susceptibility to TMZ of glioma cells underexpressingRLIP76is due to reduced RLIP76protein expression and a concomitant decrease inRLIP76-mediated TMZ efflux.