MGr1-antigen/37kDa Laminin Receptor Precursor Contributes To Cellular Prion Protein Induced Multidrug-resistance Of Gastric Cancer

Gastric cancer is still one of the most common malignancies in China. Adjuvant treatments, which usually comprise chemotherapy, are the main methods for treating advanced gastric cancer patients now. But many of patients died of treatment failure, which could be ascribed to a phenomenon known as multidrug resistance(MDR). As one of the fatal steps of tumor progressing, MDR could be a good breakthrough point of research about gastric cancer treatment.MGr1 is a MDR related monoclonal antibody established by our lab by the hybridoma technique, using SGC7901/VCR cells as the immunogen. Immunohistochemical staining revealed a much stronger staining of MGr1 in SGC7901/VCR cells compared with its parental cells SGC7901. Subsequent researches showed that MGr1-Ag shared the same gene coding sequence of 37 LRP, 37 kDa laminin receptor precursor protein. The resistance of SGC7901/VCR to various chemotherapeutics could be reversed obviously by interfering MGr1-Ag/37 LRP. MGr1-Ag/37 LRP may be involved in various MDR mechanisms such as hypoxia pathway. It could participate in gastric cancer MDR through regulating P-gp, MRP, Bcl-2/Bax, FAK and PI3 K. PrPC(cellular prion protein) was previously verified to participate in multidrug-resistance of gastric cancer by our laboratory. PrPC is ubiquitously expressed in gastric cancer cell lines and gastric cancer tissues. And it was found to be highly expressed in drug-resistant gastric cancer cell line compared with its parental cells. PrPC contributes to both P-gp dependent and P-gp independent MDR in gastric cancer cell line SGC7901. Besides PI3K/Akt pathway, classic MDR molecules P-gp and apoptosis related protein Bcl-2 and Bax were also regulated by PrPC in gastric cancer MDR. Subsequently studies revealed that PrPC had notable effects on promoting proliferation, invasion, metastasis and other malignant phenotypes in gastric cancer.As a glycosyl-phosphatidylinositol-anchored membrane bound glycoprotein, how could PrPC trigger an intracellular signaling cascade resulting in MDR phenotype? 37 LRP was thought to be a salient ligand molecule of PrP which played an important role in internalization, propagation of PrPSc(scrapie form of PrP) and infection of scrapie disease in neuronal cells. PrPC and MGr1-Ag/37 LRP are all found to be involved in hypoxia mediated gastric cancer MDR. Moreover, they were verified to alter the tumorous drug resistant phenotypes through similar signal pathways. Thus, we started to explore whether MGr1-Ag/37 LRP was involved in PrPC mediated MDR phenotype and their relationship in gastric cancer MDR. 【Objectives】1. To clone and identify MGr1 hybridoma cell line. To purify and titer MGr1 mouse ascites. To investigate the effects of purified MGr1 on drug sensitivity and cell proliferation in drug resistant gastric cancer cells. 2. To detect and evaluate the co-expression and co-localization of MGr1-Ag/37 LRP and PrPC in gastric cancer. 3. To investigate if PrPC could regulate MGr1-Ag/37 LRP in gastric cancer cell lines and to study whether MGr1-Ag/37 LRP is participated in PrPC mediated gastric cancer MDR. 4. To reveal the underlying mechanisms via which MGr1-Ag/37 LRP contributes to PrPC mediated gastric cancer MDR. 【Methods】1. Cloning and purification of MGr1, effects of purified MGr1 on drug sensitivity and cell proliferation in drug resistant gastric cancer cells: Limited dilution assay was used to cloning MGr1 hybridoma cells and immunocytochemistry was used to detecting MGr1 secretion ability of hybridoma cells. Titer of MGr1 mouse ascites prepared with selected subclone cells was evaluated by Western blot. Octanoic acid-ammonium sulphate precipitation, xMagTM ProteinG and rProteinG was employed to purify MGr1 mouse ascites. Effects of purified MGr1 on 5-FU sensitivity in drug resistant gastric cancer cell line SGC7901/VCR was detect with ArrayScan® VTI HCS Reader using Hoechst staining and drug sensitive assay. Effects of purified MGr1 on cell proliferation in SGC7901/VCR were detected with ArrayScan® VTI HCS Reader using Hoechst staining.2. Co-expression, co-localization of and interaction between MGr1-Ag/37 LRP and PrPC in gastric cancer: Co-expression of MGr1-Ag/37 LRP and PrPC in gastric cancer cell lines SGC7901, SGC7901/VCR, MKN28 and AGS was evaluated by Western blot. Co-expression of MGr1-Ag/37 LRP and PrPC in gastric cancer tissues was investigated by immunohistostaining. Co-localization of MGr1-Ag/37 LRP and PrPC protein in gastric cancer cell lines and gastric cancer tissues was tested by double-immunofluorescence analysis using laser scanning confocal fluorescence assay. Co-immunoprecipitation was performed to investigate the interaction of MGr1-Ag/37 LRP and PrPC in gastric cancer cell line SGC7901/VCR.3. Whether MGr1-Ag/37 LRP is participated in PrPC mediated gastric cancer MDR: Western blot analysis was engaged to evaluate the expression levels of MGr1-Ag/37 LRP protein in PrPC up-regulated(SGC7901/PrP) and down-regulated(SGC7901/PrPi) gastric cancer cells. SiRNA targeting MGr1-Ag/37 LRP was employed as a tool for interfering the expression of MGr1-Ag/37 LRP in drug resistant gastric cancer cell line SGC7901/VCR. The protein level of MGr1-Ag/37 LRP was detected by Western blot. MTT assay was employed to evaluate whether MGr1-Ag/37 LRP could influence the drug resistance activity of PrPC. In vitro effects of drugs on the growth of SGC7901/PrP-37 LRPi compared with SGC7901/PrP-ic and SGC7901/VCR-37 LRPi compared with SGC7901/VCR-ic were detected. SGC7901/PrP incubated with 20μg/ml purified MGr1(SGC7901/PrP-MGr1) was also detected to investigate whether MGr1 could interfere in the MDR activity of PrPC in gastric cancer cell compared with SGC7901/PrP incubated with 20μg/ml mouse IgG(SGC7901/PrP-IgG).4. Underlying mechanisms via which MGr1-Ag/37 LRP contributes to PrPC mediated gastric cancer MDR: ADR intracellular accumulation and releasing assay were detected to further investigate the effects of MGr1-Ag/37 LRP siRNA on drug releasing index in SGC7901/VCR and SGC7901/PrP. Influence of MGr1-Ag/37 LRP siRNA on drug induced apoptosis in SGC7901/PrP cells was tested using Hoechst staining. Annexin â…¤/PI double staining and flow cytometry were used to evaluate the influence of MGr1-Ag/37 LRP siRNA on VCR induced apoptosis in SGC7901/VCR and SGC7901/PrP. Influence of MGr1-Ag/37 LRP siRNA on VCR induced caspase 3 activity in SGC7901/PrP was tested with Cellomics® Caspase 3 Activation Kit using ArrayScan® VTI HCS Reader. The expression level of Akt/pAkt protein in SGC7901/PrP cells transiently transfected with siRNA targeting MGr1-Ag/37 LRP was evaluated by Western blot. 【Results】1. Cloning and purification of MGr1, effects of purified MGr1 on drug sensitivity and cell proliferation in drug resistant gastric cancer cells: After cloning by limiting dilution assay and consequently immunocytochemistry assay, one MGr1 hybridoma subcloning cells MGr1-5 which could secret MGr1 efficiently was obtained. The ascites prepared with MGr1-5 cells could detect the target protein even at dilution of 1:2000 and could be purified by xMagTM ProteinG and rProtein G Agarose without decreasing the activity of antibody. Hoechst staining demonstrated that purified MGr1 could significantly increase the 5-FU sensitivity of SGC7901/VCR at the concentration of 20μg/ml(p<0.05). Hoechst staining demonstrated that purified MGr1(20μg/ml) and 5-FU(2μg/ml) could significantly suppress the proliferation of SGC7901/VCR cells compared with untreated SGC7901/VCR cells. Neither significant differences of effects between purified MGr1 and 5-FU nor differences between mouse IgG(20μg/ml) and untreated SGC7901/VCR cells were observed. Unfortunately, there were no significant difference of effects between purified MGr1 and mouse IgG, and also no significant difference of effects detected between 5-FU and mouse IgG. Thus we cannot make a definite conclusion about the influence of purified MGr1 on proliferation in SGC7901/VCR cell line.2. Co-expression, co-localization of and interaction between MGr1-Ag/37 LRP and PrPC in gastric cancer: Western blot analysis showed a significant correlative expression(p<0.05) between MGr1-Ag/37 LRP and PrPC in gastric cancer cell lines SGC7901/VCR, SGC7901, AGS and MKN28. Immunohistostaining showed a similar expression pattern of MGr1-Ag/37 LRP and PrPCin gastric cancer tissue serial sections. MGr1-Ag/37 LRP could be detected positively in 52% of the 50 patients,while PrPC could be detected positively in 66% of the patients. Statistical analysis results showed a significant expression correlation between these two proteins(p<0.05, r=0.3786). Partially co-localization of MGr1-Ag/37 LRP and PrPC protein both in the endochylema and on the surface but rare in nucleus of gastric cancer cell lines SGC7901 and AGS was observed by immunofluorescence analysis using laser scanning confocal fluorescence assay employing rabbit anti-human PrPC antibody and MGr1 mouse monoclonal antibody. Co-localization of MGr1-Ag/37 LRP and PrPC protein at the gland of gastric cancer tissues was observed by immunofluorescence analysis using laser scanning confocal fluorescence assay employing rabbit anti-human PrPC antibody and MGr1. Co-immunoprecipitation performed in vitro displayed coexistence of both PrPC and MGr1-Ag/37 LRP in anti-PrPC immunoprecipitate sample of SGC7901/VCR, demonstrating that these two proteins might coexistent in protein complex and interacted in SGC7901/VCR.3. Whether MGr1-Ag/37 LRP is participated in PrPC mediated gastric cancer MDR: Western blot analysis showed that the expression of MGr1-Ag/37 LRP protein were increased in SGC7901/PrP compared with SGC7901/PCDNA3.1 and decreased in SGC7901/PrPi compared with SGC7901/Psilencer3.1, indicating a regulation of protein expression of MGr1-Ag/37 LRP by PrPCin gastric cancer cells. SiRNA targeting MGr1-Ag/37 LRP was employed as a tool for interfering the expression of MGr1-Ag/37 LRP. Western blot analysis showed that the MGr1-Ag/37 LRP protein expression was significantly decreased in the cells transiently transfected with siRNA targeting MGr1-Ag/37 LRP compared with cells transfected with control oligonucleotide(p<0.05), indicating a effectively suppression of MGr1-Ag/37 LRP protein expression by siRNA. MTT assay showed that siRNA targeting to MGr1-Ag/37 LRP could reverse the drug resistance of SGC7901/PrP and SGC7901/VCR to P-gp related drugs ADR, VCR, VP-16 and P-gp non-related drugs 5-FU and CDDP(p<0.05). Purified MGr1 had similarly effects to 37 LRP siRNA at the concentration of 20μg/ml. SGC7901/PrP-MGr1 showed significantly decreased IC50 values to drugs compared to SGC7901/PrP(p<0.05), either. All these data suggested that MGr1-Ag/37 LRP might contribute to PrPC induced MDR phenotypes in gastric cancer cells possibly through both P-gp related and P-gp non-related pathways.4. Underlying mechanisms via which MGr1-Ag/37 LRP contributes to PrPC mediated gastric cancer MDR: Transiently transfected with siRNA targeting to MGr1-Ag/37 LRP, SGC7901/PrP-37 LRPi and SGC7901/VCR-37 LRPi had a significant decreased ADR releasing index compared to their control cell lines(p<0.05), which demonstrated that MGr1-Ag/37 LRP might influence the drug intake and pumping capability of SGC7901-derived gastric cancer cells. Hoechst staining showed that apoptotic cells were significantly increased in SGC7901/PrP cells transiently transfected with siRNA targeting to MGr1-Ag/37 LRP compared with control cells after treated with VCR. Annexin â…¤/PI double staining showed that apoptosis rate of SGC7901/PrP-37 LRPi and SGC7901/VCR-37 LRPi were much higher than that of their control cells under the same VCR induced condition(p<0.05). These results revealed a better resistance to VCR-induced apoptosis of SGC7901/PrP-37 LRPi and SGC7901/VCR-37 LRPi compared with their controls respectively. Caspase 3 activation assay demonstrated that MGr1-Ag/37 LRP siRNA significantly increased caspase 3 activation in SGC7901/PrP compared with control group,indicating another potential pathway of MGr1-Ag/37 LRP in promoting PrPC mediated gastric cancer MDR through regulating protein which could influence caspase 3 activation. Western blot results showed that pAkt were significantly decreased in SGC7901/PRP when transiently transfected with MGr1-Ag/37 LRP siRNA compared to the siRNA control group. 【Conclusions】MGr1-5 is a subcloning hybridoma cell line that can effectively produce MGr1 mouse monoclonal antibody, which could be purified by x MagTM ProteinG and rProtein G Agarose without cripple the activity of antibody. Purified MGr1 could significantly increase the 5-FU sensitivity of SGC7901/VCR at the concentration of 20μg/ml. Purified MGr1 could not have direct inhibition effect on cell proliferation of SGC7901/VCR cell line. MGr1-Ag/37 LRP and PrPC were found correlatively expressed and co-localized in gastric cancer cell lines and gastric cancer tissues. The protein expression of MGr1-Ag/37 LRP might be regulated by PrPC in gastric cancer cells, and siRNA targeting MGr1-Ag/37 LRP could notably increase the drug sensitivity of PrPC up-regulated SGC7901/PrP cells and multidrug resistant SGC7901/VCR cells. Cells incubated with purified MGr1 at the concentration of 20μg/ml showed similar effect of drug sensitization compared to control cells. According to our results, MGr1-Ag/37 LRP might decrease the drug sensitivity of SGC7901-derived cells through altering the pumping rate of drugs. The resistance to apoptosis induced by chemical drugs would be another potential mechanism of the MDR function of MGr1-Ag/37 LRP. MGr1-Ag/37 LRP might promote PrPC induced gastric cancer multi-drug-resistance through the PI3K/Akt pathway. Combined with the earlier findings gained by our colleagues and other researchers, we suppose MGr1-Ag/37 LRP to be an important regulatory molecule in PrPC mediated gastric cancer MDR partially through increasing drug releasing and resistance to drug induced apoptosis. Although needed to be further confirmed in vivo, our findings deepen the understanding of underlying mechanisms in gastric cancer MDR. Our study also provides new strategies to reverse the clinical drug resistance.