shRNA-mediated silencing of sorcin increases drug chemosensitivity in myeloma KM3/DDP and U266/ADM cell lines

Sorcin is a penta-EF hand calcium binding protein, which is involved in the resistance to chemotherapeutics in cancer cells, and is overexpressed in various cancer cells. However, tumor relapse combined with the development of drug resistance remains a significant problem. Here, we demonstrated that silencing of Sorcin in chemotherapy resistance myeloma U266/ADM and KM3/DDP cell lines resulted in reduced cell proliferation, cell cycle arrest and cell apoptosis. Sorcin siRNA successfully silenced Sorcin mRNA and protein expression. Silencing of Sorcin also significantly reduced the mRNA and protein expression levels of MDR1, MRP1, GST-π, Survinvin, Livin, Bcl-2, Cyclin-D1, phospho-Src, C-myc, p21, NF-κB and phospho-AKT, while p53 expression and caspase-3 and caspase-8 activity significantly increased when compared with control group. Silencing of Sorcin significantly increased the sensitivity of KM3/DDP cells to cisplatin and the sensitivity of U266/ADM to adriamycin, compared to cells untransfected and transfected with negative control shRNA. In addition, intracellular accumulation of Rhodamine 123 significantly increased in KM3/DDP and U266/ADM cells. In summary, our studies indicate that drug resistance can be effectively reversed in cisplatin-resistance and adriamycin-resistant myeloma cells through delivery of siRNAs targeting Sorcin. Assessment of potential as a target for human myeloma treatment is clearly warranted.     

EFEMP2 is upregulated in gliomas and promotes glioma cell proliferation and invasion

Gliomas are the most common and aggressive form of primary brain tumor. Although EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2), an extracellular matrix (ECM) glycoprotein, is regarded as a candidate oncogene, little is known about the association of EFEMP2 and gliomas. Here, the expression of EFEMP2 was significantly increased in glioma tissues (n=60) compared to non-tumorous brain tissues (n=25). Silencing of EFEMP2 expression through RNA interference in two glioma cell lines (U87 and U373) remarkably inhibited cell proliferation and G1/S transition. More importantly, EFEMP2 silencing significantly induced cell apoptosis via increasing the ratio of Bax and Bcl-2. Additionally, knockdown of EFEMP2 significantly inhibited the invasive ability of both glioma cells, which was associated with the downregulated expression of metalloproteinase-2 (MMP-2) and MMP-9. In conclusion, expression of EFEMP2 was associated with the oncogenic potential of gliomas and silencing of its expression can suppress cancer cell growth and metastasis. Inhibition of EFEMP2 may be a therapeutic strategy for gliomas.     

miR-130b在胶质瘤对替莫唑胺耐药中的作用

目的:探讨微小RNA-130b(microRNA-130b,miR-130b)在胶质瘤细胞中的表达及其调控体外胶质瘤细胞对替莫唑胺(temozolomide,TMZ)耐药中的作用。方法:利用RT-qPCR法检测miR-130b在胶质瘤细胞株U251、SHG-44和U87中的表达水平;计算TMZ对不同胶质瘤细胞株(U251、SHG-44和U87)的半数抑制浓度(IC_(50));通过不同浓度梯度的TMZ作用于体外U251细胞,从而获得相对稳定的对TMZ耐药的U251(U251/TMZ resistance,U251/TR)细胞,计算TMZ对U251/TR细胞的IC_(50)及耐药指数(resistance factor,RF);使用miR-130b模拟物(miR-130b mimics)和miR-130b抑制物(miR-130b inhibitor)瞬时转染体外胶质瘤细胞;CCK-8法检测体外胶质瘤细胞的活力;流式细胞术检测胶质瘤细胞凋亡;通过生物信息学工具分析miR-130b可能的靶基因,萤光素酶报告基因实验测定萤光素酶活性;电泳迁移率变动分析检测核因子κB(nuclear factor-κB,NF-κB)的活性;Western blot法测定肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、Bcl-2、X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)和survivin蛋白在胶质瘤细胞中的表达水平。结果:TMZ对不同胶质瘤细胞株(U251、SHG-44和U87)的IC_(50)分别为54.8、94.8和149.6μmol/L;TMZ对U251/TR细胞的IC_(50)为(446.5±61.3)μmol/L,其RF为8.1;转染miR-130b mimics可明显增强TMZ对U251/TR细胞的生长抑制和凋亡诱导作用;转染miR-130b inhibitor可显著抑制TMZ对U251/TR细胞的生长抑制和凋亡诱导作用;萤光素酶报告基因实验验证TNF-α是miR-130b的直接作用靶点;与mimics NC组相比,转染miR-130b mimics后U251/TR细胞中的NF-κB活性及TNF-α、Bcl-2、XIAP和survivin蛋白表达均明显下调;与inhibitor NC组相比,转染miR-130b inhibitor后U251细胞中的NF-κB活性及TNF-α、Bcl-2、XIAP和survivin蛋白表达均显著上调(P0.05);NF-κB抑制剂Bay 11-7082可增强TMZ对U251/TR细胞凋亡的诱导作用。结论:miR-130b在耐药型胶质瘤细胞中表达下调,并通过靶向调控TNF-α/NF-κB通路增强TMZ对体外胶质瘤细胞的作用。     

Influence of far upstream element binding protein 1 gene on chemotherapy sensitivity in human U251 glioblastoma cells

Introduction

The aim of this study was to determine the influence of the far upstream element binding protein 1 gene (FUBP1) on chemotherapy sensitivity in human U251 glioblastoma cells.

Material and methods

Real-time polymerase chain reaction (PCR) was used to determine the expression of the FUBP1 gene in 43 cases of human brain gliomas. Western blot analysis was used to determine the inhibitory effect of RNA interference on FUBP1 gene expression. Methyl thiazolyl tetrazolium assay (MTT) and flow cytometry methods were used to determine the growth inhibitory rate and apoptosis rate of the U251 cells with FUBP1 silencing. The growth inhibitory rate and apoptosis rate were further determined after treatment of those U251 cells with cisplatin (DDP).

Results

The expression of FUBP1 mRNA was up-regulated significantly in gliomas, 177.65% as much as in peri-cancerous tissues (p < 0.05). The expression of FUBP1 protein was inhibited significantly with siRNA-FUBP1 (p < 0.05). In FUBP1-silenced cells, the growth inhibitory rate increased from 1.4% to 29.5%, and the apoptosis rate increased from 2.68% to 5.84% (p < 0.05 for both). After treating with DDP at various concentrations (1, 3, 5 µg/ml), the growth inhibitory rate of FUBP1-silenced cells increased from 14.42%, 17.46% and 23.55% to 21.69%, 27.51% and 37.57%; the apoptosis rate increased from 8.85%, 14.37% and 18.21% to 13.25%, 18.46% and 26.52%.

Conclusions

The up-regulation of FUBP1 relates to the carcinogenesis of gliomas. FUBP1 silencing increases the growth inhibitory rate and apoptosis rate of the U251 cells, and enhances the chemotherapy sensitivity of U251 cells to DDP.     

Arsenic trioxide induces autophagy and apoptosis in human glioma cells in vitro and in vivo through downregulation of survivin

Gliomas are the most aggressive of all human malignancies. Survivin is overexpressed in gliomas, and overexpression of survivin is associated with the progression of gliomas and the poor prognosis of glioma patients. Arsenic trioxide (ATO) is used in patients with acute promyelocytic leukemia and is active in vitro in several solid tumor cell lines. In the present study, the human glioma cell line U118-MG was used to investigate the anti-cancer effect of ATO in vitro and in vivo. The molecular mechanisms of the relationship between cell death (autophagy and apoptosis) and survivin were analyzed. ATO reduced cell viability through an increase in mitotic cells in a concentration-dependent manner. The mechanisms of ATO-induced autophagy and apoptosis were mediated by the inhibition of PI3K/Akt and the activation of MAPK signaling pathways. The ATO treatment of U118-MG cells pre-treated with specific chemical inhibitors of PI3K/AKT and MAPK significantly changed the cytotoxicity and the expression of survivin, suggesting that survivin plays a pivotal role in ATO-induced cell death. When U118-MG cells were transfected with survivin shRNA, the results demonstrated a significant increase in apoptotic and autophagic cells. In in vivo studies, the ATO treatment of SCID mice showed a significant tumor growth delay time and the decreased expression of survivin in tumor tissue. An important result from the current study is the finding that survivin could suppress both autophagy and apoptosis in glioma cells. This study suggests that ATO treatment or survivin inhibition could be a novel therapeutic strategy in malignant gliomas.     

Influence of the corticospinal tract on the cutaneous silent period: a study in patients with pyramidal syndrome

Celecoxib is a cyclooxygenase 2-selective nonsteroidal anti-inflammatory drug (NSAID) that exhibited therapeutic activity in cancer. In this study three malignant glioma, U87-MG, U251 and A172, were treated with celecoxib, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Single treatment with celecoxib (25-100muM) for 24h resulted in a concentration-dependant decrease of cellular viability in U87-MG, U251 and A172. Combining subtoxic concentrations of celecoxib with TRAIL strongly increased cell death in human malignant glioma cells. After 8h treatment with celecoxib we found down-regulation of the inhibitor of apoptosis protein survivin that was mediated by proteasomal degradation. In addition, over-expression of survivin not only attenuated celecoxib-induced cytotoxicity but also cytotoxicity induced by the combination of celecoxib and TRAIL. Taken together, in malignant glioma survivin is a key regulator in celecoxib- and TRAIL-celecoxib-mediated cell death.     

LRIG1 Enhances Chemosensitivity by Modulating BCL-2 Expression and Receptor Tyrosine Kinase Signaling in Glioma Cells

Purpose

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) are an inhibitor of receptor tyrosine kinases (RTKs) that was discovered in recent years, and many studies showed that LRIG1 is a tumor suppressor gene and may be related to tumor drug resistance. In this study, we explored whether LRIG1 protein expression can improve the chemosensitivity of glioma cells and what was its mechanism.

Materials and Methods

We collected 93 cases of glioma tissues and detected the expression of LRIG1 and BCL-2. We constructed a multidrug resistance cell line U251/multidrug resistance (MDR) and examined the change of LRIG1 and BCL-2 at mRNA and protein expression levels. LRIG1 expression was upregulated in U251/MDR cells and we detected the change of multidrug resistance. Meanwhile, we changed the expression of LRIG1 and BCL-2 and explored the relationship between LRIG1 and BCL-2. Finally, we also explored the relationship between LRIG1 and RTKs.

Results

LRIG1 was negatively correlated with BCL-2 expression in glioma tissue and U251/MDR cells, and upregulation of LRIG1 can enhance chemosensitivity and inhibit BCL-2 expression. Furthermore, LRIG1 was negatively correlated with RTKs in U251/MDR cells.

Conclusion

These results demonstrated that LRIG1 can improve chemosensitivity by modulating BCL-2 expression and RTK signaling in glioma cells.     

Evaluation of IRES-mediated, cell-type-specific cytotoxicity of poliovirus using a colorimetric cell proliferation assay

PVS-RIPO is a recombinant oncolytic poliovirus designed for clinical application to target CD155 expressing malignant gliomas and other malignant diseases. PVS-RIPO does not replicate in healthy neurons and is therefore non-pathogenic in rodent and non-human primate models of poliomyelitis. A tetrazolium salt dye-based cellular assay was developed and qualified to define the cytotoxicity of virus preparations on susceptible cells and to explore the target cell specificity of PVS-RIPO. In this assay, PVS-RIPO inhibited proliferation of U87-MG astrocytoma cells in a dose-dependent manner. However, HEK293 cells were much less susceptible to cell killing by PVS-RIPO. In contrast, the Sabin type 1 live attenuated poliovirus vaccine strain (PV(1)S) was cytotoxic to both HEK293 and U87-MG cells. The correlation between expression of CD155 and cytotoxicity was also explored using six different cell lines. There was little or no expression of CD155 and PVS-RIPO-induced cytotoxicity in Jurkat and Daudi cells. HEK293 was the only cell line tested that showed CD155 expression and resistance to PVS-RIPO cytotoxicity. The results indicate that differential cytotoxicity measured by the colorimetric assay can be used to evaluate the cytotoxicity and cell-type specificity of recombinant strains of poliovirus and to demonstrate lot to lot consistency during the manufacture of viruses intended for clinical use.     

RNA干扰沉默Aurora A基因表达对胶质瘤细胞增殖及凋亡的影响

目的 探讨RNA干扰技术抑制人胶质瘤U251细胞中Aurora A基因的表达,研究其对U251细胞增殖及凋亡的影响。 方法 设计并合成特异性针对Aurora A基因的siRNA,转染至U251细胞中,采用RT-PCR和Western blotting检测Aurora A mRNA和蛋白的表达情况,同时利用四甲基偶氮唑盐(MTT)实验和流式细胞仪观察转染后U251细胞增殖抑制率和细胞凋亡,透射电镜观察细胞凋亡超微结构改变。 结果 转染Aurora A siRNA后,U251细胞Aurora A mRNA表达受到抑制(P<0.01),蛋白表达水平降低;U251细胞增殖的抑制率和细胞凋亡率显著增高 (P<0.01),且U251细胞发生了显著的凋亡形态改变。 结论 体外合成的针对Aurora A基因的特异性siRNA成功地抑制了U251细胞中Aurora A基因的表达,并能抑制胶质瘤细胞增殖、促进凋亡,提示Aurora A可能成为胶质瘤基因治疗的新靶点。     

Zeste基因增强子同源物2通过调控Wnt/β-catenin信号通路影响脑胶质瘤细胞凋亡

目的:探讨zeste基因增强子同源物2(EZH2)通过调控Wnt/β-catenin信号通路对脑胶质瘤细胞凋亡的影响。方法:以RT-q PCR和Western blot检测胶质瘤U87、H4和U251细胞及正常人脑星形细胞(NHA)中EZH2的表达水平。在H4细胞中转染EZH2 siRNA和siRNA control,MTT测定细胞活力,流式细胞术测定细胞凋亡,分光光度计法检测caspase-3的活性,Western blot检测Wnt/β-catenin信号通路中关键蛋白β-连环蛋白(β-catenin)和下游靶分子c-Myc的蛋白表达。用Wnt/β-catenin信号通路激活剂处理转染EZH2 siRNA的H4细胞,流式细胞术测定细胞凋亡,Western blot测定β-catenin和c-Myc的表达。结果:胶质瘤U87、H4和U251细胞中EZH2的mRNA和蛋白水平均明显高于NHA(P0.05),并且H4细胞中EZH2 mRNA和蛋白水平高于U87和U251细胞(P0.05)。EZH2 siRNA可以明显下调H4细胞中EZH2的mRNA和蛋白水平。EZH2表达下调后的H4细胞从48 h开始细胞活力降低,并且细胞凋亡率也明显升高,细胞中caspase-3活性也明显升高(P0.05),同时EZH2表达下调还可以抑制β-catenin和c-Myc的表达。Wnt/β-catenin信号通路的激活剂可以减少EZH2诱导的H4细胞凋亡,降低细胞中caspase-3的活性。结论:EZH2在脑胶质瘤细胞中过度表达,下调其表达可以通过抑制Wnt/β-catenin信号通路的激活而诱导胶质瘤细胞凋亡。     

慢病毒介导的靶向骨桥蛋白的siRNA对人脑胶质瘤细胞侵袭和凋亡的影响

目的 研究骨桥蛋白(osteopontin,OPN)下调后对胶质瘤细胞侵袭和凋亡的影响.方法 在体外以慢病毒介导的OPN小干扰RNA(small interference,siRNA)感染胶质瘤细胞系U251细胞,实时PCR和Western印迹检测OPN表达.通过Transwell实验、流式细胞仪检测,观察OPN表达下调后对胶质瘤细胞侵袭和细胞凋亡的影响.结果 慢病毒介导的OPN siRNA感染能显著降低U251细胞OPNmRNA水平及蛋白表达,有效抑制U251细胞侵袭能力,并诱导其凋亡,OPN siRNA感染组中Bcl-2、尿激酶型纤溶酶原激活剂(uPA)、MMP-2和MMP-9明显减少,Bax显著增多.结论 慢病毒介导的OPNsiRNA可显著抑制U251细胞的侵袭,并促进其凋亡.     

CBX8过表达或沉默对人神经胶质瘤细胞增殖和凋亡的影响

目的:探究染色质结构域蛋白8(chromodomain protein 8,CBX8)对人神经胶质瘤细胞增殖与凋亡的作用。方法:Western blot和RT-qPCR检测组织及细胞系中CBX8的表达。构建过表达CBX8和沉默CBX8载体,转染神经胶质瘤细胞T98G和U87MG,分别用MTT法和BrdU实验检测细胞增殖,流式细胞术检测细胞凋亡。结果:与正常脑组织和星形胶质细胞相比,神经胶质瘤组织及细胞中的CBX8蛋白和mRNA水平明显上升。在T98G和U87MG细胞中,过表达CBX8均促进细胞增殖,抑制细胞凋亡,并上调Rb/E2F1的表达水平,而沉默CBX8则作用相反。sh-E2F1转染细胞之后,cyclin D1的表达以及Bcl-2/Bax的比值降低。结论:CBX8可能通过Rb/E2F1通路调节胶质瘤细胞的增殖和凋亡。     

Expression of complement membrane regulators membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) in human malignant gliomas.

Gliomas are malignant brain tumors, which, despite recent progress in surgical and radiological treatment, still have a poor prognosis. Since gliomas apparently resist immunological clearance mechanisms, we became interested in examining bow gliomas resist killing by the human complement system. The resistance of human cells to complement-mediated damage is, in large part, mediated by specific inhibitors of complement:membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59). In the present study we examined the expression of complement regulators in 14 human glioma tumors and in 7 glioma cell lines (U251, U87, HS683, U373, U138, U118, and H2). Protectin was found to be strongly expressed by all glioma tumors and cell lines. Northern blotting analysis demonstrated the typical pattern of four to five protectin mRNAs in the glioma cells. Except for blood vessels, the expression of decay-accelerating factor was weak or absent in the tumors in situ, whereas in the cell lines its expression varied, ranging from negative to intermediate. Membrane cofactor protein was moderately expressed by all the cell lines but only weakly in the tumors. Cell-killing experiments demonstrated that the glioma cell lines were exceptionally resistant to C-mediated lysis. Five of the seven cell lines (U373, HS683, U118, U138, and H2) resisted complement lysis under conditions where most other cell lines were sensitive to killing. Neutralization experiments using specific monoclonal antibodies indicated that protectin was functionally the most important complement regulator in the glioma cells. The killing of the U87 and U251 cells could be significantly increased by a blocking anti-protectin monoclonal antibody, whereas for the other cell lines only moderate or no response was observed. The H2 cell line resisted killing by all antibodies and by complement. These results show that protectin is the most important complement regulator on human glioma cells. The exceptional complement resistance of some glioma cell lines suggests that they may utilize other, hitherto less well characterized, mechanisms to resist complement killing.     

胃癌多药耐药细胞株BGC823/5-FU的建立及其耐药机制的研究

目的：建立5-氟尿嘧啶（5-FU）诱导的胃癌多药耐药细胞株BGC823/5-FU，探讨凋亡相关蛋白Survivin、Bcl-2、Bax及caspase-3与其耐药性产生的关系。 方法:采用反复短期暴露并逐渐增加5-FU浓度的方法建立胃癌耐药细胞株BGC823/5-FU，MTT法检测此耐药细胞株对5-FU的耐药倍数及其对临床常用化疗药物阿霉素、丝裂霉素和顺铂的交叉耐药性，流式细胞术检测细胞P-糖蛋白的表达和柔红霉素积累量；Western blotting法检测耐药胃癌细胞株BGC823/5-FU与其亲代药物敏感胃癌细胞株BGC823凋亡相关蛋白Survivin、Bcl-2、Bax及caspase-3的表达。 结果:成功诱导出胃癌多药耐药细胞株BGC823/5-FU，较其亲代细胞BGC823对5-FU、阿霉素、丝裂霉素和顺铂的耐药性分别提高10.82、2.50、22.23和2.00倍。其P-糖蛋白表达较BGC823细胞增高（P<0.01），柔红霉素积累量较BGC823细胞减低（P<0.01）。与亲代药物敏感BGC823细胞相比，耐药细胞株BGC823/5-FU细胞Survivin表达上升（P<0.05），Bcl-2表达升高（P<0.05），Bax表达下降（P<0.05），caspase-3表达减低（P<0.05）。结论:胃癌细胞株BGC823在5-FU的诱导下可形成多药耐药细胞株BGC823/5-FU，P-糖蛋白、凋亡相关蛋白Survivin、Bcl-2、Bax及caspase-3可能参与其耐药性的形成。     

沉默miR-21对子宫内膜癌顺铂耐药细胞株Ishikawa/DDP的影响

目的:观察沉默miR-21对子宫内膜癌顺铂耐药细胞株Ishikawa/DDP的影响.方法:以Lipofectamine 2000介导miR-21抑制剂转染Ishikawa/DDP细胞株,同时设置阴性组和耐药组.采用反转录PCR检测miR-21、多药耐药基因MDR1、促凋亡基因Bax和抗凋亡基因Bcl-2的表达.采用蛋白印迹法检测多药耐药蛋白P-gp、促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达.采用噻唑蓝比色法检测细胞对顺铂的敏感性.采用流式细胞术检测细胞凋亡情况.结果:与耐药组和阴性组比较,抑制剂组miR-21,MDR1和Bcl-2 mRNA表达显著下调(P＜0.0l),而Bax mRAN表达显著上调(P＜0.00l);抑制剂组P-gp和Bcl-2蛋白表达显著低下调(p＜0.05),而Bax蛋白表达显著上调(P＜0.001).与耐药组和阴性组比较,顺铂对抑制剂组的IC50值显著(P＜0.001);顺铂对抑制剂组细胞的诱导凋亡率显著增加(P＜0.00l).结论:沉默miR-21可显著提高Ishikawa/DDP细胞株对顺铂的敏感性,并促进细胞凋亡,其具体机制可能与下调MDR1,P-gp和Bcl-2表达,以及上调Bax表达有关.     

Inhibition of tissue factor/protease-activated receptor-2 signaling limits proliferation,migration and invasion of malignant glioma cells

Tissue factor (TF) is upregulated in several malignant diseases, including gliomas. Here, we demonstrate pronounced differences in the expression of TF and its interactors factor VII and protease-activated receptor 2 (PAR-2) in nine human glioma cell lines (U87, U251, U343, U373, MZ-18, MZ-54, MZ-256, MZ-304, Hs 683) as detected by RT-PCR and Western blot analysis. Inhibition of TF signaling by a neutralizing monoclonal antibody (mAb TF9-10H10) led to significantly reduced proliferation in high-grade astroglial (MZ-18 and MZ-304) and oligodendroglial (Hs 683) cell lines abundantly expressing TF, but not in U373 cells expressing low amounts of TF. Scratch migration assays and Boyden chamber assays indicated that mAb TF9-10H10 and lentiviral knockdown of TF significantly reduced cell migration and invasion of MZ-18, MZ-304 and Hs 683 cells, both under normoxic and hypoxic conditions. Of note, all three cell lines displayed increased cell migration and invasion under hypoxic conditions (1% O₂), which was associated with enhanced expression of TF and increased phosphorylation of p44/42 mitogen-activated protein kinase (ERK1/2). Silencing of TF blocked activation of the ERK pathway, induction of TF expression and the potentiating effect of hypoxia on cell migration and invasion. RNA interference against PAR-2 abrogated the autocrine effects of TF on cell proliferation, migration and invasion, indicating that TF signals via PAR-2 in glioma cells. Our results suggest an important role for the TF/FVIIa/PAR-2/ERK axis in tumor growth and invasion of glioma and suggest that TF may be a suitable target for the development of novel therapies against high-grade glioma.     

MDR1-P-glycoprotein behaves as an oncofetal protein that promotes cell survival in gastric cancer cells

P-glycoprotein (P-gp), traditionally linked to cancer poor prognosis and multidrug resistance, is undetectable in normal gastric mucosa and overexpressed in gastric cancer (GC). We propose that P-gp may be involved in Helicobacter pylori (Hp)-related gastric carcinogenesis by inhibiting apoptosis. Aim of the study was to evaluate the expression of P-gp in fetal stomach and in Hp-related gastric carcinogenesis, the epigenetic control of the multi-drug resistance-1 (MDR1) gene, the localization and interaction between P-gp and Bcl-x(L) and the effect of the selective silencing of P-gp on cell survival. P-gp and Bcl-xl expression was evaluated by immunohistochemistry on 28 spontaneously abortive human fetuses, 66 Hp-negative subjects, 138 Hp-positive chronic gastritis (CG) of whom 28 with intestinal metaplasia (IM) and 45 intestinal type GCs. P-gp/Bcl-x(L) colocalization was investigated by confocal immunofluorescence microscopy and protein-protein interaction by co-immunoprecipitation, in basal conditions and after stress-induced apoptosis, in GC cell lines AGS and MKN-28 and hepatocellular carcinoma cell line Hep-G2. The role of P-gp in controlling apoptosis was evaluated by knocking down its expression with a specific small interfering RNAs in stressed AGS and MKN-28 cell lines. P-gp is expressed in the gastric mucosa of all human fetuses while, it is undetectable in adult normal mucosa and re-expressed in 30/110 Hp-positive non-IM-CG, 28/28 IM-CG and 40/45 GCs. P-gp expression directly correlates with that of Bcl-x(L) and with the promoter hypomethylation of the MDR1 gene. In GC cell lines, P-gp is localized on the plasma membrane and mitochondria where it colocalizes with Bcl-x(L). Co-immunoprecipitation confirms the physical interaction between P-gp and Bcl-x(L) in AGS, MKN-28 and Hep-G2, at both basal level and after stress-induced apoptosis. The selective silencing of P-gp sensitizes GC cells to stress-induced apoptosis. P-gp behaves as an oncofetal protein that, by cross-talking with Bcl-x(L), acts as an anti-apoptotic agent in Hp-related gastric carcinogenesis.     

MiR-16 modulate temozolomide resistance by regulating BCL-2 in human glioma cells

Temozolomide (TMZ) with radiotherapy is the current standard of care for newly diagnosed glioma. However, glioma patients who are treated with the drug often develop resistance to it and some other drugs. Recently studies have shown that microRNAs (miRNAs) play an important role in drug resistance. In present study, we first examined the sensitivity to temozolomide in six glioma cell lines, and established a resistant variant, U251MG/TR cells from TMZ-sensitive glioma cell line, U251MG. We then performed a comprehensive analysis of miRNA expressions in U251MG/TR and parental cells using cancer microRNA PCR Array. Among the downregulated microRNAs was miR-16, members of miR-15/16 family, whose expression was further validated by qRT-PCR in U251MG/TR and U251MG cells. The selective microRNA, miR-16 mimics or inhibitor was respectively transfected into U251MG/TR cells and AM38 cell. We found that treatment with the mimics of miR-16 greatly decreased the sensitivity of U251MG/TR cells to temozolomide, while sensitivity to these drugs was increased by treatment with the miR-16 inhibitor. In addition, the downregulation of miR-16 in temozolomide-sensitive AM38 cells was concurrent with the upregulation of Bcl-2 protein. Conversely, overexpression of miR-16 in temozolomide-resistant cells inhibited Bcl-2 expression and decreased temozolomide resistance. In conclusion, MiR-16 mediated temozolomide-resistance in glioma cells by modulation of apoptosis via targeting Bcl-2, which suggesting that miR-16 and Bcl-2 would be potential therapeutic targets for glioma therapy.