Comparative proteomic analysis reveals differentially expressed proteins regulated by a potential tumor promoter, BRE, in human esophageal carcinoma cells

Esophageal tumorigenesis is a complex and cascading process, involving the interaction of many genes and proteins. In this study, we have used the comparative proteomic approach to identify tumor-associated proteins and explore the carcinogenic mechanisms. Two-dimensional electrophoresis (2-DE) and MALDI-TOF MS analysis of esophageal carcinoma and control cells revealed 10 proteins that were upregulated. A further 10 proteins were downregulated. Among these 20 differentially expressed proteins, brain and reproductive organ-expressed (BRE) protein was identified as a potential tumor promoter. It was high expressed by the esophageal carcinoma cells, as confirmed by RT-PCR and immunoblotting. BRE has been reported to be a stress-responsive protein. To gain further insight into its function, BRE expression was silenced in esophageal carcinoma cells using BRE-specific small interference RNA. It was discovered that silencing BRE expression downregulated prohibitin expression, but upregulated tumor-suppressor p53 expression. Furthermore, cyclin A and CDK2 expressions were suppressed suggesting that BRE inhibited cell proliferation. These results implied that BRE plays a significant role in mediating antiapoptotic and proliferative responses in esophageal carcinoma cells.     

BRE enhances in vivo growth of tumor cells

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice. In vitro growth of the BRE stable transfectants was, however, not accelerated. No significant difference in metastasis between the transfectants and the parental D122 was detected. Thus, overexpression of BRE promotes local tumor growth but not metastasis. We conclude that the enhanced tumor growth is more likely due to the antiapoptotic activity of BRE than any direct effect of the protein on cell proliferation.     

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110. The expression of p110 siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110 siRNA induced CDK inhibitor p27^KIP1 levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110 siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110 and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27^KIP1 levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.     

抑制VEGF基因表达对乳腺癌细胞细胞周期的影响

目的：研究针对VEGF基因的siRNA（small interferenceRNA）对乳腺癌MCF-7细胞细胞周期的影响。方法：依据Promega公司在网上提供的设计软件,设计针对VEGF基因的siRNA,合成DNA模板,体外转录合成siRNA。脂质体转染法将合成的siRNA转染入MCF-7细胞,以未转染细胞以及错义序列siRNAscr转染细胞为对照。用细胞计数法检测siRNA对MCF-7细胞增殖的影响：流式细胞法检测细胞周期变化,RT—PCR法比较转染前后p21、CyclinDl表达水平的变化,Westemblot检测转染前后磷酸化ERK的表达。结果：细胞计数法结果显示,转染24h后siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。siRNA转染后能有效地抑制MCF-7细胞的增殖,阻滞细胞周期于G0／G1期,S期细胞明显减少,G0／G1期细胞比例逐渐增多;p21mRNA表达显著上调,抑制CyclinD1mRNA及磷酸化ERK蛋白的表达。结论：体外转录合成的siRNA可能通过上调细胞周期蚤白激酶抑制剂p21的表达,下调CyclinDl及磷酸化ERK的表达,将细胞周期阻滞于G0／G1期,从而显著抑制MCF-7细胞的增殖。     

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.     

p53siRNA therapy reduces cell proliferation, migration and induces apoptosis in triple negative breast cancer cells

p53 protein is probably the best known tumor suppressor. Earlier reports proved that human breast cancer cells expressing mutant p53 displayed resistance to apoptosis. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block p53 expression, as well as its subsequent effect on cell growth, apoptosis and migration on a triple negative human breast cancer cell line (Hs578T). p53siRNA significantly reduced cell index (CI) compared to the control and we observed an inhibition of cellular migration in the interval of time between 0 and 30 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. Also, by using PCR-array technology, a panel of 84 key genes involved in apoptosis was investigated. Our studies indicate that the knockdown of p53 expression by siRNA modulates several genes involved in cell death pathways and apoptosis, showing statistically significant gene expression differences for 22 genes, from which 18 were upregulated and 4 were downregulated. The present research also emphasizes the important role of BCL-2 pro-apoptotic family of genes (Bim, Bak, and Bax) in activating apoptosis and reducing cell proliferation by p53siRNA treatment. Death receptors cooperate with BCL-2 pro-apoptotic genes in reducing cell proliferation. The limited success may be due to the activation of the antiapoptotic gene Mcl-1, and it may be associated with the resistance of triple negative breast cancer cells to cancer treatment. Thus, targeting p53siRNA pathways using siRNA may serve as a promising therapeutic strategy for the treatment of breast cancers.     

Anti-apoptotic protein BRE/BRCC45 attenuates apoptosis through maintaining the expression of caspase inhibitor XIAP in mouse Lewis lung carcinoma D122 cells

Brain and Reproductive Organ Expressed (BRE), or BRCC45, is a death receptor-associated antiapoptotic protein, which is also involved in DNA-damage repair, and K63-specific deubiquitination. BRE overexpression attenuates both death receptor- and stress-induced apoptosis, promotes experimental tumor growth, and is associated with human hepatocellular and esophageal carcinoma. How BRE mediates its antiapoptotic function is unknown. Here we report based on the use of a mouse Lewis lung carcinoma cell line D122 that BRE has an essential role in maintaining the cellular protein level of XIAP, which is the most potent endogenous inhibitor of the caspases functioning in both extrinsic and intrinsic apoptosis. shRNA-mediated exhaustive depletion of BRE sensitized D122 cells to apoptosis induced not only by etopoxide, but also by TNF-α even in the absence of cycloheximide, which blocks the synthesis of antiapoptotic proteins by TNF-α-activated NF-κB pathway. In BRE-depleted cells, protein level of XIAP was downregulated, but not the levels of other antiapoptotic proteins, cIAP-1, 2, and cFLIP, regulated by the same NF-κB pathway. Reconstitution of BRE restored XIAP levels and increased resistance to apoptosis. XIAP mRNA level was also reduced in the BRE-depleted cells, but the level of reduction was less profound than that of the protein level. However, BRE could not delay protein turnover of XIAP. Depletion of BRE also increased tumor cell apoptosis, and decreased both local and metastatic tumor growth. Taken together, these findings indicate that BRE and its XIAP-sustaining mechanism could represent novel targets for anti-cancer therapy.     

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.     

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.     

Knockdown of Cripto-1 inhibits the proliferation,migration, invasion,and angiogenesis in prostate carcinoma cells

Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto-1/FRL1/Cryptic gene family that plays a key role in the various malignant cancers. However, the role of CR-1 in prostate carcinoma (PCa) remains limited. The expression of CR-1 was down-regulated by small interfering RNA (siRNA). Western blot measured the expression levels of CR-1 and some related proteins. We performed Cell Counting Kit-8, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and flow cytometry to detect the cellular proliferation and cycle. The transwell assay was used to observe cellular migration and invasion. The ability of angiogenesis was evaluated by tube formation assay. Our results showed that CR-1 knockdown markedly inhibited cell proliferation and induced cycle arrest in G1 phase, as p21 and p27 were up-regulated, whereas cyclin D1 and cyclin E1 were diminished. Moreover, silencing of CR-1 dramatically inhibited cell migration and invasion, repressed matrix metalloproteinases, and disturbed epithelial-mesenchymal transition. CR-1 siRNA suppressed the secreted level of vascular endothelial growth factor, and reduced protein level of Vascular endothelial growth factor receptor 2. We further found that decreased CR-1 expression inhibited FAK/Src/PI3K and Wnt/β-catenin signalling in PCa cells. These results suggested CR-1 might be served as an effective therapeutic target in PCa.     

小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提     

Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells

The ubiquitin specific peptidase 22 (USP22) is a positive regulator of the growth of tumors. However, little is known about the impact of USP22 knockdown on the growth of human bladder cells. In the present study, we designed a series of asymmetric interfering RNAs (aiRNAs) and compared the efficacy of aiRNA and conventional symmetric interfering RNA (siRNA) in the silencing of USP22 expression and the growth of human bladder EJ cells in vitro and in vivo. In comparison with transfection with the USP22-specific siRNA, transfection with 15/21 aiRNA was more potent in down-regulating the USP22 expression and inhibiting EJ cell proliferation in vitro. Furthermore, transfection with 15/21 aiRNA induced higher frequency of EJ cells arrested at the G0/G1 phases, but did not trigger EJ cell apoptosis. Moreover, transfection with either the siRNA or 15/21 aiRNA up-regulated the expression of p53 and p21, but down-regulated the expression of cyclin E and Mdm2 in EJ cells. The up-regulated p53 expression induced by the specific siRNA or aiRNA was abrogated by induction of Mdm2 over-expression. In addition, treatment with the specific siRNA or aiRNA inhibited the growth of implanted human bladder tumors in mice and the aiRNA had more potent anti-tumor activity in vivo. Therefore, our data suggest that knockdown of USP22 expression by the aiRNA may down-regulate the expression of Mdm2 and cyclin E, resulting in the up-regulated expression of p53 and p21 and leading to cell cycling arrest and inhibition of human bladder EJ cell proliferation. Our findings indicate that the USP22-specific aiRNA may be a novel approach for the intervention of human bladder tumors.     

Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1

The ubiquitin (Ub)-proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub-specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain-containing Ub aldehyde-binding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2-mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53-dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1(D88A)) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild-type Otub1 and its catalytic mutant (Otub1(C91S)), but not Otub1(D88A), bind to the MDM2 cognate E2, UbcH5, and suppress its Ub-conjugating activity in vitro. Overexpression of Otub1(D88A) or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.