GRIM-19 inhibits the STAT3 signaling pathway and sensitizes gastric cancer cells to radiation

Gastric cancer is one of the most common malignancies, and radiation resistance is one of the key obstacles in gastric cancer treatment. In this study, we demonstrate that “genes associated retinoid–IFN induced mortality-19” (GRIM-19) expression was lower in patients with radiotherapy-resistant tumors compared to patients with radiotherapy-sensitive tumors. In order to further investigate the effects of GRIM-19 expression on the radiation response in gastric cancer cells, we established BGC-803 clones stably expressing exogenous GRIM-19. We found that the percentage of apoptotic cells was higher in cells expressing GRIM-19 than untransfected cells post-radiation treatment. Furthermore, caspase-3, -8, and -9 activity was significantly increased in GRIM-19-expressing cells compared to untransfected cells after radiation. Finally, we demonstrate that expression of GRIM-19 in BGC-803 cells suppresses accumulation of STAT3. Collectively, these data show that GRIM-19 expression sensitizes BGC-803 cells to radiation, and this is likely due to suppression of STAT3 accumulation. In summary, our results indicate that GRIM-19 expression might be a useful therapy to enhance apoptosis in gastric cancer cells in response to radiation treatment.     

A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus

STAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protein. Tyrosine phosphorylation allows the normally cytoplasmic STAT proteins to enter the nucleus and bind to their recognition sequences in responsive promoters. The ability of LMP1 to activate STAT3 was further established by immunofluorescence assays in which coexpression of LMP1 in transfected cells was sufficient to mediate nuclear relocalization of Flag-STAT3 and by an electrophoretic mobility shift assay which showed that LMP1 expression in CNE2-LNSX cells was associated with increased endogenous STAT3 DNA binding activity. In addition, the activity of a downstream target of STAT3, c-Myc, was upregulated in HeLa-Bx1 and CNE2-LMP1 cells. A linkage was established between interleukin-6 (IL-6)- and LMP1-mediated STAT3 activation. Treatment with IL-6 increased phosphorylated STAT3 levels in CNE2-LNSX cells, and conversely, treatment of CNE2-LMP1 cells with IL-6 neutralizing antibody ablated STAT3 activation and c-Myc upregulation. The previous observation that STAT3 activated the LMP1 terminal repeat promoter in reporter assays was extended to show upregulated expression of endogenous LMP1 mRNA and protein in HeLa-Bx1 cells transfected with a constitutively activated STAT3. A model is proposed in which EBV infection of an epithelial cell containing activated STATs would permit LMP1 expression. This in turn would establish a positive feedback loop of IL-6-induced STAT activation, LMP1 and Qp-EBNA1 expression, and viral genome persistence.     

Inhibitory effects and underlying mechanism of 7-hydroxyflavone phosphate ester in HeLa cells

Chrysin and its phosphate ester have previously been shown to inhibit cell proliferation and induce apoptosis in Hela cells; however, the underlying mechanism remains to be characterized. In the present study, we therefore synthesized diethyl flavon-7-yl phosphate (FP, C(19)H(19)O(6)P) by a simplified Atheron-Todd reaction, and explored its anti-tumor characteristics and mechanisms. Cell proliferation, cell cycle progression and apoptosis were measured by MTS, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling techniques, respectively in human cervical cancer HeLa cells treated with 7-hydroxyflavone (HF) and FP. p21, proliferating cell nuclear antigen (PCNA) and cAMP levels in Hela cells were analyzed by western blot and radioimmunoassay. Both HF and FP inhibited proliferation and induced apoptosis in HeLa cells via induction of PCNA/p21 expression, cleaved caspase-3/poly (ADP-ribose) polymerase (PARP)-1, elevation of cAMP levels, and cell cycle arrest with accumulation of cells in the G0/G1 fraction. The effects of FP were more potent than those of HF. The interactions of FP with Ca(2+)-calmodulin (CaM) and Ca(2+)-CaM-phosphodiesterase (PDE)1 were explored by electrospray ionization-mass spectrometry and fluorescence spectra. FP, but not HF, formed non-covalent complexes with Ca(2+)-CaM-PDE1, indicating that FP is an inhibitor of PDE1, and resulting in elevated cellular cAMP levels. It is possible that the elevated cAMP levels inhibit growth and induce apoptosis in Hela cells through induction of p21 and cleaved caspase-3/PARP-1 expression, and causing down-regulation of PCNA and cell cycle arrest with accumulation of cells in the G0/G1 and G2/M fractions. In conclusion, FP was shown to be a Ca(2+)-CaM-PDE inhibitor, which might account for its underlying anti-cancer mechanism in HeLa cells. These observations clearly demonstrate the special roles of phosphorylated flavonoids in biological processes, and suggest that FP might represent a potential new drug for the therapy of human cervical carcinoma.     

Metformin-induced stimulation of adenosine 5' monophosphate-activated protein kinase (PRKA) impairs progesterone secretion in rat granulosa cells

Metformin is an anti-diabetic drug commonly used to treat cycle disorders and anovulation in women with polycystic ovary syndrome. However, the effects and molecular mechanism of metformin in the ovary are not entirely understood. We investigated the effects of this drug on steroidogenesis and proliferation in rat granulosa cells. Metformin (10 mM) treatment for 48 h reduced progesterone and estradiol (E2) production in both basal conditions and under FSH stimulation. It also decreased the levels of the HSD3B, CYP11A1, STAR, and CYP19A1 proteins in response to FSH (10(-8) M) and of HSD3B in the basal state only. Metformin treatment (10 mM, 24 h) also reduced cell proliferation and the levels of CCND2 and CCNE proteins without affecting cell viability, both in the basal state and in response to FSH. Furthermore, metformin treatment for 1 h simultaneously increased the Thr172 phosphorylation of PRKAA (adenosine 5' monophosphate-activated protein kinase alpha) and the Ser79 phosphorylation of ACACA (acetyl-Coenzyme A carboxylase alpha). The adenovirus-mediated production of dominant-negative PRKAA totally abolished the effects of metformin on progesterone secretion, HSD3B and STAR protein production, and MAPK3/1 phosphorylation. Conversely, total inhibition of PRKAA Thr172 phosphorylation with the dominant-negative PRKAA adenovirus did not restore the decrease in E2 production and cell proliferation induced by metformin. Our results therefore strongly suggest that metformin reduces progesterone production via a PRKAA-dependent mechanism, whereas PRKAA activation is not essential for the decrease in E2 production and cell growth induced by metformin in rat granulosa cells.     

GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

Background

Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis.

Methodology/Principal Findings

The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model.

Conclusion/Significance

The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data suggest that GRIM-19 can block E6/E6AP complex; and synergistically suppress cervical tumor growth with p53.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

Long noncoding RNA H19 participates in metformin-mediated inhibition of gastric cancer cell invasion

Recent research suggests that the first-line oral antidiabetes drug metformin may prevent gastric cancer progression and improve prognosis. Many studies have also shown that long noncoding RNAs (lncRNAs) play important roles in many biological processes. Therefore, we aimed to explore whether lncRNAs participate in the mechanisms by which metformin affects gastric cancer cells. In the current study, we found that metformin significantly inhibited the cellular functions of gastric cancer cells through Cell Counting Kit-8 and invasion assays. We found that lncRNA H19 was greatly downregulated in gastric cancer cells treated with metformin using lncRNA microassays. Based on bioinformatics analyses of the Oncomine and The Cancer Genome Atlas databases, H19 is shown to be overexpressed in gastric cancer tissues, with increased expression of H19 relating to advanced pathological tumor stage and pathological tumor node metastasis stage, indicating that H19 may be associated with the invasive ability of gastric cancer. We knocked down H19 in AGS and SGC7901 cell lines and found that knocked-down H19 could decrease gastric cancer cell invasion and that metformin could not further decrease invasion after the knock down. Moreover, H19 depletion increased AMPK activation and decreased MMP9 expression, and metformin could not further activate AMPK or decrease MMP9 in H19 knocked-down gastric cancer cells. In summary, metformin has a profound antitumor effect on gastric cancer cells, and H19 is a key component in the process of metformin suppressing gastric cancer cell invasion.     

Ⅰ型磷酸酶抑制亚基-1对人宫颈癌HeLa细胞增殖的抑制作用

PPI1(Inhibitor-1 ofprotein phosphatase 1)是I型磷酸酶的抑制亚基之一,其活化依赖于35位苏氨酸蛋白激酶(PKA)的磷酸化而发挥抑制作用.本研究目的在于探讨PPIl持续活化型突变体的表达对人宫颈癌细胞株增殖的影响及其可能的作用机制.利用PPI1野生型和活化型突变体表达质粒分别转染HeLa细胞,首先通过H~3TdR掺入实验、迁移实验观察PPI1基因对HeLa细胞增殖能力的影响,研究结果表明:在活化型突变体表达的HeLa细胞株中,细胞~3H掺入量和迁移能力明显受抑.其次通过流式细胞术、Giemsa染色法分析PPIl对HeLa细胞的细胞周期的影响;FACS分析表明HeLa细胞G2/M期比例明显升高;经脱氧胸苷同步化后,该组细胞进入有丝分裂期明显滞后.最后利用Western blot分析PPI1对MAPK信号转导通路的影响,Western blot分析显示该组细胞的ERK磷酸化水平明显下降.研究表明PPI1活化型突变体的表达可抑制人宫颈癌细胞株的增殖,这与其诱导HeLa细胞G2/M期停滞、有丝分裂的进入延缓有关,其中涉及到MAPK信号转导通路的活化受抑制.     

GRIM-19: A master regulator of cytokine induced tumor suppression,metastasis and energy metabolism

Cytokines induce cell proliferation or growth suppression depending on the context. It is increasingly becoming clear that success of standard radiotherapy and/or chemotherapeutics to eradicate solid tumors is dependent on IFN signaling. In this review we discuss the molecular mechanisms of tumor growth suppression by a gene product isolated in our laboratory using a genome-wide expression knock-down strategy. Gene associated with retinoid-IFN-induced mortality −19 (GRIM-19) functions as non-canonical tumor suppressor by antagonizing oncoproteins. As a component of mitochondrial respiratory chain, GRIM-19 influences the degree of “Warburg effect” in cancer cells as many advanced and/or aggressive tumors show severely down-regulated GRIM-19 levels. In addition, GRIM-19 appears to regulate innate and acquired immune responses in mouse models. Thus, GRIM-19 is positioned at nodes that favor cell protection and/or prevent aberrant cell growth.     

泛素偶联酶2C促进肺癌细胞增殖和迁移与抑制细胞衰老

泛素偶联酶2C与多种肿瘤细胞的增殖密切相关,但其与肺癌发生和发展的关系尚不明确。 本研究以肺癌A549细胞为材料,通过RT-PCR、Western印迹、免疫荧光、SA-β-Gal细胞衰老染色、细胞划痕和Trans-well实验,阐明UBE2C与肺癌细胞的增殖、衰老和迁移能力的关系。结果显示,UBE2C在肺癌细胞中的表达明显高于正常细胞。利用基因修饰技术瞬时过表达或靶向沉默UBE2C后,在肺癌A549细胞中,UBE2C的mRNA和蛋白质水平显著增加3.5倍或减少0.5倍,显著促进或抑制细胞增殖,进而减少或增加细胞的凋亡率。过表达UBE2C后,显著抑制细胞衰老;但沉默UBE2C后,则增加细胞衰老。此外,过表达UBE2C后,下调转移相关基因E-钙黏着蛋白的mRNA和蛋白质表达水平,且上调波形蛋白基因的表达水平,进而促进肺癌细胞的迁移。但靶向敲除UBE2C后,上调E-钙黏着蛋白,同时下调波形蛋白表达水平,进而抑制肺癌细胞的迁移。本研究的开展将明确UBE2C在肺癌中的作用及其机制,为以UBE2C为靶点,提高病人生存期提供了理论基础。     

髓样分化蛋白2基因沉默对高糖诱导的大鼠心肌细胞增殖抑制、凋亡及炎症反应的影响*

目的:研究髓样分化蛋白2(MD2)基因沉默对高糖(HG)诱导的大鼠心肌细胞增殖抑制、凋亡及炎症反应的影响及其机制。方法:体外大鼠心肌细胞系H9C2细胞随机分为4组(n=3):LG组、HG组、HG + NC组、HG + si-MD2组,分别转染MD2基因小干扰RNA(si-MD2)或阴性对照24 h后进行低糖或高糖处理48 h。RT-qPCR检测MD2及细胞内炎症细胞因子TNF-α、IL-1β、IL-6的表达水平,MTS法、流式细胞术检测细胞增殖能力、细胞周期和细胞凋亡率,Western blot法检测细胞内相关蛋白的表达水平及磷酸化水平。结果:转染si-MD2后,H9C2细胞中MD2的表达水平明显下降(P＜0.01)。与低糖(LG)组比较,高糖处理后的H9C2细胞中TNF-α、IL-1β、IL-6的mRNA水平显著升高,细胞增殖能力下降并发生G1期阻滞,细胞凋亡率和Cleaved Caspase-3蛋白水平升高(P＜ 0.01)。而MD2基因沉默可拮抗高糖对H9C2细胞增殖、细胞周期、凋亡及细胞中TNF-α、IL-1β、IL-6 mRNA水平的影响(P＜0.05)。Western blot测定结果表明高糖处理后的H9C2细胞中细胞外信号调节激酶(ERK1/2)、P38丝裂原活化蛋白激酶(P38 MAPK)和C-Jun氨基末端激酶(JNK)蛋白的磷酸化水平明显升高,而MD2基因沉默可抑制高糖诱导下的ERK1/2、P38 MAPK和JNK蛋白激活(P＜0.01)。结论:MD2基因沉默可能通过抑制ERK、P38 MAPK和JNK信号通路的激活来减少高糖诱导的大鼠心肌细胞炎症细胞因子表达,减少心肌细胞凋亡,促进细胞增殖。     

二氢杨梅素对高糖诱导的H9C2心肌细胞损伤的影响

目的探讨二氢杨梅素（DHM）对高糖（HG）诱导的心肌细胞H9C2损伤的影响及机制。 方法细胞处理分为对照组、35 mmol/L HG组、35mmol/L HG+50 μmol/L DHM组及50 μmol/L DHM组。CCK-8法检测细胞活力,化学比色法检测丙二醛（MDA）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）水平,流式细胞术检测ROS水平;荧光定量PCR法及Elisa法分别检测TNFα、IL1β、IL6 mRNA和含量,Western Blotting检测p-IκBα、IκBα蛋白及核蛋白NF-κB p65的表达水平。采用单因素方差分析进行组间比较。 结果对照组、35mmol/?L HG组、35?mmol/L HG+50?μmol/L DHM组、35?mmol/L HG+100?μmol/L DHM组的细胞活力分别是（100±0.00） ﹪、（52.23±5.69） ﹪、（74.58±6.12） ﹪和（86.04±3.76）﹪,差异具有统计学意义（F?= 40.61,P?< 0.01）。对照组、35?mmol/L HG组和35?mmol/L HG+100?μmol/L DHM组的MDA和ROS水平,SOD和CAT活性分别是（0.44±0.06）?nmol/?ml,（2.33±0.40）?nmol/?ml,（1.48±0.41）?nmol/ml、（156.0±9.00）U/ml,（325.3±10.69）U/ml,（244.0±9.54）?U/ml,（10.62± 1.59）?U/?ml,（5.18±0.34）U/ml,（7.75±0.53）U/ml,（11.31±0.98）?U/ml,（5.20±1.12）?U/?ml和（8.06±0.66）U/ml,差异具有统计学意义（F?= 30.34,29.75,14.72,P均< 0.01）。DHM预处理可明显拮抗HG对H9C2心肌细胞TNFα、IL1β和IL6 mRNA及含量的上调作用,差异存在统计学意义（P?均< 0.01）。DHM可抑制HG对H9C2心肌细胞p-IκBα/?IκBα蛋白和核蛋白NF-κB p65表达的增加作用,差异存在统计学意义（P均< 0.01）。 结论DHM可拮抗HG诱导的H9C2心肌细胞损伤,这可能与其抑制NF-κB信号通路有关。