Silencing of ATG4D suppressed proliferation and enhanced cisplatin-induced apoptosis in hepatocellular carcinoma through Akt/Caspase-3 pathway

ATG4D, a member of autophagy-related protein 4 (ATG4) family, plays an interplay role between autophagy and apoptosis in cancers. However, the role of ATG4D in hepatocellular carcinoma (HCC) has not been defined. Herein, this study aimed to investigate the role and the underlying mechanism of ATG4D in regulating HCC cell apoptosis. ATG4D was silenced in MHCC-97L HCC cells, and then cell proliferation and apoptosis were examined. ATG4D expression was significantly upregulated in HCC tissues when compared with paired non-tumor tissues. In vitro assays revealed that silencing of ATG4D significantly suppressed cell proliferation, promoted cell apoptosis, and enhanced sensitivity to cisplatin of MHCC-97L cells. Furthermore, silencing of ATG4D decreased the phosphorylation of Akt and increased the protein level of caspase-3. Taken together, ATG4D may play an oncogenic role in HCC progression. These findings suggest that ATG4D may serve as a therapeutic target for HCC therapy.

     

Overexpression of RhoGDI,a novel predictor of distant metastasis,promotes cell proliferation and migration in hepatocellular carcinoma

RhoGDI (Rho GDP-dissociation inhibitor alpha, or RhoGDIα) was identified as a regulator of Rho GTPases, but its role in cancer remains controversial. In this study, increased expression of RhoGDI was detected in hepatocellular carcinoma (HCC) cell lines and tissues with highly metastatic potential. RhoGDI overexpression correlated with postoperative distant metastasis. Enforced expression of RhoGDI in HCC cells significantly enhanced cell proliferation and migration. Conversely, knockdown of RhoGDI caused an inhibition of the aggressive phenotypes of HCC cells. Furthermore, RhoGDI up-regulated Rho, but not Rac, and enhanced PI3K/AKT and MAPK pathway activity. Our findings suggest that RhoGDI overexpression is a predictor of distant metastasis and plays an important role in the progression of HCC.     

基于CRISPR转录激活系统构建内源性着丝粒蛋白F稳定过表达的肝细胞癌细胞模型

临床研究表明着丝粒蛋白F(centromere protein F,CENPF)与肝细胞癌(hepatocellular carcinoma,HCC)的发生相关。由于CENPF蛋白的分子量高达358 kDa,目前有关CENPF致癌机制研究使用的体内外模型主要基于CENPF表达敲低,尚缺乏将CENPF过表达以探究其与癌变因果关系的研究,CENPF过表达改变到底是“因”还是“果”仍不清楚。本研究旨在利用包括lentiMPHv2和lentiSAMv2两个载体的CRISPR/dCas9转录激活协同激活介体(synergistic activation mediator,SAM)系统构建内源性CENPF稳定过表达的细胞模型,为阐明CENPF过表达与HCC发生发展的因果关系提供有效工具。首先设计并合成能够特异性识别CENPF基因转录起始位点的单向导核糖核酸(single guide RNAs,sgRNA),并将其插入lentiSAMv2质粒中。利用慢病毒载体将lentiMPHv2质粒导入Huh-7和HCCLM3细胞株中,用潮霉素B筛选后再用慢病毒载体将连接好的携带sgRNA序列的lentiSAMv2质粒导入细胞中,并用杀稻瘟菌素S继续筛选。对2种抗生素筛选获得的Huh-7和HCCLM3细胞株样品进行CENPF mRNA和蛋白表达水平的检测,结果显示sgRNA1和sgRNA4序列均能够激活内源CENPF的表达,其中sgRNA4诱导转录效果更加明显。本研究利用CRISPR/dCas9系统成功构建内源性CENPF稳定过表达的HCC细胞模型,为研究CENPF过表达与肿瘤发生的因果关系奠定基础,并为构建大分子量蛋白过表达的细胞模型提供参考方案。     

DLC-1与肝癌关系研究

DLC-1基因是一种肿瘤抑制基因,位于人类染色体8p21．3-22。它是RhoA特异性GTP酶的激动蛋白,与调控细胞增殖和粘附的信号传导通路关系密切,在人类多种肿瘤中呈低表达或表达缺失。研究发现DLC-1基因在原发性肝癌(HCC)及肝癌细胞系中表达缺失,提示该基因在原发性肝癌中抑制了肝癌细胞的增殖。DLC-1表达的恢复引起了caspase-3介导的细胞凋亡,抑制肝癌细胞的生长和癌细胞的浸润,从而在肝细胞癌的转移、侵袭及肿瘤细胞的生物特性方面发挥作用。因其与肝癌发生,转移乃至复发关系密切,使其在肝癌早期发现,早期预测肝癌的转移复发及肝癌的预后方面发挥重要角色。     

Delineation of the effects of angiotensin type 1 and 2 receptors on HL-1 cardiomyocyte apoptosis

Angiotensin II (Ang II) exerts its effects by activating its receptors, primarily type 1 (AT1R) and type 2 (AT2R). While the role of AT1R activation in cardiomyocyte physiology is well known, the role of AT2R in cardiomyocyte apoptosis remains controversial. To define the precise role of AT1R and AT2R in this process, we transfected HL-1 cardiomyocytes with AT1R or AT2R cDNA, and examined markers of apoptosis. We found that AT1R overexpression was associated with upregulation of endogenous AT2R expression, but AT2R overexpression did not affect endogenous AT1R expression. Caspase-3 staining indicated that overexpression of AT1R as well as AT2R resulted in cardiomyocyte apoptosis with appropriate alterations in annexin V, Bax and Bcl2 expression. Overexpression of AT1R and AT2R markedly increased IL-1β (AT2R>AT1R), iNOS (AT2R>AT1R) and eNOS expression. AT2R-induced cell apoptosis could be blocked by the iNOS selective inhibitor 1,400?W, and did not require exogenous Ang II. These findings suggest that AT2R overexpression induces cardiomyocyte apoptosis, most likely via iNOS upregulation. AT1R-mediated cardiomyocyte apoptosis may be partially mediated by upregulation of endogenous AT2R.     

Potentiation of the antihypertensive action of losartan by peripheral overexpression of the ANG II type 2 receptor

Our previous studies demonstrated that peripheral overexpression of angiotensin II (ANG II) type 2 receptors (AT(2)R) prevents hypertension-induced cardiac hypertrophy and remodeling without altering high blood pressure. This, coupled with the observations that AT(2)R play a role in the antihypertensive actions of ANG II type 1 receptor (AT(1)R) blockers (ARBs), led us to propose that peripheral overexpression of AT(2)R would improve the antihypertensive action of losartan (Los) in Sprague-Dawley (SD) rats made hypertensive via chronic infusion of ANG II. Here we utilized adenoviral vector-mediated AT(2)R gene transfer to test this hypothesis. A single intracardiac injection of adenoviral vector containing genomic AT(2)R (G-AT(2)R) DNA and enhanced green fluorescent protein (EGFP) gene controlled by cytomegalovirus (CMV) promoters (Ad-G-AT(2)R-EGFP; 5 x 10(9) infectious units) into adult SD rats produced robust AT(2)R overexpression in cardiovascular tissues (kidney, lung, heart, aorta, mesenteric artery, and renal artery) that persisted for 3-5 days postinjection. By 7 days post viral injection, the overexpressed AT(2)R are reduced toward basal values in certain tissues (lung, kidney, and heart) and are undetectable in others (kidney and blood vessels). In two separate protocols, we demonstrated that the hypotensive effect of Los (0.125, 0.5, and 1.0 mg/kg iv) was significantly greater in the AT(2)R-overexpressing animals (-40.7 +/- 4.3, -41.8 +/- 4.8, and -48.1 +/- 2.6 mmHg, respectively) compared with control vector (Ad-CMV-EGFP)-treated rats (-12.4 +/- 2.2, -20.2 +/- 3.4, and -27.3 +/- 3.4 mmHg, respectively). These results provide support for a depressor role of AT(2)R and the proposal that combined AT(2)R agonist and ARB treatment may be an improved therapeutic strategy for controlling hypertension.     

Mitochondria-dependent caspase-9 activation is necessary for antigen receptor-mediated effector caspase activation and apoptosis in WEHI 231 lymphoma cells

Engagement of the B cell Ag receptor (BCR) on immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. Previously, we have shown that cross-linking CD40 on B cells stimulates the expression of A1, an antiapoptotic member of the Bcl-2 family, and that transduction of the murine B lymphoma line WEHI 231, a model for immature B cells, with A1 protected the cells against BCR-induced apoptosis. Here we demonstrate that A1 strongly interferes with activation of caspase-7, the major effector caspase activated after BCR cross-linking on WEHI 231 lymphoma cells. The pathway leading to activation of the effector caspase cascade including caspase-7 is unclear. Using retrovirally transduced WEHI 231 cell populations, we show that a catalytically inactive mutant of caspase-7 is cleaved almost as efficiently as the wild-type form, arguing against autocatalysis as the sole activating process. In contrast, overexpression of catalytically inactive caspase-9 strongly interferes with caspase-7 processing, poly(ADP-ribose) polymerase cleavage, and DNA laddering, suggesting a role for caspase-9 and hence for the mitochondrial pathway. The importance of the mitochondrial/caspase-9 pathway for BCR-triggered apoptosis is highlighted by our finding that both A1 and the mutant caspase-9 attenuate BCR-induced apoptosis. Thus, our data suggest that the BCR-mediated apoptotic signal in immature B cells spreads via a mitochondrial/caspase-9 pathway.     

甘氨鹅脱氧胆酸钠通过MAPK/ERK1/2介导Bcl-2在Ser70位点的磷酸化引起人肝细胞癌的抗药

B细胞淋巴瘤-2（Bcl-2）是一种重要的抗凋亡蛋白质,在多种人类肿瘤中普遍过表达。甘氨鹅脱氧胆酸钠（GCDA）与消化道肿瘤的发生发展密切相关,并能介导肝癌细胞对化疗药物的抵抗。本文旨在探讨在GCDA介导的人肝细胞癌（HCC）耐药性中Bcl-2的作用及其机制。本研究以肝癌细胞系为研究对象,Western印迹结果显示,Bcl-2在多种肝癌细胞系中均有表达。设计靶向Bcl-2的siRNA沉默HCC细胞系内源性Bcl-2的表达,发现Bcl-2沉默之后促进了化疗药物5-FU介导的HCC细胞凋亡。机制上,GCDA可介导Bcl-2在Ser70位点的磷酸化,而Ser70位点的磷酸化能够被PD98059（MAPK/ERK1/2抑制剂）所抑制。构建huBcl2-WT和huBcl2-S70A真核表达载体,脂质体转染HCC细胞系。用Annexin V-FITC/PI流式细胞术检测凋亡细胞。结果显示,huBcl2-WT过表达能抑制5 FU介导的凋亡,S70位点失活突变成A后,Bcl-2的过表达不能抑制5-FU介导的凋亡。本研究提示,GCDA通过MAPK/ERK1/2通路介导的Bcl-2 Ser70位点的磷酸化,在肝癌细胞的存活和抗药中发挥重要作用。抑制Bcl-2能够促进化疗药物5-FU介导的HCC细胞凋亡,该结果为治疗GCDA介导的耐药性肝癌提供新的思路。     

Estradiol and Estrogen Receptor Agonists Oppose Oncogenic Actions of Leptin in HepG2 Cells

Obesity is a significant risk factor for certain cancers, including hepatocellular carcinoma (HCC). Leptin, a hormone secreted by white adipose tissue, precipitates HCC development. Epidemiology data show that men have a much higher incidence of HCC than women, suggesting that estrogens and its receptors may inhibit HCC development and progression. Whether estrogens antagonize oncogenic action of leptin is uncertain. To investigate potential inhibitory effects of estrogens on leptin-induced HCC development, HCC cell line HepG2 cells were treated with leptin in combination with 17 β-estradiol (E2), estrogen receptor-α (ER-α) selective agonist PPT, ER-β selective agonist DPN, or G protein-coupled ER (GPER) selective agonist G-1. Cell number, proliferation, and apoptosis were determined, and leptin- and estrogen-related intracellular signaling pathways were analyzed. HepG2 cells expressed a low level of ER-β mRNA, and leptin treatment increased ER-β expression. E2 suppressed leptin-induced HepG2 cell proliferation and promoted cell apoptosis in a dose-dependent manner. Additionally E2 reversed leptin-induced STAT3 and leptin-suppressed SOCS3, which was mainly achieved by activation of ER-β. E2 also enhanced ERK via activating ER-α and GPER and activated p38/MAPK via activating ER-β. To conclude, E2 and its receptors antagonize the oncogenic actions of leptin in HepG2 cells by inhibiting cell proliferation and stimulating cell apoptosis, which was associated with reversing leptin-induced changes in SOCS3/STAT3 and increasing p38/MAPK by activating ER-β, and increasing ERK by activating ER-α and GPER. Identifying roles of different estrogen receptors would provide comprehensive understanding of estrogenic mechanisms in HCC development and shed light on potential treatment for HCC patients.     

Lipocalin-2 Induces Apoptosis in Human Hepatocellular Carcinoma Cells Through Activation of Mitochondria Pathways

Lipocalin 2 (LCN2) is a secreted, iron-binding glycoprotein that is abnormally expressed in some malignant human cancers. However, the roles of LCN2 in hepatocellular carcinoma (HCC) cells are unknown. In this study, we suggested the LCN2 and LCN2R were weak detected in the HCC cell lines, LCN2 and LCN2R were found to be down-regulated in tumor tissues in 16 HCC patients. MTT, DAPI, TUNEL, and flow cytometry analyses revealed that LCN2 overexpression dramatically inhibited cell viability, induced apoptosis features of cell-cycle arrest in sub-G1 phase, in DNA fragmentation, and in condensation of chromatin in Huh-7 and SK-Hep-1 cells. Western blots were used to detect the activation of caspase, pro-apoptosis, and anti-apoptosis protein expression in overexpress-LCN2 HCC cells. LCN2-induced apoptosis was characterized by cleavage of caspase-9, -8, -3, and PARP protein, and a reduction in the mitochondrial membrane potential (MMP). Furthermore, LCN2 also enhanced the down-regulated Bcl-2 and up-regulated the expression of Bax. In addition, our experiments with caspase inhibitors LEHD-FMK and IETD-FMK prevent LCN2-induced apoptosis. We also demonstrated that treatment of overexpress-LCN2 HCC cells with the LCN2 neutralized antibody also significantly attenuated LCN2-induced cell apoptosis. These findings indicate that LCN2 overexpression can effectively induce apoptosis of HCC cells and may be used as a potent therapy against human HCC.     

Long Non-coding RNA URHC Regulates Cell Proliferation and Apoptosis via ZAK through the ERK/MAPK Signaling Pathway in Hepatocellular Carcinoma

Long non-coding RNAs (lncRNAs) have previously been implicated in human disease states, especially cancer. Although the aberrant expression of lncRNAs has been observed in cancer, the biological functions and molecular mechanisms underlying aberrantly expressed lncRNAs in hepatocellular carcinoma (HCC) have not been widely established. In the present study, we investigated a novel lncRNA, termed URHC (up-regulated in hepatocellular carcinoma), and evaluated its role in the progression of HCC. Expression profiling using a lncRNA microarray revealed that URHC was highly expressed in 3 HCC cell lines compared to normal hepatocytes. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses confirmed that URHC expression was increased in hepatoma cells and HCC tissues. Moreover, using qRT-PCR, we confirmed that URHC expression was up-regulated in 30 HCC cases (57.7%) and that its higher expression was correlated with poor overall survival. We further demonstrated that URHC inhibition reduced cell proliferation and promoted apoptosis. We hypothesize that URHC may function by regulating the sterile alpha motif and leucine zipper containing kinase AZK (ZAK) gene, which is located near URHC on the same chromosome. We found that ZAK mRNA levels were down-regulated in HCC tissues and the expression levels of ZAK were negatively correlated with those of URHC in the above HCC tissues. Next, we confirmed that URHC down-regulated ZAK, which is involved in URHC-mediated cell proliferation and apoptosis. Furthermore, ERK/MAPK pathway inactivation partially accounted for URHC-ZAK-induced cell growth and apoptosis. Thus, we concluded that high URHC expression can promote cell proliferation and inhibit apoptosis by repressing ZAK expression through inactivation of the ERK/MAPK pathway. These findings may provide a novel mechanism and therapeutic targets for the treatment of HCC.     

Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT(1)R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation of mouse iPS cells with AT(1)R may enhance LIF-induced DNA synthesis, by augmenting the generation of superoxide and activating JAK/STAT3, and that AT(1)R stimulation may enhance Col IV-induced differentiation into mesodermal progenitor cells via p38 MAPK activation.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.     

Modulatory efficacy of dieckol on xenobiotic-metabolizing enzymes,cell proliferation,apoptosis, invasion and angiogenesis during NDEA-induced rat hepatocarcinogenesis

Altered microRNA expression is associated with tumor proliferation, metastasis, and tumorigenesis. In this study, we studied the role of miR-3117 in hepatocellular carcinoma (HCC) cell proliferation and found that miR-3117 was upregulated in HCC tissues and cells. MTT assay, soft agar growth assay, BrdU assay, and cell cycle assay revealed that miR-3117 overexpression promoted HCC HepG2 cell proliferation and that knockdown of miR-3117 suppressed HepG2 proliferation. Mechanism analysis suggested PH domain and leucine-rich repeat protein phosphatase-like (PHLPPL) as the target of miR-3117. Luciferase reporter assay suggested that miR-3117 directly binds to the 3′UTR of PHLPPL. Double knockdown of miR-3117 and PHLPPL copied the phenotypes caused by miR-3117 overexpression, suggesting that miR-3117 contributes to the proliferation of HepG2 by targeting PHLPPL. Our study provided a target for HCC therapy.     

长链非编码RNA对HepG2 肝癌细胞增殖及凋亡的影响

目的:探讨长链非编码RNA RP13-514E23.1在肝癌细胞系中的表达,并观察上调该基因后其下游基因MAPK10的变化及对肝癌细胞Hep G2增殖、凋亡的影响。方法:通过荧光定量PCR(q RT-PCR)检测RP13-514E23.1在正常肝细胞与肝癌细胞系中的表达差异,进一步构建质粒转染肝癌Hep G2细胞上调RP13-514E23.1的表达,以Mock组(只加转染试剂)和NC组(转染空载体pc DNA3.1-NC)作为对照评估转染效率及对MAPK10表达的影响。用CCK-8实验和流式细胞术检测转染前后Hep G2细胞的增殖、凋亡的变化。结果:Lnc RNA RP13-514E23.1在大部分肝癌细胞系(Hep G2,SMMC,Huh7,Hep3B)中的表达明显低于正常肝细胞(0.58±0.05 vs 1.00,P0.05);转染pc DNA-RP13-514E23.1后,q RT-PCR检测Hep G2细胞的RP13-514E23.1和MAPK10m RNA表达量显著升高(分别为29.90±1.40、2.42±0.25,P0.05),western blot检测MAPK10蛋白表达量较对照组也升高(2.10±0.16,P0.05);CCK-8结果显示Hep G2细胞在各个时间段增殖均受到抑制(P0.01),Mock组、NC组和实验组的凋亡率分别为(5.53±1.17)%、(6.40±2.84)%和(46.87±3.45)%(P0.01)。结论:Lnc RNA RP13-514E23.1在肝癌细胞系中表达异常降低,上调其表达后MAPK10的表达升高,且Hep G2细胞增殖受到抑制、凋亡增加。     

Focal adhesion kinase,RhoA, and p38 mitogen-activated protein kinase modulates apoptosis mediated by angiotensin II AT2 receptors

Apoptosis plays an important role in cellular processes such as development, differentiation, and homeostasis. Although the participation of angiotensin II (Ang II) AT₂ receptors (AT ₂R) in cellular apoptosis is well accepted, the signaling pathway involved in this process is not well established. We evaluated the participation of signaling proteins focal adhesion kinase (FAK), RhoA, and p38 mitogen-activated protein kinase (p38MAPK) in apoptosis induced by Ang II via AT ₂R overexpressed in HeLa cells. Following a short stimulation time (120 to 240 minutes) with Ang II, HeLa-AT ₂ cells showed nuclear condensation, stress fibers disassembly and membrane blebbing. FAK, classically involved in cytoskeleton reorganization, has been postulated as an early marker of cellular apoptosis. Thus, we evaluated FAK cleavage, detected at early stimulation times (15 to 30 minutes). Apoptosis was confirmed by increased caspase-3 cleavage and enzymatic activity of caspase-3/7. Participation of RhoA was evaluated. HeLa-AT ₂ cells overexpressing RhoA wild-type (WT) or their mutants, RhoA V14 (constitutively active form) or RhoA N19 (dominant-negative form) were used to explore RhoA participation. HeLa-AT ₂ cells expressing the constitutively active variant RhoA V14 showed enhanced apoptotic features at earlier times as compared with cells expressing the WT variant. RhoA N19 expression prevented nuclear condensation/caspase activation. Inhibition of p38MAPK caused an increase in nuclear condensation and caspase-3/7 activation, suggesting a protective role of p38MAPK. Our results clearly demonstrated that stimulation of AT ₂R induce apoptosis with participation of FAK and RhoA while p38MAPK seems to play a prosurvival role.     

NADPH oxidase is involved in angiotensin II-induced apoptosis in H9C2 cardiac muscle cells: effects of apocynin

Angiotensin II stimulates NADPH oxidase activity in vascular cells. However, it is not fully understood whether angiotensin II, which plays an important role in heart failure, stimulates NADPH oxidase activation and expression in cardiac myocytes. Previous studies have shown that angiotensin II induces myocyte apoptosis, but whether the change is mediated via NADPH oxidase remains to be elucidated. In this study we proposed to determine whether angiotensin II stimulated NADPH oxidase activation and NADPH oxidase subunit p47-phox expression in H9C2 cardiac muscle cells. If so, we would determine whether the NADPH oxidase inhibitor apocynin prevented angiotensin II-induced apoptosis. The results showed that angiotensin II increased NADPH oxidase activity, p47-phox protein and mRNA expression, intracellular reactive oxygen species, and apoptosis in H9C2 cells. Angiotensin II elevated p38 mitogen-activated protein kinase (MAPK) activity, decreased Bcl-2 protein, and increased Bax protein and caspase-3 activity. Apocynin treatment inhibited angiotensin II-induced NADPH oxidase activation and increases in p47-phox expression, intracellular reactive oxygen species, and apoptosis. The effect of apocynin on apoptosis was associated with reduced p38 MAPK activity, increased Bcl-2 protein, and decreased Bax protein and caspase-3 activity. These results suggest that angiotensin II-induced apoptosis is mediated via NADPH oxidase activation probably through p38 MAPK activation, a decrease in Bcl-2 protein, and caspase activation.     

Overexpression of Ras Homologous C (RhoC) Induces Malignant Transformation of Hepatocytes In Vitro and in Nude Mouse Xenografts

Ras homologous C (RhoC) is expressed in various cancers, including hepatocellular carcinoma (HCC). In this study, we first analyzed RhoC expression in 46 HCC tissue specimens and found that RhoC expression was significantly increased in HCC tissues compared to the adjacent normal liver tissues. Next, we investigated the role of RhoC in malignant transformation of normal hepatocytes. The HL7702 cell line was stably transfected with a RhoC expression vector and then subjected to cell proliferation, differentiation, colony formation, migration and invasion assays, as well as nude mouse xenograft assays. Gene expressions in these cells were determined using RT-PCR and Western blot. Overexpression of RhoC significantly promoted proliferation and anchorage-independent growth of HL7702 cells, but suppressed cell differentiation, as compared with the parental cells and the empty vector-transfected control cells. Moreover, RhoC overexpression induced migration and invasion of HL7702 cells in vitro. Molecularly, RhoC increased the expression of cell cycle-related genes, matrix metalloprotease 2 (MMP2), MMP9 and vascular endothelial growth factor (VEGF). In addition, RhoC-transfected cells formed tumors in nude mice, whereas vector-transfected HL7702 cells did not form any tumors in nude mice. This study demonstrated the role of RhoC overexpression in malignant transformation of normal human hepatocytes, suggesting that RhoC may function as an oncogene in hepatocytes.     

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation,migration and malignant signalling events in hepatoma cells

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of β1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.