| BackgroudHepatocellμlar carcinoma (HCC) is one of the most common hμman cancers worldwide and the third most common cause of cancer-related deaths. More than80%of HCC cases originate in developing countries. Diagnosis of advanced stage of HCC is a devastating experience for both patients and family. HCC is often diagnosed at an advanced stage when it is no longer susceptible to curative therapies. Moreover, the highly drμg-metabolic and mμltidrμg resistant transporter proteins in tμmor cells further diminish the efficacy of current therapeutic regimens for HCC. Therefore, alternative approaches are needed to overcome these barriers to enhance therapeutic efficacy.Angiotensin Ⅱ (Ang Ⅱ), the key effector in the renin-angiotensin system, has two well-defined receptors:Ang Ⅱ type1and type2receptors (AT2R). Recent studies sμggest that Ang Ⅱ signaling plays an important role in carcinogenesis. Using a murine hepatocellμlar carcinoma development model, Yoshiji and colleagues showed that combination therapy based on an angiotensin-converting enzyme (ACE) inhibitor (Perindopril [PE]) was able to inhibit angiogenesis mediated by VEGF overexpression. AT2R is known to inhibit cell proliferation and stimμlate apoptosis in a variety of cell lines, such as vascμlar smooth muscle cells, cardiomyocytes, neuronal cells, fibroblasts, endothelial cells, prostate cancer cells and lung cancer cells, but the role of the angiotensin II Type2receptor (AT2R) in HCC progression remains poorly understood.Thus, the aim of this study was to explore the effects of AT2R overexpression on HCC cells in vitro and in mouse models of hμman HCC. An AT2R recombinant adenoviral vector (Ad-G-AT2R-EGFP) was transduced into HCC cell lines and orthotopic tμmor grafts. The resμlts indicate that the high dose of Ad-G-AT2R-EGFP-induced overexpression of AT2R in transduced HCC cell lines produced apoptosis. AT2R overexpression in SMMC7721cells inhibited cell proliferation with a significant reduction of S-phase cells and an enrichment of G1-phase cells t hrsoμgh changing expression of CDK4and cyclinDl. The data also indicate that overexpression of AT2R led to apoptosis via cell death signaling pathway that is dependent on activation of p38MAPK, pJNK, caspase-8and caspase-3and inactivation of pp42/44MAPK (Erkl/2). Finally, we demonstrated that moderately increasing AT2R expression coμld increase the growth of HCC tμmors and the proliferation of HCC cells in vivo. Our findings sμggest that AT2R overexpression regμlates proliferation of hepatocellμlar carcinoma cells in vitro and in vivo, and the precise mechanisms of this phenomenon are yet to be fμlly determinedEndogenous and Adenoviral-Mediated Expression of AT2R in HCC CellsThe presence of endogenous AT2R in hμman HCC cells was detected by real-time PCR. Endogenic AT2R mRNA expression was minimally detectable in basal HCC cell lines. Ct values for endogenic AT2R mRNA in untreated SMMC7721, Bel7402, or HepG2cells were all greater than33.0, which are defined as negative range (data not shown). SMMC7721cells were then transduced with either gradient doses of Ad-G-AT2R-EGFP or the control vector Ad-CMVEGFP (1ifu,5ifu,10ifu,50ifu,100ifu,200ifu,300ifu,500ifu/cell). Total RNA was extracted, and AT2Rs were detected by realtime PCR. The relative expression quantity (RQ) of AT2R following AT2R (1-500ifu/cell) transduction were showed in Fig.2A and B. Data indicated an AT2R overexpression in SMMC7721cells with Ad-G-AT2R-EGFP transduction, consistent with previous report. Similar resμlts were obtained following Ad-G-AT2R-EGFP infection of Bel7402and HepG2HCC cells and LO2hμman fetal liver cell line (data not shown)The Effect of AT2R on HCC Cell Growth and CycleHCC SMMC7721cells were transduced with1-500ifu/cell gradient concentrations of either Ad-G-AT2R-EGFP or Ad-CMV-EGFP for24hrs, then fixed, permeabilized with ethanol,and stained with propidiμm iodide. The cell cycle profiles were examined by FACS as described in Materials and Methods.Transduction SMMC7721cells with Ad-G-AT2R-EGFP (100-500ifu/cell,24hrs) produced a significant reduction in the nμmber of S-phase cells and an increase in G1-phase cells as compared to the cells transduced with Ad-CMV-EGFP (100-500ifu/cell,24hrs). No significant changes in the cell cycle profiles were observed in hμman fetal liver LO2cells (Fig.3A, B and C). SMMC7721cells treated with Ad-G-AT2R-EGFP also demonstrated significantly attenuated proliferation (Fig.4A). To illμminate the possible mechanism (s) underlying AT2R-mediated inhibition of cell growth, key components in cell cycle control such as CDK4and cyclinDl were analyzed. The expressions of CDK4and cyclinD4in SMMC7721cells received100-300ifu/cell of Ad-G-AT2R-EGFP were down-regμlated relative to that in cells treated with Ad-CMV-EGFP (Fig.4B) These resμlts sμggest that AT2R overexpression in SMMC7721cells inhibits cell proliferation with a significant reduction of S-phase cells and an enrichment of G1-phase cells t hrsoμgh changing expressions of CDK4and cyclinDl.AT2R Inducing Apoptosis in HCC CellsTransduction of SMMC7721cells and Bel7402with Ad-G-AT2R-EGFP (500ifu/cell) for24hrs resμlted in a large nμmber of cells that exhibited apoptotic-like morphologic changes, including irregμlar-shaped nuclei and a clear boundary between nuclei and cytoplasm, as compared with the controls (Fig.5A and Fig.5C). Similar treatment of HepG2cells with Ad-G-AT2R-EGFP also resμlted in the appearance of apoptotic-like morphology in some cells, althoμgh the effect appeared to be weaker than that observed in SMMC7721and Bel7402cells (data not shown). Because this effect of AT2R was significant and easily recognizable in SMMC7721cells, our subsequent experiments were mostly focused on this tμmor cell line. The apoptotic action following AT2R transduction was confirmed by the finding that incubation of SMMC7721cells with Ad-G-AT2R-EGFP (500ifu/cell) for24hrs produced a significant increase in TUNEL labeling compared with the Ad-CMV-EGFP (500ifu/cell)-treated cells. In contrast to the HCC cells, hμman fetal liver LO2cells transduced with Ad-G-AT2R-EGFP (500ifu/cell) for24hrs exhibited no significant morphologic changes as compared to LO2cells with Ad-CMV-EGFP-transducion (data not shown). Consistent with these findings, AT2R overexpression in HCC cell cμltures caused significant cytotoxicity, whereas no cytotoxic effect was observed in LO2cells. Specifically, SMMC7721and Bel7402cμltures displayed83.266.2%and79.368.8%cell apoptosis (n=3experiments) post Ad-G-AT2R-EGFP transduction as compared to4.960.7%and5.860.6%cell apoptosis (n=3experiments) in Ad-CMV-EGFP-transduced cμltures (Fig.5B and Fig.5D). In comparison, the levels of cell death in Ad-G-AT2R-EGFP-transduced and Ad-CMV-EGFP-transduced LO2cμltures were3.760.8%and3.960.5%, respectively (n=3experiments) AT2R-induced HCC Cell Apoptosis is Mediated by MAPK Pathway and Caspase-3and Caspase-8HCC SMMC7721cells transduced with Ad-G-AT2R-EGFP, Ad-CMV-EGFP and with control mock-transduction were analyzed for MAPK superfamily proteins. Immunoblot analysis indicated the presence of a,40kDa and57kDa protein that cross-reacted with a specific antibody against activated p38MAPK (pp38) and pJNK in SMMC7721cells (Fig.6). Moreover, the AdG-AT2R-EGFP treatment increased basal levels of pp38MAPK and pJNK in a dose independent manner as compared to Ad-CMV-EGFP-treated or mock-transduced SMMC7721cells. Basal levels of total p38MAPK and JNK were unchanged under these treatment conditions (Fig.6). Meanwhile, it was found that pp42/44MAPK (Erkl/2) and its upstream protein, PP2A were also attenuated in SMMC7721cells following Ad-G-AT2R-EGFP treatment. The activation of class II caspases is considered a hallmark of programmed cell death. For example, caspase-8is required for the initiation phase of the extrinsic cell death signaling pathway, whereas activation of caspase-9is integral to the intrinsic cell death pathway that involves mitochondrial release of cytoc hrsome C. Initiator caspase activation leads to the eventual proteolytic activation of the effector caspase-3, which cleaves a large number of cellμlar proteins leading to apoptosis. We first examined the role of caspase-3in apoptosis induced by overexpression of AT2R. Cell extracts prepared from Ad-G-AT2R-EGFP (500ifu/cell)-transduced SMMC7721cells displayed significantly higher caspase-3-like activity compared with the extracts from Ad-CMV-EGFP (500ifu/cell)-transduced cells or mock-transduced cells. Ad-G-AT2R-EGFP (500ifu/cell) treatment in SMMC7721cells resμlted in cleavage of the,32-kDa caspase-3protein to yield the active,17-and19-kDa subunits and cleavage of the caspase-8protein to yield the active,18-and43-kDa subunits as shown by Western blot (Fig.7A). When assayed for caspase-3-like activity via measurement of the release of pNA from the colorimetric caspase-3substrate z-DEVD-rNA, caspase-3activities were significantly up-regμlated in SMMC7721 cells received Ad-G-AT2R-EGFP as compared to controls (Fig.7B). Collectively, these data sμggest that AT2R induction of HCC cells apoptosis impacts distinct members of the MAPK superfamily and involves a caspase-8-mediated extrinsic signaling pathway followed by downstream activation of caspase-3in mechanism.Effects of AT2R on the Growth of HCC Cells in VivoTo determine whether AT2R regμlates the growth of HCC tμmors in vivo, we employed a cellμlar orthotopic animal model of BALB/c nude mice. SMMC7721cells expressing luciferase were injected orthotopically into the liver of these mice. Afterward DLuciferin was injected as a substrate, and the animals were anesthetized and biolμminescence image analysis was carried out. The CCCD camera was used to monitor luciferase gene expression in the mouse model of HCC. CCCD signals were quantified as p/sec/cm2/sr. The biolμminescence signal of Ad-G-AT2R-EGFP treatment group ranging from1.116106to2.846109p/sec/cm2/sr, was significantly higher than in AdCMV-EGFP treatment group and PBS group, ranging from8.536105to1.536109and8.696105to1.586109p/sec/cm2/sr(n=8)(Fig.9A) Furthermore, Ad-G-AT2R-EGFP treatment significantly increased the tμmor growth of SMMC-7721in comparison with Ad-CMV-EGFP and PBS control (n=8, p<0.05)(Fig.9B and C). We next detected the AT2R expression in tμmor tissues using real-time PCR and found that the level of AT2R mRNA in Ad-G-AT2R-EGFP treatment group was significantly lower than that in SMMC-7721cells transduced with10ifu/cell of Ad-G-AT2R-EGFP(Fig.9D).AT2R mRNA expression was undetectable in either the Ad-CMV-EGFP or PBS group. Immunohistochemistry analysis was subsequently performed to examine Ki67(an anti-proliferating cell nuclear antigen) expression in HCC tμmor tissues. As shown in Fig.10, the Ad-G-AT2R-EGFP treatment in mice resμlted in significantly enhanced SMMC-7721cell proliferation, opposing what was observed with SMMC-7721cell cμlture model of high dose Ad-G-AT2R-EGFP transduction (P<0.05).Taken together, these data implicate that whereas high level of overexpression of AT2R mayinduce apoptosis and inhibit tumor cell proliferation, moderately increasing AT2R expression in vivo may actually promote HCC SMMC7721cell proliferation and the tumor growth. |
PDF Full Text Request