Antitumor effects of hyaluronic acid inhibitor 4-methylumbelliferone in an orthotopic hepatocellular carcinoma model in mice

Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2-3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis.     

Notch1 signaling contributes to the oncogenic effect of HBx on human hepatic cells

Hepatocellular carcinoma (HCC) is a malignant tumor and hepatitis B virus X protein (HBx) plays a crucial role in its pathogenesis. The Notch1 signaling pathway is involved in various malignant tumors including liver cancers and down-regulation of Notch-1 may exert anti-tumor effects. Here, we demonstrate that inhibition of Notch1 by plasmid-based shRNA suppresses growth of human hepatic cells transfected with HBx through G0/G1 cell cycle arrest and apoptosis inhibition, possibly linked to the promoted expression of cyclin-dependent kinase inhibitor, P16, and decreased expression of apoptosis inhibitor, Bcl-2. The anti-proliferative and pro-apoptotic effects of Notch1 shRNA in HBx-transformed L02 cell may be partly mediated by down-regulation of nuclear factor-kappaB (NF-κB) binding activities, demonstrating possible cross-talk between Notch-1 and NF-κB signaling pathways. The oncogene HBx may therefore induce malignant transformation of human hepatic cells via Notch1 pathway, indicating that Notch1 plays a crucial role in HBx-related liver cancer and could be an effective therapeutic target for HCC.     

Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related mortality in the world. Hepatitis C virus (HCV) is a major etiologic agent of HCC. A majority of HCV infections lead to chronic infection that can progress to cirrhosis and, eventually, HCC and liver failure. A common pathogenic feature present in HCV infection, and other conditions leading to HCC, is oxidative stress. HCV directly increases superoxide and H₂O₂ formation in hepatocytes by elevating Nox protein expression and sensitizing mitochondria to reactive oxygen species generation while decreasing glutathione. Nitric oxide synthesis and hepatic iron are also elevated. Furthermore, activation of phagocytic NADPH oxidase (Nox) 2 of host immune cells is likely to exacerbate oxidative stress in HCV-infected patients. Key mechanisms of HCC include genome instability, epigenetic regulation, inflammation with chronic tissue injury and sustained cell proliferation, and modulation of cell growth and death. Oxidative stress, or Nox proteins, plays various roles in these mechanisms. Nox proteins also function in hepatic fibrosis, which commonly precedes HCC, and Nox4 elevation by HCV is mediated by transforming growth factor β. This review summarizes mechanisms of oncogenesis by HCV, highlighting the roles of oxidative stress and hepatic Nox enzymes in HCC.     

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.     

Involvement of ADAMs in tumorigenesis and progression of hepatocellular carcinoma: Is it merely fortuitous or a real pathogenic link?

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and one of the most frequent types of cancer worldwide. It normally develops in patients with chronic liver disease, especially cirrhosis, although some cases without an apparent underlying liver disease have been reported. The pathogenesis of HCC is multi-factorial and complex. Hepatitis viruses are the main factors favoring the development of HCC. In fact, chronic inflammation associated with hepatitis C or B virus infection can lead to progressive liver fibrosis, cirrhosis and ultimately HCC. Chronic inflammation and liver fibrosis cause a continuous remodeling of the extracellular matrix (ECM), a dynamic process that involves several molecules including integrins and matrix processing enzymes. An increasing body of evidence indicates that ADAMs are involved in promoting tumor formation and progression of HCC. A Disintegrin And Metalloproteases (ADAMs) are a group of proteins belonging to the zinc protease superfamily. ADAMs are usually transmembrane proteins that contain disintegrin and metalloprotease domains and are, therefore, able to carry out both cell adhesion and protease activities. Soluble isoforms of ADAMs have also been discovered and characterized. In this review, we focus on the contribution of ADAM proteins to HCC tumorigenesis and cancer progression. The potential role of ADAMs as key modulators of tumor–stroma interactions during tumor progression, by means of the activities of their constituent domains, is also discussed.     

Depletion of Aurora A leads to upregulation of FoxO1 to induce cell cycle arrest in hepatocellular carcinoma cells

Aurora A kinase has drawn considerable attention as a therapeutic target for cancer therapy. However, the underlying molecular and cellular mechanisms of the anticancer effects of Aurora A kinase inhibition are still not fully understood. Herein, we show that depletion of Aurora A kinase by RNA interference (RNAi) in hepatocellular carcinoma (HCC) cells upregulated FoxO1 in a p53-dependent manner, which induces cell cycle arrest. Introduction of an RNAi-resistant Aurora A kinase into Aurora A-knockdown cells resulted in downregulation of FoxO1 expression and rescued proliferation. In addition, silencing of FoxO1 in Aurora A-knockdown cells allowed the cells to exit cytostatic arrest, which, in turn, led to massive cell death. Our results suggest that FoxO1 is responsible for growth arrest at the G₂/M phase that is induced by Aurora A kinase inhibition.     

Effect of Co-culturing both placenta-derived mesenchymal stem cells and their condition medium in the cancer cell (HepG2) migration,damage through apoptosis and cell cycle arrest

Human placental-derived mesenchymal stem cells (hPMSCs) are a promising candidate to inhibit the proliferation of hepatocellular carcinoma (HCC) cell lines such as HepG2. The effects of hPMSCs and their conditioned media on HepG2 are, however, still a mystery. As a result, the goal of this study was to look into the effects of hPMSCs and their conditioned media on HepG2 and figure out what was going on. Fluorescence-activated cell sorting and the MTT test were used to determine the percentage of cells that died (early apoptosis, late apoptosis). The DIO and DID colors were used to detect cell fusion and cell death in both cells. HepG2 cells were co-treated with hPMSCs or hPMSCs-conditioned medium (hPMSCs-CM) to reduce growth and promote apoptosis. Morphological changes were also seen in the 30 percent, 50 percent, and 60 percent cases. The secretion of cytokine was determined by the ELISA. Flow cytometry, caspase 9 immunofluorescence, qPCR (detection of Bax, Bcl-2, and β-catenin genes), western blot, and immunophenotyping revealed that treatment with hPMSCs or hPMSCs-CM caused HepG2 cell death through apoptosis (detection of caspase 9, caspase 3 protein). HepG2 cell cycle arrest could be induced by hPMSCs and hPMSCs-CM. Following treatment with hPMSCs or hPMSCs-CM, HepG2 cell development was stopped in the G0/G1 phase. These treatments also inhibited HepG2 cells from migrating, with the greatest effect when the highest ratio/concentration of hPMSCs (70%) and hPMSCs-CM were used (90%). Our findings indicated that hPMSCs and hPMSCs-CM could be promising treatment options for liver cancer. To elucidate the proper effect, more research on liver cancer-induced rat/mice is needed.     

细胞因子与酒精性肝病

酒精性肝病(alcoholic liver disease,ALD)的发生发展过程与体内多种细胞因子有关,尤其是肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和转化生长因子-β(transforming growth factor-β,TGF-β)在调节肝细胞的凋亡过程中具有重要作用。TNF-α可引起肝细胞凋亡与炎症反应等,抗TNF-α治疗能明显减轻酒精引起的肝损害;TGF-β具有增加细胞外基质的合成和抑制细胞外基质降解的作用,TGF-β1升高与肝纤维化密切相关。细胞因子可能是防治酒精性肝病的有效分子靶点。     

T‐LAK cell‐originated protein kinase is essential for the proliferation of hepatocellular carcinoma SMMC‐7721 cells

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and typically has poor prognosis. Like most cancers, altered gene expression was always associated with the induction and maintenance of HCC. Here, we reported that the expression level of T‐LAK cell‐originated protein kinase (TOPK) is significantly up‐regulated in human HCC samples and cell lines. The suppression of TOPK by short hairpin RNA in HCC cell line SMMC‐7721 caused cell cycle arrest and reduced cell growth and colony formation ability. Moreover, the tumor formation ability of the TOPK‐suppression cells was significantly impaired compared with the control cells in nude mice. In addition, the knockdown expression of TOPK reduced the AKT phosphorylation. Taken together, we unveiled a novel role of TOPK which acts as an important positive regulator in human HCC cell proliferation. Copyright © 2013 John Wiley & Sons, Ltd.     

Longikaurin A,a natural ent-kaurane,induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.     

肝细胞癌中抑癌基因的发现与研究进展

肝细胞癌（HCC）的发病率高、治疗效果差。HCC的发病机制复杂,主要有2类：肝炎、酒精、黄曲霉素、代谢紊乱引起的肝损伤,进而导致的肝硬化;致癌基因和抑癌基因的突变或对应染色体区域的扩增或缺失。细胞内一些信号通路参与了HCC的发生发展,包括RAF／MEK／ERK、P13K／AKT／mTOR、WNT/β-catenin、胰岛素样生长因子、肝细胞生长因子／c-MET、生长因子调节的血管新生等6类信号通路。抑癌基因通过调节信号通路而调节细胞增殖、细胞周期、细胞凋亡等对肿瘤的发生、发展起重要作用的过程。我们简要概述HCC相关的肿瘤抑制分子及其所在的信号通路及作用的分子机制。     

Intercellular communication among liver cells in the perisinusoidal space of the injured liver: Pathophysiology and therapeutic directions

Hepatic stellate cells (HSCs) in the perisinusoidal space are surrounded by hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and other resident immune cells. In the normal liver, HSCs communicate with these cells to maintain normal liver functions. However, after chronic liver injury, injured hepatocytes release several proinflammatory mediators, reactive oxygen species, and damage-associated molecular patterns into the perisinusoidal space. Consequently, such alteration activates quiescent HSCs to acquire a myofibroblast-like phenotype and express high amounts of transforming growth factor-β1, angiopoietins, vascular endothelial growth factors, interleukins 6 and 8, fibril forming collagens, laminin, and E-cadherin. These phenotypic and functional transdifferentiation lead to hepatic fibrosis with a typical abnormal extracellular matrix synthesis and disorganization of the perisinusoidal space of the injured liver. Those changes provide a favorable environment that regulates tumor cell proliferation, migration, adhesion, and survival in the perisinusoidal space. Such tumor cells by releasing transforming growth factor-β1 and other cytokines, will, in turn, activate and deeply interact with HSCs via a bidirectional loop. Furthermore, hepatocellular carcinoma-derived mediators convert HSCs and macrophages into protumorigenic cell populations. Thus, the perisinusoidal space serves as a critical hub for activating HSCs and their interactions with other cell types, which cause a variety of liver diseases such as hepatic inflammation, fibrosis, cirrhosis, and their complications, such as portal hypertension and hepatocellular carcinoma. Therefore, targeting the crosstalk between activated HSCs and tumor cells/immune cells in the tumor microenvironment may also support a promising therapeutic strategy.     

Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions

Background aimsHuman mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations.MethodsMSC aggregation and sphere formation was studied with the use of hanging drop cultures with medium containing fetal bovine serum or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with the use of gene expression and protein secretion measurements and with functional studies with the use of macrophages and cancer cells.ResultsMSCs did not condense into tight spheroids and express a full complement of therapeutic genes in α-minimum essential medium or several commercial stem-cell media. However, we identified a chemically defined xeno-free media, which, when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (prostaglandin E2, TSG-6 TNF-alpha induced gene/protein 6) and anti-cancer molecules (TRAIL TNF-related apoptosis-inducing ligand, interleukin-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage system and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death.ConclusionsWe demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed in the present study for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications.     

Induction of apoptosis coupled to endoplasmic reticulum stress in human prostate cancer cells by n-butylidenephthalide

Background

N-butylidenephthalide (BP) exhibits antitumor effect in a variety of cancer cell lines. The objective of this study was to obtain additional insights into the mechanisms involved in BP induced cell death in human prostate cancer cells.

Methods/Principal Findings

Two human prostate cancer cell lines, PC-3 and LNCaP, were treated with BP, and subsequently evaluated for their viability and cell cycle profiles. BP caused cell cycle arrest and cell death in both cell lines. The G0/G1 phase arrest was correlated with increase levels of CDK inhibitors (p16, p21 and p27) and decrease of the checkpoint proteins. To determine the mechanisms of BP-induced growth arrest and cell death in prostate cancer cell lines, we performed a microarray study to identify alterations in gene expression induced by BP in the LNCaP cells. Several BP-induced genes, including the GADD153/CHOP, an endoplasmic reticulum stress (ER stress)-regulated gene, were identified. BP-induced ER stress was evidenced by increased expression of the downstream molecules GRP78/BiP, IRE1-α and GADD153/CHOP in both cell lines. Blockage of IRE1-α or GADD153/CHOP expression by siRNA significantly reduced BP-induced cell death in LNCaP cells. Furthermore, blockage of JNK1/2 signaling by JNK siRNA resulted in decreased expression of IRE1-α and GADD153/CHOP genes, implicating that BP-induced ER stress may be elicited via JNK1/2 signaling in prostate cancer cells. BP also suppressed LNCaP xenograft tumor growth in NOD-SCID mice. It caused 68% reduction in tumor volume after 18 days of treatment.

Conclusions

Our results suggest that BP can cause G0/G1 phase arrest in prostate cancer cells and its cytotoxicity is mediated by ER stress induction. Thus, BP may serve as an anticancer agent by inducing ER stress in prostate cancer.     

Molecular characterization of apoptosis induced by CARF silencing in human cancer cells

Collaborator of ARF (CARF) was cloned as an ARF-interacting protein and shown to regulate the p53-p21(WAF1)-HDM2 pathway, which is central to tumor suppression via senescence and apoptosis. We had previously reported that CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis, however, the mechanisms governing this phenomenon remained unknown. Thus, we examined various cell death and survival pathways including the mitochondrial stress, ataxia telangiectasia mutated (ATM)-ATR, Ras-MAP kinase and retinoblastoma cascades. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, it protected the cells against genotoxicity; CARF knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and γH2AX, leading to induction of mitotic arrest and eventual apoptosis. We also show that the CARF-silencing-induced apoptosis in vitro translates to in vivo. In a human tumor xenograft mouse model, treatment of developing tumors with short hairpin RNA (shRNA) against CARF via an adenovirus carrier induced complete suppression of tumor growth, suggesting that CARF shRNA is a strong candidate for an anticancer reagent. We demonstrate that CARF has a vital role in genome preservation and tumor suppression and CARF siRNA is an effective novel cancer therapeutic agent.     

MicroRNA200a enhances antitumor effects in combination with doxorubicin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is often treated with doxorubicin. MicroRNAs have been shown to have important regulatory roles in cancer and serve as a target in chemoresistance. In this study, we investigated the effects of specific microRNA-200a (miR-200a) on HCC tumor cell growth and effect of doxorubicin-mediated cytotoxicity. Our results show miR-200a is downregulated in human HCC and HCC tumor cell lines. Increasing miR-200a expression inhibited HCC growth and synergized with the antitumor effects of doxorubicin. Inhibiting endogenous miR-200a promoted tumor growth and chemotherapeutic resistance. Increasing miR-200a expression inhibited tumor metabolism (ATP production, mitochondrial respiration, glycolysis), while inhibition of endogenous miR-200a reversed these effects. MiR-200a expression also increased autophagy and synergized with doxorubicin-mediated cytotoxicity. This study identifies a novel role of miR-200a in potentiating doxorubicin-mediated therapeutic effects in HCC.