Growth Of Human Hepatocellular Carcinoma Cells Research Articles

Methionine Sulfoxide Reductase B1 Regulates Hepatocellular Carcinoma Cell Proliferation and Invasion via the Mitogen-Activated Protein Kinase Pathway and Epithelial-Mesenchymal Transition.

Methionine sulfoxide reductase B1 (MsrB1) is a member of the selenoprotein family, which contributes to the reduction of methionine sulfoxides produced from reactive oxygen species (ROS) by redox processes in energy pathways. However, few studies have examined the role of MsrB1 in human hepatocellular carcinoma (HCC). We observed that MsrB1 is highly expressed in HCC tissues and that its expression correlated with the prognoses of patients with HCC after hepatectomy. In vitro, knockdown of MsrB1 inhibits HCC cell growth by MTT and EdU proliferation assay, and MsrB1 interference enhances H2O2/trx-induced apoptosis. We observed that phosphorylation of the key proteins of the MAPK pathway, namely, ERK, MEK, and p53, was inhibited, but PARP and caspase 3 were increased, thus infecting mitochondrial integrity. In vivo, MsrB1 knockdown effectively inhibited tumor growth. Furthermore, MsrB1 knockdown reduced HCC cell migration and invasion in a transwell assay through inhibition of cytoskeletal rearrangement and spread. This change was linked to epithelial-mesenchymal transition (EMT) inhibition resulting from increases in E-cadherin expression and decreases in expression in TGF-β1, Slug, MMP-2/9, and so on. MsrB1 regulates HCC cell proliferation and migration by modulating the MAPK pathway and EMT. Thus, MsrB1 may be a novel therapeutic target with respect to the treatment of HCC.

Identification of WT1 as determinant of heptatocellular carcinoma and its inhibition by Chinese herbal medicine Salvia chinensis Benth and its active ingredient protocatechualdehyde.

Candidates from Chinese herbal Medicine might be preferable in drug discovery as the abundant experiences of traditional use usually hint the clinical efficacy. In this study, we screened the anti-tumour effect of several commonly used Chinese herbal Medicines on human hepatocellular carcinoma cells (HCC). We identified that Salvia chinensia Benth. (Shijianchuan in Chinese, SJC) exhibited prominent in vitro inhibition of HCC cells and suppressed the orthotopic growth of HCC in the liver of mice and repressed the lung metastasis of tumour cells. Using a pathway-specific PCR array and Gene Ontology analysis, we identified that Wnt/β-catenin pathway was associated with the suppressive effect of SJC on HCC cell proliferation and cell cycle progression. SJC repressed transcription activity of Wnt/β-catenin pathway and reduced expression of β-catenin in GSK-3β-independent but promoter-specific transcription inhibition mechanism. The suppressive effect of SJC on β-catenin expression and its transcription activity was associated with Wilms’ tumor 1 (WT1) protein. WT1 was overexpressed in HCC tissues, and was negatively correlated to the overall survival of HCC patients. WT1 promoted proliferation and invasion of HCC cells, as well as β-catenin-dependent transcription activation of Wnt products, while knockdown of WT1 had the opposite effect. Docking experiment revealed that protocatechualdehyde (PCA) might be the active component of the herb. PCA suppressed transcription activity of Wnt/β-catenin pathway in WT1-dependent manner. Our study sheds light on the potential of PCA from commonly used anti-cancer Chinese herbal Medicine SJC as a lead compound targeting WT1 in the discovery of anti-HCC drugs.

Silencing HOXD10 by promoter region hypermethylation activates ERK signaling in hepatocellular carcinoma

BackgroundHepatocellular carcinoma is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. Dysregulation of HomeoboxD10 (HOXD10) was found to suppress or promote cancer progression in different cancer types. The function and regulation of HOXD10 remain unclear in human hepatocellular carcinoma (HCC).MethodsPrimary HCC samples (117), normal liver tissue samples (15), and 13 HCC cell lines (SNU182, SNU449, HBXF344, SMMC7721, Huh7, HepG2, LM3, PLC/PRF/5, BEL7402, SNU387, SNU475, QGY7703, and Huh1) were included in this study. Methylation-specific PCR, flow cytometry, western blot, transwell, siRNA, and chromatin immunoprecipitation assays were employed.ResultsHOXD10 was methylated in 76.9% (90/117) of human primary HCC samples. HOXD10 methylation was significantly associated with vessel cancerous embolus, tumor cell differentiation, and the 3-year overall survival rate (all P < 0.05). The expression of HOXD10 was regulated by promoter region methylation. HOXD10 suppressed colony formation, cell proliferation, cell invasion and migration, and induced G2/M phase arrest and apoptosis in HCC cells. HOXD10 suppressed HCC cell xenograft growth in mice. HOXD10 suppresses HCC growth by inhibiting ERK signaling.ConclusionHOXD10 is frequently methylated in human HCC, and the expression of HOXD10 is regulated by promoter region methylation. HOXD10 suppresses HCC cell growth both in vitro and in vivo. HOXD10 suppresses human HCC by inhibiting ERK signaling.

One-Step Multigram-Scale Biomimetic Synthesis of Psiguadial B.

A gram-scale synthesis of psiguadial B, a purported inhibitor of human hepatoma cell growth, has been achieved in one step by a biomimetic three-component coupling of caryophyllene, benzaldehyde, and diformylphloroglucinol. This cascade reaction is catalyzed by N,N'-dimethylethylenediamine, and proceeds at ambient temperature to generate four stereocenters, two rings, one C-O bond, and three C-C bonds. Combined computational and experimental investigations suggest the biosynthesis of the natural product is non-enzyme mediated, and is the result of a Michael addition between caryophyllene and a reactive ortho-quinone methide, followed by two sequential intramolecular cationic cyclization events.

One‐Step Multigram‐Scale Biomimetic Synthesis of Psiguadial B

AbstractA gram‐scale synthesis of psiguadial B, a purported inhibitor of human hepatoma cell growth, has been achieved in one step by a biomimetic three‐component coupling of caryophyllene, benzaldehyde, and diformylphloroglucinol. This cascade reaction is catalyzed by N,N′‐dimethylethylenediamine, and proceeds at ambient temperature to generate four stereocenters, two rings, one C−O bond, and three C−C bonds. Combined computational and experimental investigations suggest the biosynthesis of the natural product is non‐enzyme mediated, and is the result of a Michael addition between caryophyllene and a reactive ortho‐quinone methide, followed by two sequential intramolecular cationic cyclization events.

Capsaicin exerts synergistic antitumor effect with sorafenib in hepatocellular carcinoma cells through AMPK activation.

In this study, we investigated the antitumoral effects of combined treatment using sorafenib and capsaicin in hepatocellular carcinoma (HCC) cells. Here we showed that the combination of the two drugs had a much stronger inhibitory effect on both HepG2 and Huh-7 human HCC cells growth than either drug alone. The isobolograms demonstrated that the combinations investigated in this study produced a synergistic interaction. In the combination treatment using capsaicin and sorafenib, increased apoptosis, followed by the activation of caspase-9 and PARP, was observed. In addition, the present study demonstrated that sorafenib treatment induces activation of Akt, probably as a mechanism of resistance, whereas capsaicin inhibits Akt providing a possible pathway whereby capsaicin sensitizes to sorafenib in HCC cells. Moreover, capsaicin singly and the combination of capsaicin and sorafenib induce AMPK activation and Acetyl CoA carboxylase phosphorylation in HCC cells. Knocking down of AMPK by selective siRNA abrogates capsaicin-induced Akt inhibition, suggesting the involvement of AMPK in the antiproliferative effect. In vivo experiments further showed that that the anti-tumor effect of sorafenib was enhanced by its combination with 2.5 mg/Kg of capsaicin. Overall, these results show that combined treatment with capsaicin and sorafenib might improve sorafenib sensitivity and therefore it represents a promising and attractive strategy for the treatment of HCC.

Knockdown of long non-coding RNA MALAT1 inhibits growth and motility of human hepatoma cells via modulation of miR-195.

The metastasis-associated lung adenocarcinoma transcription 1 (Malat1) is a long non-coding RNA (lncRNA), exerts oncogenic role in multiple cancers, including hepatocellular carcinoma (HCC). This study was aimed to investigate its posttranscriptional regulation in HCC cells. RT-PCR was performed to monitor the expression levels of Malat1 in normal liver and HCC cell lines. The expression of Malat1, microRNA (miR)-195, and epidermal growth factor receptor (EGFR) in HepG2 and MHCC97 cells was respectively or synchronously altered by transfection. Then the changes in cell viability, apoptotic cell rate, cell cycle distribution, migration, and invasion were respectively assessed. As a result, we found that Malat1 was highly expressed in HCC cell lines when compared to normal liver cells. Malat1 silence suppressed HCC cells viability, migration and invasion, induced apoptosis, and arrested more cells in G0/G1 phase. Malat1 acted as a circular endogenous RNA (ceRNA) for miR-195. Malat1 silence could not suppress HCC cell growth and motility when miR-195 was knocked down. EGFR was a direct target of miR-195. miR-195 overexpression could not suppress HCC cell growth and motility when the 3'UTR site of EGFR was overexpressed. Furthermore, Malat1 silence blocked the activation of PI3K/AKT and JAK/STAT pathways, while EGFR overexpression activated them. Our study demonstrates Malat1-miR-195-EGFR axis plays a critical role in HCC cells which provided a better understanding of Malat1 in HCC.

RYBP Expression Is Regulated by KLF4 and Sp1 and Is Related to Hepatocellular Carcinoma Prognosis

The expression of Ring1- and YY1-binding protein (RYBP) is reduced in several human cancers, but the molecular mechanism(s) have remained elusive. In this study, we used human hepatocellular carcinoma (HCC) cell lines and tissue specimens to study the mechanism and herein report several new findings. First, we cloned and characterized the basal promoter region of the human RYBP gene. We found that the decreased RYBP expression in HCC tissues was not due to promoter sequence variation/polymorphisms or CpG dinucleotide methylation. We identified two transcription factors, KLF4 and Sp1, which directly bind the promoter region of RYBP to induce and suppress RYBP transcription, respectively. We mapped the binding sites of KLF4 and Sp1 on the RYBP promoter. Studies in vitro showed that KLF4 suppresses whereas Sp1 promotes HCC cell growth through modulating RYBP expression. Deregulated KLF4 and Sp1 contributed to decreased expression of RYBP in HCC tumor tissues. Our studies of human HCC tissues indicated that a diminished RYBP level in the tumor (in association with altered KLF4 and Sp1 expression) was statistically associated with a larger tumor size, poorer differentiation, and an increased susceptibility to distant metastasis. These findings help to clarify why RYBP is decreased in HCC and indicate that deregulated KLF4, Sp1, and RYBP may lead to a poorer prognosis. Our findings support the idea that RYBP may represent a target for cancer therapy and suggest that it may be useful as a prognostic biomarker for HCC, either alone or in combination with KLF4 and Sp1.

Resveratrol suppresses human hepatocellular carcinoma via targeting HGF-c-Met signaling pathway.

Resveratrol, one of the major polyphenols found in red wine, is suggested to have a role as a chemo-prevention or chemotherapy agent in various human cancer models. Herein, we report that resveratrol has a profound antitumor effect on human hepatocellular carcinoma (HCC) cells by down-regulation of the HGF-c-Met signaling pathway. Resveratrol inhibited anchorage-dependent and -independent growth of HCC cells in a dose-dependent manner. Short-term resveratrol exposure substantially decreased HGF-induced c-Met signaling pathway activation, and long-term exposure to resveratrol markedly inhibited c-Met expression on the cell membrane. Additionally, resveratrol suppressed HGF-induced cell invasion, and knockdown of c-Met decreased the sensitivity of HCC cells to resveratrol treatment. Finally, the antitumor activity of resveratrol was validated in xenograft model and resveratrol prominently restrained tumor growth invivo. In summary, our results suggested that c-Met offers a candidate molecular target for hepatocellular carcinoma management.

Effects of Genistein and Synergistic Action in Combination with Tamoxifen on the HepG2 Human Hepatocellular Carcinoma Cell Line

Introduction:The flavonoids comprise a diverse group of polyphenolic compounds with antioxidant activity that is present in edible plants like soybeans and soy products. In vivo studies have concentrated on the effects of flavonoids on cancer and genistein (GE), a soy-derived isoflavone, has been reported to reduce prostate, colon, hepatic and breast adenocarcinoma risk. Tamoxifen (TAM) is an important drug for cancer treatment worldwide, which can induce apoptosis in various cancers, including examples in the liver, breast and ovaries. The aim of the present study was to evaluate the effects of GE and TAM, alone and in combination, on proliferation and apoptosis in the human hepatocellular carcinoma (HCC) HepG2 cell line.Materials and Methods:HepG 2 cells were treated with GE, TAM and GE/TAM and then MTT and flow cytometry assays were conducted to determine effects on viability and apoptosis, respectively.Results:GE and TAM inhibited cell proliferation and induced apoptosis in the HepG 2 cell lines.Discussion:Our findings clearly indicated that GE and TAM may exert inhibitory and apoptotic effects in liver cancer cells.Conclusion:GE and TAM can significantly inhibit growth of HCC cells and play a significant role in apoptosis.

Targeting endogenous DLK1 exerts antitumor effect on hepatocellular carcinoma through initiating cell differentiation.

Cancer stem cells (CSCs) are responsible for tumor initiation and progression. We previously showed that Delta-like homolog 1 (DLK1) may be a therapeutic target against the CSCs of human hepatocellular carcinoma (HCC). However, the therapeutic efficacy and underlying mechanism remain unclear. Here we demonstrated that knockdown of DLK1 using a tet-inducible short hairpin RNA (shRNA) system significantly inhibited proliferation, spheroid formation and in vivo xenograft tumor growth of human HCC cells. Furthermore, in an orthotopic xenograft mouse model, adenovirus-mediated DLK1 knockdown could significantly reduce tumor size, as shown by in vivo imaging approach. Subsequently, an adenoviral vector harboring mouse Dlk1 shRNA was applied. The results showed that Dlk1 knockdown also could inhibit tumor progression in a diethylnitrosamine (DEN) induced mouse HCC model. At cellular mechanism, DLK1 knockdown delayed the cell cycle G1-S transition, along with the decreased expression of cyclin E1 and D1. Significantly, DLK1 knockdown resulted in the decrease of molecular markers such as AFP and EpCAM for hepatic progenitor cells, but the increase of KRT18 and KRT19 for the differentiated hepatocytes. The collective data indicated that targeting endogenous DLK1 may exert antitumor effect on HCCs possibly through initiating cell differentiation.

TTF1-NPs Induce ERS-Mediated Apoptosis and Inhibit Human Hepatoma Cell Growth In Vitro and In Vivo.

Previous studies have shown that 5,2′,4′-trihydroxy-6,7,5′-trimethoxyflavone (TTF1) is the primary anticancer constituent of the traditional Chinese medicinal plant Sorbaria sorbifolia (SS), which has been applied to treat cancer in China. In this study, we investigated the in vitro and in vivo antitumor effects and biological mechanisms of small-molecule TTF1 nanoparticles (TTF1-NPs). The effects of TTF1-NPs on cell growth and apoptosis were investigated using human hepatoma cells. The molecular changes associated with the effects of TTF1-NPs were analyzed by immunocytochemistry and Western blot analysis. The in vivo effect of TTF1-NPs was investigated using the HepG2 tumor xenograft model. We found that TTF1-NPs exhibited antitumor effects in vitro accompanied by induction of apoptosis in human hepatoma cells. Mechanistically, our data showed that TTF1-NPs induced apoptosis via endoplasmic reticulum stress (ERS) pathway in hepatoma cells. Moreover, inhibition of ERS activation blocked TTF1-NP-induced apoptosis in HepG2 cells. Finally, TTF1-NPs inhibited the growth of HepG2 xenograft tumors. Taken together, our results demonstrated that TTF1-NP-induced apoptosis was mediated at least in part by the ERS pathway and thus inhibited hepatoma tumor growth.

Aberrantly activated Gli2-KIF20A axis is crucial for growth of hepatocellular carcinoma and predicts poor prognosis.

Glioma-associated oncogene 2 (Gli2), a primary transcriptional regulator of Hedgehog (Hh) signaling, is essential for hepatocellular carcinoma (HCC) growth and survival. However, the underlying molecular mechanism and crucial downstream targets of Gli2 in human HCC are not fully understood. Here, we report the identification of kinesin family member 20A (KIF20A) as a novel downstream target of Gli2, which is important for HCC proliferation and tumor growth. Inhibition of Hh signaling leads to a remarkable decrease of KIF20A expression in HCC cells, whereas overexpression of Gli2 elevates KIF20A expression by activating Forkhead Box M1 (FoxM1)-MMB complex-mediated transcription of this kinesin gene. Gli2-induced HCC cell growth requires enhanced expression of KIF20A, and knockdown of Gli2 or KIF20A represses the proliferation of HCC cells in vitro and in vivo. Correlated with these results, analyses of clinical HCC samples show that Gli2, FoxM1 and KIF20A are highly elevated in primary HCC samples and represent significant risk factors for HCC recurrence and survival. Conclusion: KIF20A is an important downstream target gene of Hh signaling. And, the Gli2-KIF20A axis is essential for the proliferation and growth of human HCC cells. Our study also suggests Gli2-KIF20A axis as a potential target for future therapeutic intervention and as an independent prognostic biomarker for HCC.

Methylation-regulated miR-124-1 suppresses tumorigenesis in hepatocellular carcinoma by targeting CASC3.

This study was to investigate the roles and mechanisms of miR-124-1 in hepatocellular carcinoma (HCC). We analyzed the expression of miR-124-1 in human HCC tissues and cell lines. Luciferase reporter assays were used to analyze the target of miR-124-1. Human HCC cell lines were transduced with lentiviruses expressing miR-124-1, and proliferation and colony formation were analyzed. The growth of human HCC cells overexpressing miR-124-1 was assessed in nude mice. The expression of p38-MAPK, JNK, ERK and related signaling molecules was detected by western blotting and immunohistochemistry. Our results showed that miR-124-1 levels were reduced in HCC tissues and cell lines compared with those in adjacent non-cancer tissues and normal liver cell lines respectively. Downregulation of miR-124-1 in HCC cell lines were attributed to hypermethylation of its promoter region. Overexpression of miR-124-1 inhibited HCC cell proliferation in vitro, whereas miR-124-1 was correlated with clinicopathological parameters of HCC patients. HCC cell-mediated overexpression of miR-124-1 in nude mice suppressed tumor growth. Cancer susceptibility candidate 3 (CASC3) was identified as a direct target of miR-124-1 by computational analysis and experimental assays. MiR-124-1-mediated downregulation of CASC3 resulted in the inactivation of p38-MAPK, JNK and ERK. Our findings provide potential new targets for the prevention or treatment of HCC.

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Noscapine inhibits human hepatocellular carcinoma growth through inducing apoptosis in vitro and in vivo.

Noscapine, a phthalideisoquinoline alkaloid derived from opium, has been demonstrated as a promising anti-tumor compound against various cancers. However, the anti-cancer activity of noscapine in hepatocellular carcinoma has not been defined. In this study, we investigate the inhibitive effects of noscapine on human hepatocellular carcinoma (HCC) using both in vitro and in vivo models. In vitro proliferation assay showed that noscapine suppressed HepG2 and Huh7 cells in dose- and time-dependent manners. With a mouse xenograft model, noscapine showed notable inhibition on HCC tumor growth in vivo without suppression of body weight. Moreover, apoptotic induction and regulation of related signalings by noscapine were examined by nuclear DNA staining, TUNEL, and western blotting assays. Results showed that noscapine induced apoptosis in HCC cells both in vitro and in vivo. Further studies indicated that noscapine induced antive-capsase-3, cleavage PARP, and decreased the ratio of Bcl-2/Bax. Hence, these data indicates that noscapine selectively suppresses HCC cell growth through apoptosis induction, providing evidence for application of noscapine as a novel agent against human hepatocellular carcinoma.

STAT1 inhibits human hepatocellular carcinoma cell growth through induction of p53 and Fbxw7.

Background Aberrant STAT1 signaling is observed in human hepatocellular carcinoma (HCC) and has been associated with the modulation of cell proliferation and survival. However, the role of STAT1 signaling in HCC and its underlying mechanism remain elusive.MethodsWe transiently transfected pcDNA3.1-STAT1 and STAT1 siRNA into SMMC7721 and HepG2 cells. Western blot and qRT-PCR examined the expression of protein and RNA of target genes. Cell viability was assessed using MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry.ResultsWe found that STAT1 overexpression increased protein expression of p53 and Fbxw7, and downregulated the expression of cyclin A, cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. These changes led to growth inhibition and induced G0/G1 cell cycle arrest and apoptosis in SMMC7721 and HepG2 cells. Conversely, ablation of STAT1 had the opposite effect on p53, Fbxw7, Hes-1, NF-κB p65, cyclin A, cyclin D1, cyclin E and CDK2, and improved the viability of SMMC7721 and HepG2 cells.ConclusionsOur data indicate that STAT1 exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis, and may provide a basis for the design of new therapies for the intervention of HCC in the clinic.

Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC-7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC-7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC-7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase-8 and caspase-9 were activated in MUC1 gene-silenced SMMC-7721 cells. The pro-apoptotic protein Bcl-2-associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti-apoptotic protein B-cell lymphoma 2 was decreased in MUC1 gene-silenced cells. In addition, results from the co-immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase-8 and these interactions were reduced upon MUC1 gene silencing in SMMC-7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC-7721 cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

MicroRNA-122 affects cell aggressiveness and apoptosis by targeting PKM2 in human hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is a highly invasive tumor with frequent distant metastasis, which is the main cause for the poor prognosis. However, the mechanisms for metastasis remain poorly investigated. MicroRNAs (miRNAs) have been implicated in HCC progression. MicroRNA-122 (miR-122) is considered as a tumor suppressor in human cancer. In the present study, miR-122 expression was found to be significantly lower in HCC than the level in normal tumor-adjacent tissues. miR-122 was clearly silenced or downregulated in five HCC cell lines (HepG2, Hep3B, MHCC97H, Huh7 and SMMC-7721) compared with normal hepatocytes (LO2). HCC patients with low expression of miR-122 had a poor 3-year survival. Univariate analysis and multivariate Cox regression analysis indicated that miR-122 is an independent prognostic factor in HCC. Downregulation of miR-122 promoted proliferation and inhibited apoptosis in Hep3B cells. We found that the public miRNA database (TargetScan) predicted that PKM2 may be a target for miR-122, and the 3'-untranslated region (3'-UTR) of PKM2 contains a highly conserved binding site for miR-122. To identify this, pre-miR-122/anti-miR-122 were respectively transfected into the Hep3B cell line. We found that miR-122 overexpression significantly reduced the level of PKM2. Moreover, knockdown of PKM2 significantly increased miR-122 inhibitor-mediated Hep3B cell apoptosis and reduced miR-122 inhibitor-mediated Hep3B cell migration and invasion. Moreover, re-expression of PKM2 partially abrogated miR-122-induced HCC cell growth arrest and apoptosis in vivo. In conclusion, miR-122 serves as a prognostic biomarker and induces apoptosis and growth arrest by downregulating PKM2 in HCC.

Abstract 4645: Decursin, a coumarin compound, inhibits the growth of human hepatoma cells involving cell cycle arrest, apoptosis, and autophagy

Abstract Although recent advances have been made for the treatment of liver cancer, it still remains the sixth most diagnosed cancer worldwide and second most cause of mortality related to cancer. Decursin, a naturally occurring coumarin compound isolated from Angelica gigas Nakai is known for its hepatoprotective activity. For the first time we show that decursin has strong cytotoxic and growth inhibitory effects against liver cancer cells. Decursin (1-25 μM) resulted in a dose-dependent inhibition of Hep3B cell proliferation by upto 65-85% (p&amp;lt;0.005-0.001) in 24-48 h. Similarly in HepG2 cells, decursin (25-100 μM) decreased cell number by upto 61-74% (p&amp;lt;0.005-0.001) at 24-48 h. Growth inhibition was not due to the cell death in HepG2 cells, whereas, Hep3B cells showed an increased cell death (9-45%; p&amp;lt;0.001). Decursin enhanced sub-G1 population of cells in Hep3B but not in HepG2. Further, decursin induced G1 arrest at lower doses, however, at the higher doses cell death prevailed over cell cycle arrest. A consistent G2/M phase arrest in HepG2 cells at all the time points, however, at 100 μM, there was an induction of S-phase arrest. Decursin strongly decreased the colony forming efficiency of Hep3B cells. Decursin induced both apoptotic and autophagic cell death. In flow cytometry analyses, an increase in the acidic vesicular organelle development was observed at 24 and 48 h in Hep3B cells. Interestingly, in decursin treated HepG2 cells, autophagic induction occurred much later (48 h). This was further validated by MDC staining. Mechanistic studies for cell cycle regulators showed that decursin mediated G1 arrest could be via an increase in p21/cip1. Concomitantly, we observed reduced levels of Cyclin A and B1 and their respective kinases. PCNA, a marker of proliferation showed decreased in expression. Decursin mediated induction of apoptosis involved cleavage of caspase-3 and PARP and reduction in survivin level. This was accompanied by an enhanced Bax/Bcl-2 ratio. Additionally, a potent decrease in the phosphorylation of IGF-IR and its downstream effector Akt, was observed. Decursin increased Erk1/2 phosphorylation, however, it was not associated with cell growth or survival. Collectively, these findings suggest that decursin strongly inhibits liver cancer growth and proliferation involving induction of cell cycle arrest, apoptosis and autophagy and could be further explored and developed as novel treatment option for the hepatic malignancies. Citation Format: Praveen K. Kujur, Dongsool Yim, Rana P. Singh. Decursin, a coumarin compound, inhibits the growth of human hepatoma cells involving cell cycle arrest, apoptosis, and autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4645. doi:10.1158/1538-7445.AM2015-4645