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Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

ABSTRACT: The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-?B activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

SUBMITTER: Monga J

PROVIDER: S-EPMC3722203 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

Monga Jitender J Pandit Saurabh S Chauhan Rajinder Singh RS Chauhan Chetan Singh CS Chauhan Shailender Singh SS Sharma Manu M

PloS one 20130724 7

The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was ...[more]

PMID: 23894334

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Project description:BackgroundSulfatase 2 (SULF2) removes the 6-O-sulfate groups from heparan sulfate proteoglycans (HSPG) and consequently alters the binding sites for various signaling molecules. Here, we elucidated the role of SULF2 in the differentiation of hepatic stellate cells (HSCs) into carcinoma-associated fibroblasts (CAFs) in the hepatocellular carcinoma (HCC) microenvironment and the mechanism underlying CAF-mediated HCC growth.MethodsThe clinical relevance of SULF2 and CAFs was examined using in silico and immunohistochemical (IHC) analyses. Functional studies were performed to evaluate the role of SULF2 in the differentiation of HSCs into CAFs and elucidate the mechanism underlying CAF-mediated HCC growth. Mechanistic studies were performed using the chromatin immunoprecipitation, luciferase reporter, and RNA immunoprecipitation assays. The in vitro findings were verified using the nude HCC xenograft mouse model.ResultsThe Cancer Genome Atlas (TCGA) database and IHC analyses revealed that the expression of CAF markers, which was positively correlated with that of SULF2 in the HCC tissues, predicted unfavorable postsurgical outcomes. Co-culturing HSCs with HCC cells expressing SULF2 promoted CAF differentiation. Additionally, CAFs repressed HCC cell apoptosis by activating the SDF-1/CXCR4/PI3K/AKT signaling pathway. Meanwhile, SULF2-induced CAFs promoted epithelial-to-mesenchymal transition (EMT) of HCC cells by modulating the SDF-1/CXCR4/OIP5-AS1/miR-153-3p/SNAI1 axis. Studies using HCC xenograft mouse models demonstrated that OIP5-AS1 induced EMT by upregulating SNAI1 and promoted HCC growth in vivo.ConclusionThese data indicated that SULF2 secreted by the HCC cells induced the differentiation of HSCs into CAFs through the TGFβ1/SMAD3 signaling pathway. SULF2-induced CAFs attenuated HCC apoptosis by activating the SDF-1/CXCR4/PI3K/AKT signaling pathway and induced EMT through the SDF-1/CXCR4/OIP5-AS1/miR-153-3p/SNAI1 axis. This study revealed a novel mechanism involved in the crosstalk between HCC cells and CAFs in the tumor microenvironment, which can aid in the development of novel and efficient therapeutic strategies for primary liver cancer.

Project description:Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIPS) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIPS is down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIPS, consequently causes FLIPS to form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIPS levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIPS regulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.

Dataset Information

Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

Publications

Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

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