ginsenoside-m1 and Carcinoma--Hepatocellular

Epidemiological studies have shown that hepatocellular carcinoma (HCC) is a main cause of tumor death worldwide. Accumulating data indicate that ginsenoside CK is an effective compound for preventing HCC growth and development. However, improvement of pharmaceutical effect of the ginsenoside CK is still needed. In our study, we performed acetylation of ginsenoside CK (CK-3) and investigated the antitumor effects of the derivative in vitro and in vivo. The cytotoxicity analysis revealed that compared with CK, CK-3 could inhibit the proliferation of multiple tumor cell lines at a lower concentration. Treating with CK-3 on HCC cells arrested cell cycle in G2/M phase and induced cell apoptosis through AO/EB staining, TUNEL analysis and flow cytometry. Meanwhile, CK-3 significantly inhibited tumor growth in an HCC xenograft model and showed no side effect on the function of the main organs. Mechanistically, whole transcriptome analysis revealed that the antitumor effect of CK-3 was involved in the Hippo signaling pathway. The immunoblotting and immunofluorescence results illustrated that CK-3 directly facilitated the phosphorylation of YAP1 and decreased the expression of the main transcription factor TEAD2 in HCC cell lines and tumor tissue sections. Collectively, our results demostrate the formation of a new derivative of ginsenoside CK and its regulatory mechanism in HCC, which could activate the Hippo-YAP1-TEAD2 signaling pathway to regulate HCC progression. This research could provide a new direction for traditional Chinese medicine in the therapy of tumors.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Ginsenosides; Humans; Liver Neoplasms

Since the AKT/mammalian target of rapamycin (mTOR)/c-Myc signaling plays a pivotal role in the modulation of aerobic glycolysis and tumor growth, in the present study, the role of AKT/mTOR/c-Myc signaling in the apoptotic effect of Compound K (CK), an active ginseng saponin metabolite, was explored in HepG2 and Huh7 human hepatocellular carcinoma cells (HCCs). Here, CK exerted significant cytotoxicity, increased sub-G1, and attenuated the expression of pro-Poly (ADP-ribose) polymerase (pro-PARP) and Pro-cysteine aspartyl-specific protease (pro-caspase3) in HepG2 and Huh7 cells. Consistently, CK suppressed AKT/mTOR/c-Myc and their downstreams such as Hexokinase 2 (HK2) and pyruvate kinase isozymes M2 (PKM2) in HepG2 and Huh7 cells. Additionally, CK reduced c-Myc stability in the presence or absence of cycloheximide in HepG2 cells. Furthermore, AKT inhibitor LY294002 blocked the expression of p-AKT, c-Myc, HK2, PKM2, and pro-cas3 in HepG2 cells. Pyruvate blocked the ability of CK to inhibit p-AKT, p-mTOR, HK2, and pro-Cas3 in treated HepG2 cells. Overall, these findings provide evidence that CK induces apoptosis via inhibition of glycolysis and AKT/mTOR/c-Myc signaling in HCC cells as a potent anticancer candidate for liver cancer clinical translation.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Ginsenosides; Glycolysis; Hep G2 Cells; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Ginsenoside compound K (M1) is the active form of major ginsenosides deglycosylated by intestinal bacteria after oral administration. However, M1 was reported to selectively accumulate in liver and transform to fatty acid esters. Ester of M1 was not excreted by bile as M1 was, which means it was accumulated in the liver longer than M1. This study reported a synthetic method of M1-O, a mono-octyl ester of M1, and evaluated the anticancer property against murine H22 cell both in vitro and in vivo. As a result, both M1 and M1-O showed a dose-dependent manner in cytotoxicity assay in vitro. At lower dose of 12.5 μm, M1-O showed moderate detoxification. Instead, M1-O exhibited significantly higher inhibition in H22-bearing mice than M1. M1-O induced murine H22 tumor cellular apoptosis in caspase-dependent pathway given that pan-caspase inhibitor, Z-VAD-FMK, could reverse the cytotoxicity induced by M1-O. Additionally, pro- and anti-apoptosis proteins, Bcl-2 and Bax, altered and consequently induced increased expression of cleaved caspase-3. Interestingly, cyclophosphamide regimen significantly induced atrophy of spleen and thymus, main immune organs, while M1-O treatment greatly alleviated this atrophy. Collectively, we propose M1-O as a candidate for live cancer treatment.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Ginsenosides; Liver Neoplasms; Mice; Proto-Oncogene Proteins c-bcl-2

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Survival; Chitosan; Deoxycholic Acid; Drug Carriers; Drug Liberation; Ginsenosides; Hep G2 Cells; Humans; Liver Neoplasms; Nanoparticles

An intestinal bacterial metabolite of ginseng protopanaxadiol saponin, 20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol (compound K), has been reported to induce apoptosis in a variety of cancer cells. However, the precise mechanisms induced by compound K in human hepatocellular carcinoma (HCC) cells remain unclear. In order to examine possible apoptotic mechanisms, we investigated the anticancer effect of compound K in MHCC97-H. MTT assay showed that compound K inhibited the proliferation of MHCC97-H cells with a relatively low toxicity in normal hepatoma cells. Cell cycle progression and cell staining showed an increase in apoptotic sub-G1 fraction. Treatment of MHCC97-H with compound K also induced a reduction in mitochondrial membrane potential (Δψm) and DNA damage. Further study showed that compound K upregulated Fas, FasL, Bax/Bcl-2 ratio and downregulated pro-caspase-9, pro-caspase-3 in a dose-dependent manner, and it also inhibited Akt phosphorylation. These results suggest that compound K significantly inhibits cell proliferation and induces apoptosis in MHCC97-H cells through Fas- and mitochondria-mediated caspase-dependent pathways in human HCC cells.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; DNA Damage; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Ginsenosides; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial

The intestinal metabolite of ginseng saponin, compound K (CK), has various chemopreventive and chemotherapeutic activities, including anti-tumor activity. However, the functional mechanisms through which CK attenuates metastatic growth in hepatocellular carcinoma (HCC) remain unclear. Here, using multiple IN VITRO and IN VIVO models, we reported that CK strongly attenuated colony formation, adhesion, and invasion of HCC cells IN VITRO and dramatically inhibited spontaneous HCC metastatic growth IN VIVO. At the molecular level, immunofluorescence and Western blotting analysis confirmed that inhibition of metastatic growth of HCC induced by CK treatment caused a time-dependent decrease in nuclear NF- κB p65 and a concomitant increase in cytosolic NF- κB p65, indicating that CK suppressed the activation of the NF- κB pathway. Meanwhile, our study showed that the inhibition of matrix metalloproteinase2/9 (MMP2/9) expression caused by CK treatment was associated with NF- κB p65 nuclear export. Taken together, our results not only revealed that NF- κB p65 nuclear export and the reduction of MMP2/9 expression were associated with the metastatic inhibition induced by CK, but also suggested that CK may become a potential cytotoxic drug in the prevention and treatment of HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Ginsenosides; Humans; Liver Neoplasms; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; NF-kappa B; Panax; Plant Extracts

Compound K (CK) is a major intestinal metabolite of ginsenosides derived from ginseng radix. Although antidiabetic and antihyperlipidemic activities of CK have been investigated in recent years, action mechanism of CK remains poorly understood. Therefore, we examined whether CK affects the lipid metabolism in insulin-resistant HepG2 human hepatoma cells. In this study, a significant increase in AMP-activated protein kinase (AMPK) was observed when the cells were treated with CK. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase (ACC), a substrate of AMPK. CK attenuated gene expression of sterol regulatory element-binding protein 1c (SREBP1c) in time- and dose-dependent manners. Genes for fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1), well-known target molecules of SREBP1c, were also suppressed. In contrast, gene expressions of peroxisome proliferator-activated receptor alpha (PPAR-alpha) and CD36 were increased. These effects were reversed by treatment of compound C, an AMPK inhibitor. However, there were no differences in gene expressions of SREBP2, hydroxymethyl glutaryl CoA reductase (HMGR), and low-density-lipoprotein receptor (LDLR). Taken together, AMPK mediates CK induced suppression and activation of SREBP1c and PPAR-alpha, respectively, and these effects seem to be one of antidiabetic and/or antihyperlipidemic mechanisms of CK in insulin-resistant HepG2 human hepatoma cells.

Topics: AMP-Activated Protein Kinases; Carcinoma, Hepatocellular; Cell Line, Tumor; Enzyme Activation; Gene Expression; Ginsenosides; Humans; Intestinal Mucosa; Lipid Metabolism; Liver; Liver Neoplasms

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), a novel intestinal bacterial metabolite of ginseng protopanaxadiol saponins, is reported to induce apoptosis in a variety of cancer cells. We purified the compound and measured its in vitro anti-tumor activity. IH-901 inhibited cell growth of human hepatocellular carcinoma SMMC7721 cells in a dose- and time-dependent manner. We also found that IH-901 induced apoptotic cell death concurrent with cell cycle arrest in G0-G1 phase in SMMC7721 cells. At the molecular level, we show that IH-901 upregulates cytochrome c, p53, and Bax expression, and downregulates pro-caspase-3 and pro-caspase-9 expressions in a dose-dependent manner, while the levels of Bcl-2 and Bcl-X(L) were unchanged in IH-901-treated SMMC7721 cells. These results provide significant insight into the anticarcinogenic action of IH-901.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cell Proliferation; Cytochromes c; Flow Cytometry; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sapogenins; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Recently, a novel intestinal bacterial metabolite of ginseng protopanaxadiol saponins, i.e., 20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), has been reported to induce apoptosis in a variety of cancer cells. Here we show a differential effect of IH-901 on several cell types. Exposure to IH-901 for 48 hours at a supposedly subapoptotic concentration of 40 mumol/L led to both apoptotic cell death and G1 arrest in Hep3B cells, but only resulted in G1 arrest in MDA-MB-231, Hs578T, and MKN28 cells. Additionally, the treatment of MDA-MB-231, but not of Hep3B, with IH-901 up-regulated cyclooxygenase-2 (COX-2) mRNA (2 hours) and protein (6 hours), and enhanced the production of prostaglandin E2. In MDA-MB-231 cells, IH-901 induced the sustained activation of extracellular signal-regulated kinase (ERK), whereas inhibition of mitogen-activated protein/ERK kinase blocked IH-901-mediated COX-2 induction and resulted in apoptosis, suggesting the involvement of an ERK-COX-2 pathway. Combined treatment with IH-901 and nonsteroidal anti-inflammatory drugs inhibited COX-2 enzyme and induced apoptosis in MDA-MB-231 and Hs578T cells. Adenovirus-mediated COX-2 small interfering RNAs also effectively inhibited COX-2 protein expression and enhanced IH-901-mediated apoptosis without inhibiting ERK 1/2 phosphorylation, thus providing direct evidence that COX-2 is an antiapoptotic molecule. Moreover, IH-901-mediated G1 arrest resulted from an increase in p27Kip1 mRNA and protein expression followed by a decrease in CDK2 kinase activity that was concurrent with the hypophosphorylation of Rb and p130. In conclusion, IH-901 induced both G1 arrest and apoptosis, and this apoptosis could be inhibited by COX-2 induction.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Carrier Proteins; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Membrane Proteins; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Proteins; Retinoblastoma Protein; Retinoblastoma-Like Protein p130; RNA, Messenger; RNA, Small Interfering; Sapogenins; Tumor Cells, Cultured

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 microM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Caspase 8; Caspases; Cell Division; Cell Line, Tumor; Cytosol; DNA Fragmentation; Enzyme Activation; Flow Cytometry; Humans; Liver Neoplasms; Membrane Potentials; Mitochondria; Panax; Sapogenins; Saponins; Signal Transduction