sapogenins and Liver-Neoplasms

Diol-type ginsenosides, such as protopanaxadiol (PPD), exhibit antioxidation, anti-inflammation, and antitumor effects. However, the antitumor effect of these ginsenosides and the mechanism of PPD remain unclear. In this work, the antitumor effects of several derivatives, including PPD, Rg5, Rg3, Rh2, and Rh3, were evaluated in five different cancer cell lines. PPD demonstrated the best inhibitory effects on the proliferation and migration of the five cancer cell lines, especially the hepatocellular carcinoma (HCC) cell lines. Therefore, the mechanism of action of PPD in HCC cells was elucidated. PPD inhibited the proliferation, migration, and invasion ability of HepG2 and PLC/PRF/5 cells in a dose-dependent manner. Western blot and immunofluorescence assay showed that PPD can alter the expression of epithelial-mesenchymal transition markers, increase E-cadherin expression, and decrease vimentin expression. Docking and biacore experiments revealed that STAT3 is the target protein of PPD, which formed hydrogen bonds with Gly583/Leu608/Tyr674 at the SH2 domain of STAT3. PPD inhibited the phosphorylation of STAT3 and its translocation from the cytosol to the nucleus, thereby inhibiting the expression of Twist1. PPD also inhibited tumor volume and tumor lung metastasis in PLC/PRF/5 xenograft model. In conclusion, PPD can inhibit the proliferation and metastasis of HCC cells through the STAT3/Twist1 pathway.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Hep G2 Cells; Humans; Liver Neoplasms; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Sapogenins; Signal Transduction; STAT3 Transcription Factor; Transfection; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related mortality with poor prognosis and treatment. More effective strategies should be studied in HCC.. After treated with ruscogenin, the cell proliferation was assessed by CCK-8 method. Cell migration and invasion were estimated using wound healing and transwell assays. Pathological changes of lung tissue were observed by HE staining and IHC methods. MMP-2, MMP-9, uPA, VEGF and HIF-1α levels were measured using ELISA, RT-qPCR and WB tests. PI3K/Akt/mTOR pathway related molecules were detected using WB analysis.. The results indicated the hypotoxicity of ruscogenin. Meanwhile, ruscogenin showed obvious interruption on the cancer cell migration and invasion, and inhibition on the metastatic foci in pulmonary tissue. Significantly, ruscogenin decreased the levels of MMP-2, MMP-9, uPA, VEGF and HIF-1α, down-regulated the phosphorylation of Akt, mTOR.. The present study indicated a novel use of ruscogenin in suppressing HCC metastasis by reducing the expression of MMP-2, MMP-9, uPA, VEGF and HIF-1α via regulating the PI3K/Akt/mTOR signaling pathway.

Topics: Animals; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sapogenins; Signal Transduction; Spirostans; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

20(S)-Protopanaxadiol (PPD), a metabolite of ginsenosides, has been demonstrated to possess cytotoxic effects on several cancer cell lines. The molecular mechanism is, however, not well understood. In this study, we have shown that PPD inhibits cell growth and induces apoptosis in human hepatocarcinoma HepG2 cells. PPD-treated cells showed a massive cytoplasmic vacuolization and a dramatic change of endoplasmic reticulum (ER) morphology. The induction of ER stress is associated with the upregulation of ER stress-associated genes and proteins. PPD activates the unfolded protein response (UPR) through the phosphorylation of PERK and eIF2α, the splicing of XBP1 mRNA, and the cleavage of AFT6. PPD also induces the intrinsic and extrinsic apoptotic pathways. It activates DR5, caspase-8, -9, -3, and promotes the cleavage of PARP while it downregulates Bcl-2, Bcl-x(L) and mitochondrial membrane potential. Knockdown of one of the three UPR limbs by specific siRNAs did not affect PPD-induced apoptosis, which was however, significantly suppressed by the downregulation of CHOP. Western blot analysis showed that PPD-stimulated downregulation of Bcl-2 protein, increase of DR5 protein, activation of caspase-8 and cleavage of PARP were significantly inhibited in CHOP siRNA-transfected cells. Taken together, we have identified ER as a molecular target of PPD and our data support the hypothesis that PPD induces HepG2 cell apoptosis through the ER stress pathway.

Topics: Apoptosis; Endoplasmic Reticulum Stress; Extracellular Signal-Regulated MAP Kinases; Ginsenosides; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitochondria; p38 Mitogen-Activated Protein Kinases; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sapogenins; Transcription Factor CHOP; Unfolded Protein Response; Up-Regulation; Vacuoles

To investigate the antioxidative effects of ginsenosides [protopanaxadiol derivatives (PD):protopanaxatriol derivatives (PT) = 1:1] from the roots of Korean ginseng, cell viability, malondialdehyde (MDA) production, antioxidant enzyme activities, and expressions of apoptosis were analyzed after pretreatment of human hepatoma HepG2 cells with H(2)O(2). Cell death was increased through H(2)O(2) treatment dose dependently, and a dose of ginseng extract (PD:PT = 1:1) of 18.6 microg/mL was enough to derive it in reverse. MDA production was reduced through the administration of ginseng extracts even with more intensive H(2)O(2) treatments. Through the use of even low levels of ginseng extract (e.g., 1.86 microg/mL), catalase (CAT) activity was easily reduced from the plateau induced by H(2)O(2). The glutathione peroxidase activity was no better than that of CAT. We assume that ginseng extract acts as an antioxidant even when effective levels of ginseng differ. A ginseng extract dose of 18.6 microg/mL increased the apoptotic expression of oxidative stressed signals, such as c-Jun-N-terminal kinase and stress-activated protein kinase expressions, and mitochondrial cytochrome c released caspase-3 activation; however, these expressions changed with higher doses of ginseng.

Topics: Antioxidants; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Catalase; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Korea; Liver Neoplasms; Malondialdehyde; Mitogen-Activated Protein Kinases; Oxidants; Panax; Plant Extracts; Sapogenins; Saponins

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

Saikosaponin a, a purified ingredient of Chinese herb with known antitumor activity, can inhibit cell growth and DNA synthesis of hepatoma cell line HepG2. Both mRNA and protein of the CDK inhibitor p-16(INK4a) and p-15(INK4b) in HepG2 were greatly induced by saikosaponin a while that of p-21(CIP), p-27(KIP) and other cell cycle related genes were not. In addition, reduced phosphorylation of RB protein is observed in saikosaponin a-treated HepG2. Staurosporin, one of the PKC inhibitors, significantly prevented the saikosaponin a induced growth inhibition suggesting PKC pathway be involved. On the other hand, the phorbol ester tumor promoter TPA (12-O-Tetredecanolyphorbol 13-acetate) also inhibited HepG2 growth and specifically induced p-16(INK4a) and p-15(INK4b) mRNA expression. The results suggest that both saikosaponin a and TPA-induced HepG2 growth inhibition are associated with p-15(INK4a) and p-16(INK4b) gene expression and might be mediated by PKC signaling pathway.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Liver Neoplasms; Oleanolic Acid; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sapogenins; Saponins; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins

Saikosaponin A (SSA) induced cell death in the human hepatoma cell line (HuH-7) was investigated. Shortly after exposure to SSA, a DNA histogram showed a 'sub-G1 peak', which was recently reported as suggestive of apoptosis by other researchers. However, the electrophoresis of DNA indicated that such was not the case in the present experiment. The decreased intake of rhodamine 123 and the swelling of mitochondria were remarkable shortly after SSA exposure. These data seem to support the idea that the sub-G1 peak represents necrosis rather than apoptosis. Interestingly, the DNA electrophoresis revealed the smear pattern of small DNA fragments shortly after SSA-exposure, although it did not show the apoptotic ladder pattern. This finding and the appearance of the 'sub-G1 peak' is thought to have been a result of the degradation of the nuclear DNA in the early stages of cell death. Because these findings are different not only from apoptosis, but also from typical necrosis, a distinct mechanism of cell death caused by SSA is suggested.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Death; Cell Nucleus; DNA, Neoplasm; Electrophoresis, Agar Gel; Flow Cytometry; Humans; Liver Neoplasms; Mitochondrial Swelling; Necrosis; Oleanolic Acid; Sapogenins; Saponins; Tumor Cells, Cultured

The present study was undertaken to investigate the effects and the mechanism of the components of Sho-saiko-to (baicalein, baicalin, saikosaponin-a, saikosaponin-c, ginsenoside Rb1, ginsenoside Rg1) on cultured human hepatoma cells (HuH-7). Cell cycle analysis was carried out with flow cytometry and the bromodeoxyuridine (BrdU)-labelling method. The results showed that baicalein, baicalin and saikosaponin-a inhibited cell proliferation dose-dependently but independently of the cell cycle. Furthermore, it was found that saikosaponin-a possesses a strong cell-killing effect. On the other hand, saikosaponin-c, ginsenoside Rb1 and ginsenoside Rg1 had no effect on cell proliferation.

Topics: alpha-Fetoproteins; Antineoplastic Agents; Astringents; Carcinoma, Hepatocellular; Cell Cycle; Cell Death; Cell Division; DNA, Neoplasm; Drugs, Chinese Herbal; Flavanones; Flavonoids; Ginsenosides; Glycyrrhetinic Acid; Glycyrrhizic Acid; Humans; Liver Neoplasms; Oleanolic Acid; Panax; Plants, Medicinal; Sapogenins; Saponins; Tumor Cells, Cultured