wogonin and Carcinoma--Hepatocellular

Wogonin has been reported to exhibit pharmacological effects against cancer by regulating cell proliferation, metastasis and apoptosis, however, the role of wogonin in hepatocellular carcinoma (HCC) remains poorly elucidated.. The current study aimed to illustrate whether wogonin influences HCC cell cycle progression and apoptosis by regulating Hippo signaling.. The effects of wogonin on HCC cell viability, cell cycle progression and apoptosis were analyzed by utilizing CCK-8 and flow cytometry. RNA-seq was employed to analyze the expression profiles between wogonin-treated and control HCC cells, and the selected RNA-seq transcripts were validated by Reverse Transcription-quantitative realtime Polymerase Chain Reaction (RT-qPCR). Immunofluorescence staining was performed to detect the distribution of YAP/TAZ in the nucleus and cytoplasm in HCC cells. Western blotting and human apoptosis array were performed to examine the expression of the indicated genes.. We demonstrated that wogonin induced cell cycle arrest and apoptosis of HCC cell lines SMMC7721 and HCCLM3. RNA-seq analysis showed enrichment in genes associated with cell cycle progression and apoptosis following incubation with wogonin in HCC cells, and the pathways analysis further identified that Hippo signaling pathways highly altered in wogonin-treated cells. Specifically, wogonin increased the phosphorylation of MOB1 and LATS1, promoted translocation of endogenous YAP and TAZ from the nucleus to the cytoplasm, and facilitated phosphorylation of YAP and TAZ. Notably, overexpression of YAP or TAZ partially abrogated the wogonin-mediated HCC cell cycle arrest and apoptosis, and reversed wogonin-mediated suppression of Claspin.. Wogonin induced HCC cell cycle arrest and apoptosis probably by activating MOB1-LATS1 signaling to inhibit the activation of YAP and TAZ, and then decrease the expression of Claspin, suggesting that the understanding of the molecular mechanisms underlying wogonin-induced cell cycle arrest and apoptosis may be useful in HCC therapeutics.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line; Cell Line, Tumor; Cell Proliferation; Flavanones; Hippo Signaling Pathway; Humans; Liver Neoplasms; Protein Serine-Threonine Kinases; Transcription Factors

Wogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far.. The aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression.. Herein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC.. Our results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased.. In summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Down-Regulation; Enzyme Activation; Flavanones; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Molecular Docking Simulation; Phosphorylation; Xenograft Model Antitumor Assays

Screening active components from Chinese traditional medicine is an effective approach to discover new drugs or active structures. Cell membrane chromatography (CMC), developed rapidly because of its high sensitivity and effectiveness, has achieved a wide application in screening active components on pathological cells or tissues. However, it is hard to clarify the selectivity between pathological and normal tissues through simply using pathological cells. In this study, a novel comparative two-dimensional (2D) cell membrane chromatography system was established. Briefly, hepatic carcinoma HepG2 CMC columns and normal hepatic L02 CMC columns were simultaneously loaded to screen potential selective antitumor components from Scutellariae Radix by comparing the retention behaviors on two kinds of cells. Totally 13 components in Scutellariae Radix retained on both HepG2/ CMC and L02/ CMC columns. Among them, three components, oroxylin A, wogonin and chrysin, were screened out to perform stronger affinity on HepG2 columns, and in further cell proliferation assay, IC

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Membrane; Cell Proliferation; Chromatography, High Pressure Liquid; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Flavanones; Flavonoids; Hep G2 Cells; Humans; Inhibitory Concentration 50; Liver Neoplasms; Mass Spectrometry; Scutellaria baicalensis

Natural flavonoids from plants have been demonstrated to possess promising chemopreventive activities against various diseases. 7-{4-[Bis-(2-hydroxy-ethyl)-amino]-butoxy}-5-hydroxy-8-methoxy-2-phenyl-chromen-4-one (V8), a newly synthesized derivative of wogonin may have antioxidant, antiviral, anti-inflammatory and anti-tumor potentials as wogonin. Based on the recent findings of V8, the anti-tumor activities and fundamental mechanisms by which V8 inhibits growth of hepatocellular carcinoma were further investigated in this study. After the treatment of V8, a significant inhibition of HepG2 cell proliferation was observed in a dose-dependent manner with the IC50 value of 23 μM using MTT assay. The exposure to V8 also resulted in apoptosis induction and an accumulation of ROS and Ca(2+). Meanwhile, a release of cytochrome c (Cyt-c), activation of BH-3 only proteins and Bax, decrease in mitochondrial membrane potential ΔΨ, as well as a suppression of Bcl-2, pro-caspase9 and pro-caspase3 expression were shown. Moreover, knocking down CHOP partly decreased the effect of V8-mediated apoptosis and activation of GRP78, p-PERK, p-eIF2α, ATF4 and CHOP modulated ER stress triggered by V8. In vivo, V8 inhibited the transplanted mice H22 liver carcinomas in a dose-dependent manner. Compared with wogonin, V8 exhibited stronger anti-proliferative effects both in vitro and in vivo. The underlying mechanism of activating PERK-eIF2α-ATF4 pathway by which V8 induces apoptosis was verified once again in vivo. The apoptosis induction via the mitochondrial pathway by modulating the ROS-mediated ER signaling pathway might serve to provide support for further studies of V8 as a possible anticancer drug in the clinical treatment of cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Dose-Response Relationship, Drug; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Flavanones; Flavones; Hep G2 Cells; Humans; Inhibitory Concentration 50; Liver Neoplasms; Membrane Potential, Mitochondrial; Mice; Reactive Oxygen Species; Signal Transduction; Transcription Factor CHOP; Xenograft Model Antitumor Assays

Combination therapies may increase the antitumor effects and reduce the adverse effects for the treatment of hepatocellular carcinoma. In this study, we determined the effects of 5-fluorouracil alone or in combination with wogonin in vitro and in vivo, and we investigated the possible mechanisms. The combination of these 2 drugs led to a decrease in survival and a significant synergistic inhibitory effect on high COX-2 expression in SMMC-7721 hepatocellular carcinoma (HCC) cells. Furthermore, the results show that this combination inhibits COX-2 expression and increases sensitivity to chemotherapeutic agents partly through regulating the PI3K/Akt signaling pathway. Moreover, the combination treatment caused a significant growth inhibition of human tumor xenografts in vivo. In conclusion, wogonin may increase the cytotoxicity of some antineoplastic agents and it can be used in combination with these agents as a novel therapeutic regimen for HCC treatment.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Drug Synergism; Female; Flavanones; Fluorouracil; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Plant Extracts; Proto-Oncogene Proteins c-akt; Signal Transduction

The molecular mechanisms of wogonin-induced apoptosis of human hepatoma SMMC-7721 cells are reported. Wogonin treatment resulted in significant inhibition of SMMC-7721 cells in a time-dependent and concentration-dependent manner. Typical morphological changes and apoptotic blebbing in SMMC-7721 cells were observed after treatment with 1x10(-4) mol/l wogonin for a period of 0-48 h. Flow cytometry and Annexin-V/propidium iodide double-staining experiments revealed a dramatic increase in the number of apoptotic and G0/G1 phase cells after wogonin treatment. The proapoptotic activity of wogonin is attributed to its ability to modulate the expression of bcl-2 and bax proteins. It is observed that the expression of bax protein is dramatically increased whereas the synthesis of bc1-2 protein is significantly decreased when cells are treated with wogonin. The results presented in this paper suggested an important relationship between gene regulation and wogonin-induced apoptosis, and indicated the possibility of developing naturally occurring monoflavonoids as novel anticancer agents for better management of human cancers.

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Shape; Colorimetry; DNA Fragmentation; DNA, Neoplasm; Flavanones; Humans; Propidium; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles

The effects of the flavonoids from Scutellaria baicalensis Georgi (baicalein, baicalin and wogonin) in cultured human hepatoma cells (Hep G2, Hep 3B and SK-Hep1) were compared by MTT assay and flow cytometry. All three flavonoids dose-dependently decreased the cell viabilities accompanying the collapse of mitochondrial membrane potential and the depletion of glutathione content. However, the influence of baicalein, baicalin or wogonin on cell cycle progression was different. All three flavonoids resulted in prominent increase of G2/M population in Hep G2 cells, whereas an accumulation of sub G1 (hypoploid) peak in Hep 3B cells was observed. In SK-Hep1 cells, baicalein and baicalin resulted in dramatic boost in hypoploid peak, but wogonin made mainly in G1 phase accumulation. These data, together with the previous findings in other hepatoma cell lines, suggest that baicalein, baicalin and wogonin might be effective candidates for inducing apoptosis or inhibiting proliferation in various human hepatoma cell lines.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; DNA, Neoplasm; Drugs, Chinese Herbal; Flavanones; Flavonoids; Flow Cytometry; Glutathione; Humans; Lamiaceae; Medicine, Chinese Traditional; Mitochondria; Molecular Structure; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Wogonin and fisetin are flavonoids, which are widely distributed in plants. Our recent study demonstrated that, among seven structurally related flavonoids, wogonin and fisetin showed the most potent apoptosis-inducing activities in human promyeloleukemic cells HL-60. In the present investigation, we performed molecular studies to assess the apoptotic effects of wogonin and fisetin on hepatocellular carcinoma cells SK-HEP-1. Both wogonin and fisetin showed dose-dependent cytotoxic effects on SK-HEP-1 cells, accompanied by DNA fragmentation. Microscopic observation under Giemsa staining showed that wogonin and fisetin, at the dose of 80 microM, induced cellular swelling and the appearance of apoptotic bodies, characteristics of apoptosis, in SK-HEP-1 cells. Furthermore, flow cytometry analysis showed an increase of hypodiploid cells in wogonin- and fisetin-treated SK-HEP-1 cells. These data demonstrated that wogonin and fisetin were effective inducers of apoptosis in SK-HEP-1 cells. Treatment with an apoptosis-inducing concentration of wogonin or fisetin caused induction of caspase 3/CPP32 activity, but not of caspase 1 activity. In addition, a caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor Ac-YVAD-CHO, reversed the cytotoxic effects of wogonin and fisetin on SK-HEP-1 cells. Further, cleavage of caspase 3 substrates including poly(ADP-ribose) polymerase (PARP) and D4-GDI protein, and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated SK-HEP-1 cells. Increase of p53 protein was associated with wogonin- and fisetin-induced apoptosis; however, a p53-controlled gene, p21(Waf/Cip-1), was only induced in wogonin- (not fisetin-) treated SK-HEP-1 cells. Serum starvation elevated p21(Waf/Cip-1) protein expression, and enhanced the apoptotic induction activity of wogonin (not fiseitn) in SK-HEP-1 cells. Our study has provided molecular evidence to demonstrate that wogonin and fisetin had effective cytotoxic effects through apoptosis induction in hepatocellular carcinoma cells SK-HEP-1; activation of caspase 3 cascade, induction of p53 protein and alternative expression of p21(Waf/Cip-1) protein were involved.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 1; Caspase 3; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; Flavanones; Flavonoids; Flavonols; Flow Cytometry; Humans; Tumor Cells, Cultured; Tumor Suppressor Protein p53