shogaol and Liver-Neoplasms

Currently, chemoresistance seriously attenuates the curative outcome of liver cancer. The purpose of our work was to investigate the influence of 6-shogaol on the inhibition of 5-fluorouracil (5-FU) in liver cancer. The cell viability of cancer cells was determined by MTT assay. Liver cancer cell apoptosis and the cell cycle were examined utilizing flow cytometry. Moreover, qRT-PCR and western blotting was used to analyse the mRNA and protein expression levels, respectively. Immunohistochemistry assays were used to examine multidrug resistance protein 1 (MRP1) expression in tumour tissues. In liver cancer cells, we found that 6-shogaol-5-FU combination treatment inhibited cell viability, facilitated G0/G1 cell cycle arrest, and accelerated apoptosis compared with 6-shogaol or 5-FU treatment alone. In cancer cells cotreated with 6-shogaol and 5-FU, AKT/mTOR pathway- and cell cycle-related protein expression levels were inhibited, and MRP1 expression was downregulated. AKT activation or MRP1 increase reversed the influence of combination treatment on liver cancer cell viability, apoptosis and cell cycle arrest. The inhibition of AKT activation to the anticancer effect of 6-shogaol-5-FU could be reversed by MRP1 silencing. Moreover, our results showed that 6-shogaol-5-FU combination treatment notably inhibited tumour growth in vivo. In summary, our data demonstrated that 6-shogaol contributed to the curative outcome of 5-FU in liver cancer by inhibiting the AKT/mTOR/MRP1 signalling pathway.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Catechols; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Fluorouracil; Humans; Liver Neoplasms; Multidrug Resistance-Associated Proteins; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

Liver cancer is a gastrointestinal malignant tumor with high lethality. The prognosis of liver cancer remains poor. Compounds derived from natural products have been confirmed to alleviate the progression of various diseases, including cancers. Additionally, 6-Shogaol has been reported to induce apoptosis in liver cancer cells. However, the mechanism by which 6-shogaol regulates apoptosis in liver cancer cells remains unclear. To investigate the function of 6-shogaol in liver cancer, RT-qPCR and western blotting were used to detect the expression of TLR4 and FOXO3a in liver cancer cells, respectively. The OD value of liver cancer cells was measured using the MTT assay. Flow cytometry was used to measure cell apoptosis. 6-Shogaol inhibited the growth of liver cancer cells. TLR4 and Wnt/[Formula: see text]-catenin were upregulated in liver cancer cells, and FOXO3a was inactivated, but 6-Shogaol reversed the expression of TLR4, Wnt/[Formula: see text]-catenin and FOXO3a in liver cancer cells. Additionally, TLR4 overexpression partially reversed the inhibitory effect of 6-shogaol on the progression of liver cancer cells via Wnt/[Formula: see text]-catenin signaling. Furthermore, the 6-shogaol-induced increase in FOXO3a expression in liver cancer was notably suppressed by TLR4 or Wnt/[Formula: see text]-catenin upregulation. Thus, 6-Shogaol suppresses the progression of liver cancer by mediating Wnt/[Formula: see text]-catenin signaling and is a potential agent for the treatment of liver cancer.

Topics: Apoptosis; beta Catenin; Catechols; Catenins; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Toll-Like Receptor 4; Wnt Signaling Pathway

Tumor necrosis factor (TNF)‑related apoptosis‑inducing ligand (TRAIL) is a member of the TNF superfamily and is an antitumor drug that induces apoptosis in tumor cells with minimal or no effects on normal cells. Here, it is demonstrated that 6‑shogaol (6‑sho), a bioactive component of ginger, exerted anti‑inflammatory and anticancer properties, attenuated tumor cell propagation and induced TRAIL‑mediated cell death in liver cancer cells. The current study identified a potential pathway by revealing that TRAIL and 6‑sho or chloroquine acted together to trigger reactive oxygen species (ROS) production, to upregulate tumor‑suppressor protein 53 (p53) expression and to change the mitochondrial transmembrane potential (MTP). Treatment with N‑acetyl‑L‑cysteine reversed these effects, restoring the MTP and attenuated ROS production and p53 expression. Interestingly, treatment with 6‑sho increased p62 and microtubule‑associated proteins 1A/1B light chain 3B‑II levels, indicating an inhibited autophagy flux. In conclusion, attenuation of 6‑sho‑induced autophagy flux sensitized cells to TRAIL‑induced apoptosis via p53 and ROS, suggesting that the administration of TRAIL in combination with 6‑sho may be a suitable therapeutic method for the treatment of TRAIL‑resistant Huh7 liver cells.

Topics: Acetylcysteine; Apoptosis; Autophagy; Caspases, Initiator; Catechols; Cell Line, Tumor; Chloroquine; Drug Synergism; Drug Therapy, Combination; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mutagens; Plant Extracts; Reactive Oxygen Species; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53; Zingiber officinale

The epithelial-mesenchymal transition (EMT) is an important cellular process during which polarized epithelial cells become motile mesenchymal cells, which promote cancer metastasis. Ginger, the rhizome of Zingiber officinale, is extensively used in cooking worldwide and also as a traditional medicinal herb with antioxidant, anti-inflammatory and anticancer properties. Several pungent compounds have been identified in ginger, including zingerone, which has anticancer potential. However, the role of zingerone in EMT is unclear. We investigated the synergistic effect of zingerone and its derivative on EMT. Transforming growth factor-beta 1 (TGF-β1) induces the EMT to promote hepatocellular carcinoma metastasis, including migration and invasion. To understand the repressive role of the combination of zingerone and its derivative (ZD 2) in hepatocellular carcinoma metastasis, we investigated the potential use of each compound of ginger, such as zingerone, ZD 2 and 6-shogaol, or the mixture of zingerone and ZD 2 (ZD 2-1) as inhibitors of TGF-β1 induced EMT development in SNU182 hepatocellular carcinoma cells in vitro. We show that ZD 2-1, but not zingerone, ZD 2 and 6-shogaol significantly increased expression of the epithelial marker E-cadherin and repressed Snail upregulation and expression of the mesenchymal marker N-cadherin during initiation of the TGF-β1 induced EMT. In addition, ZD 2-1 inhibited the TGF-β1 induced increase in cell migration and invasion of SNU182 hepatocellular carcinoma cells. Furthermore, ZD 2-1 significantly inhibited TGF-β1 regulated matrix metalloproteinase-2/9 and activation of Smad2/3. We also found that ZD 2-1 inhibited nuclear translocation of NF-κB, activation of p42/44 MAPK/AP1 signaling pathway in the TGF-β1 induced EMT. Our findings provide new evidence that combined treatment with ZD 2, novel zingerone derivative, and zingerone synergistically suppresses hepatocellular carcinoma metastasis in vitro by inhibiting the TGF-β1 induced EMT.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Synergism; Epithelial-Mesenchymal Transition; Guaiacol; Humans; Liver Neoplasms; Neoplasm Invasiveness; Transforming Growth Factor beta1

Shogaols are a group of the active constituents of ginger that have been identified to have various biological activities. The aim of the current study was to investigate the antitumor activity of 6-shogaol in hepatocellular carcinoma (HCC) and the possible involvement of reactive oxygen species as a putative mechanism of action. HCC cell lines, HepG2 and Huh-7, were used to study the in vitro anti-cancer activity of 6-shogaol via the application of various molecular biology techniques. Results showed that 6-shogaol effectively inhibited the cell viability, caused cell cycle arrest at G2/M phase and induced apoptosis in HCC cells as indicated by MTT assay, DAPI nuclear staining, annexin V assay, cell cycle analysis, and activation of caspase-3. Western blot analysis revealed the ability of 6-shogaol to target cancer survival signaling pathways mediated by mitogen-activated protein kinase (MAPK), 5' AMP-activated protein kinase (AMPK) and Akt. In addition, 6-Shogaol induced alteration of cyclin proteins expression and caused cleavage of protein kinase C delta. Furthermore, 6-Shogaol was able to induce the production of reactive oxygen species and endoplasmic reticulum (ER) stress-associated proteins and the consequent activation of autophagy in HepG2 cells. Taken together, the current study highlights evidences that 6-shogaol induces apoptosis, modulates cyclins expression and targets cancer survival signaling pathways in HCC cell lines, at least in part, via the production of reactive oxygen species. These findings support 6-shogaol's clinical promise as a potential candidate for HCC therapy.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Catechols; Cell Proliferation; Endoplasmic Reticulum Stress; G2 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Liver Neoplasms; M Phase Cell Cycle Checkpoints; Phosphorylation; Reactive Oxygen Species; Signal Transduction

The aim of this study was to investigate the protective effects of 6-shogaol on tert-butyl hydroperoxide (tBHP)-induced oxidative stress leading to apoptosis in human hepatoma cell line HepG2. The cells were exposed to tBHP (100 μmol/l) after pretreatment with 6-shogaol (2.5 and 5 μmol/l), and then cell viability was measured. 6-Shogaol fully prevented HepG2 cell death caused by tBHP. Treatment of tBHP resulted in apoptotic cell death as assessed by TUNEL assay and the expression of apoptosis regulator proteins, Bcl-2 family, caspases and cytochrome c. Cells treated with 6-shogaol showed rapid reduction of apoptosis by restoring these markers of apoptotic cells. In addition, 6-shogaol significantly recovered disruption of mitochondrial membrane potential as a start sign of hepatic apoptosis induced by oxidative stress. In line with this observation, antioxidative 6-shogaol inhibited generation of reactive oxygen species and depletion of reduced glutathione in tBHP-stimulated HepG2 cells. Taken together, these results for the first time showed antioxidative and antiapoptotic activities of 6-shogaol in tBHP-treated hepatoma HepG2 cells, suggesting that 6-shogaol could be beneficial in hepatic disorders caused by oxidative stress.

Topics: Apoptosis; Carcinoma, Hepatocellular; Catechols; Cell Survival; Dose-Response Relationship, Drug; Glutathione; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Oxidative Stress; Reactive Oxygen Species; tert-Butylhydroperoxide

We previously demonstrated that 6-shogaol and 6-gingerol, two active compounds in ginger (Zingiber officinale), possess antiinvasive activity against highly metastatic hepatoma cells. The aims of this study were to evaluate the inhibitory effect and molecular mechanism underlying the transcription and translation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) in Hep3B cells as well as the antiangiogenic activity of 6-gingerol and 6-shogaol.. By gelatin zymography and luciferase reporter gene assays, we found that 6-gingerol and 6-shogaol regulate MMP-2/-9 transcription. Moreover, 6-gingerol directly decreased expression of uPA, but the 6-shogaol-mediated decrease in uPA was accompanied by up-regulation of plasminogen activator inhibitor (PAI)-1. 6-Gingerol and 6-shogaol concentrations of ≥ 10 μM and ≥ 2.5 μM, respectively, significantly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling, the activation of NF-κB, and the translocation of NF-κB and STAT3. Incubation of 6-gingerol or 6-shogaol with human umbilical vein endothelial cells or rat aortas significantly attenuated tube formation.. 6-Shogaol and 6-gingerol effectively inhibit invasion and metastasis of hepatocellular carcinoma through diverse molecular mechanisms, including inhibition of the MAPK and PI3k/Akt pathways and NF-κB and STAT3 activities to suppress expression of MMP-2/-9 and uPA and block angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Hepatocellular; Catechols; Fatty Alcohols; Humans; Interleukin-8; Liver Neoplasms; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases; NF-kappa B; Phosphatidylinositol 3-Kinases; Plasminogen Activator Inhibitor 1; Proto-Oncogene Proteins c-akt; Rats; STAT3 Transcription Factor; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A

6-Shogaol is an active compound isolated from Ginger (Zingiber officinale Rosc). In this work, we demonstrated that 6-shogaol induces apoptosis in human hepatocellular carcinoma cells in relation to caspase activation and endoplasmic reticulum (ER) stress signaling. Proteomic analysis revealed that ER stress was accompanied by 6-shogaol-induced apoptosis in hepatocellular carcinoma cells. 6-shogaol affected the ER stress signaling by regulating unfolded protein response (UPR) sensor PERK and its downstream target eIF2α. However, the effect on the other two UPR sensors IRE1 and ATF6 was not obvious. In prolonged ER stress, 6-shogaol inhibited the phosphorylation of eIF2α and triggered apoptosis in SMMC-7721 cells. Salubrinal, an activator of the PERK/eIF2α pathway, strikingly enhanced the phosphorylation of eIF2α in SMMC-7721 cells with no toxicity. However, combined treatment with 6-shogaol and salubrinal resulted in significantly increase of apoptosis and dephosphorylation of eIF2α. Overexpression of eIF2α prevented 6-shogaol-mediated apoptosis in SMMC-7721 cells, whereas inhibition of eIF2α by small interfering RNA markedly enhanced 6-shogaol-mediated cell death. Furthermore, 6-shogaol-mediated inhibition of tumor growth of mouse SMMC-7721 xenograft was associated with induction of apoptosis, activation of caspase-3, and inactivation of eIF2α. Altogether our results indicate that the PERK/eIF2α pathway plays an important role in 6-shogaol-mediated ER stress and apoptosis in SMMC-7721 cells in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Catechols; Cell Line, Tumor; Cell Survival; Cinnamates; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; eIF-2 Kinase; Electrophoresis, Gel, Two-Dimensional; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Humans; Liver Neoplasms; Mice; Neoplasm Proteins; Phosphorylation; RNA, Small Interfering; Thiourea; Time Factors; Transcription Factor CHOP; Unfolded Protein Response; Up-Regulation; Xenograft Model Antitumor Assays

Hepatocellular carcinoma is the most common type of liver cancer and is highly metastatic. Metastasis is considered to be the major cause of death in cancer patients. Ginger is a natural dietary rhizome with anti-oxidative, anti-inflammatory, and anti-carcinogenic activities. The aims of this study were to evaluate the anti-invasion activity of 6-shogaol and 6-gingerol, two compounds found in ginger, on hepatoma cells.. The migratory and invasive abilities of phorbol 12-myristate 13-acetate (PMA)-treated HepG2 and PMA-untreated Hep3B cells were both reduced in a dose-dependent manner by treatment with 6-shogaol and 6-gingerol. Upon incubation of PMA-treated HepG2 cells and PMA-untreated Hep3B cells with 6-shogaol and 6-gingerol, matrix metalloproteinase (MMP)-9 activity decreased, whereas the expression of tissue inhibitor metalloproteinase protein (TIMP)-1 increased in both cell types. Additionally, urokinase-type plasminogen activator activity was dose-dependently decreased in Hep3B cells after incubation with 6-shogaol for 24 h. Analysis with semi-quantitative reverse transcription-PCR showed that the regulation of MMP-9 by 6-shogaol and 6-gingerol and the regulation of TIMP-1 by 6-shogaol in Hep3B cells may on the transcriptional level.. These results suggest that 6-shogaol and 6-gingerol might both exert anti-invasive activity against hepatoma cells through regulation of MMP-9 and TIMP-1 and that 6-shogaol could further regulate urokinase-type plasminogen activity.

Topics: Carcinoma, Hepatocellular; Catechols; Fatty Alcohols; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Zingiber officinale

Mahlavu cells, poorly differentiated and p53 mutants of a human hepatoma subline, are known to be highly refractory to a number of chemotherapeutic agents and radiotherapy due to their high expressions of multidrug resistance gene-1 (MDR-1) and Bcl-2 proteins. Thus, it is desirable to search for an alternative strategy for effective eradication of this type of cancer cells. We present evidence here for the first time that 6-shogaol (6-SG), an alkanone isolated from the rhizomes of ginger, can effectively induce apoptotic cell death of Mahlavu cells via an oxidative stress-mediated caspase-dependent mechanism. The cascade of events in 6-SG-induced apoptosis of these cells involved an initial overproduction of reactive oxygen species (ROS) followed by a severe depletion of intracellular glutathione (GSH) contents. Both events consequently entailed a significant drop in mitochondrial transmembrane potential (DeltaPsim), which ultimately activated the activities of caspases 3/7 resulting in the DNA fragmentation. Interestingly, we also found that N-acetylcysteine (NAC), an antioxidant and a precursor of GSH biosynthesis, could offer a near complete protection of apoptotic cell death exerted by 6-SG. Similarly, exogenously added GSH could also provide protection with an equal efficacy. However, it was paradoxical that both Boc-Asp(OMe)-fmk (a broad caspases inhibitor) and cyclosporin A (an mitochondrial permeability transition opening inhibitor) could only partially protect these cells from 6-SG-induced apoptosis. Taking these data into consideration, it is obvious that GSH depletion is the major contributing factor in arbitrating 6-SG-induced apoptosis of Mahlavu cells. In conclusion, we provide here a novel modality that can help to eradicate a p53 mutant of human hepatoma cells by using a natural consistent isolated form of ginger. These data also provide evidence to reaffirm the notion that consumption of certain foodstuffs can be beneficial to health because some of the constituents contained in them may be anticarcinogenic.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspases; Catechols; Cell Line, Tumor; DNA Damage; Humans; Liver Neoplasms; Mutation; Oxidative Stress; Plant Extracts; Plant Roots; Reactive Oxygen Species; Zingiber officinale