Chih-Hsiung Wu

Nicotine-induced human breast cancer cell proliferation attenuated by garcinol through down-regulation of the nicotinic receptor and cyclin D3 proteins

Previous studies have demonstrated that the persistent exposure of human bronchial epithelial cells to nicotine (Nic) through nicotinic acetylcholine receptors increases cyclin D1 promoter activity and protein expression. The main purpose of this study is to elucidate the carcinogenic role of cyclin D3, which is involved in breast tumorigenesis when induced by Nic. Real-time PCR analysis revealed that cyclin D3 is highly expressed at the mRNA level in surgically dissected breast tumor tissue, compared to the surrounding normal tissue (tumor/normal fold ratioxa0=xa017.93, nxa0=xa074). To test whether Nic/nicotinic acetylcholine receptor (nAChR) binding could affect cyclin D3 expression in human breast cancer cells, the transformed cell line MCF-10A-Nic (DOX) was generated from normal breast epithelial cells (MCF-10A) with inducible α9-nAChR gene expression, using the adenovirus tetracycline-regulated Tet-off system. Tet-regulated overexpression of α9-nAChR in MCF-10A-Nic (DOX) xenografted BALB/c-nu/nu mice resulted in a significant induction of cyclin D3. In contrast, cyclin D3 expression was down-regulated in α9-nAChR knock-down (siRNA) MDA-MB-231-xenografted tumors in NOD.CB17-PRKDC(SCID)/J(NOD-SCID) mice. Furthermore, we found that Nic-induced human breast cancer (MDA-MB-231) cell proliferation was inhibited by 1xa0μM of garcinol (Gar), isolated from the edible fruit Garcinia indica, through down-regulation of α9-nAChR and cyclin D3 expression. These results suggest that α9-nAChR-mediated cyclin D3 overexpression is important for nicotine-induced transformation of normal human breast epithelial cells. The homeostatic regulation of cyclin D3 has the potential to be a molecular target for antitumor chemotherapeutic or chemopreventive purposes in clinical breast cancer patients.

Glucose-Regulated Protein 78 Is a Novel Contributor to Acquisition of Resistance to Sorafenib in Hepatocellular Carcinoma

BackgroundSorafenib is a newly established cancer drug found to be an effective systemic treatment for advanced hepatocellular carcinoma (HCC). However, little is known about any potential effectors that modify tumor cell sensitivity towards sorafenib. Here, we present the first evidence that glucose-regulated protein 78 (GRP78) is intimately associated with acquisition of resistance towards sorafenib.MethodsThe role of GRP78 in acquisition of resistance towards sorafenib was determined using HepJ5 (a GRP78-overexpressing subline) and HepG2 as its pair-matched control. RNA interference in cancer cells was applied to determine the influence of GRP78 expression on sensitivity to sorafenib treatment.ResultsWe found that HepG2 cells exhibited higher sensitivity toward sorafenib, with 50% inhibition concentration (IC50) >20 μΜ for HepJ5 and 4.8xa0μM forxa0HepG2. Specifically, when HepG2 cells received 20xa0μM sorafenib treatment for 24xa0h, over 80% of cells underwent apoptosis compared with only 32% of HepJ5 cells under similar experimental conditions. Similarly, GRP78 knockdown in HepJ5 cells by small interfering RNA (siRNA) technique enhanced the efficacy of sorafenib-mediated cell death. This was reflected by a shift of IC50 values from >20xa0μM to 4.8xa0μM.ConclusionsGRP78 is a positive modifier for sorafenib resistance acquisition in HCC and represents a prime target for overcoming sorafenib resistance.

Apple polyphenol phloretin potentiates the anticancer actions of paclitaxel through induction of apoptosis in human hep G2 cells.

Phloretin (Ph), which can be obtained from apples, apple juice, and cider, is a known inhibitor of the type II glucose transporter (GLUT2). In this study, real‐time PCR analysis of laser‐capture microdissected (LCM) human hepatoma cells showed elevated expression (>5‐fold) of GLUT2 mRNA in comparison with nonmalignant hepatocytes. In vitro and in vivo studies were performed to assess Ph antitumor activity when combined with paclitaxel (PTX) for treatment of human liver cancer cells. Inhibition of GLUT2 by Ph potentiated the anticancer effects of PTX, resensitizing human liver cancer cells to drugs. These results demonstrate that 50–150 µM Ph significantly potentiates DNA laddering induced in Hep G2 cells by 10 nM PTX. Activity assays showed that caspases 3, 8, and 9 are involved in this apoptosis. The antitumor therapeutic efficacy of Ph (10 mg/kg body weight) was determined in cells of the SCID mouse model that were treated in parallel with PTX (1 mg/kg body weight). The Hep G2‐xenografted tumor volume was reduced more than fivefold in the Phu2009+u2009PTX‐treated mice compared to the PTX‐treated group. These results suggest that Ph may be useful for cancer chemotherapy and chemoprevention.

Glucose-Regulated Protein 78 (GRP78) Silencing Enhances Cell Migration but Does Not Influence Cell Proliferation in Hepatocellular Carcinoma

BackgroundGRP78 plays an essential role in embryonic development and in the therapeutic treatment and progression of cancer. However, little is known about the role of GRP78 in hepatocellular carcinoma (HCC).MethodsIn this study, we characterized five different HCC cell lines to examine GRP78 expression patterns and found that only HepJ5 cells ectopically overexpress GRP78. We knocked down GRP78 expression in HepJ5 cells using a small interfering RNA (siRNA), and the proliferation assay and migration assay were performed.ResultsUsing siRNA technique, we could successfully reduce GRP78 expression levels in HepJ5 cells. In a cell growth study, we found that GRP78-siRNA caused no significant changes in cellular proliferation in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and cell cycle distribution. In a cell migration study, we found that GRP78-siRNA HepJ5 cells had dramatically increased migration ability in Transwell assay.ConclusionsWe conclude that ectopically expressed GRP78 does not contribute to the increased proliferation of HepJ5 cells, but does correlate with the migration of HCC cells under normoxic conditions.

MicroRNA-200a and -200b mediated hepatocellular carcinoma cell migration through the epithelial to mesenchymal transition markers

BackgroundMicroRNAs (miRNAs) play an essential role in mediating gene expression in both normal and malignant cells. However, little is known about specific miRNAs during the development of hepatocellular carcinoma (HCC) from well-differentiated to poorly differentiated cells.MethodsWe performed miRNA array analysis of three different HCC cell lines: HepG2, HepJ5, and skHep-1. The expression patterns of miR-200 family members were confirmed by real-time polymerase chain reaction (PCR). We overexpressed miR-200 family members by using a lentivirus system and selected for stably transduced cells using antibiotics. The migration ability of the cells was tested using the Transwell migration assay system.ResultsOur miRNA array and real-time PCR results indicated a decrease in the expression of miR-200 family members in poorly differentiated skHep-1 cells compared with well-differentiated HepG2 cells. We overexpressed miR-200a and miR-200b in both HepJ5 and skHep-1 cells and found that the overexpression of the miR-200 family members did not influence proliferation, although migration was decreased in these cells. We found that overexpression of miR-200 family members led to an upregulation of E-cadherin expression in both HepJ5 and skHep-1 cells. Furthermore, we silenced E-cadherin expression by shRNA in miR200a-HepJ5 cells and found that the migratory ability of these cells was enhanced upon the decrease in E-cadherin expression.ConclusionsMembers of the miR-200 family (miR-200a and miR-200b) play important roles in HCC migration by regulating E-cadherin expression.

Knockdown of Thrombomodulin Enhances HCC Cell Migration through Increase of ZEB1 and Decrease of E-cadherin Gene Expression

BackgroundThrombomodulin (TM) is a key molecule mediating circulation homeostasis through its binding to thrombin. The TM–thrombin complex can activate protein C and thrombin-activatable fibrinolysis inhibitor to form a tight clot. In many cancer tissues, decrease of TM expression may correlate with cancer metastasis. However, the role of TM in hepatocellular carcinoma (HCC) progression is still unclear.MethodsWe characterized TM expression in HCC cells (HepJ5 and skHep-1 cells) using real-time polymerase chain reaction (PCR) and Western blotting. We then manipulated TM expression using both TM-specific short hairpin RNA (shRNA) and overexpressing it in HCC cells. Transwell migration assay was performed to monitor the migratory ability of HCC cells under different levels of TM expression.ResultsWe found that TM was ectopically highly expressed in skHep-1 at both transcriptional and translational levels. After silencing TM expression in skHep-1 cells, we found that metastatic capability was dramatically increased. Conversely, overexpression of TM in HepJ5 cells decreased metastatic ability. We investigated the possible mechanism and found that decreased TM-mediated enhancement of cell migration was dependent on upregulation of ZEB1, a repressor of E-cadherin.ConclusionsTM may be a modulator of cancer metastasis in HCC. Downregulation of TM expression may increase ZEB1 and decrease E-cadherin levels.

Silencing of Glucose-Regulated Protein 78 (GRP78) Enhances Cell Migration Through the Upregulation of Vimentin in Hepatocellular Carcinoma Cells

BackgroundGlucose-regulated protein 78 (GRP78) plays an important role in embryonic development and cancer progression. However, there is little information regarding the regulation of GRP78 in hepatocellular carcinoma (HCC) metastasis.MethodsWe used RNA silencing and cDNA expression vectors to manipulate target gene expression in HCC cells. The transwell migration assay and xCelligence biosensor system were applied to determine the proliferatory and migratory ability of the HCC cells.ResultsIn this study, we found that GRP78 silencing enhanced cell migration in both HepJ5 and Mahlavu cells. Overexpressed GRP78 in skHep1 cells suppressed the migratory ability. In the insight mechanism dissection for GRP78-mediated cancer migration, we found that downregulation of GRP78 caused the increase of vimentin expression on HCC cells. Suppressed vimentin expression also decreased the migratory ability on HCC, indicating that vimentin expression levels modulated the cell migratory ability.ConclusionWe found that silencing GRP78 in HCC cells may enhance cell migration through the increase of vimentin expression.

From Smoking to Cancers: Novel Targets to Neuronal Nicotinic Acetylcholine Receptors

Cigarette smoking bears a strong etiological association with many neovascularization-related diseases, including cancer, cardiovascular disease, and age-related macular degeneration. Cigarette smoke is a complex mixture of many compounds, including nicotine, which is the major active and addictive component of tobacco. Nicotine and its specific metabolized carcinogens directly bind to nicotinic acetylcholine receptors (nAChRs) on cell membranes and trigger the nAChR signal cascade. The nAChRs were originally thought to be ligand-gated ion channels that modulate physiological processes ranging from neurotransmission to cancer signaling. For several decades, the nAChRs served as a prototypic molecule for neurotransmitter receptors; however, they are now important therapeutic targets for various diseases, including Alzheimers and Parkinsons diseases, schizophrenia, and even cancer. This paper describes recent advances in our understanding of the assembly, activity, and biological functions of nicotinic receptors, as well as developments in the therapeutic application of nicotinic receptor ligands.

NNK enhances cell migration through α7-nicotinic acetylcholine receptor accompanied by increased of fibronectin expression in gastric cancer.

BackgroundIn this study, we intended to dissect the mechanism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-enhanced migration of gastric cancer. Smoking has been defined as a risk factor for gastric cancer. Tobacco-specific carcinogen, NNK, was reported to enhance cancer progression in gastric cancer. Currently, metastasis is the major issue for clinical cancer therapy, but the influence of NNK on the migration of gastric cancer remains to be determined.MethodsThe expression of nicotinic receptor in gastric cancer cells was identified by real-time polymerase chain reaction and Western blotting. The influence of NNK on migration of gastric cancer cells was evaluated by the transwell migration assay system. Receptor-mediated migration was studied by both inhibitor and small interfering RNA.ResultsAlpha7 nicotinic acetylcholine receptor, alpha7-nicotinic acetylcholine receptor (nAChR), was identified higher than alpha9-nAChR in gastric cancer cell lines, AGS cells. NNK enhanced significantly gastric cancer cell migration in transwell assay. We used inhibitor and siRNA to demonstrate that alpha7-nAChR mediated NNK-enhanced gastric cancer cell migration and upregulation of fibronectin were involved in NNK-enhanced migration of gastric cancer cells. Finally, we found that silenced fibronectin expression level inhibited the migratory ability in AGS cells.ConclusionsNNK enhanced gastric cancer metastasis through alpha7-nAChR and fibronectin—one of the hallmarks of epithelial mesenchymal transition.

Antitumor Activity of Combination Treatment of Lentinus edodes Mycelium Extracts with 5-Fluorouracil against Human Colon Cancer Cells Xenografted in Nude Mice

AIM: 5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs targeting various cancers, but its chemoresistance remains as a major obstacle in clinical settings. In this study, we evaluated the in vivo efficacy of Lentinus edodes mycelium extracts (designated as LEM), an edible mushroom extracts, as a 5-FU adjuvant agent. Furthermore, we intended to study the underlying mechanisms to account for the role of LEM. METHODS: Human colon cancer COLO 205 cells were treated with 5-FU, LEM, or combination of 5-FU with LEM. Induction of apoptosis and cell cycle arrest was demonstrated by DNA ladder electrophoresis and flow cytometry, respectively. Additionally, COLO 205 cells were transplanted into athymic nude mice as a tumor model for evaluation of the antitumor effect of combination treatment with LEM plus 5-FU. The mechanisms for altered cell cycle progression were investigated by immunoblotting analyses of the G 0 /G 1 -phase regulatory proteins. RESULTS: COLO 205 cells were markedly sensitized to apoptosis and G 0 /G 1 -phase arrest by combination treatment of 5-FU with LEM when compared with 5-FU alone. Our results furthermore indicated that LEM markedly enhanced the 5-FU-mediated upregulation of the p53, p21/Cip1 and p27/Kip1 proteins in COLO 205 cells-xenografted tumor tissues. In contrast, although the expression levels of cyclins B and D3 proteins were down regulated in the 5-FU-treated tumor tissues, no significant potentiation effect was observed in the tumors with 5-FU and LEM combination treatment. CONCLUSION: Our results suggest that combination of 5-FU with LEM may represent a novel chemotherapeutic strategy in colon cancers and that p53, p21/Cip1 and p27/Kip1 may play some important roles for the involvement in antitumor activity.