ABSTRACT
BACKGROUND: Long-term cigarette smoking increases the risk of colorectal cancer mortality. Tobacco's addictive toxin, nicotine, was reported to increase DNA synthesis of colon cancer cells. Because metastasis is the major cause of cancer death, the influence of nicotine on the migration of colon cancer cells remains to be determined. METHODS: The influence of nicotine on the migration of colon cancer cells was evaluated using transwell assay. Nicotine receptor-mediated migration was studied by using both inhibitors and small interfering RNA (siRNA). The role of COX-2 signal was studied using pharmacological inhibitors. The expression of epithelial mesenchymal transition (EMT) marker and COX-2 signal was evaluated using real-time polymerase chain reaction (PCR). RESULTS: Nicotine enhanced DLD-1 and SW480 cell migration in a dose-dependent manner. We used inhibitors and siRNA to demonstrate that α7-nAChR mediates nicotine-enhanced colon cancer cell migration and upregulates fibronectin expression, which is involved in nicotine-enhanced migration. Furthermore, COX-2 signal was induced by nicotine treatment and is involved in nicotine-enhanced fibronectin expression. CONCLUSIONS: Nicotine, tobacco's additive toxin, enhances colon cancer metastasis through α7-nAChR and fibronectin--a mesenchymal marker for epithelial mesenchymal transition. Furthermore, COX-2 signal was involved in the induction of fibronectin. Therefore, smoking may play role in the progression of colon cancer.
Subject(s)
Cell Movement/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Fibronectins/metabolism , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Blotting, Western , Colonic Neoplasms/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Epithelial-Mesenchymal Transition , Humans , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
BACKGROUND: Thrombomodulin (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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: TM may be a modulator of cancer metastasis in HCC. Downregulation of TM expression may increase ZEB1 and decrease E-cadherin levels.
Subject(s)
Cadherins/metabolism , Carcinoma, Hepatocellular/pathology , Cell Movement , Homeodomain Proteins/metabolism , Liver Neoplasms/pathology , Thrombomodulin/metabolism , Transcription Factors/metabolism , Blotting, Western , Cadherins/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Homeodomain Proteins/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Thrombomodulin/antagonists & inhibitors , Thrombomodulin/genetics , Transcription Factors/genetics , Tumor Cells, Cultured , Zinc Finger E-box-Binding Homeobox 1ABSTRACT
BACKGROUND: GRP78 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). METHODS: In 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. RESULTS: Using 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. CONCLUSIONS: We 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.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/genetics , Cell Movement/genetics , Gene Silencing , Heat-Shock Proteins/genetics , Liver Neoplasms/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation , Endoplasmic Reticulum Chaperone BiP , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathologyABSTRACT
BACKGROUND: Sorafenib 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. METHODS: The 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. RESULTS: We found that HepG2 cells exhibited higher sensitivity toward sorafenib, with 50% inhibition concentration (IC(50)) >20 microMu for HepJ5 and 4.8 microM for HepG2. Specifically, when HepG2 cells received 20 microM sorafenib treatment for 24 h, 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 IC(50) values from >20 microM to 4.8 microM. CONCLUSIONS: GRP78 is a positive modifier for sorafenib resistance acquisition in HCC and represents a prime target for overcoming sorafenib resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Benzenesulfonates/pharmacology , Carcinoma, Hepatocellular/drug therapy , Drug Resistance, Neoplasm , Heat-Shock Proteins/metabolism , Liver Neoplasms/drug therapy , Pyridines/pharmacology , Apoptosis/drug effects , Blotting, Western , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Caspases/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cytochromes c/metabolism , Endoplasmic Reticulum Chaperone BiP , Flow Cytometry , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/genetics , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Membrane Potential, Mitochondrial/drug effects , Niacinamide/analogs & derivatives , Phenylurea Compounds , RNA, Small Interfering/pharmacology , Sorafenib , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To study the mechanism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-enhanced migration of colon cancer cells. BACKGROUND DATA: Long-term cigarette smoking increases the risk of colorectal cancer mortality. Tobacco-specific carcinogen, NNK, was reported to increase DNA synthesis of colon cancer cells. Since metastasis is the major cause of cancer death, the influence of NNK on the migration of colon cancer cells remains to be determined. METHODS: Receptor for NNK in colon cancer cells was identified by polymerase chain reaction (PCR) and real-time PCR. The influence of NNK on migration of colon cancer cells was evaluated by transwell and wound-healing assay. Receptor-mediated migration was studied by both inhibitor and small interfering RNA. RESULTS: alpha7 nicotinic acetylcholine receptor, alpha7-nAChR, was identified in 2 colon cancer cell lines, HT29 and DLD-1. NNK enhanced HT29 cell migration in both transwell and wound-healing assays. NNK also enhanced DLD-1 cell migration in dose dependent manner. We used inhibitor and siRNA to demonstrate that alpha7-nAChR mediated NNK-enhanced colon cancer cell migration and downregulation of E-cadherin were involved in NNK-enhanced migration of colon cancer cells. Furthermore, Snail and ZEB1, 2 major transcription repressors of E-cadherin in colon cancers, were induced by NNK treatment. CONCLUSIONS: Tobacco specific carcinogen, NNK, enhanced colon cancer metastasis through alpha7-nAChR and E-cadherin--one of the hallmarks of epithelial mesenchymal transition--and its transcription repressors. Therefore, smoking should be avoided in the patients with colorectal cancer.
Subject(s)
Adenocarcinoma/pathology , Carcinogens/pharmacology , Colonic Neoplasms/pathology , Nitrosamines/pharmacology , Receptors, Nicotinic/physiology , Adenocarcinoma/metabolism , Cadherins/metabolism , Cell Culture Techniques , Cell Movement/drug effects , Colonic Neoplasms/metabolism , HT29 Cells/drug effects , Homeodomain Proteins/metabolism , Humans , Snail Family Transcription Factors , Transcription Factors/metabolism , Zinc Finger E-box-Binding Homeobox 1 , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
This paper examines the evolution and current role of Medicaid in improving access to preconception care for low-income women. The authors review Medicaid's eligibility policy and benefits of relevance to women of reproductive age and discuss various approaches to promote preconception care in Medicaid. The challenges facing the program and potential opportunities to use the program to promote preconception care to low-income women are discussed.
