ABSTRACT
The aim of this study was to construct an RNA-interference plasmid (p-HIF-1α RNAi) targeting the human HIF-1α gene and assess its effects on HIF-1α expression and its anti-tumour functions in vitro. p-HIF-1α RNAi was constructed and confirmed by polymerase chain reaction (PCR) and DNA sequencing. Reverse transcriptase PCR (RT-PCR) and western blot were performed to detect HIF-1α expression in HCT116 cells following transfection of p-HIF-1α RNAi and p-control. The anti-tumour effects and mechanism of action of p-HIF-1α RNAi in HCT116 cells were further investigated. p-HIF-1α RNAi significantly inhibited HIF-1α expression in the HCT116 cell line. p-HIF-1α RNAi inhibited cell viability and reduced VEGF but not bFGF expression in the supernatant of HCT116 cells, down-regulated b-catenin and VEGF expression, and altered ß-catenin location in the HCT116 cell nucleus. The plasmid p-HIF-1α RNAi can effectively and specifically inhibit HIF-1α expression, inhibit cell proliferation, and alter the expression of key components in the Wnt/ß-catenin signaling pathway. Thus, p-HIF-1α RNAi is a novel and extremely promising therapeutic inhibitor of HIF-1α.
Subject(s)
Cell Proliferation , Colorectal Neoplasms/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Neovascularization, Pathologic , RNA Interference , Blotting, Western , Cell Survival , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Culture Media, Conditioned/metabolism , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Wnt Signaling Pathway , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Curcumin, the biologically active compound from the rhizome of Curcuma longa, could inhibit cell growth and induce apoptosis in gastric carcinoma. However, the underlying mechanism of curcumin on gastric carcinoma cells still needs further investigation. In this study, morphological observation indicated that curcumin inhibited the proliferation of AGS cells in a dose-dependent manner. According to the flow cytometric analysis, curcumin treatment resulted in G2/M arrest in AGS cells, accompanied with an increased expression of cyclin B1 and a decreased expression of cyclin D1. In addition, DNA ladders were observed by gel electrophoresis. Meanwhile, the activities of caspase-3, -8, and -9 were also enhanced in curcumin-treated AGS cells. Nevertheless, the increased activities could be inhibited by benzyloxycarbonyl-Val-Ala-Asp (OME)-fluoromethylketone (z-VAD-fmk), which suggested that the apoptosis was caspase-dependent. Furthermore, downregulation of rat sarcoma (Ras) and upregulation of extracellular-signal-regulated kinase (ERK) were also observed in AGS cells treated with curcumin by Western blot. U0126, an ERK inhibitor, blocked curcumin-induced apoptosis. The results suggested that curcumin inhibited the growth of the AGS cells and induced apoptosis through the activation of Ras/ERK signaling pathway and downstream caspase cascade, and curcumin might be a potential target for the treatment of gastric carcinoma.
Subject(s)
Curcumin/pharmacology , Stomach Neoplasms/drug therapy , Amino Acid Chloromethyl Ketones/chemistry , Apoptosis/drug effects , Caspase 3/metabolism , Caspases/metabolism , Cell Cycle/drug effects , Cell Cycle Checkpoints/drug effects , Curcuma/chemistry , Extracellular Signal-Regulated MAP Kinases/drug effects , Humans , Molecular Structure , Oligopeptides , Signal Transduction/drug effectsABSTRACT
The study aims to investigate the effect of microRNA-497 (miR-497) expression and bufalin treatment in regulating colorectal cancer invasion and metastasis. The expression of miR-497 in colorectal cancer cells with prior treatment with bufalin was determined using real-time quantitative PCR. The nude mouse abdominal aortic ring assay and the human umbilical vein endothelial cell (HUVEC) migration assays were used to measure the angiogenic effect of bufalin. The effect of both bufalin treatment and miR-497 overexpression on colorectal cancer metastasis was measured using an animal tumor model together with in vivo imaging. These results suggested: (1) In the HCT116 cells and HUVECs, proliferation was inhibited in a time-dependent and/or concentration-dependent manner following the administration of bufalin; (2) Bufalin inhibited cell migration in a concentration-dependent manner by cell motility assays; (3) In the aortic ring assay, administration bufalin to the aortic ring significantly promoted micro-angiogenesis of nude mouse abdominal aorta in a concentration-dependent and time-dependent manner; (4) miR-497 was upregulated in human colorectal cancer HCT116 cells treated with different concentrations of bufalin in a concentration-dependent manner; and (5) Combined application of bufalin and miR-497 significantly reduced metastatic lesions and reduced weight loss compared with bufalin alone and control groups in vivo. This study revealed that bufalin inhibited angiogenesis and regulated miR-497 expression and that bufalin and miR-497 acted in synergy to inhibit colorectal cancer metastasis, resulting in improved quality of life in a nude mouse model.
Subject(s)
Antineoplastic Agents/pharmacology , Bufanolides/pharmacology , Colorectal Neoplasms/genetics , MicroRNAs/genetics , Neoplasm Invasiveness/genetics , Animals , Blotting, Western , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Colorectal Neoplasms/pathology , Humans , Male , Mice , Mice, Nude , MicroRNAs/metabolism , Neoplasm Invasiveness/pathology , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
Golgi phosphoprotein 2 (GOLPH2) is a very important biomarker in a variety of diseases. Its biological function is not clear, particularly in gastric cancer. To investigate the role of GOLPH2 in human gastric cancer, and determine its effect on the Th1 lymphocyte response, its expression and that of IL-12A were measured by real-time PCR and immunohistochemistry. The relationship between GOLPH2 and IL-12A was analysed statistically. The effect of GOLPH2 on the Th1 lymphocyte response was investigated with an in vitro co-culture system. The results showed that in human gastric cancer, the expression of GOLPH2 was significantly higher and the expression of IL-12A was lower than in normal gastric mucosal tissues, and the expression levels of GOLPH2 and IL-12A were negatively correlated. In addition, obvious down-regulation of the Th1 response was observed when lymphocytes were co-cultured with gastric cancer SGC7901 cells over-expressing GOLPH2. GOLPH2 down-regulated the expression of IL-12A, and inhibited the expression of TNF-α and IFN-γ. The results indicated that GOLPH2 down-regulates the Th1 response via suppression of IL-12A in human gastric cancer, and this might provide a target for the prevention and treatment.
