ABSTRACT
8-hydroxydeoxyguanosine (8-OHdG) is generated consequent to oxidative stress, but its paradoxical anti-oxidative, anti-inflammatory, and anti-mutagenic effects via Rho-GTPase inhibition were noted in various models of inflammation and cancer. Metastasis occurs through cell detachment, epithelial-mesenchymal transition (EMT), and cell migration; during these processes, changes in cell morphology are initiated through Rho-GTPase-dependent actin cytoskeleton polymerization. In this study, we explored the anti-metastatic mechanisms of 8-OHdG in Panc-1 pancreatic cancer cells. 8-OHdG inhibits cell migration by inactivating ERM and Rho-GTPase proteins, and inhibiting focal adhesion kinase (FAK) and matrix metalloproteinases (MMPs). At 15min, 8-OHdG significantly inactivated ERM (p < 0.05) and led to a significant retardation of wound healing; siERM and H1152 (ROCK inhibitor) had similar effects (p < 0.05). However, FAK inhibitor 14, DPI (NOX inhibitor), and NAC (antioxidant) significantly delayed wound healing without inhibiting ERM or CD44 (p < 0.05). In the experiments on cell migration, siERM, siCD44, DPI, and 8-OHdG significantly inhibited MMPs. 8-OHdG significantly decreased DCF-DA activation in Panc-1 pancreatic cancer cells and down-regulated NOXs (nox-1, nox-2, and nox-3). Finally, all of these anti-migration actions of 8-OHdG resulted in significant inhibition of EMT, as evidenced by the up-regulation of ZO-1 and claudin-1 and down-regulation of vimentin. We found significant inhibition of lung metastasis of Panc-1 cells by 8-OHdG. In conclusion, exogenous 8-OHdG had potent anti-metastasis effects mediated by either ERM or Rho GTPase inhibition in metastasis-prone pancreatic cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Deoxyguanosine/analogs & derivatives , Epithelial-Mesenchymal Transition/drug effects , GTPase-Activating Proteins/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/drug therapy , Pancreatic Neoplasms/drug therapy , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , 8-Hydroxy-2'-Deoxyguanosine , Acetylcysteine/pharmacology , Animals , Cell Line, Tumor , Cell Movement/drug effects , Claudin-1/agonists , Claudin-1/genetics , Claudin-1/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Deoxyguanosine/pharmacology , Epithelial-Mesenchymal Transition/genetics , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/metabolism , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Imidazoles/pharmacology , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Mice , Mice, Nude , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pyrroles/pharmacology , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Vimentin/antagonists & inhibitors , Vimentin/genetics , Vimentin/metabolism , Xenograft Model Antitumor Assays , Zonula Occludens-1 Protein/agonists , Zonula Occludens-1 Protein/genetics , Zonula Occludens-1 Protein/metabolism , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
Enhanced migratory potential and invasiveness of cancer cells contribute crucially to cancer progression. These phenotypes are achieved by precise alteration of invasion-associated genes through local epigenetic modifications which are recognized by a class of proteins termed a chromatin reader. ZMYND8 [zinc finger MYND (myeloid, Nervy and DEAF-1)-type containing 8], a key component of the transcription regulatory network, has recently been shown to be a novel reader of H3.1K36Me2/H4K16Ac marks. Through differential gene expression analysis upon silencing this chromatin reader, we identified a subset of genes involved in cell proliferation and invasion/migration regulated by ZMYND8. Detailed analysis uncovered its antiproliferative activity through BrdU incorporation, alteration in the expression of proliferation markers, and cell cycle regulating genes and cell viability assays. In addition, performing wound healing and invasion/migration assays, its anti-invasive nature is evident. Interestingly, epithelial-mesenchymal transition (EMT), a key mechanism of cellular invasion, is regulated by ZMYND8 where we identified its selective enrichment on promoters of CLDN1/CDH1 genes, rich in H3K36Me2/H4K16Ac marks, leading to their up-regulation. Thus, the presence of ZMYND8 could be implicated in maintaining the epithelial phenotype of cells. Furthermore, syngeneic mice, injected with ZMYND8-overexpressed invasive breast cancer cells, showed reduction in tumor volume and weight. In concert with this, we observed a significant down-regulation of ZMYND8 in invasive ductal and lobular breast cancer tissues compared with normal tissue. Taken together, our study elucidates a novel function of ZMYND8 in regulating EMT and invasion of cancer cells, possibly through its chromatin reader function.
Subject(s)
Breast Neoplasms/metabolism , Cadherins/agonists , Claudin-1/agonists , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/metabolism , Receptors, Cell Surface/metabolism , Animals , Antigens, CD , Breast/metabolism , Breast/pathology , Breast Neoplasms/pathology , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Survival , Claudin-1/genetics , Claudin-1/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness/pathology , Neoplasm Proteins/agonists , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Transplantation , Promoter Regions, Genetic , RNA Interference , Receptors for Activated C Kinase , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Tumor Suppressor ProteinsABSTRACT
We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (Papp) of fluorescein in Caco-2 cells pretreated with 20 µM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any Papp reduction, purpurogallin pretreatment resulted in a significant Papp reduction similar to that of TF-3'-O-gallate (TF3'G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the Papp reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3'-O-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3'G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells.