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By Jamshed Arslan Pharm.D.
Gastric carcinoma is the second leading cause of cancer-related deaths worldwide. One of the key features of gastric carcinoma is acidosis, which promotes growth and metastasis of gastric cancer cells by inducing HIF-1alpha, TGF-beta, and epithelial-mesenchymal transition (EMT) signaling pathways. Gastric cancer cells have a reduced expression of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a pseudoreceptor that negatively regulates TGF-beta signaling by inhibiting the formation of an active receptor complex. The intricacies of the BAMBI-TGF-beta relationship in gastric carcinoma are yet to be explored.
To shed light on the interaction between BAMBI and TGF-beta/EMT signaling, researchers from Guangxi Medical University, China, analyzed growth and metastasis of gastric cancer using cell lines and tumor-bearing mice. The team found that induced BAMBI overexpression in gastric cancer cells increased intracellular pH, downregulated DNA methylation of regions encoding TGF-beta/EMT signaling, but reduced the upregulation of Smad6 and Smad7 (TGF-beta target genes), and inhibited growth of gastric tumors in mice. In other words, BAMBI overexpression inhibits gastric cancer via TGF-beta/EMT signaling pathway.
Researchers first observed a downregulation of BAMBI in gastric cancer cells in line with the previous findings. To elucidate the function of BAMBI, they cloned BAMBI gene into eukaryotic expression vector (pBAMBI) and transfected into gastric cancer cells using lipofectamine. This endogenous BAMBI overexpression, inhibited growth and migratory ability of gastric cancer cells, enhanced protein expression of proliferation markers (Ki67 and IPO-38), and increased intracellular pH; BAMBI knockdown by siRNA had the opposite effects. BAMBI-overexpressing cells showed a downregulation in protein levels of EMT markers (SOX4, MMP9, TWIST1), EMT transcription regulators (VIM, SNAI1, ACTA2), TGF-beta, and regulatory molecules of TGF-beta (N-Cadherin, Collagen1/CT-I and fibronectin). Both BAMBI knockdown and TGF-beta treatment reversed these inhibitory actions of BAMBI overexpression.
Method’s Highlight To demonstrate the role of BAMBI on TGF-beta signaling, investigators relied on antibodies to N-Cadherin and Collagen-I from Novus Biologicals. |
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To explore the epigenetic mechanism behind this BAMBI overexpression-induced downregulation of TGF-beta/EMT signaling, DNA methylation was studied through bisulfite pyrosequencing. BAMBI overexpression led to reduced DNA methylation of molecules associated with TGF-beta signal transduction. This implies an increased TGF-beta signal transduction, but western blotting showed a decrease in BAMBI-induced upregulation of Smad6 and Smad7 (TGF-beta target genes). In other words, BAMBI overexpression reduced TGF-beta signaling in gastric cancer cells, partly by regulating DNA methylation of regions encoding TGF-beta/EMT signaling.
The next step was to validate these effects of BAMBI overexpression in a mammalian model.
Immunocytochemistry/Immunofluorescence: Ki-67/MKI67 Antibody [NB500-170] - Ki67 Antibody [NB500-170] - Confocal immunofluorescent analysis of MCF7 cells using Ki67 antibody (NB500-170, 1:5). An Alexa Fluor 488-conjugated Goat to rabbit IgG was used as secondary antibody (green). Actin filaments were labeled with Alexa Fluor 568 phalloidin (red). DAPI was used to stain the cell nuclei (blue).
Researchers found a decrease in tumors in mice bearing BAMBI overexpressed gastric cancer cells relative to the controls. The tumors from these BAMBI overexpressing mice showed higher intracellular pH, lower expression of regulatory factors in TGF-beta/EMT signaling (HIF-alpha and Ki67), downregulation in the mediators of both EMT (TWIST1, MMP9, SOX4) and TGF-beta (N-cadherin, CT-I and fibronectin) signaling pathways.
All in all, BAMBI overexpression was found to reduce gastric cancer growth in vitro and in vivo by regulating TGF-beta/EMT signaling.
This study highlights the mechanism behind BAMBI’s protective effects in gastric cancers, thereby paving the way for targeted therapies. This investigation describes a critical role of BAMBI in pH regulation, which is crucial information in pharmacotherapy since highly acidic environments in stomach can render many anticancer drugs ineffective.
Learn more about TGF-beta signaling
Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.
References
Yuan, Chun-Ling, et al. "Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor Overexpression Inhibits Gastric Tumor Cell Invasion via the Transforming Growth Factor-b/Epithelial-Mesenchymal Transition Signaling Pathway." Experimental and Therapeutic Medicine, vol. 15, no. 6, 2018, pp. 5422 – 5430. doi: 10.3892/etm.2018.6083.
