Bufalin prevents the migration and invasion of T24 bladder carcinoma cells through the inactivation of matrix metalloproteinases and modulation of tight junctions.

Bufalin, a cardiotonic steroid extracted from toad venom, has generally been known to possess a range of biological activities; however, only a few studies have reported the anti-metastatic activity of bufalin. In the present study, we investigated the inhibitory effects of bufalin on cell migration and invasion, two critical cellular processes that are often deregulated during metastasis, using the human bladder cancer cell line, T24. Within the concentration range that was not cytotoxic, bufalin markedly inhibited the cell motility and invasiveness of T24 cells. The inhibitory effects of bufalin on cell invasiveness were associated with the tightening of tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance (TER). Bufalin treatment also repressed the levels of claudin proteins (claudin-2, -3 and -4) and the major components of TJs that play key roles in the control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)‑2 and -9 in T24 cells were dose‑dependently inhibited by treatment with bufalin and this also correlated with a decrease in their mRNA and protein expression levels; however, the mRNA and protein levels of the tissue inhibitor of metalloproteinase (TIMP)‑1 and -2 were increased. In addition, these effects were related to the increased phosphorylation of the extracellular signal-regulated protein kinase (ERK) pathway. The inhibition of ERK (PD98059) significantly prevented the bufalin‑induced suppression of T24 cell migration. These findings suggest that bufalin inhibits the migration and invasion of T24 cells by modulating the activity of TJs and MMPs, possibly in association with the activation of ERK.

[1]  Jai-Sing Yang,et al.  Bufalin‐inhibited migration and invasion in human osteosarcoma U‐2 OS cells is carried out by suppression of the matrix metalloproteinase‐2, ERK, and JNK signaling pathways , 2014, Environmental toxicology.

[2]  T. Gress,et al.  Claudin-4 as therapeutic target in cancer. , 2012, Archives of biochemistry and biophysics.

[3]  Seok-Jun Kim,et al.  Galectin-3 increases the motility of mouse melanoma cells by regulating matrix metalloproteinase-1 expression , 2012, Experimental & Molecular Medicine.

[4]  In-Chul Lee,et al.  Anti-inflammatory functions of purpurogallin in LPS-activated human endothelial cells. , 2012, BMB reports.

[5]  N. Takai,et al.  Bufalin, a traditional oriental medicine, induces apoptosis in human cancer cells. , 2012, Asian Pacific journal of cancer prevention : APJCP.

[6]  V. Mechírová,et al.  Matrix Metalloproteinases and Their Role in Oncogenesis: A Review , 2012, Oncology Research and Treatment.

[7]  Yung-Hyun Choi,et al.  Bufalin induces apoptosis through activation of both the intrinsic and extrinsic pathways in human bladder cancer cells. , 2011, Oncology reports.

[8]  P. Riesz,et al.  7117 POSTER Claudins and Ki-67 – Potential Markers to Differentiate Low and High Grade Transitional Cell Carcinomas of the Urinary Bladder , 2011 .

[9]  S. Ergün,et al.  Matrix metalloproteinases and their clinical relevance in urinary bladder cancer , 2011, Nature Reviews Urology.

[10]  N. Ohashi,et al.  Occludin is regulated by epidermal growth factor receptor activation in brain endothelial cells and brains of mice with acute liver failure , 2011, Hepatology.

[11]  K. Yagi,et al.  Claudin as a target for drug development. , 2011, Current medicinal chemistry.

[12]  Wei Tang,et al.  Antitumor activity of extracts and compounds from the skin of the toad Bufo bufo gargarizans Cantor. , 2011, International immunopharmacology.

[13]  Yung-Hyun Choi,et al.  Anti-invasive activities of anthocyanins through modulation of tight junctions and suppression of matrix metalloproteinase activities in HCT-116 human colon carcinoma cells. , 2011, Oncology reports.

[14]  Cheng Jiang,et al.  Bufadienolide compounds sensitize human breast cancer cells to TRAIL-induced apoptosis via inhibition of STAT3/Mcl-1 pathway , 2011, Apoptosis.

[15]  Ying Zhang,et al.  Effects of bufalin on the proliferation of human lung cancer cells and its molecular mechanisms of action , 2010, Cytotechnology.

[16]  E. Leygue,et al.  Claudin 1 in Breast Tumorigenesis: Revelation of a Possible Novel “Claudin High” Subset of Breast Cancers , 2010, Journal of biomedicine & biotechnology.

[17]  Z. Werb,et al.  Matrix Metalloproteinases: Regulators of the Tumor Microenvironment , 2010, Cell.

[18]  P. Timpson,et al.  Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo , 2009, Cell adhesion & migration.

[19]  Y. Yoo,et al.  Anti-invasive activity of sanguinarine through modulation of tight junctions and matrix metalloproteinase activities in MDA-MB-231 human breast carcinoma cells. , 2009, Chemico-biological interactions.

[20]  W. Zhong,et al.  CD147, MMP-1, MMP-2 and MMP-9 Protein Expression as Significant Prognostic Factors in Human Prostate Cancer , 2008, Oncology.

[21]  K. Nakanishi,et al.  Expression of occludin and claudins 1, 3, 4, and 7 in urothelial carcinoma of the upper urinary tract. , 2008, American journal of clinical pathology.

[22]  T. Ueda,et al.  Bufalin induces growth inhibition, cell cycle arrest and apoptosis in human endometrial and ovarian cancer cells. , 2008, International journal of molecular medicine.

[23]  P. Morin.  Claudin Proteins in Ovarian Cancer , 2007, Disease markers.

[24]  A. Yu,et al.  Claudins and paracellular transport: an update , 2007, Current opinion in nephrology and hypertension.

[25]  C. Dinney,et al.  Bladder cancer angiogenesis and metastasis—translation from murine model to clinical trial , 2007, Cancer and Metastasis Reviews.

[26]  L. Matrisian,et al.  The other side of MMPs: Protective roles in tumor progression , 2007, Cancer and Metastasis Reviews.

[27]  Wei-wei Su,et al.  Bufalin induces apoptosis in human osteosarcoma U-2OS and U-2OS methotrexate300-resistant cell lines , 2007, Acta Pharmacologica Sinica.

[28]  F. Hollande,et al.  DNA-methylation-dependent alterations of claudin-4 expression in human bladder carcinoma. , 2007, Carcinogenesis.

[29]  A. Newby,et al.  Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates. , 2006, Cardiovascular research.

[30]  G. Daley,et al.  Novel role for PDEF in epithelial cell migration and invasion. , 2005, Cancer research.

[31]  P. Morin,et al.  Claudin proteins in human cancer: promising new targets for diagnosis and therapy. , 2005, Cancer research.

[32]  P. Morin,et al.  Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase-2 activity. , 2005, Cancer research.

[33]  E. Lo,et al.  Downregulation of matrix metalloproteinase-9 and attenuation of edema via inhibition of ERK mitogen activated protein kinase in traumatic brain injury. , 2002, Journal of neurotrauma.

[34]  Z. Werb,et al.  New functions for the matrix metalloproteinases in cancer progression , 2002, Nature Reviews Cancer.

[35]  M. Birrer,et al.  Alterations in differentiation and apoptosis induced by bufalin in cJun overexpressing U-937 cells. , 1998, International journal of oncology.

[36]  R. Dickson,et al.  Matrix metalloproteinase inhibitors , 1997, Investigational New Drugs.

[37]  Y. Jing,et al.  Differentiation of promyelocytic leukemia cells HL-60 induced by daidzein in vitro and in vivo. , 1993, Anticancer research.

[38]  T. Yoshida,et al.  Bufalin as a potent inducer of differentiation of human myeloid leukemia cells. , 1991, Biochemical and biophysical research communications.

[39]  Abderrahman Ouban,et al.  Claudins in human cancer: a review. , 2010, Histology and histopathology.

[40]  I. Căruntu,et al.  Matrix metalloproteinases involvement in pathologic conditions. , 2010, Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie.

[41]  Kyle J Hewitt,et al.  The claudin gene family: expression in normal and neoplastic tissues , 2006, BMC Cancer.

[42]  A. Nusrat,et al.  Tight junctions and cell-cell interactions. , 2006, Methods in molecular biology.

[43]  H. Klocker,et al.  Type IV collagenase (matrix metalloproteinase-2 and -9) in prostate cancer , 2004, Prostate Cancer and Prostatic Diseases.

[44]  X. Jin,et al.  [Studies on the chemical constituents from the skin of Bufo bufo gargarizans Cantor]. , 1992, Yao xue xue bao = Acta pharmaceutica Sinica.

[45]  R. Visse,et al.  This Review Is Part of a Thematic Series on Matrix Metalloproteinases, Which Includes the following Articles: Matrix Metalloproteinase Inhibition after Myocardial Infarction: a New Approach to Prevent Heart Failure? Matrix Metalloproteinases in Vascular Remodeling and Atherogenesis: the Good, the Ba , 2022 .