E. COLI HEAT STABLE ENTEROTOXIN ON COLO-205 CELLS 475 Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 475–483 DOI: 10.1002/jat JOURNAL OF APPLIED TOXICOLOGY J. Appl. Toxicol. 2008; 28: 475–483 Published online 10 September 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jat.1297 Downregulation of human colon carcinoma cell (COLO- 205) proliferation through PKG-MAP kinase mediated signaling cascade by E. coli heat stable enterotoxin (STa), a potent anti-angiogenic and anti-metastatic molecule Subhrajit Saha, Pinki Chowdhury, Amit Pal and Manoj K. Chakrabarti* Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Calcutta 700010, India Received 9 February 2007; Revised 9 July 2007; Accepted 13 July 2007 ABSTRACT: It was reported earlier that Escherichia coli heat stable enterotoxin (STa), a major causative agent of secretory diarrhea, can also inhibit the proliferation of colon carcinoma cells with the involvement of cGMP mediated calcium influx. In the present study it is shown that E. coli STa inhibits cell proliferation in the colonic carcinoma cell line COLO-205 by the PKG-ERK44/42 mediated signaling pathway. This enterotoxin negatively regulates cell prolifera- tion by downregulating the activity of ERK44/42(MAPK) and subsequently the activity of a transcription regulatory protein cMyc. The antiproliferative effect of STa was reversed by LY83583, a guanylate cyclase (GC) inhibitor and KT5823, a PKG inhibitor. Thus suggesting the involvement of cGMP dependent protein kinase (PKG) in the downregula- tion of ERK44/42 and subsequent inactivation of cMyc activity. Moreover, it has been shown that a specific ERK44/42 inhibitor, PD98059, also inhibits cMyc activation and cell proliferation, which further confirms the involvement of ERK44/ 42 in the activation of cMyc. It is also shown that E. coli STa significantly inhibits the vascular endothelial growth factor (VEGF, a potent angiogenic factor) expression in COLO-205 cells and also downregulates vascular cell adhesion molecule-1 (VCAM-1, a potent metastatic factor) expression on the COLO-205 cell surface. So it is reported for the first time that E. coli STa inhibits the proliferation of the colonic carcinoma cell line COLO-205 by the PKG-ERK44/42 mediated pathway and it may have a role against the development of colon carcinoma. Copyright © 2007 John Wiley & Sons, Ltd. KEY WORDS: heat-stable enterotoxin; calcium; MAP kinase; VEGF; VCAM-1 * Correspondence to: Dr Manoj K. Chakrabarti, Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Calcutta 700010, India. E-mail: mkc_niced@yahoo.co.in Contract/grant sponsor: Indian Council of Medical Research; University Grants Commission. Pitari et al. that heat stable enterotoxin (STa), produced by enterotoxigenic Escherichia coli (ETEC) a major causative agent of secretory diarrhea, inhibits cell prolif- eration of colon carcinoma cells (Pitari et al., 2003). According to them in the T84 colonic cell line STa induces a rise in the intracellular cGMP level which stimulates calcium influx. This rise in intracellular calcium level then inhibits DNA synthesis and cell proliferation (Pitari et al., 2003). However, previous studies on the mecha- nism of action of E. coli STa have shown that although STa binds and accumulates intracellular cGMP (Guarino et al., 1987; Visweswariah et al., 1994), no elevation of intracellular calcium was found in the T84 colonic cell line. It has been reported that in another colonic cell line COLO-205, E. coli STa not only binds and accumulates cGMP but also involved IP 3 mediated calcium mobiliza- tion from the intracellular calcium store (Bhattacharya and Chakrabarti, 1998). It was shown in another study that in the COLO-205 cell line E. coli STa stimulates IP 3 mediated calcium mobilization, which then increases the intracellular cGMP level with the involvement of protein kinase C-α (PKC-α) (Datta Gupta et al., 2005). So, the reported mechanism of action of E. coli STa in the T84 Introduction Colorectal cancer is the fourth leading cause of cancer and cancer related mortality among cancer patients and is most prevalent in industrialized developed nations, whereas the underdeveloped countries are less prone to the incidence of colorectal cancer (Ferlay et al., 2001). These geogra- phical imbalances suggest an environmental contribution to the resistance of endemic populations to intestinal neoplasia. The epidemiological studies of the U.S. Depart- ment of Health and Human Services, Atlanta on colorectal cancer and diarrheal diseases have shown an inverse re- lationship between colorectal cancer and enterotoxigenic bacteria associated diseases (Centers for Diseases Control and Prevention. Health Information for International Travel 1999–2000, Atlanta 2001). It was reported by