Long-term hydrogen peroxide exposure potentiates anoikis resistance and anchorage-independent growth in lung carcinoma cells Hasseri Halim* and Pithi Chanvorachote 1{ * Pharmaceutical Technology (International) Program, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand { Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Abstract Hydrogen peroxide (H 2 O 2 ) is upregulated in tumour microenvironments and may contribute to effects on metastatic cancer cells. This study demonstrates that treatment of lung carcinoma and melanoma cells with H 2 O 2 for 14 days results in an induction of anoikis resistance and growth in an anchorage-independent condition. H 2 O 2 exposure increased the Cav-1 (caveolin-1) level through an increase of Cav-1 mRNA with minimal effect on protein degradation. Upregulation of Cav-1 induced anoikis resistance and facilitated growth in a detached manner. The findings show a novel role of hydrogen peroxide in the regulation of metastatic potential of cancer cells. Keywords: anoikis; caveolin-1; hydrogen peroxide; long-term; lung cancer 1. Introduction Among the steps of metastasis, anoikis resistance and ability to grow in an anchorage-independent manner are associated with a high degree of metastasis and advanced stage of cancer (Hanahan and Weiberg, 2000; Shanmugathasan and Jothy, 2000; Mori et al., 2009). Cav-1 (caveolin-1) is the major protein found in caveolae that is associated with cancer progression (Glenney and Zokas 1989; Rothberg et al., 1992; Scherer et al., 1996; Galbiati et al., 2001; Fiucci et al., 2002; Ravid et al., 2005). Although Cav-1 is a tumour suppressor protein (Engelman et al., 1998) and its level is down- regulated in some cancers (Lee et al., 1998; Racine et al., 1999; Bender et al., 2000), increasing evidence supported the reverse role of Cav-1 as a cancer-potentiating protein in many cancers (Yang et al., 1998; Kato et al., 2002; Suzuoki et al., 2002). Moreover, we and others have provided supporting data indicating that Cav-1 expression is tightly related to anoikis resistance and facilitates metastasis of lung cancer (Ho et al., 2002; Moon et al., 2005; Cassani et al., 2009; Yeh et al., 2009). Regulation of the Cav-1 level is mainly through proteasome-mediated degradation (Chanvorachote et al., 2009), and several findings have indicated the tight correlation between the Cav-1 level during cell detachment and cancer cell resistance to anoikis (Hanahan and Weiberg, 2000; Ravid et al., 2005, 2006). According to the widely accepted concept, the cancer micro- environment is critical in the facilitation of metastasis (Rofstad, 2000; Isaiah, 2002) and causes significant impacts on cancer cell behaviour, such as chemotherapeutic resistance, invasion, and migration (Rennebeck et al., 2005; Wu, 2006; Brabek et al., 2010). However, the information regarding effects of microenviron- ment-associated substances on Cav-1 regulation as well as the consequences on cancer cell anoikis are largely unknown. Elevation of H 2 O 2 levels in the lungs of lung cancer patients compared to normal subjects has been reported (Zieba et al., 2000; Chung-Man et al., 2001; Chan et al., 2009), as has a high H 2 O 2 production in many cancer cells including lung cancer (Szatrowski and Nathan; 1991; Burdon, 1995; Lim et al., 2005; Liou and Storz, 2010). An inhibitory effect of endogenous H 2 O 2 generated during cell detachment on anoikis resistance in lung cancer cells has been shown. Also, exogenous H 2 O 2 obtained during cell detachment stabilized Cav-1 protein by inhibiting its degradation through the ubiquitin-proteasomal pathway and contributed to anoikis resist- ance (Rungtabnapa et al., 2011). However, there is a lack of knowledge regarding the effect of hydrogen peroxide on attached cancer cells, as well as longer periods of hydrogen peroxide exposure as found in tumour environments (Szatrowski and Nathan, 1991; Droge, 2002; Storz, 2005; Lopez-Lazaro, 2007; Chan et al., 2009), which has become the main focus of the present study. We have investigated the role of H 2 O 2 in controlling lung carcinoma cell anoikis and anchorage-independent growth, and identified the underlying mechanism. A novel mechanism of anoikis regulation by H 2 O 2 through Cav-1 regulation exists, which could be important in the understanding of anoikis resistance in metastatic cancers. 2. Materials and methods 2.1. Cell cultures and reagents Human non-small cell lung cancer cells (NCI-H460) and human melanoma cells (G361) were obtained from American Type Culture Collection (Manassas, VA). H460 cells were cultured in 1 To whom correspondence should be addressed (email pithi_chan@yahoo.com). Abbreviations: Cav-1, caveolin-1; DCFH 2 -DA, dichlorofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MTT, [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PI, propidium iodide; poly-HEMA, poly-2-hydroxyethylmethacrylate; ROS, reactive oxygen species; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. Cell Biol. Int. (2012) 36, 1055–1066 (Printed in Great Britain) Research Article E The Author(s) Journal compilation E 2012 Internation Federation for Cell Biology Volume 36 (11) N pages 1055–1066 N doi:10.1042/CBI20120111 N www.cellbiolint.org 1055