The International Journal of Biochemistry & Cell Biology 66 (2015) 75–84 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology jo ur nal home page: www.elsevier.com/locate/biocel Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21 Waf1/Cip1 mediated inhibition of Hh-Gli signaling Purusottam Mohapatra, Shakti Ranjan Satapathy, Sumit Siddharth, Dipon Das, Anmada Nayak, Chanakya Nath Kundu ∗ Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa, 751024, India a r t i c l e i n f o Article history: Received 3 March 2015 Received in revised form 10 July 2015 Accepted 21 July 2015 Available online 23 July 2015 Keywords: MCF-10A-Tr Curcumin Resveratrol Combination therapy Hh-Gli Signaling p21 Waf/Cip1 a b s t r a c t Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggres- sive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of cur- cumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC 50 value was noted in cells treated with the combination of resveratrol (3 M) and curcumin (3 M) in comparison to 30 M of resveratrol or curcumin alone. Resveratrol + curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and cas- pase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, -catenin and cleaved notch-1 val1744, respectively. Furthermore, the combina- tion also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21 Waf/Cip1 in vitro and in vivo. A significant reduc- tion of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21 Waf/Cip1 knockout cells suggests this combination caused apoptosis through p21 Waf/Cip1 . Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2 Waf/Cip1 mediated inhibition of Hedgehog-Gli cascade. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Despite of the advancement in its diagnostic and therapeu- tic approaches, breast cancer remains the leading cause of cancer related death among women worldwide. Many environmental, hormonal and dietary factors are directly associated with the Abbreviations: CSC, cigarette smoke condensate; MCF-10A-Tr, Cigarette smoke condensate mediated MCF-10A transformed cells; MTT, [3-(4, 5-dimethylthiazol- 2yl-)-2, 5-diphenyl tetrazolium bromide]; DAPI, 4 ′ * 6-Diamidino-2-phenylindole; FITC, Fluorescein isothiocyanate; PBS, Phosphate buffered saline; Res, Resver- atrol; Cmn, Curcumin; Agf, Andrographolide; BCNU, 1,3-Bis (2-chloroethyl)-1- nitrosourea; Hh-Gli, Hedgehog-Gli; PTCH1, Patched-1; SMO, Smoothened; SUFU, Super fused. ∗ Corresponding author. Tel.: +91 0674 272 5466; fax: +91 0674 237 8776. E-mail addresses: cnkundu@gmail.com, cnkundu@kiitbiotech.ac.in (C.N. Kundu). development and progression of breast cancer. Among the environ- mental factors cigarette smoke is a chief cause of mammary cancer development in adolescent girls and women globally (Narayan et al., 2004; Kundu et al., 2007; Mohapatra et al., 2014a,b). The chemi- cal carcinogens present in both the cigarette smoke and tar provoke the transformation of normal cells to attain malignancy. Hecht et al. and Phillips et al. have reported that cigarette smoke car- cinogen can stimulate mammary tumor growth in animal models (Phillips et al., 2001; Hecht, 2002). However, the unavailability of proper in vitro model system was hindering the studies related to cigarette smoke induced breast cancers. Recently, we have established a novel cigarette smoke condensate (CSC) induced- transformed breast epithelial cell line (MCF-10A-Tr) (Mohapatra et al., 2014a) that could be useful in various investigations related to anticancer research. Further in a related study, we have demon- strated that resveratrol (Res) causes apoptosis in MCF-10A-Tr cells through inhibition of LP-BER activity by p21 Waf/Cip1 dependent manner (Mohapatra et al., 2014b). http://dx.doi.org/10.1016/j.biocel.2015.07.009 1357-2725/© 2015 Elsevier Ltd. All rights reserved.