Molecular Targets of NOASA in Cancer

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Apart from effects on cell kinetics, NO-ASA has multiple pleiotropic effects that involve NF-kB, Wnt, NOS, mitogen-activated protein kinase (MAPK), COX-2, PPAR, drug-metabolizing enzymes, reactive oxygen species, and pro- and anti-inflammatory cytokines.

1. Cell Kinetics

The effects of NO-ASA on cell renewal and cell death, two determinants of cell growth, have shown that NO-ASA inhibits cell proliferation (diminished expression of the proliferation marker PCNA) and enhances cell death by inducing apoptosis. It also blocks transitions of the cells through the cell cycle, mainly between G1 and S (Kashfi et al., 2002, 2005; Williams et al., 2001).

p-NO-ASA profoundly affects the NF-kB-DNA interaction in cultured colon cancer cells decreasing it as early as 60 min after treatment, with similar results obtained with pancreatic cancer cell lines (Williams, Ji, Ouyang, Liu, and & Rigas, 2008; Williams et al., 2003). In vivo, compared to controls, p-NO-ASA decreased NF-kB activation in xenografts of estrogen receptor negative, ER(-), breast cancer cells (Kashfi & Nazarenko, 2006), and in intestinal epithelial cells of APCmin+/_ mice (Williams, Ji, Ouyang, Liu, & Rigas, 2008).

3. Wnt/p-Catenin Signaling p-NO-ASA modulates the p-catenin signaling pathway in three different cancer cell lines of different origin (Gao, Liu, & Rigas, 2005; Nath, Kashfi, Chen, & Rigas, 2003; Nath, Labaze, Rigas, & Kashfi, 2005; Nath, Vassell, Chattopadhyay, Kogan, & Kashfi, 2009). One of the significant downstream genes dependent on p-catenin/T cell factor (TCF)-4 signaling is cyclin D1, which has been implicated in carcinogenesis (Moon, Kohn, De Ferrari, & Kaykas, 2004). Inhibition of this signaling pathway by low concentrations of p-NO-ASA was associated with reduced cyclin D1 expression suggesting that it may be an important disruptor of carcinogenesis. In vivo, NO-indomethacin and m-NO-ASA inhibited p-catenin expression in a standard AOM rat model of colorectal cancer (Rao et al., 2006).

4. Nitric Oxide Synthase iNOS may have an important role in tumor development since increased expression occurs in breast (Thomsen et al., 1995), CNS (Cobbs, Brenman, Aldape, Bredt, & Israel, 1995), pancreas (Kasper, Wolf, Drebber, Wolf, & Kern, 2004), astrocytic gliomas (Hara & Okayasu, 2004), prostate (Aaltoma, Lipponen, & Kosma, 2001), acute myeloid leukaemia (Brandao, Soares, Salles, & Saad, 2001), and colon (Ambs et al., 1998) tumors. iNOS regulates COX-2 (Landino, Crews, Timmons, Morrow, & Marnett, 1996; Perez-Sala & Lamas, 2001) and its activity may be correlated with p53 mutations (Chiarugi, Magnelli, & Gallo, 1998), although this has not been replicated (Brandao et al., 2001). Importantly, iNOS inhibitors prevent colon cancer (Rao, Kawamori, Hamid, & Reddy, 1999; Takahashi et al., 2006). Various NO-NSAIDs inhibit the induction of iNOS in a macrophage cell line (Cirino et al., 1996). In HT-29 colon cancer cells, p-NO-ASA inhibits iNOS induction by cytokines (Williams et al., 2003) and also potently inhibits expression and enzymatic activity of iNOS (Spiegel et al., 2005). An NO chimera (GT-094), a nitrate containing an NSAID and disulfide pharmacophores, reduced iNOS levels in the AOM rat model of colorectal cancer (Hagos et al., 2007).

The role of COX-2 and its inhibition in colon and other cancers has been the subject of considerable debate (Kashfi & Rigas, 2005; Soh & Weinstein, 2003). NO-NSAIDs, in general, and NO-ASA, in particular, were more potent than their corresponding traditional NSAIDs in inhibiting the growth of cultured HT-29 (expressing both COX-1 and COX-2) and HCT 15 (COX null) colon cancer cells (Williams et al., 2001; Yeh et al., 2004). Similar observations were made with the pancreatic cancer cell lines BxPC-3 (COX-1, COX-2 positive) and MIA PaCa-2 (COX null) (Kashfi et al., 2002). This raises an important and interesting question about the role of COX in cancer, since NO-ASA was equieffective and equipotent in both COX positive and COX negative cell lines. In HT-29 cells, p-NO-ASA at concentrations around its IC50 value for growth inhibition increased COX-2 expression by nearly ninefold. The induced enzyme was catalytically active (Williams et al., 2003). Similar findings were obtained with the DLD-1 colon cancer, BxPC-3 pancreatic cancer (Williams et al., 2003), and MCF-7 breast cancer cell lines (Nath, Vassell, Chattopadhyay, Kogan, & Kashfi, 2009). However, NO-indomethacin and m-NO-ASA also inhibited total COX including COX-2 activity and formation of PGE2 in the AOM rat model of colon cancer (Rao et al., 2006). These results give caution in extrapolating cell culture data to in vivo. The mechanism(s) by which NO-ASA induces COX-2 is unclear, but may, in part, be Protein kinase C (PKC) dependent (Nath, Vassell, Chattopadhyay, Kogan, & Kashfi, 2009).

6. Peroxisome Proliferator-Activated Receptor d

In matched normal and tumor samples from the colon, PPAR-8 mRNA was upregulated in colorectal carcinomas and endogenous PPAR-8 was transcriptionally responsive to PGI2 (Gupta et al., 2000). Elevation of PPAR-8 expression in colorectal cancer cells was repressed by APC, an effect mediated by p-catenin/TCF-4-responsive elements in the PPAR-8 promotor (He, Chan, Vogelstein, & Kinzler, 1999). Sulindac blocked PPAR-8 from binding to its recognition sequences (He et al., 1999), and in SW480 colon cancer cells, sulindac sulfone decreased PPAR-8 expression more potently than the sulfide metabolite (Siezen et al., 2006). These data suggest that NSAIDs may, in part, inhibit tumorigenesis through inhibition of PPAR-8. In Min mice, m- and p-NO-ASAs inhibited the expression of PPAR-8 in both histologically normal and tumor tissues. m-NO-ASA suppressed PPAR-8 expression in normal mucosa by 23% and in neoplastic tissue by 41%; p-NO-ASA suppressed PPAR-8 expression in normal mucosa by 27% and in neoplastic tissue by 55% (Ouyang et al., 2006).

7. Mitogen-Activated Protein Kinase

The MAPKs are a family of kinases that transduce signals from the cell membrane to the nucleus in response to a variety of stimuli modulating gene transcription and leading to biological response (Bode & Dong, 2004). MAPKs required for specialized cell functions, controlling cell proliferation, differentiation, and death are deregulated in several malignancies, including colon cancer, and may be involved in their pathogenesis. p-NO-aspirin treatment of colon cancer cells (HT-29 and SW480) activated c-Jun N-terminal kinase (JNK) and p38 along with their respective downstream transcription factors, cJun and ATF-2. NO-ASA stimulation of p38 was biphasic, with an initial increase in phosphorylation within the 60 min of treatment, and a second much stronger increase at 4 h (Hundley & Rigas, 2006).

8. Xenobiotic-Metabolizing Enzymes

Modulation of drug-metabolizing enzymes, leading to facilitated elimination of endogenous and environmental carcinogens, represents a successful strategy for cancer chemoprevention (Kwak, Wakabayashi, & Kensler, 2004) and is exemplified by dithiolethiones, that induce phase II metabolizing enzymes. These compounds inhibit tumorigenesis of environmental carcinogens in various animal models and in clinical trials, modulate the metabolism of the carcinogen, aflatoxin B1 (Kwak et al., 2004). In general, induction of phase II enzymes is an adequate strategy for protecting mammals against carcinogens and other forms of electrophile and oxidant toxicity. Chemopreventive agents induce the expression of phase II genes through their effects on the Keap1-Nrf2 complex (Lee & Surh, 2005). In the nucleus, the transcription factor Nrf2, a member of the NF-E2 family, dimerizes with Maf protein and binds to the antioxidant response element, a cis-acting regulatory element in the promoter region of phase II enzymes. A cytoplasmic actin-binding protein, Keap1, is an inhibitor of Nrf2 that sequesters it in the cytoplasm. Inducers dissociate this complex, allowing Nrf2 to translocate to the nucleus. Studies evaluating the effects of NO-ASA on xenobiotic-metabolizing enzymes have shown that m-NO-ASA induced the activity and expression of NAD(P)H:quinone oxireductase (NQO) and glutathione S-transferases (GSTs) in mouse hepatoma Hepa 1c1c7 and HT-29 human colon cancer cells (Gao, Kashfi, Liu, & Rigas, 2006). In Min mice, m-NO-ASA also induced the activities of NQO and GST in liver cytosolic and small intestine fractions but had no effect on the activity of these enzymes in the kidney, showing some degree of tissue of specificity (Gao et al., 2006). Expression of GST P1-1, GST A1-1, and NQO1 was induced in liver cytosols from Min mice; however, the expression of two phase I metabolizing enzymes, CypP450-1A1 and CypP450-2E1, were unaffected, suggesting that m-NO-ASA is a monofunctional inducer of phase II enzymes (Gao et al., 2006). m-NO-ASA also induced the translocation of Nrf2 into the nucleus, an effect that paralleled the induction of NQO1 and GST P1-1 (Gao et al., 2006).

9. Oxidative Stress

It has become evident that anticancer agents act, at least in part, by inducing reactive oxygen and nitrogen species (RONS). At low concentrations, RONS may protect the cell and at high concentrations can initiate biological damage, including cell death (Rigas & Sun, 2008). In evaluating the effects of p-NO-ASA in SW480 colon cancer cells, it was determined that the spacer in p-NO-ASA formed a conjugate with glutathione, depleting glutathione stores and thus induced a state of oxidative stress that led to apoptosis via activation of the intrinsic apoptosis pathway (Gao, Liu, et al., 2005). p-NO-ASA through induction of RONS also oxidized thioredoxin-1 (Sun & Rigas, 2008), an oxidoreductase that is involved in redox regulation of cell signaling (Arner & Holmgren, 2006; Maulik & Das, 2008).

10. Modulation of Proinflammatory Cytokines m-NO-ASA inhibits cytokine production from endotoxin-stimulated human monocytes and macrophages (Fiorucci, Santucci, Cirino, et al., 2000) and when administered to mice, decreased IL-1p, IL-8, IL-12, IL-18, TNF-a, and INF-g production and protected against concanavalin A-induced acute hepatitis (Fiorucci, Santucci, Antonelli, et al., 2000). The effect exerted by NO-ASA on cytokine production was COX-independent

(Santucci, Fiorucci, Di Matteo, & Morelli, 1995). Locally generated NO contributes to limit inflammation by inhibiting generation of proinflamma-tory cytokines and/or by enhancing the production of anti-inflammatory cytokines, IL-10 and TGFp, resulting in downregulation of downstream mediators of inflammation including COX and NOS isoenzymes (Fiorucci, 2001; Fiorucci, Santucci, Antonelli, et al., 2000).

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