Signal transduction events elicited by cancer prevention compounds.

Many chemopreventive agents have been shown to modulate gene expression including induction of phase II detoxifying enzymes, such as glutathione S-transferases (GST) and quinone reductases (QR). Induction of phase II enzymes in general leads to protection of cells/tissues against exogenous and/or endogenous carcinogenic intermediates. The antioxidant or electrophile response element (ARE/EpRE) found at the 5'-flanking region of these phase II genes may play important role in mediating their induction by xenobiotics including chemopreventive agents. Members of the basic leucine zipper (bZIP) transcription factor, Nrf2 which heterodimerizes with Maf G/K, are found to bind to the ARE, and transcriptionally-activated ARE. Recently, we showed that the mitogen-activated protein kinases (MAPK) were activated by phase II gene inducers such as phenolic antioxidant butylated hydroxyanisol (BHA) and isothiocyanate sulforaphane (SUL), and involved in the transcription activation of ARE-mediated reporter gene. Transfection studies with wild-type and dominant negative mutants of Nrf2 and MAPK showed synergistic response during co-transfection as well as to phase II gene inducers. However, increasing the concentrations of these compounds such as BHA, the activities of cell death signaling molecules, caspases, were stimulated and resulted in apoptotic cell death. At these concentrations, BHA stimulated loss of mitochondrial membrane potential, cytochrome c release, and activation of caspase 3, 8 and 9 preceding apoptosis. Further increase in concentrations led to rapid cell necrosis. A model is proposed for BHA and SUL, in that at low concentrations, these potential chemopreventive agents may modulate MAPK pathway leading to transcription activation of Nrf2 and ARE with subsequent induction of cellular defensive enzymes including phase II detoxifying enzymes as well as other defensive genes, which may protect the cells against cellular injury, which is a homeostatic response. At higher concentrations, these agents may activate the caspase pathways, leading to apoptosis, a potential beneficial effect if occurs at preneoplastic/neoplastic tissues, but a potential cytotoxic response if occurs in normal tissues. On the other hand, some phenolic compounds such as resveratrol inhibits TPA- or UV-induced AP-1-mediated activity through the inhibition of c-Src non-receptor tyrosine kinase and MAPK pathways. It is possible that in proliferating or stimulated cells, these chemopreventive compounds may block proliferation by inhibiting these signaling kinases, whereas in non-proliferating or quiescent cells, some of these compounds may activate these signaling kinases leading to gene expression of cellular defensive enzymes such as phase II detoxifying enzymes. The studies of these and other signaling pathways may yield insights into the development of potential chemopreventive compounds.

Resveratrol inhibits phorbol ester and UV-induced activator protein 1 activation by interfering with mitogen-activated protein kinase pathways.

Resveratrol, a phenolic compound found in grapes and other food products, prevents chemical-induced carcinogenesis in a number of animal models of cancers. To better understand its chemopreventive property, we examined effects of resveratrol on the activity of activator protein 1 (AP-1), a dimeric transcription factor that plays a critical role in the carcinogenesis and tumor transformation. Pretreatment of HeLa cells with resveratrol inhibited the transcription of AP-1 reporter gene by UVC and phorbol 12-myristate 13-acetate (PMA). Pretreatment with resveratrol also inhibited the activation of extracellular signal-regulated protein kinase 2 (ERK2), c-jun N-terminal kinase 1 (JNK1), and p38. Selectively blocking mitogen-activated protein kinase (MAPK) pathways by overexpression of dominant-negative mutants of kinases attenuated the AP-1 activation by PMA and UVC. Interestingly, resveratrol had little effect on the induction of AP-1 reporter gene by active Raf-1, MEKK1, or MKK6, suggesting that it inhibited MAPK pathways by targeting the signaling molecules upstream of Raf-1 or MEKK1. Indeed, incubation of resveratrol with the isolated c-Src protein tyrosine kinase and protein kinase C diminished their kinase activities. Furthermore, inhibition of protein tyrosine kinases and protein kinase C with their selective inhibitors impaired the activation of MAPKs as well as the induction of AP-1 activity by PMA and UVC. In addition, modulation of estrogen receptor activity with 17beta-estradiol had no effect on the inhibition of AP-1 by resveratrol. Taken together, these results suggest that the effects of resveratrol on AP-1 and MAPK pathways may involve the inhibition of both protein tyrosine kinases and protein kinase C.

Induction of xenobiotic enzymes by the MAP kinase pathway and the antioxidant or electrophile response element (ARE/EpRE).

Cellular responses to xenobiotic-induced stress can signal proliferation, differentiation, homeostasis, apoptosis, or necrosis. To better understand the underlying molecular mechanisms after exposure to xenobiotics or drugs, we studied the signal transduction pathways, the mitogen-activated protein kinase (MAPK), and the basic leucine zipper transcription factor Nrf2, activated by different agents in the induction of Phase II drug metabolizing enzymes (DMEs). The MAPKs, characterized as proline-directed serine/threonine kinases, are essential components of signaling pathways that convert various extracellular signals into intracellular responses through serial phosphorylation cascades. Once activated, MAPKs can phosphorylate many transcription factors, such as c-Jun, ATF-2, and ultimately lead to changes in gene expression. Two classes of Phase II gene inducers, which are also cancer chemopreventive agents, were studied: (1) the phenolic antioxidants, namely butylated hydroxyanisole (BHA) and its active de-methylated metabolite t-butylhydroquinone (tBHQ), and phenolic flavonoids such as green tea polyphenols (GTP) and (-)-epigallocatechin-3-gallate (EGCG); and (2) the naturally occurring isothiocyanates, namely phenethyl isothiocyanate (PEITC), and sulforaphane. BHA and tBHQ are both well-known phenolic antioxidants used as food preservatives, and strongly activate c-Jun N-terminal kinase 1 (JNK1), extracellular signal-regulated protein kinase 2 (ERK2), or p38, in a time- and dose-dependent fashion. Free radical scavengers N-acetyl-L-cysteine (NAC), or glutathione (GSH), inhibited ERK2 activation and, to a much lesser extent, JNK1 activation by BHA/tBHQ, implicating the role of oxidative stress. Under conditions where MAPKs were activated, BHA or GTP also activated ARE/EpRE (antioxidant/electrophile response element), with the induction of Phase II genes such as NQO. Transfection studies with various cDNAs encoding wild-type or dominant-negative mutants of MAPKs and/or transcription factor Nrf2, substantially modulated ARE-mediated luciferase reporter activity in the presence or absence of phenolic compounds. Other phytochemicals including PEITC, and sulforaphane, also differentially regulated the activities of MAPKs, Nrf2, and ARE-mediated luciferase reporter gene activity and Phase II enzyme induction. A model is proposed where these xenobiotics (BHA, tBHQ, GTP, EGCG, PEITC, sulforaphane) activate the MAPK pathway via an electrophilic-mediated stress response, leading to the transcription activation of Nrf2/Maf heterodimers on ARE/EpRE enhancers, with the subsequent induction of cellular defense/detoxifying genes including Phase II DMEs, which may protect the cells against toxic environmental insults and thereby enhance cell survival. The studies of these signaling pathways may yield insights into the fate of cells upon exposure to xenobiotics.

Pharmacodynamics of tamoxifen and its 4-hydroxy and N-desmethyl metabolites: activation of caspases and induction of apoptosis in rat mammary tumors and in human breast cancer cell lines.

The antiestrogen tamoxifen (TAM) is extensively metabolized by cytochrome P-450 in humans and rodents. The active, estrogen receptor-binding metabolites, 4-hydroxy TAM (OHT) and N-desmethyl TAM (DMT) have been well characterized. We showed that the s.c. injection of 1 mg/kg TAM in adult female Sprague Dawley rats bearing carcinogen-induced mammary tumors resulted in rapid serum decline of parent TAM but higher exposure of the metabolites, OHT and DMT. We found for the first time that the administration of TAM for a short time resulted in a delayed induction of caspase activity and apoptosis within the mammary tumors. When TAM, OHT, or DMT was added to human breast cancer cell lines in culture, each elicited a time- and dose-dependent induction of caspase activity, preceding apoptosis. Importantly, pretreatment of the cells with a pharmacological inhibitor of caspases [benzyloxy Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk)] blocked apoptosis induced by all three of the compounds, implicating a critical role of caspases in TAM-, OHT-, or DMT-induced apoptosis. The results obtained from these studies suggest that one possible mechanism of inhibition of mammary carcinogenesis and tumor growth in vivo may be the induction of caspase-dependent apoptosis, and that the metabolites OHT and DMT may contribute to the antitumor effect of TAM.

Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism.

Antioxidant response element (ARE) regulates the induction of a number of cellular antioxidant and detoxifying enzymes. However, the signaling pathways that lead to ARE activation remain unknown. Here, we report that the expression of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1), transforming growth factor-beta-activated kinase (TAK1), and apoptosis signal-regulating kinase (ASK1) in HepG2 cells activated the ARE reporter gene, whereas the expression of their dominant-negative mutants impaired ARE activation by the chemicals sodium arsenite and mercury chloride. Coexpression of downstream kinases, MAP kinase kinase 4, MAP kinase kinase 6, and c-Jun NH(2)-terminal kinase-1, but not MAP kinase kinase 3 and p38, augmented ARE activation by MEKK1, TAK1, and ASK1. The coexpression of a basic leucine zipper transcription factor Nrf2 but not c-Jun also greatly enhanced the activation of reporter gene by MEKK1, TAK1, and ASK1; however, a dominant-negative mutant of Nrf2 (NF-E2-related factor 2) blocked this event. Furthermore, when overexpressed, MEKK1, TAK1, and ASK1 induced the expression of heme oxygenase-1, a gene regulated by ARE, and the cotransfection with the dominant-negative mutant of Nrf2 abolished the induction. Taken together, these results suggest that MAP kinase pathways that are activated by MEKK1, TAK1, and ASK1 may link chemical signals to Nrf2, leading to the activation of ARE-dependent genes.