Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion markers in CD-1 mouse skin by oleandrin

Abstract

Oleandrin, derived from the leaves of Nerium oleander, has been shown to possess anti-inflammatory and tumor cell growth-inhibitory effects. Here, we provide evidence that oleandrin could possess anti-tumor promoting effects. We determined the effect of topical application of oleandrin to CD-1 mice against l2-O-tetradecanoylphorbol-13-acetate (TPA), a widely studied skin tumor promoter, -induced conventional and novel markers of skin tumor promotion. Topical application of oleandrin (2 mg per mouse) 30 min before TPA (3.2 nmol per mouse) application onto the skin afforded significant inhibition, in a time-dependent manner, against TPA-mediated increase in cutaneous edema and hyperplasia, epidermal ornithine decarboxylase (ODC) activity and ODC and cyclooxgenase-2 (COX-2) protein expression. In search for novel markers of skin tumor promotion, we found that TPA application to mouse skin resulted, as an early event, in an increased expression of phosphatidyinositol 3-kinase (PI3K), phosphorylation of Akt at threonine³⁰⁸ and activation of nuclear factor kappa B (NF-κB). Topical application of oleandrin before TPA application to mouse skin resulted in significant reduction in TPA-induced expression of PI3K and phosphorylation of Akt, and inhibition of NF-κB activation. NF-κB is a eukaryotic transcription factor that is critically involved in regulating the expression of specific genes that participate in inflammation, apoptosis and cell proliferation. Employing Western blot analysis, we found that oleandrin application to mouse skin resulted in inhibition of TPA-induced activation of NF-κB, IKKα and phosphorylation and degradation of IκBα. Our data suggest that oleandrin could be a useful anti-tumor promoting agent because it inhibits several biomarkers of TPA-induced tumor promotion in an in vivo animal model. One might envision the use of chemopreventive agents such as oleandrin in an emollient or patch for chemoprevention or treatment of skin cancer.

Introduction

In the United States alone more than one million new cases of nonmelanoma skin cancers (NMSC) are diagnosed each year, exceeding the incidence of all other types of cancer combined (Jemal et al., 2002). These NMSCs are caused by exposure of the skin to toxic chemicals and ultraviolet radiation present in the environment. It is highly desirable to define agents that can prevent the occurrence of these cancers. An ideal agent will be that which can restore most, if not all, dysregulated cellular and molecular pathways of multistage carcinogenesis. This can be achieved through use of multiple agents, a difficult undertaking. Alternatively, this could be achieved by a single agent capable of interfering at multiple pathways in the carcinogenic process, a very desirable goal. Those agents, which have the ability to intervene at more than one critical pathway in the carcinogenic process, will have greater advantage over other single-target agents.

Oleandrin (Fig. 1), a polyphenolic cardiac glycoside derived from the leaves of Nerium oleander, has been shown to possess anti-inflammatory and tumor cell growth-inhibitory effects Hung, 1999, Stenkvist, 1999. A recent study has shown that oleandrin is a potent inhibitor of nuclear factor kappa B (NF-κB) activation by various tumor promoters in a wide variety of different cell types (Manna et al., 2000). NF-κB, a widely distributed transcription factor, is associated with many physiological processes including inflammation, cellular proliferation and cancer Baeuerle and Baltimore, 1996, Barnes and Karin, 1997, Karin and Lin, 2002. NF-κB plays a key role in the regulation of many genes that are involved in tumorigenesis Garg and Aggarwal, 2002, Pahl, 1999. Therefore, NF-κB has emerged as one of the most promising molecular target, and agents that can suppress NF-κB activation have the potential to suppress carcinogenesis. Protein kinase B (PKB/Akt), a serine or threonine kinase, is a core component of the phosphatidylinositol 3-kinase (PI3K) signaling pathway that is activated through phosphorylation of Ser-473/474 and Thr-308/309 (Harlan et al., 1995). Studies have shown that Akt activates the transcription of a wide range of genes, especially those involved in immune activation, cell proliferation, apoptosis and cell survival (Karin and Lin, 2002). Mechanisms by which Akt suppresses apoptosis include the phosphorylation and inactivation of many proapoptotic proteins such as Bad (Datta et al., 1997), caspase 9 (Cardone et al., 1998) and activation of NF-κB (Romashkova and Makarov, 1999).

The multistage mouse skin carcinogenesis model, although an artificial one, is an ideal system to study many biochemical alterations, changes in cellular functions and histological changes that take place during the different stages of chemical carcinogenesis DiGiovanni, 1991, Katiyar and Mukhtar, 1997, Slaga et al., 1982. Studies have shown that skin applications of most tumor promoting agents result in inflammatory responses, such as development of edema, hyperplasia, induction of pro-inflammatory cytokine interleukin-1 alpha, induction of epidermal ornithine decarboxylase (ODC) and cyclooxygenase (COX) activities and expression of ODC and COX-2 protein expression and activation of NF-κB Chun et al., 2002, Katiyar et al., 1995, Katiyar et al., 1996, La et al., 1999, Seo et al., 2002. In the present study, we evaluated the effect of topical application of oleandrin to CD-1 mice against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced markers of skin tumor promotion.