Regulation of p53, nuclear factor κB and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin

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Abstract

Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-κB), we also investigated the effect of bromelain on Cox-2 and NF-κB expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-κB by blocking phosphorylation and subsequent degradation of IκBα. In addition, bromelain treatment attenuated DMBA–TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-κB-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA–TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects.

Introduction

Carcinogenesis is a multistep process in which genetic and epigenetic events determine the transition from a normal to a malignant cellular state. The rate of the process of tumor progression is accelerated by mutagenic agents (tumor initiators) and by mitogenic agents (tumor promoters) (Zoumpourlis et al., 2003). Reversal of aberrant epigenetic events, including those that modulate the transcriptional activity of genes associated with various signaling pathways, holds the prospect of influencing multiple stages of tumorigenesis (Mallikarjuna et al., 2004). Mouse skin has provided a paradigm for studies of multistage chemical carcinogenesis in epithelial cells. This study has provided insight into the mechanism of carcinogen–DNA interactions and the nature of mutations in the critical target genes (Zoumpourlis et al., 2003, Saleem et al., 2005).

The dietary components as chemopreventive agents have received much attention among the researchers. A greater understanding of the pivotal events associated with carcinogenesis will facilitate the use of dietary intervention as a key strategy to prevent cancer development. Experimental studies have revealed that the dietary components regulate the molecules in several cell signal transduction pathways including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), Akt and p53 pathways. Dietary agents, among other mechanisms, activate cell death signals and induce apoptosis in precancerous or cancer cells, resulting in the inhibition of cancer development and/or progression (Sarkar and Li, 2004). Recently, bromelain, an extract from pineapple stem (Ananas cosmosus) has been used clinically for a wide variety of maladies including edema, thrombophlebitis, sinusitis, inflammation, rheumatic arthritis and as adjuvant in cancer treatment (Yuan et al., 2006, Wallace, 2002). Although poorly understood, the pleiotropic effects of bromelain are considered to be due to the complex mixture of closely related cysteine proteinases, proteinase inhibitors, phosphatases, glucosidases, peroxidases and other undefined compounds (Beuth and Braun, 2005, Stopper et al., 2003). It has also been shown that bromelain may play a role in the differentiation of malignant cells (Mynott et al., 1999). Bromelain also acts as an immunomodulator by raising the impaired immunocytotoxicity of monocytes against tumor cells from patients (Maurer, 2001). In addition, bromelain has shown both antiproliferative and antimetastatic effects in tumor models in vitro and in vivo (Beuth and Braun, 2005, Tysnes et al., 2001). It has also been shown to possess anticancer properties in hairless mouse skin (Goldstein et al., 1975).

In view of the anti-inflammatory, antiproliferative and antimetastatic activities of bromelain, we tested antitumorigenic potential of bromelain in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin carcinogenesis model.

Section snippets

Materials

DMBA, TPA, purified bromelain and antibody specific for β-actin (clone AC-74) were purchased from Sigma (St Louis, USA). The mouse monoclonal phospho-extracellular signal-regulated protein kinase (ERK1/2), MAPK (Thr202/Tyr204) phospho-Akt (Ser473), phospho-SAPK/JNK1 (Thr183/Tyr185), NF-κB (Ser536) phospho-IκBα (Ser32), caspase 3 and caspase 9 antibody were procured from Cell Signaling Technology (Beverly, USA). Anti-p53 mouse monoclonal antibody specific for wild type protein (clone PAb 1620,

Antitumorigenic potential of bromelain

Results of the present investigation revealed delay in the onset of tumorigenesis in the animals treated with bromelain. The first day of papilloma incidence in the positive control (DMBA–TPA) Gr. II was on the 52nd day; however, it was 70th day in the bromelain-treated Gr. III. About 22% of animals of Gr. III remained papilloma-free until the termination of the experiment (Fig. 1a). However, by the end of the 15th week, all the animals of the positive control Gr. II developed papilloma. Some

Discussion

Chemoprevention has become a practical approach in identifying potentially useful inhibitors of malignant transformation and also offers opportunities to study the mechanisms of anti-carcinogenesis (Arora et al., 2006, Chun et al., 2003). The present study was aimed at unraveling the basis of antitumorigenic effect of bromelain in DMBA–TPA induced mouse skin papillomas. DMBA, like other polycyclic aromatic hydrocarbons, requires metabolic activation to become an ultimate carcinogen. Many

Acknowledgments

Authors are thankful to Dr. Ashwani Kumar, Director Industrial Toxicology Research Centre, Lucknow for his keen interest in the study. Authors are also thankful to the Indian Council of Medical Research (India) for providing fellowship to Ms. Neetu Kalra and to Mr. Sahdeo Prasad.

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