Cancer Letters

Cancer Letters

Volume 282, Issue 2, 18 September 2009, Pages 167-176
Cancer Letters

Bromelain inhibits COX-2 expression by blocking the activation of MAPK regulated NF-kappa B against skin tumor-initiation triggering mitochondrial death pathway

https://doi.org/10.1016/j.canlet.2009.03.003 Get rights and content

Abstract

Chemoprevention impels the pursuit for either single targeted or cocktail of multi-targeted agents. Bromelain, potential agent in this regard, is a pharmacologically active compound, present in stems and fruits of pineapple (Ananas cosmosus), endowed with anti-inflammatory, anti-invasive and anti-metastatic properties. Herein, we report the anti tumor-initiating effects of bromelain in 2-stage mouse skin tumorigenesis model. Pre-treatment of bromelain resulted in reduction in cumulative number of tumors (CNT) and average number of tumors per mouse. Preventive effect was also comprehended in terms of reduction in tumor volume up to a tune of ∼65%. Components of the cell signaling pathways, connecting proteins involved in cell death were targeted. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in Bcl-2. A marked inhibition in cyclooxygenase-2 (Cox-2) expression and inactivation of nuclear factor-kappa B (NF-κB) was recorded, as phosphorylation and consequent degradation of Iκ Bα was blocked by bromelain. Also, bromelain treatment curtailed extracellular signal regulated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and Akt activity. The basis of anti tumor-initiating activity of bromelain was revealed by its time dependent reduction in DNA nick formation and increase in percentage prevention. Thus, modulation of inappropriate cell signaling cascades driven by bromelain is a coherent approach in achieving chemoprevention.

Introduction

Cancer is a hyperproliferative disorder which involves morphological cellular transformation, uncontrolled cellular proliferation, deregulation of apoptosis, invasion, angiogenesis, and metastasis. Propensity of an organism towards the disease cannot be ascribed to a single factor but may be a corollary of several contributing and/or diverse factors. Exposure to chemical carcinogens can cause damage to nuclear chromatin, the most vulnerable part of the cell, by inducing DNA damage, chromosomal abnormalities and mutations, which foreshadow the danger of cancer development [1]. Approximately 20% of all human cancers in adults result from chronic inflammatory states which are triggered by infectious agents or exposure to other environmental aspects, or by a combination thereof [2].

Due to the fact that malignant conversion of normal cells requires several steps and often proceeds over considerable time periods, primary prevention of this process should include several approaches, with optimization of nutrition and diet being among most viable. Diet derived agents may find widespread, long term use among populations, owing to their expected safety and because they are not perceived as ‘medicine’. During past decades, several chemicals have been studied in terms of their potential chemopreventive role in cancer development and more than 50 promising agents as well as agent combinations are in vanguard for clinical evaluation as chemopreventive drugs [3].

Bromelain is a bioactive agent possessing remarkable therapeutic properties. It is a complex natural mixture of plant cysteine proteolytic enzymes that is derived from pineapple (Ananas cosmosus) stems. Bromelain is popular in the west and is commonly sold in the health food stores as a nutritional supplement to ‘promote digestive health’ [4], also majorly used in vaccine formulation. It is known to possess pleiotropic therapeutic effects, i.e., interference with growth of malignant cells, antiedematous, anti-inflammatory, anti-metastatic, antithrombotic, etc.

Although, it is established that consumption of fruits and vegetables can combat cancer, yet it is unclear that which of their constituents do so, and how? Implication of pivotal cell signaling pathways that converge on specific molecules viz. nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (Cox-2), mitogen-activated protein kinases (MAPKs), Akt and p53 undeniably alludes to tumor initiation, growth and development. These molecules can be deemed as major culprits and therefore, therapeutic targets in cancer. For that reason, prevention hinges on the regulation of these key molecules by diet derived agents, since they possess the ability to restore the deregulated molecular pathways of multistage carcinogenesis. The data from published literature indicates that these agents regulate transcription, translation and activation of the aforementioned molecules [5]. The modulation of initiation phase and/or inhibition of cancer development by these dietary agents may involve discernable mechanisms including alterations to the profile of drug metabolizing enzymes; altered rates of DNA repair; scavenging of reactive oxygen species and activation of cell death signals to induce apoptosis.

Although few studies have reported bromelain to exhibit anti-proliferative and anti-metastatic effects in tumor models both in vitro and in vivo as early as 1985 [6], yet there has been a lacuna to address its potency against the explicitly understood process of carcinogenesis. To fill the gap we earlier elucidated the anti-tumorigenic role of bromelain against skin tumors in mouse skin [7], deciphering the underlying mechanisms. The aforementioned study investigated the effects of bromelain when given after exposure of mouse skin to tumor initiator and addressed to the inhibition of tumor promotion by bromelain. On the grounds of above finding which established bromelain to be discernibly potent anti tumor-promoting agent, we hypothesized that bromelain may also possess ability to combat against tumor initiation, even before exposure to the carcinogen occurs. Henceforth, we tested anti tumor-initiating potential of bromelain in 2-stage mouse skin carcinogenesis model.

Section snippets

Materials

7,12-Dimethyl-benzanthracene (DMBA), 12-O-tetradecanoylphorbol-13-acetate (TPA), purified bromelain and antibody specific for β-actin (clone AC-74) were purchased from Sigma (St. Louis, USA). The mouse monoclonal p38 MAPK, Akt, extracellular signal regulated protein kinase (ERK1/2) [total and phospho forms]; phospho-c-jun terminal kinase 1 (JNK1); procaspase and caspase (3 and 9); and NF-κB antibody were procured from Cell Signaling Technology (Beverly, USA). Anti-p53 mouse monoclonal antibody

Effect of bromelain on tumor-initiation

Anti tumor-initiating potential assessed by animal bioassay revealed delay in onset of tumorigenesis in the animals pre-treated with bromelain. First day of papilloma incidence in the positive control (DMBA–TPA) Gr. II was on 52nd day, while 76th day in bromelain pre-treated Gr. III. 32% of animals of Gr. III remained tumor free till the termination of the experiment without attainment of 100% tumorigenesis (Fig. 1a, Table 1). However, by the end of the 15th week, 100% tumorigenesis was

Discussion

Initiation of carcinogenesis, involving damage to the DNA can be almost completely prevented or reduced by blocking agents, which are particularly effective if administered before the carcinogen. The present investigation therefore undertakes both, long term and short term studies to illustrate the basis of preventive and hence anti tumor-initiating potential of bromelain by employing mouse model.

Studies, however few in number, have reported bromelain to demonstrate anti-proliferative,

Conflict of interest

None declared.

Acknowledgements

Authors are thankful to Dr. Ashwani Kumar, Director Indian Institute of Toxicology Research, Lucknow, for his keen interest in the study. Authors are also thankful to Indian Council of Medical Research (India) for providing fellowship to Kulpreet Bhui and Jasmine George. Sincere thanks are also due to Ms. Babita Singh for her computer aided support.

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