Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy

Gastrointestinal malignancies account for more than one third of all deaths from cancer worldwide [1]. Representing the most common gastrointestinal cancers [2], colorectal and gastric cancers have the potential to disseminate throughout the peritoneal cavity. Although known as a manifestation of advanced disease, peritoneal carcinomatosis is a locoregional cancer spread, with the peritoneum serving as the first line of defense against the progression of carcinomatosis [3]. Hence, cytoreductive surgery in combination with hyperthermic intraperitoneal chemotherapy [4],[5] has brought about long-term survival to patients with peritoneal surface malignancies [6] and is a promising approach to peritoneal carcinomatosis of gastrointestinal origin. However, disruption of the peritoneal barrier is believed to contribute to the recurrence [3]. Novel modalities are thus needed to complement the current standard of care through enhancement of microscopic cytoreduction. To this end, our research team at St George Hospital (Sydney, Australia), with an established Peritoneal Surface Malignancy Program since 1996 [7],[8], has sought to develop novel locoregional approaches to peritoneal malignancies. As such, a variety of agents have been tested for their potential value in such a strategy, among which bromelain and N-acetylcysteine (NAC) as two natural agents with good safety profiles have shown promise in our investigations. We previously described the efficacy of bromelain in combination with NAC for in situ lysis of the mucin secreted in pseudomyxoma peritonei [9]. In the present article, we report for the first time to our knowledge that bromelain and NAC, on their own and more potently in combination, inhibit growth and proliferation of gastrointestinal cancer cells and promote cell death in vitro.

Bromelain is a crude, aqueous extract from the pineapple comprised of sulfhydryl-containing proteolytic enzymes as the main constituents, and a variety of non-proteolytic enzymes and components [10],[11]. Being available as a pharmaceutical product since 1956 [12], bromelain has shown benefits in digestion, wound healing, burn debridement, and enhancement of antibiotic absorption. Immunomodulatory, anti-coagulative and anti-inflammatory activities of bromelain with potential applications in various pathological conditions, including infections, cardiovascular and respiratory diseases and musculoskeletal injuries, have been reported [13],[14]. Moreover, anticancer properties of bromelain have been shown in different studies [15]. N-acetylcysteine (NAC), known as a sulfhydryl group donor naturally found in Allium plants, is the acetylated derivative of the amino acid L-cysteine and a precursor to the powerful antioxidant glutathione. Initial studies in 1960s and 1970s reported clinical benefits of NAC as an effective mucolytic agent in cystic fibrosis [16] and an antidote for acetaminophen overdose hepatotoxicity [17]. Since then, its utility in different pathological conditions, e.g. chronic obstructive pulmonary disease, contrast-induced nephropathy, cardiovascular diseases, neuropsychiatric disorders and cancer, has been investigated [18],[19]. Both drugs are considered as inexpensive, well-tolerated agents with good safety profiles.

We found in the present study that bromelain and NAC exert cytotoxic effects on a panel of human gastrointestinal cancer cells of different origin, chemosensitivity and phenotype. MKN45 and KATO-III are gastric cancer cell lines classified as poorly differentiated and signet ring carcinoma cells, respectively. HT29-5F12 and HT29-5M21 are two subpopulations of HT29 colon adenocarcinoma cell line resistant to 5-fluorouracil and methotrexate, respectively. LS174T is a colon adenocarcinoma cell line with a goblet cell-like phenotype. Furthermore, apart from the unified expression of the membrane-associated mucin MUC1, these cells exhibit different expression phenotypes with respect to the gastrointestinal secretory mucins. While MKN45 and KATO-III both represent a predominantly gastric phenotype and specifically express MUC5AC [20], HT29-5F12 and HT29-5M21 mainly express MUC2 and MUC5AC, respectively [21], and LS174T expresses the goblet cell-specific mucins MUC2, MUC5AC and MUC6 [22].

Anticancer properties of bromelain or NAC, individually, have been evaluated in some in vitro and in vivo models before. Bromelain has shown cytotoxic and/or cytostatic effects on murine lung carcinoma, mammary adenocarcinoma, leukemia, lymphoma, sarcoma, melanoma and ascitic tumor cell lines [23]-[26], as well as on human cell lines derived from gastric and colon carcinoma [23],[27],[28], glioma [29], breast cancer [30]-[32], epidermoid carcinoma, melanoma [33] and malignant peritoneal mesothelioma [34]. Bromelain was also found to induce differentiation of leukemia cell lines in vitro [35] and to exert chemopreventive effects on skin [36]-[38] and colon [28] tumorigenesis in vivo. Clinically, however, benefits of bromelain in cancer have been explored in few studies [39]-[42]. NAC has been reported to inhibit growth, proliferation and/or invasive behavior of human cancer cells, including colorectal [43], bladder [44],[45], prostate [46], tongue [47] and lung [48] carcinoma cell lines, in vitro. In addition, anticarcinogenic [49],[50] and chemoprotective [51] properties of NAC and its potential value as a chemopreventive agent [52]-[55] or a chemoprotectant [56]-[61] has been investigated. NAC has also been shown to interact with and enhance cytotoxic effects of chemotherapeutic drugs [62],[63], interferon ? [64], copper [65] and epigallocatechin-3-gallate [66] on cancer cells. In contrast, few contradictory reports are also available. Tysnes et al. [29] observed that bromelain significantly and reversibly reduced adhesion, migration and invasion of glioma cells, but did not affect cell viability in vitro. In a recent study by Sceneay et al. [67], while NAC targeted hypoxic response of breast cancer cells in vitro, it did not inhibit, but even enhanced, tumor growth in vivo. Together, these findings imply that bromelain and NAC might function in a cell- and/or context-dependent manner. Examining the efficacy of the two agents in combination, we interestingly found in the present study the synergistic and additive interactions between bromelain and NAC with resultant potentiation of cytotoxicity in combination therapy. Here, we report for the first time cytotoxic effects of bromelain and NAC on gastrointestinal cancer cells with added value in combination therapy.

Mechanistically, we found that bromelain and NAC, in particular in combination, inhibit cell cycle progression and induce both apoptotic and autophagic cell death in the gastrointestinal carcinoma cells. Orderly progression of the cell cycle from one phase to another is coordinated by cyclins sequentially activating their partner proteins, cyclin-dependent kinases (CDKs) [68]. This is initiated by the expression of cyclin D in early G1 which drives the cell cycle through to late G1 and followed by subsequent induction of cyclin E, cyclin A and cyclin B at late G1, early S and late S phases, respectively. Thus, our results indicating diminution of cyclins D, A and B suggest that treatment halts cell cycle progression in early G1. This was found to be associated with activation of intrinsic and extrinsic caspase-dependent apoptotic pathways and inhibition of pro-survival pathways which collectively reduce cell viability. Finally, autophagy was shown to apparently contribute to cell death when deregulation of autophagy-related proteins and, more importantly, an increase in the expression of LC3-II, known as the most reliable marker of autophagosomes [69], were observed. Mechanistic basis for the growth-inhibitory effects of bromelain or NAC on malignant cells have been explored in a number of studies. Kalra et al. and Bhui et al. have demonstrated in their investigations on mouse skin tumors that bromelain treatment is associated with upregulation of p53 and Bax, activation of caspase 3 and caspase 9, attenuation of Erk and Akt phosphorylation and decrease in Bcl-2 [37],[38]. They later reported that bromelain treatment of human epidermoid carcinoma and melanoma cells, in vitro, resulted in cell cycle arrest at G(2)/M phase (by modulation of cyclin B1, phospho-cdc25C, Plk1, phospho-cdc2 and myt1) and subsequent induction of apoptosis through modulation of Bax-Bcl-2 ratio, apoptotic protease activating factor 1 (Apaf-1), caspase-9, and caspase-3 [33]. In a separate study on breast cancer cells in vitro, they demonstrated induction of apoptosis and autophagy in response to bromelain where autophagy preceded and facilitated apoptotic response [30]. Bromelain-induced apoptosis in breast [31],[32] and colon cancer cells [28] has also been supported by other investigators.

As reviewed by De Flora et al. [70], NAC utilizes a variety of mechanisms to inhibit carcinogenesis. NAC has been shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of cyclin D1 and DNA synthesis [71], to diminish DNA synthesis in human astrocytoma cells [72], to inhibit p38 mitogen-activated protein kinase (MAPK) activation and abnormal cell cycle progression in human lymphoma cells [73], and to induce CDK inhibitors and G1 cell cycle arrest in murine papilloma cells [74]. Moreover, despite a large body of evidence establishing anti-apoptotic effects of NAC protecting normal cells against cytotoxic stimuli, there are reports arguing for a pro-apoptotic role of NAC. As an initial observation, Tsai et al. [75] reported that NAC induce apoptosis in aortic smooth muscle cells but not in endothelial cells. In agreement, Liu et al. [76] and Havre et al. [77] indicated that NAC selectively induced p53-mediated apoptosis in several oncogenically-transformed fibroblasts, but not in normal cells. In a study by Nargi et al. [43], it was demonstrated in a range of colorectal cancer (CRC) cell lines that NAC differentially induced cell cycle arrest or apoptosis in CRC cells and identified CDK inhibitor p21(WAF1/Cip1), functional p53, Ras status and basal levels of reactive oxygen species (ROS) in CRC cells as important determinants of susceptibility to apoptosis. Cho et al. also found that NAC reduced ROS production and Akt phosphorylation in breast cancer cells, resulting in apoptotic cell death [78]. NAC has also been shown to enhance H₂O₂-, UV- and MK886-induced apoptosis of murine hybridoma [79], human melanoma [80] and human T-cell leukemia cells [81], respectively. These reports and our findings conclusively support the notion that NAC can exert pro-apoptotic effects in a cell type- and context-dependent manner.

In summary, we found in the present study that bromelain and NAC, on their own and more potently in combination, inhibit growth, proliferation and survival of human gastrointestinal cancer cells of different phenotypes and characteristics. Our data suggests that bromelain in combination with NAC could be of potential value in novel therapeutical approaches to cancer. With special regard to peritoneal malignancies and carcinomatosis, this formulation holds promise for enhancement of microscopic cytoreduction in locoregional strategies and thus represents an interesting avenue for future research.