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

Abstract

Background: Bromelain and N-acetylcysteine are two natural, sulfhydryl-containing compounds with good safety profiles which have been investigated for their benefits and application in health and disease for more than fifty years. As such, the potential values of these agents in cancer therapy have been variably reported in the literature. In the present study, the efficacy of bromelain and N-acetylcysteine in single agent and combination treatment of human gastrointestinal carcinoma cells was evaluated in vitro and the underlying mechanisms of effect were explored.

Methods: The growth-inhibitory effects of bromelain and N-acetylcysteine, on their own and in combination, on a panel of human gastrointestinal carcinoma cell lines, including MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T, were assessed by sulforhodamine B assay. Moreover, the influence of the treatment on the expression of a range of proteins involved in the regulation of cell cycle and survival was investigated by Western blot. The presence of apoptosis was also examined by TUNEL assay.

Results: Bromelain and N-acetylcysteine significantly inhibited cell proliferation, more potently in combination therapy. Drug-drug interaction in combination therapy was found to be predominantly synergistic or additive. Mechanistically, apoptotic bodies were detected in treated cells by TUNEL assay. Furthermore, Western blot analysis revealed diminution of cyclins A, B and D, the emergence of immunoreactive subunits of caspase-3, caspase-7, caspase-8 and cleaved PARP, withering or cleavage of procaspase-9, overexpression of cytochrome c, reduced expression of anti-apoptotic Bcl-2 and pro-survival phospho-Akt, the emergence of the autophagosomal marker LC3-II and deregulation of other autophagy-related proteins, including Atg3, Atg5, Atg7, Atg12 and Beclin 1. These results were more prominent in combination therapy.

Conclusion: We report for the first time to our knowledge the growth-inhibitory and cytotoxic effects of bromelain and N-acetylcysteine, in particular in combination, on a panel of gastrointestinal cancer cell lines with different phenotypes and characteristics. These effects apparently resulted from cell cycle arrest, apoptosis and autophagy. Towards the development of novel strategies for the enhancement of microscopic cytoreduction, our results lay the basis for further evaluation of this formulation in locoregional approaches to peritoneal surface malignancies and carcinomatosis.

Figure 1

Sulforhodamine B assay on MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T cells after single agent treatment with bromelain and NAC. As assayed 72 hours post treatment, concentration-dependent inhibition of cell proliferation was observed with escalating concentrations of bromelain (left panel) and NAC (right panel). Cisplatin was used as the positive control of the experiment (small graphs a-e). Significant changes (p <0.05) are marked by asterisks.

Figure 2

Concentration-response curves for single agent treatment of MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T cells with bromelain and NAC. These curves plot growth percentage versus drug concentration after 72 h treatment of the cancer cells with bromelain (left panel) and NAC (right panel). Half maximal inhibitory concentration (IC₅₀) values are demonstrated for each curve individually.

Figure 3

Combination treatment of MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T cells with bromelain and NAC. Cells were treated for 72 hours with three concentrations of each agent on their own and in combination. In general, growth-inhibitory effects of combination therapy were significantly more potent than those induced by single agent treatment. Significant changes (p <0.05) are marked by bold (*) and non-bold (*) asterisks when single agent bromelain and NAC are considered as control, respectively.

Figure 4

Concentration-response curves and drug-drug interaction analysis of combination treatment of MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T cells. Left panel demonstrates left-sided shift of concentration-response curves of NAC after being used in combination with bromelain. Drug-drug interaction analysis (right panel) revealed synergism and additivity as the predominant patterns of interaction between bromelain and NAC in combination therapy.

Figure 5

Fluorometric TdT-mediated dUTP nick-end labeling (TUNEL) assay on treated MKN45 cells. After 48 hours of treatment with bromelain (100 and 200 μg/mL), NAC (5 and 10 mM) or the combination, cells were assayed for TUNEL reactivity and viewed under laser scanning confocal microscope. Green (fluorescein-12-dUTP) and red (propidium iodide) fluorescence correspond to fragmented DNA and the nucleus, respectively, indicating the presence of apoptotic cells in all treatment groups as compared with DNase I-treated cells used as the positive control. Scale bar: 50 μm.

Figure 6

Western blot analysis of the expression of proteins involved in the regulation of cell cycle and survival in MKN45 cells. After 48 hours of treatment with single agent or combined bromelain (100 and 200 μg/mL) and NAC (5 and 10 mM), activation of caspase system (represented by caspase proteins, cytochrome c and PARP), inhibition of anti-apoptotic and pro-survival processes (represented by Bcl-2 and phospho-Akt), diminution of the cell cycle regulators cyclin A, cyclin B and cyclin D along with the emergence of the autophagosomal marker LC3-II and deregulation of other autophagy-related proteins, including Atg3, Atg5, Atg7, Atg12 and Beclin 1, were found. As seen, results were more prominent in combination therapy. Our data implies that growth arrest, apoptosis and autophagy are likely mechanisms underlying cytotoxic effects of bromelain and NAC on gastrointestinal cancer cells. GAPDH was used as the loading control.