Combination of cisplatin and bromelain exerts synergistic cytotoxic effects against breast cancer cell line MDA-MB-231 in vitro
© The Author(s) 2016
Received: 13 September 2016
Accepted: 4 November 2016
Published: 15 November 2016
Bromelain, which is a cysteine endopeptidase commonly found in pineapple stems, has been investigated as a potential anti-cancer agent for the treatment of breast cancer. However, information pertaining to the effects of combining bromelain with existing chemotherapeutic drugs remains scarce. This study aimed to investigate the possible synergistic cytotoxic effects of using bromelain in combination with cisplatin on MDA-MB-231 human breast cancer cells.
MDA-MB-231 cells were treated with different concentrations (0.24–9.5 µM) of bromelain or cisplatin alone, as well as four different combinations of these two agents to assess their individual and combination effects after 24 and 48 h. Cell viability was analyzed using an MTT assay. The induction of apoptosis was assessed using cell cycle analysis and an Annexin V-FITC assay. The role of the mitochondrial membrane potential in the apoptotic process was assessed using a JC-1 staining assay. Apoptotic protein levels were assessed by western blot analysis and proteome profiling using an antibody array kit.
Single-agent treatment with cisplatin or bromelain led to dose- and time-dependent decreases in the viability of the MDA-MB-231 cells at 24 and 48 h. Furthermore, most of the combinations evaluated in this study displayed synergistic effects against MDA-MB-231 cells at 48 h, with combination 1 (bromelain 2 µM + cisplatin 1.5 µM) exhibiting the greatest synergistic effect (P = 0.000). The results of subsequent assays indicated that combination 1 treatment induced apoptosis via mitochondria-mediated pathway. Combination 1 also resulted in significant decreases in the levels of several apoptotic proteins such as Bcl-x and HSP70, compared with bromelain (P = 0.002 and 0.000, respectively) or cisplatin (P = 0.000 and 0.001, respectively) single treatment. Notably, MDA-MB-231 cells treated with combination 1 showed increased levels of the pro-apoptotic proteins Bax compared with those treated with bromelain (P = 0.000) or cisplatin single treatment (P = 0.043).
Bromelain in combination with cisplatin synergistically enhanced the induction of apoptosis in MDA-MB-231 cells.
Breast cancer is the most frequently diagnosed invasive non-skin malignancy and the leading cause of cancer-related deaths in women throughout the world . In Asia, the incidence and mortality rates of breast cancer have been steadily rising for several years, with unhealthy diet, physical inactivity and obesity being identified as the main contributing factors . Although there are many different strategies available for the clinical treatment of breast cancer, the effectiveness of these approaches can be limited by the occurrence of adverse side effects and the development of drug resistance.
cis-Diamminedichloroplatinum (CDDP), which is more commonly known as cisplatin, is one of the most effective anti-cancer agents currently used in clinical practice, with pronounced activity against various cancers, including breast cancer [3, 4]. Cisplatin interacts with DNA and interferes with the mechanisms responsible for its transcription and replication [5, 6]. Consequently, cisplatin treatment is associated with a number of serious side effects such as nephrotoxicity, myelosuppression, ototoxicity, anaphylactic reactions, peripheral neuropathies and hypomagnesemia [7–9]. Furthermore, the clinical application of cisplatin is limited by the development of resistance mechanisms in the cancer cells [10–12]. The combination of cisplatin with other anti-cancer agents that operate via a different mode of action could therefore be used as an effective strategy to impede the growth of human cancer cells that develop resistance to cisplatin. This strategy could also minimize the severity of the side effects associated with the individual agents, whilst maintaining or even enhancing the effectiveness of the treatment process.
Pineapple (Ananascomosus L.) has been used to treat a wide range of diseases in several different countries, including Thailand, Malaysia, Taiwan and China, as well as the state of Hawaii . Pineapple plants are commonly used in folk medicine, especially their crown leaves, which are used to treat open wounds and inflammation. The results of a previous study demonstrated that pineapple crown leaf extract exhibited several interesting biological properties, including antimicrobial, anti-edema and anti-inflammatory activities .Pineapple stems have also been reported to exhibit a broad range of promising pharmacological properties. Stem bromelain is a cysteine endopeptidase, which is commonly found at a high concentration in the crude extract of pineapple stems (Ananascomosus L.) . The results of several in vitro and in vivo studies [16–21] have demonstrated that bromelain exhibited various beneficial therapeutic effects, including anti-tumor activity. These results therefore support the potential application of stem bromelain as a therapeutic agent for the treatment of cancer. Moreover, bromelain exhibits good stability over a wide range of pH values [22, 23] and is readily adsorbed in the human intestinal tract in its functional active form when it is consumed in high concentrations (up to 12 g/day). Taken together with the fact that its consumption does not lead to any major side effects, these results further highlight the potential of bromelain as an anti-cancer agent [24, 25].
The study aimed to investigate the possible synergistic cytotoxic effects of using bromelain in combination with cisplatin for the treatment of MDA-MB-231 human breast cancer cells.
Chemicals and reagents
Unless specified otherwise, all of the chemicals used in this study, including bromelain and cisplatin, were obtained from Sigma Aldrich (St Louis, MO, USA). Stock solutions of bromelain in water were freshly prepared prior to each experiment using deionized water. The resulting aqueous solutions were filtered (0.2 µm) prior to being used in the experiments. A stock solution of cisplatin was prepared in the dark using deionized water containing 0.9% (w/w) sodium chloride. The resulting stock solution was stored at 4 °C in the absence of light prior to being used.
The MDA-MB-231 cells used in this study obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in Roswell Park Memorial Institute medium enriched with 10% fetal bovine serum and 100 units/mL penicillin–streptomycin antibiotic at 37 °C under a humidified atmosphere containing 5% CO2.
Cell growth inhibition was determined using a colorimetric MTT assay. The assay was conducted in a 96-well plate with a cell density of 8 × 103 cells per well with an incubation period of 24 h. The medium was subsequently removed and replaced with fresh medium containing the test compound, followed by an incubation period of 24 or 48 h. The cells were then incubated with MTT solution (0.5 mg/mL) for 4 h, and the resulting formazan precipitate was dissolved in 170 µL of DMSO. The absorbance of each well was then measured at 570 nm using a microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). The percentage of cell survival was calculated using the following formula: percentage (%) cell survival = [(mean absorbency in test wells)/(mean absorbency in control wells)] × 100. These experiments were conducted in triplicate. We then constructed a graph of the percentage cell viability against the concentration of the test compound. The resulting graph was used to determine the IC10, IC20, IC30, IC40 and IC50 values of bromelain and cisplatin for the single treatment of the MDA-MB-231 cells.
We also conducted a series of MTT assays using four different combinations of bromelain and cisplatin (i.e., IC40 bromelain + IC10 cisplatin, IC30 bromelain + IC20 cisplatin, IC20 bromelain + IC30 cisplatin and IC10 bromelain + IC40 cisplatin) with concentrations in the range of 0.24–9.5 µM. All of these assays were conducted in a 96-well plate with a cell density of 8 × 103 cells per well with an incubation period of 24 h. The medium was subsequently removed and replaced with fresh medium containing the test compound, followed by an incubation period of 48 h. The cells were then incubated with MTT solution (0.5 mg/mL) for 4 h, and the resulting formazan precipitate was dissolved in 170 µL of DMSO. The absorbance of each well was then measured at 570 nm using a microplate spectrophotometer (Bio-Tek Instruments). All of these experiments were conducted independently in triplicate.
Annexin V-FITC assay
The cells were seeded into a 6-well plate and incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. The medium in each well was subsequently replaced with fresh medium containing different concentrations of the test compounds. After an incubation period of 24 or 48 h, all of the detached/dead and viable cells were collected. The cells were then washed and resuspended with PBS. The harvested cells were stained with Annexin V for 30 min before being treated with PI and analyzed by flow cytometry using a FACScan system (Becton–Dickinson and Company, San Jose, CA, USA) equipped with version 3.3 of the CellQuest software (Becton–Dickinson and Company). This assay was conducted according to the manufacturer’s protocol (BD PharmingenAnnexin V-FITC Apoptosis Detection Kit 1).
Measurement of mitochondrial membrane potential (JC-1 staining assay)
The cells were seeded into a 6-well plate and incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. The medium in each well was then replaced with fresh medium containing different concentrations of the test compounds. After an incubation period of 48 h, all the detached/dead and viable cells were harvested, washed with PBS and incubated with culture medium containing JC-1 for 30 min at 37 °C in the absence of light. The cells were then washed twice with PBS, resuspended in 500 µL of PBS and immediately analyzed by flow cytometry using a FACScan system (Becton–Dickinson and Company) equipped version 3.3 of the CellQuest software.
Cell cycle analysis
The cells were seeded into a 6-well plate and incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. The medium in each well was then replaced with fresh medium containing different concentrations of the test compounds. After an incubation period of 24 or 48 h, all of the detached/dead and viable cells were collected, washed with cold PBS and resuspended in 50 mL of cold PBS before being treated with 450 µL of cold ethanol. The cells were then incubated for 24 h at 4 °C. At the end of the incubation period, the cells were centrifuged (Model 5804 R, Eppendorf, Hamburg, Germany) at 200×g for 5 min and the resulting pellet was washed with cold PBS and resuspended in 500 µL of PBS. The cells were then incubated with 5 µL of RNase (20 μg/mL final concentration) for 30 min before being incubated with PI (50 µg/mL) on ice for 1 h in the dark. The distribution of cells was then immediately analyzed by flow cytometry using a FACScan system (Becton–Dickinson and Company) equipped with version 3.3 of the CellQuest software.
Proteome Profiler™: human apoptosis array
The cells were seeded in a 6-well plate and incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. The medium in each well was then replaced with fresh medium containing different concentrations of the test compounds. After an incubation period of 48 h, the cells were washed with PBS and lysed. All of the immunodetection steps were performed using a Proteome Profiler Human Apoptosis Array Kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. Briefly, the array was washed and incubated with a mixture of biotinylated detection antibodies. Streptavidin-HRP and chemiluminescent detection reagents were used, and a signal corresponding to the amount of protein bound was produced on each capture spot. After incubation, the membranes were developed using enhanced chemiluminescence reagents and immediately viewed and analyzed using a ChemiDoc XRS + system (Bio-Rad, Hercules, CA, USA). Protein expression was normalized to a positive control, which was present in each membrane.
The cells were seeded into a 6-well plate and incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. The medium in each well was then replaced with fresh medium containing different concentrations of the test compound. Following an incubation period of 24 or 48 h, the cells were washed with PBS and lysed in RIPA lysis buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, 1% deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS)] containing protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The extracted proteins (20–60 µg) were separated by electrophoresis on SDS–polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad) and probed with respective primary antibodies against Beta-actin, Bax and Bcl-2 (Abcam, Cambridge, Massachusetts, USA). After being incubated with the corresponding secondary antibodies (Abcam, the immunoreacted proteins were detected using a chemiluminescence system (ECL Western blot substrate; Abcam, Cambridge, UK).The bands obtained were quantitated using the ImageJ software (Bio Techniques, New York, NY, USA).
All of the experiments described in this study were repeated independently for at least three times (n = 3) and measured in triplicate, unless specified otherwise. The results have been reported as the corresponding mean values ± standard deviation (SD). Statistical data for MTT assay, Annexin-V/FitC assay, JC-1 staining assay and cell cycle analysis was analyzed by the Student’s t test and one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post hoc test. Statistical analyses were performed using version 19 of the SPSS software for Windows (IBM SPSS, Chicago, IL, USA). Differences between the experimental groups were considered significant for P values of less than 0.05. Data analyses to determine the CI value of the combination treatment used were performed using the CompuSyn software (Combo SynInc, City, State, USA). CI < 1 indicates synergism, CI = 1 indicates additive effect, and CI > 1 indicates antagonism.
Inhibitory effects of cisplatin and bromelain (single-agent and combined treatment) on the growth of MDA-MB-231 cells
Viability percentage of MDA-MB-231 at 48 h treated with bromelain and cisplatin combinations
Combination dosage (µM)
% viability ± SD
Bromelain dosage (IC value)
Cisplatin dosage (IC value)
2.0 µM (IC40)
1.5 µM (IC10)
50.24% ± 1.67
0.9 µM (IC30)
4.0 µM (IC20)
70.26% ± 2.66
0.5 µM (IC20)
5.9 µM (IC30)
61.30% ± 2.43
0.24 µM (IC10)
9.5 µM (IC40)
56.95% ± 3.61
Synergistic effects of combinations of cisplatin and bromelain for the treatment of MDA-MB-231 cells
Combination index (CI) and dose reduction index (DRI) values at 48 h for bromelain and cisplatin combinations. CI < 1 indicates synergistic effect, CI = 1 indicates additive effects and CI > 1 indicates antagonistic effect
Combination dosage (µM)
Bromelain dosage (IC value)
Cisplatin dosage (IC value)
2.0 µM (IC40)
1.5 µM (IC10)
0.9 µM (IC30)
4.0 µM (IC20)
0.5 µM (IC20)
5.9 µM (IC30)
0.24 µM (IC10)
9.5 µM (IC40)
Regulation of apoptosis and cell cycle progression in MDA-MB-231 cells by bromelain, cisplatin and their combined treatment
Activation of apoptosis via an intrinsic (mitochondrial) pathway in MDA-MB-231 cells treated with a combination of bromelain and cisplatin
Modulatory effects of bromelain and cisplatin alone, as well as a combination of these two agents on the expression levels of apoptosis-related proteins
The expression levels of Bcl-2 protein were found to be significantly reduced in the combination 1-treated cells compared with the bromelain- (P = 0.002) and untreated cells (P = 0.001). The expression level of Livin, Bax, Catalase, HO-1 and XIAP were also found to be significantly reduced in the combination 1-treated cells compared with the bromelain- (P = 0.000) and untreated cells (P = 0.000). However, the expression levels of several anti-apoptotic proteins, including HSP70 increased significantly in the MDA-MB-231 cells treated with combination 1 compared with the bromelain- (P = 0.016) and cisplatin-treated cells (P = 0.001). Moreover, the expression level of Bcl-x were also found to significantly higher in the combination 1-treated cells compared with the bromelain- (P = 0.002) and cisplatin-treated cells (P = 0.000).The expression levels of pro-apoptotic proteins HSP60 were found to be significantly reduced in the cells treated with combination 1 compared with the bromelain- (P = 0.041) and untreated cells (P = 0.014).
It was envisaged that the combination of bromelain and cisplatin would lead to synergistic effects, which would enhance their anti-cancer effects towards MDA-MB-231 human breast cancer cells. The CI values and isobolograms obtained using the MTT data clearly showed that the treatment of the MDA-MB-231 cells with a combination of bromelain and cisplatin resulted in a synergistic and dose-dependent increase in the inhibitory activity for combination 1 (bromelain 2 µM + cisplatin 1.5 µM) towards the growth of these cells. We also demonstrated that the IC50 dose of cisplatin was reduced almost 13-fold in combination 1. Cisplatin is a well-known anti-cancer agent, which is used in clinical practice to treat a variety of different cancers. However, despite its effectiveness and broad range of activity, the use of cisplatin is associated with several adverse side effects [3, 4, 7–9]. In contrast, the cysteine endopeptidase bromelain is a non-toxic agent with proven anti-cancer activity against various cancers [17, 24]. The concentration ratio of bromelain to cisplatin played an important role in determining the extent of the synergistic effects, as exemplified by the differences observed for different concentrations of cisplatin and bromelain in the different combinations evaluated in this study.
Annexin V-FITC and cell cycle analyses were performed on the treated and untreated MDA-MB-231 cells to determine whether the growth inhibitory effects of combination 1 could be attributed to apoptosis. The results of these assays showed that the synergistic effects of combination 1 were caused by an augmented apoptotic response. This effect could also be attributed to a different mode of action to those employed by cisplatin and bromelain. Cisplatin exerts its activity through the formation of intra- and inter-strand DNA adducts that damage the DNA and interferes with its normal functions [6, 26]. In contrast, the inhibitory effects of bromelain are dependent on its proteolytic activity, which allow it to remove certain cell surface molecules associated with cellular migration and adherence . The proteolytic activity of bromelain has also been reported to promote apoptosis in a number of human cancer cells, particularly in breast cancer cells [18, 28].
Consistent with our results, previous studies have demonstrated the involvement of the mitochondrial pathway in the apoptosis induced by single-agent treatment with cisplatin or bromelain in cancer cells [28–31]. The treatment of the MDA-MB-231 cells with combination 1 resulted in a loss of mitochondrial membrane potential, which suggested that the mitochondrial pathway was involved in the apoptosis induced by combination 1.
The results obtained using the Proteome Profiler Human Apoptosis Array kit showed that combination 1 led to decreases in the expression levels of several anti-apoptotic proteins, including cIAP 1, Bcl-2, catalase, clusterin, HO-1, livin, XIAP, HSP27 and claspin. Notably, combination 1 led to considerable decreases in the expression levels of cIAP1, catalase, clusterin, HO-1, livin and XIAP compared with the cells treated with bromelain or cisplatin. Interestingly, we also observed pronounced increases in the expression levels of several anti-apoptotic proteins, including HSP70 and Bcl-x in the cells treated with combination 1 compared with the untreated cells. In contrast, we did not observe any increases in the regulation of any pro-apoptotic proteins (e.g., Bax). We also observed an increase in the expression of the apoptotic protein HSP60.
Taken together with the observed decreases in the anti-apoptotic proteins Bcl-2 and HSP27, the increase observed in the pro-apoptotic protein Bax provided clear evidence that combination 1 induced mitochondria-mediated apoptosis. This result was consistent with previous studies, which established the important roles played by Bcl-2, HSP27 and Bax in the regulation of apoptosis activated through the mitochondrial pathway [32–38]. We also observed significant increases in the levels of the anti-apoptotic proteins HSP70 and Bcl-x in the cells treated with combination 1. Bcl-x and HSP70 play essential roles as anti-apoptotic proteins by preventing the release of mitochondrial apoptogenic factors such as cytochrome C and apoptosis inducing factor (AIF), and consequently inhibit apoptosis [36, 39]. The elevated levels of Bcl-x and HSP70 observed in this study would be insufficient to shut down the apoptotic process induced by combination 1 in MDA-MB-231 cells. The fold-change values shown in Fig. 6 for the pro-apoptotic protein Bax to the anti-apoptotic protein Bcl-x indicated that the increase in Bax was much greater than that of Bcl-x, suggesting that the pro-apoptotic effects of upregulated Bax would overwhelm the anti-apoptotic effects of Bcl-x. It is therefore possible that some other pathway or mechanism was being activated or inhibited by combination 1 and that this process was compensating for the interference in mitochondrial mediated-apoptosis and contributing to the synergistic effect of combination 1.
We also observed pronounced decreases in the levels of the anti-apoptotic proteins cIAP 1 and XIAP in the combination 1-treated cells. This change was attributed to an increase in the expression of HSP70. Although HSP70 is well known for its cytoprotective effects, previous studies [40, 41] have also shown that elevated levels of HSP70 may have led to an increased susceptibility to apoptosis. Elevated HSP70 levels would result in an increase in the inhibition of IkB kinase activity, which would inhibit the activity of the NF-kB pathway, leading to the inhibition of NF-kB-dependent anti-apoptotic gene induction [40–42]. Given that the expression levels of cIAP and XIAP are highly dependent on the NF-kB pathway, the inhibition of the IkB kinase by elevated levels of HSP70 would lead to a reduction in the expression levels of cIAP and XIAP. This change could therefore explain the considerable decrease observed in the levels of cIAP and XIAP in this study.
Bromelain in combination with cisplatin synergistically enhanced the induction of apoptosis in MDA-MB-231 cells.
SKY, NBA, TS, NA, ARO, SAA and ALTC conceived and designed the study. AZMP, SKY and KLL performed the assays. AZMP, NA and SGT wrote the manuscript. All authors read and approved to the final manuscript.
This research was supported by Research University Grant Scheme (Project No: 05-02-12-1718RU) provided by Universiti Putra Malaysia, Malaysia.
The authors declare that they have no competing interests.
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