Z-LEHD-FMK

Pineapple bromelain induces autophagy, facilitating apoptotic response in mammary carcinoma cells

Kulpreet Bhui,1 Shilpa Tyagi,1 Bharti Prakash,2 and Yogeshwer Shukla1*

Abstract.

Bromelain, from pineapple, possesses potent anticancer effects. We investigated autophagic phenomenon in mammary carcinoma cells (estrogen receptor positive and negative) under bromelain treatment and also illustrated the relationship between autophagy and apoptosis in MCF-7 cells. MCF-7 cells exposed to bromelain showed delayed growth inhibitory response and induction of autophagy, identified by monodansylcadaverine localization. It was succeeded by apoptotic cell death, evident by sub-G1 cell fraction and apoptotic features like chromatin condensation and nuclear cleavage. 3-Methyladenine (MA, autophagy inhibitor) pretreatment reduced the bromelain-induced autophagic level, also leading to decline in apoptotic population, indicating that here autophagy facilitates apoptosis. However, addition of caspase-9 inhibitor Z-LEHDFMK augmented the autophagy levels, inhibited morphological apoptosis but did not prevent cell death. Next, we found that bromelain downregulated the phosphorylation of extracellular signal-regulated kinase 1=2 (ERK1=2), whereas that of c-jun N-terminal kinase (JNK) and p38 kinase were upregulated. Also, MA had no influence on bromelain-suppressed ERK1=2 activation, yet, it downregulated JNK and p38 activation. Also, addition of mitogen-activated protein kinase (MAPK) inhibitors enhanced the autophagic ratios, which suggested the role of MAP kinases in bromelain-induced autophagy. All three MAPKs were seen to be constantly activated over the time. Bromelain was seen to induce the expressions of autophagy-related proteins, light chain 3 protein B II (LC3BII), and beclin-1. Using ERK1=2 inhibitor, expressions of LC3BII and beclin-1 increased, whereas p38 and JNK inhibitors decreased this protein expression, indicating that bromelain-induced autophagy was positively regulated by p38 and JNK but negatively regulated by ERK1=2. Autophagyinducing property of bromelain can be further exploited in breast cancer therapy.

Keywords: bromelain, autophagy, light chain 3 protein II, MAP kinases

1. Introduction

Macroautophagy or autophagy is a catabolic process for the autophagosomic–lysosomal degradation of bulk cytoplasmic contents. This process is characterized by the sequestration of cytoplasmic material by a membrane to form an autophagosome that can receive input highlights the imperative role of autophagy in human pathologies, including cardiomyopathy and breast cancer [5].
Identification of a set of genes conserved from yeast to humans that are involved in the signaling and formation of autophagic vacuoles has led to a greater understanding of the molecular controls of autophagy [6,7]. Two conjugation systems are involved in the formation of the autophagosome [8–10]. In this complex, the protein beclin-1 is a tumor suppressor gene product [11]. Beclin-1 is localized within cytoplasmic structures including the mitochondria and can stimulate autophagy when overexpressed in mammalian cells [12]. Earlier, it has been shown that expression of beclin-1 is greatly reduced in breast tumors and breast carcinoma cell lines including MCF-7 cells, but the autophagic capacities are restored by induced expression of beclin-1 [13]. Tamoxifen (TAM) has been reported to stimulate the expression of beclin-1 in MCF-7 cells [14].
In the second conjugation system, Apg4, a cysteine protease is required for the processing of Apg8 before the conjugation reactions and before the release of Apg8 from the phospholipid anchor [15]. Several human Apg8 homologs, which can be engaged in Apg7-dependent conjugating reactions, exist in mammals [16], light chain 3 (LC3) being one of them. It associates to the autophagosome membrane, after processing [17]. As LC3 plays an important role in the autophagic process, it can serve as a good marker for autophagy.
Either apoptosis or autophagy may occur in the same tissue/cells depending on the trigger; whereas it is sometimes even possible for the two phenomena to coincide in certain tissues/cells [18,19]. Occurrence of both autophagic and apoptotic cell death was shown during deprivation of neural growth factor induced in primary sympathetic neurons [20]. Cui et al. [21] have also illustrated that oridonin, a phytochemical from traditional Chinese herb, Rabdosia rubescens, induces apoptosis following autophagy in MCF-7 cells. However, still, some autophagic cell death events can also be attributed to apoptosis and so the mutual relationship between apoptotic and autophagic death is debated [22,23].
MCF-7 cells have been routinely used to evaluate the anticancer potency of antiestrogens and other compounds/ drugs [24–27]. Thus, better understanding of the cell’s suicide mechanisms triggered by variety of compounds may eventually help to elucidate new compounds and targets for therapeutic intervention. Bromelain, referred to a group of enzymes or a single enzyme obtained from Ananas comosus is a popular drug used in the West for various maladies and has also been used against inflammation [28]. Also, it has been earlier reported by us to possess potent antitumorigenic activity, in vivo [29,30]. The pathway(s) leading to autophagic and/or apoptotic cell death are, at present, poorly understood; in this study, we have elucidated the bromelain-induced death mechanism in estrogen receptor (ER) positive MCF-7 and negative MDA-MB-231 cell lines and its regulation at molecular level, involving both autophagy and apoptosis cells in MCF-7 cells.

2. Material and methods

2.1. Chemicals

Bromelain, propidium iodide (PI), monodansylcadaverine (MDC), 40-6-diamidino-2-phenylindole (DAPI), 3-methyladenine (MA), TAM, SB203580 (p38 inhibitor), SP600125 [c-jun N-terminal kinase (JNK) inhibitor], PD98059 [extracellular signal-regulated kinase 1=2 (ERK1=2) inhibitor], thiazolyl blue (MTT), and b-actin (clone AC-74) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The mouse monoclonal antibodies against mitogen-activated protein kinases (MAPKs): ERK1=2, phospho ERK1=2, p38, phospho p38, JNK, phospho JNK; light chain protein 3B (LC3B), beclin-1, and horseradish peroxidase-conjugated secondary antibody were procured from Cell Signaling Technology (Danvers, MA, USA). Caspase-9 inhibitor: Z-LEHD-FMK was purchased from BD PharmingenTM (BD Biosciences, San Jose, CA, USA). The polyvinylidene fluoride (PVDF) membrane was obtained from Millipore (Bedford, MA, USA). Rest of the chemicals were of analytical grade of purity and procured locally.

2.2. Cell culture

The human breast cancer MCF-7 and MDA-MB-231 cell lines were obtained from National Center for Cell Sciences (Pune, India) and cultured in Minimal Essential Medium with Earl’s salts and Dulbecco’s Modified Eagle’s Medium, respectively, supplemented with 10% heat inactivated fetal bovine serum, 100 lg/mL penicillin streptomycin (all cell culture reagents are from Gibco Invitrogen Corporation, Carlsbad, CA, USA), and maintained in a humidified atmosphere of 95% air and 5% CO2 at 37C in the laboratory. The cell lines had been tested and authenticated by the cell bank before procuring.

2.3. Cell growth inhibition assay

The effect of bromelain on the growth and proliferation of MCF-7 and MDA-MB-231 cells was determined using MTT assay as described earlier [31]. The cells were plated at 1 104 cells per well in 100 lL of complete culture medium and treated with 0–100 lg/mL concentrations of bromelain. Bromelain stock solution freshly prepared at 1 mg/mL concentration was mixed with fresh medium to achieve the desired final concentrations. After incubation for 24, 48, 72, and 96 h at 37C, the effect of bromelain on growth inhibition was assessed as percent activity where vehicle (complete medium) treated cells were taken as 100% active.

2.4. Doses and treatment of cells

The cells (70% confluent) were treated with the selected concentration of bromelain (10 lg/mL) for 96 or 120 h depending on the assay with or without 1-H pretreatment of MA at 2 mM. The treatment time and dose concentration were selected on the basis of the results obtained by MTT assay. Cells that served as control were incubated with the vehicle (medium) alone. Cells treated with 5-lM TAM were used as positive control for studying autophagy. The cells were harvested, washed twice with cold phosphate buffered saline (PBS), and processed as desired, for different assays.

2.5. Observation of fluorescence changes by microscopic analysis

37C for 60 min [32]. For apoptosis observation, DAPI staining was carried out. After treatment with bromelain and/or MA and/or caspase inhibitor, cells were washed and incubated with PBS containing 0.1% triton-X for 10 min on ice. Then they were incubated in 4% PBS buffered paraformaldehyde containing 10 lg/mL DAPI. The fluorescence of the cells’ nuclei was captured at excitation wavelength of 350 nm using Olympus IX51 microscope (Olympus America, Center Valley, PA, USA) equipped with Image Pro Express software as collective system. For necrosis observation, cells treated with bromelain and/or MA and/or caspase inhibitor were fixed in 70% ethanol, stained with PI solution (50 lg/mL) containing RNase of 100 mg/mL for 30 min in dark, and fluorescence was captured at excitation wavelength of 488 nm.

2.6. Flow cytometric quantification of autophagy and cell death

In brief, after stipulated treatment times, cells with or without MA and/or caspase inhibitor were harvested by trypsinization and rinsed with PBS. For measuring autophagic ratio, the cell pellets were suspended with 0.05 mM MDC at 37C for 60 min [32]. Besides, for measuring cell cycle distribution, the collected cells were fixed in 70% ethanol, stained with PI solution (50 lg/mL) containing RNase of 100 mg/mL at 4C for 30 min. Samples from both these assays were analyzed on flow cytometer (Becton Dickinson LSR II, CA, USA) using the CellQuest software to determine the percentage of cells undergoing autophagy/cell death.

2.7. Terminal deoxynucleotidyl transferase biotin dUTP nick end labeling assay

We used Apo-BrdU terminal deoxynucleotidyl transferase biotin dUTP nick end labeling (TUNEL) kit (Molecular Probes, Invitrogen Detection Technologies, Eugene, OR) to visualize apoptosis in MCF-7 cells after 120 h of treatment. Briefly, treated and vehicle control MCF-7 cells (3 104) were fixed with 1% paraformaldehyde in PBS. Next, the cells were washed in wash buffer and labeled with DNA-labeling solution (BrdUTP) for 60 min at 37C. Following incubation, cell samples were rinsed with rinse buffer and incubated with Alexa FluorR 488 dye—labeled anti-BrdU antibody for 30 min in dark. The fluorescence was captured at excitation wavelength of 488 nm.

2.8. Western blot analysis

Cells were preincubated with or without MA, TAM, 1 lM of SB203580, SP600125, and PD98059 for 60 min, then treated with bromelain for 96 h. Western blot was performed as previously described [33]. Briefly, total cell lysates were prepared and protein concentration was estimated by using the method of Lowry et al. [34]. Protein samples (50 lg) were resolved on 8 or 18% sodium dodecyl sulfate (SDS)-polyacrylamide gels followed by electrotransfer onto PVDF membrane. The blots were blocked overnight with 5% nonfat dry milk, probed with respective primary antibodies, and detected by horseradish peroxidase-conjugated anti-mouse/ anti-rabbit IgG using chemiluminescence kit from Millipore and visualized by Versa Doc Imaging System (BioRad Model 4000, Hercules, CA). The intensity of the bands was measured using software UNSCAN-IT automated digital system version 5.1., fold change calculated in terms of relative pixel density for each band normalized to band of b-actin.

2.9. Statistical analysis

All results presented were confirmed in at least three independent experiments. The data were analyzed for mean values and expressed as means 6 SD for all treated and vehicle controls, which were subjected to statistical comparison using SPSS software (version 14). P < 0.05 were considered significant. 3. Results 3.1. Bromelain inhibits proliferation of MCF-7 and MDA-MB-231 cells A dose and time dependent but delayed cytotoxic response was observed in MCF-7 cells exposed to bromelain as compared with MDA-MB-231 cells. In MCF-7 cells, bromelain exerted inhibitory effects on MCF-7 cell growth but only after a time duration of 96 h (Fig. 1). The IC50 was achieved at 60 lg/mL (96 h); therefore, the concentration lower than the IC50 concentration was chosen to study the hypothesized autophagic response preceding the cell death. However, in MDA-MB-231 cells, IC50 was achieved much earlier at 50 lg/mL (72 h; Fig. 1). Autophagic responses were studied in both the cell lines, but due to delayed cytotoxic response to bromelain in MCF-7 cells, mechanism studies were conducted in the latter. 3.2. Bromelain induces autophagy in both MCF-7 and MDA-MB-231 cells Bromelain was speculated to induce autophagy preceding cell death. In cells treated with bromelain, MDC-labeled autophagic vacuoles appeared as distinct dot-like structures distributed in the cytoplasm or in the perinuclear regions whereas in the untreated cells, there was a homogeneous distribution of MDC (Fig. 2A). This was further quantified by estimation of % MDC fluorescent cells using flow cytometry (Fig. 2B). TAM, used as a positive control for autophagy, led to significantly higher autophagic ratio over control (P < 0.05). When the specific autophagic inhibitor MA was added, the autophagic cell number declined, when compared with bromelain alone and TAM alone treatment. 3.3. Bromelain stimulates apoptosis following autophagy in MCF-7 cells A delayed cytotoxic response was observed following bromelain treatment due to which we went on to check the effect of bromelain on cell cycle distribution and cell death after observing its effect on autophagy. On treatment with bromelain, there was an increase in the sub-G1 population (32%) over vehicle control (2%), which on was markedly reduced after pretreatment with MA (17%) as shown in Fig. 3A. Apoptosis and resulting chromatin condensation and DNA fragmentation were confirmed by DAPI staining and TUNEL assay (Figs. 3B and 3C). Similar results were obtained. As observed from the fluorescence microscopy of DAPI-stained cells as shown in Fig. 3B, bromelain induced nuclear cleavage and resulting increased fluorescence. Cell nuclei exhibited apoptotic bodies and nuclear membrane disappeared completely; when MA was introduced, this DNA change was partially recovered, nuclear membrane existed but nuclei displayed slight damage; whereas control and MA alone treated cells showed very few or no apoptotic nucleus. In control cell cultures, majority of the cells showed a homogenous morphology with nuclei lightly and evenly stained by DAPI and less than 2% cells showed apoptotic features. Ten different fields for each treatment were randomly 3.4. Blockade of apoptosis fails to inhibit selected for counting of (i) 300 cells and the percentage of autophagy cells with fragmented nuclei (DAPI stained) was calculated To study the effect of blockade of apoptosis on bromelainand (ii) TUNEL positive cells. induced autophagy, we studied the autophagic levels in presence of caspase-9 inhibitor, as MCF7 cells are caspase-3 deficient. In the presence of Z-LEHD-FMK, majority of the cells underwent autophagy (62%; Fig. 3E) and eventually died in caspase-independent manner shown by PI uptake (>55%) and necrotic morphology showing swelling/ increased diameter (Fig. 3D). Ten different fields for each treatment were randomly selected for counting and the percentage of PI positive cells was calculated.

3.5. Involvement of MAPKs in autophagic process and their effect on LC-3II and beclin-1

The role of MAPKs well known to contribute in proliferation and stress-induced growth inhibition [35] was next studied in bromelain-treated MCF-7 cells. Therefore, expression analyses of phosphorylated ERK1=2, JNK, and p38 MAPKs was studied following bromelain treatment, with or without MA, by flow cytometry. Positive control, TAM augmented the expression levels of phosphorylated p38 and JNK by 3 and 2.4 folds, respectively, while bromelain elevated them by 2.7 and 2.3 folds over vehicle control. However, activation/phosphorylation of ERK1=2 was recorded to be inhibited by TAM and bromelain by approximately 8 and 3 folds over vehicle control. Further, supplementation with MA resulted in inhibition of bromelain-induced p38 and JNK activation by 2.2 and 1.5 folds, respectively, over bromelain alone treatment while it had no influence on bromelain-suppressed ERK1=2 activation (Figs. 4Ai and 4Aii). The total protein expression levels of ERK1=2, JNK, and p38 did not show any alterations on bromelain treatment (data not shown). Altogether, this indicated that in case of suppression of autophagy by MA, the bromelain-induced phosphorylation of JNK and p38 MAPKs was also inhibited. We also studied the autophagic ratios in presence of inhibitors of MAPKs viz. SB600125, SP600125, or PD98059 and found that in presence of SB600125 and SP600125 with bromelain, autophagy decreased; however, addition of PD98059 further augmented the bromelaininduced response (Fig. 4B). Therefore, to elucidate their role, we detected the expression levels of autophagy-related proteins, LC-3II, and beclin-1 in presence of PD98059, SP600125, or SB600125. The expression of both LC-3II and beclin-1 was elevated on bromelain treatment over control levels. Although PD98059 pretreatment further raised the expression levels of these two proteins, SP600125 or SB600125 suppressed them (Fig. 4C).

3.6. Time course of induction of autophagy, apoptosis, and activation of signaling by bromelain

Bromelain-induced autophagy was seen to increase time dependently but was significantly higher at 96 h (P < 0.05; Fig. 5A). Apoptosis induction although increased with time but was seen significantly high only at 120 h (P < 0.05; Fig. 5B). The time course of activation of signaling by bromelain showed that this bromelain-induced activation was not transient but all the three MAPKs were continuously activated; p38 and JNK maximum and ERK1=2 minimum at 96 h (Fig. 5C). 4. Discussion Cell death is long known as a physiological phenomena occurring in multicellular organisms; the term apoptosis being introduced as early as 1972 based on morphological grounds. However, accumulating evidence suggests that programed cell death (PCD) is not confined to apoptosis but that cells use different pathways for active self-destruction as reflected by different morphology: condensation prominent, type I: apoptosis or type II: autophagy prominent, etc. [36]. Here, we report the induction of autophagy by bromelain in both ER positive MCF-7 and negative MDA-MB-231 cells, as shown by punctate staining of autophagolysosomes by MDC as well as increased mean fluorescence intensity (MFI) of autophagic cells (Fig. 2). Vitamin D analog EB1089 has been reported to trigger dramatic lysosomal changes and beclin-1–mediated autophagic cell death in MCF-7 cells [37]. On the same lines, here, induction of beclin-1, which although is monoallelically deleted in MCF-7 cells and elevation of light chain 3 protein B II (LC3BII) was seen in response to bromelain, confirming autophagy (Fig. 4C). As recently pointed out by Zakeri [38], autophagic and apoptotic PCD are not considered as mutually exclusive phenomena. Rather, they appear to reflect a high degree of flexibility in a cell’s response to changes of environmental conditions, either physiological or pathological. Autophagy has been shown to engage in a complex interplay with apoptosis. Sometimes, it can serve as a cell survival pathway, suppressing apoptosis, else, it can lead to death itself, either in collaboration with apoptosis or as a back-up mechanism when the former is defective. The molecular regulators of both pathways are interconnected and both pathways share several genes that are critical for their execution. The crosstalk between apoptosis and autophagy is therefore quite complex, and sometimes contradictory, but surely critical to the overall fate of the cell. Furthermore, it is a key factor in the outcome of death-related pathologies such as cancer, its development, and treatment [39]. Although autophagy has been described as a mechanism of cell death in mammary carcinoma MCF-7 cells by many researchers [24,26,27], caspase-3 deficiency does not discount the occurrence of apoptosis along with it. Apoptosis–autophagy interaction may manifest itself in various ways. Little apoptotic alterations have been observed in TAM-treated MCF-7 cells but an accumulation of autophagic vacuoles was predominantly reported, that is, a subfraction of dying cells showed autophagic cell death with an apoptotic nuclear morphology [40]. Further, the induction of autophagic death of MCF-7 cells by TAM has been unlikely to be due to the lack of caspase-3 [36]. The coexistence of autophagy and apoptosis has also been shown in MCF-7 cells on treatment with camptothecin [41], suggesting a considerable overlap or interdependence of both these programs. Therefore, several pathways resulting in active death may coexist in a given cell type. This notion is supported by our findings showing that autophagic and apoptotic PCD seem to share commonalities. By using MA, the specific inhibitor of autophagy pathway, the apoptotic level was decreased (Figs. 3A– 3C), indicating that autophagy facilitated and was an enhancer of apoptosis and bromelain-induced autophagy was indispensable for apoptosis in these settings. However, it may be speculated that inhibition of autophagy induction delayed the apoptotic response. Cui et al. [21] have also shown that autophagy induction was essential to oridonin-induced apoptosis in MCF-7 cells, indicating that execution of apoptosis is preceded by and even depends on the occurrence of autophagy. Such findings have also been reported by other researchers [42]. Although 3-MA delayed apoptosis, conversely, autophagic activity remained elevated in cells treated with the caspase inhibitor Z-LEHDFMK, which inhibited morphological apoptosis but did not prevent cell death (Figs. 3D and 3E). Therefore, this is indicative that once autophagy gets activated, it may mediate caspase-independent cell death. MAPK family proteins, playing a role in proliferation, differentiation, development, transformation, and apoptosis, are activated by diverse mechanisms. In general, ligand binding of receptors leads to guanosine 50-triphosphate (GTP) loading and activation of the small GTPase Ras, which recruits Raf to the membrane where it is activated. Raf subsequently phosphorylates the dual specificity ERK kinase (MEK1=2), which in turn phosphorylates and thereby activates ERK. ERK is a promiscuous kinase and can phosphorylate/ activate more than 100 different substrates, hence, affecting a broad array of cellular functions including proliferation, survival, apoptosis, motility, transcription, metabolism, and differentiation [35]. Other activated MAPKs translocate to nucleus, change gene expression, and thereby modulate cell survival/death or other pathways. JNK and p38 MAPK are mainly involved in cell death [19]. In liver cells, the p38 MAPK pathway controls the regulation of autophagy by cell volume but ERK1=2 does not seem to be involved in this process [1]. Likewise, here, bromelain-induced autophagy was seen to be regulated by MAPKs. Bromelain treatment increased the phosphorylation of JNK and p38 MAPKs while the phosphorylation of ERK1=2 was seen to be reduced as seen by flow cytometric protein expression analysis (Fig. 4A). Also, in presence of specific MAPK inhibitors, autophagic ratio was significantly modulated (Fig. 4B). These two observations indicated the involvement of MAPKs in bromelain-induced autophagy. Therefore, to determine the role of ERK1=2, p38, and JNK in bromelain-induced autophagy in MCF-7 cells, we evaluated the expression levels of autophagy-related proteins in cells treated with these MAPK inhibitors. The suppressed levels of autophagy markers, LC3BII and beclin-1 by the pretreatment of JNK and p38 inhibitors show that these two MAPKs positively regulate the bromelain-induced autophagy. However, on pretreatment with ERK1=2 inhibitor, autophagy levels were markedly elevated over control level, confirming the negative regulation of bromelain-induced autophagy by ERK1=2 (Fig. 4C). Conclusively, we report autophagy-inducing potential of bromelain in ER positive MCF-7 and ER negative MDA-MB-231 cells and delayed apoptotic cell death response to bromelain due to autophagy induction in MCF-7 cells, which is speculated to be tightly regulated by activation of JNK, p38, and ERK1=2 MAPKs. Also, once activated, autophagy may mediate caspasedependent or independent cell death in these settings. References [1] Pattingre, S., Bauvy, C., and Codogno, P. 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