Introduction
The endoplasmic reticulum (ER) is a dynamic organelle responsible for several specialized functions, such as protein translocation, protein folding and calcium sequestration. The disruption of ER function interferes with protein folding. This can lead to the accumulation of unfolded proteins and the activation of an evolutionarily conserved adaptive response, known as the unfolded protein response (UPR) (1). The UPR in mammalian cells is governed by three transmembrane ER stress sensors, namely PKR-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), which are kept in an inactive state by binding to the ER chaperone, BiP, preventing their oligomerization-induced activation (2). If the UPR fails to counter ER stress, caspase-mediated cell death ensues. Accordingly, an imbalance between cell survival and death decisions in response to ER stress may dictate the occurrence and development of a number of disorders, such as Alzheimer’s and Parkinson’s diseases, as well as diabetes (3).
Tunicamycin (TM) is a typical inducer of ER stress. Both bioenergetic challenge and ER stress have been shown to activate autophagy, a bulk cellular degradation process that may play either a pro- or anti-apoptotic role (4). In the present study, the pathways with which TM interferes in order to activate autophagy were identified, and the role of this process in modulating TM-induced toxicity was evaluated.
It has been found that autophagy is activated upon ER stress and that this serves as a defence mechanism, promoting cell survival (5). Autophagy is an intracellular protein degradation system required for the normal turnover of cellular components and the starvation response. Autophagy can induce the formation of a double-membrane structure known as an autophagosome, which is formed either de novo or from existing membranes and encloses the subcellular components. Upon fusion of the outer membrane of the autophagosome with the lysosomal membrane, the cytoplasm-derived contents and the inner membrane of the autophagosome are degraded. Although the contribution of the endomembrane organelles to autophagy is still under investigation, evidence is emerging that the ER produces the membrane for autophagosome formation and that autophagy is critical to ER homeostasis (6).
3-Methyladenine (3-MA) is a commonly used to inhibit autophagy. It has been reported to inhibit the activity of phosphoinositide 3-kinase (PI3K) and block the formation of pre-autophagosome, autophagosome and autophagic vacuoles (7). Increasing evidence indicates that autophagy is closely associated with tumors. It participates in the early stages of tumor development as a suppressor and acts as a proto-oncogene during the advanced stages (8,9). It is also related to cancer therapy (10). Autophagy has been reported to be increased in response to chemotherapy, which either causes the cancer cell to undergo autophagic cell death, termed programmed cell death (PCD), or mediates its adaptation to drug cytotoxicity, as in apoptosis. The anti- and pro-apoptotic roles of autophagy have prompted scientists to explore the association between autophagy and cancer, which has become a primary focus in cancer research. However, although researchers suggest that autophagy may be a therapeutic target in adjuvant chemotherapy, its exact role and the association between autophagy, autophagic cell death and apoptosis in cancer remains poorly understood and may be more complex than originally consisered (11).
B-cell lymphoma/leukemia 2 (Bcl-2) is an anti-apoptotic protein. It interacts with beclin-1 and downregulates beclin-1-dependent autophagy by preventing the beclin-1/hVps34 complex from forming (12). Class III PI3Ks, such as hVps34, are significant reglators in the initial steps of autophagy (13). In mammals, beclin-1 interacts with UVRAG, Ambra-1 and Bif-1 (also known as endophilin B1) to activate hVps34 and induce autophagy. However, beclin-1 can be present in two different complexes, one that stimulates autophagy and involves an interaction with hVps34, and another that inhibits autophagy and involves an interaction with Bcl-2 and Bcl-xL (14). Accordingly, the overexpression of Bcl-2 and Bcl-xL disrupts the interaction of hVps34 with beclin-1, suggesting that Bcl-2/Bcl-xL inhibit autophagy by sequestering beclin-1 away from hVps34 (15). Beclin-1 has a BH3-only domain that permits the interaction of this protein with the anti-apoptotic proteins, Bcl-2 and Bcl-xL. This interaction abrogates the ability of beclin-1 to induce autophagy (15). Different stimuli, including ER stress, modulate the interaction between beclin-1 and Bcl-2 family members, which is considered an important mechanism in the regulation of autophagy. The genetic ablation of Bax/Bak in mice has been shown to cause an abnormal response to TM-induced ER stress in the liver along with extensive tissue damage, a decreased expression of X-box binding protein 1 (XBP1) and reduced JNK activation (16). Furthermore, the requirement of Bax/Bak proteins for proper IRE1 signaling was confirmed in Bax/Bak double knockout (DKO) mice (16). A previous study by Klee et al showed that the reconstitution of the expression of Bak at the ER membranes in DKO cells is sufficient to re-establish IRE1-tumor necrosis factor receptor-associated factor (TRAF)2 activation and mitochondrial apoptosis instigated by the reticular forms of the BH3-only proteins, Bim and Puma (17).
Of note, as previously demonstrated, the IRE1 pathway activated by reticular BH3-only effectors is atypical as it does not lead to XBP1 splicing, possibly since other arms of the UPR required for the upregulation of the XBP1 mRNA levels, such as ATF6, are not sufficiently activated (17). However, an alternative and intriguing possibility may involve the differential regulation of IRE1 RNase activity (required for XBP1 mRNA splicing) and IRE1-TRAF2 complex formation (required to activate pro-apoptotic JNK signaling) by a different subset of pro-apoptotic proteins at the ER membrane. Clearly, further studies are required to shed more light into the mechanisms regulating IRE1 signal transduction. If Bcl-2 is phosphorylated at the mitotic phase or after microtubule disruption, it does not combine with Bax, but it is not certain that the phosphorylation of Bcl-2 induces cell death (18). Cancer cell apoptosis is a prominent feature of numerous pathological conditions. Multiple cellular signaling pathways, including mitogen-activated protein kinases (MAPKs), are involved in cancer cell apoptosis (19). MAPKs are among the most widespread signaling molecules involved in diverse cellular responses, including proliferation, differentiation, inflammation and apoptosis (20). MAPKs include the protein kinases p38 (p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in the development of cancer (21). All these data unveil an intricate association between autophagy and cancer, as well as ER stress and cancer. In the present study, we aimed to investigate the association between autophagy and ER stress, and to evaluate the effects of TM that target the cross signaling pathways between autophagy and ER stress at a molecular level.
Materials and methods
Cells and culture conditions
MCF-7 and MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection (ATCC; Mannasas, VA, USA) and maintained in DMEM containing 4 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 1.5 g/l sodium bicarbonate and 4.5 g/l glucose with 10% fetal bovine serum (FBS; HyVlone, Logan, UT, USA). Cultures were maintained in 5% CO2 and humidified in a 37°C incubator.
Cell viability assay
The cytotoxic effects of TM and 3-MA on the MCF-7 and MDA-MB-231 cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, as previously described (22). The cells were dispensed in 96-well flat-bottom microtiter plates (NUNC, Roskilde, Denmark) at a density of 1×105 cells/well. After 24 h of incubation, the cells were treated with various concentrations (1.5, 3, 6, 9 and 12 μmol/l) of TM and 3-MA for various periods of time (24, 48, or 72 h). Four hours before the end of the incubation period, 20 ml MTT solution (5.0 mg/l) was added to each well. The resulting crystals were dissolved in DMSO. Cell viability was measured by MTT assay using a plate microreader (Tecan Spectra, Wetzlar, Germany). The relative amount of inhibition of cell growth was calculated as follows: cell viability (%) = (Asample − Ablank)/(Acontrol − Ablank) ×100.
Propidium iodide (PI) uptake assay
Tumor cells were seeded at 1×105 cells/well in 24-well plates and allowed to reach the exponential growth phase for 24 h prior to treatment. The PI uptake assay was performed as previously described (22). Briefly, adherent cells and non-adherent cells were collected and washed with ice-cold PBS. The cells were then stained with 750 ml PI at 1×106/ml. Following overnight incubation at 4°C, the cells were analyzed by flow cytometry. The cells were divided into 4 groups: control, no treatment; 3-MA, treated with 2 mmol/l 3-MA; TM, treated with 3 μmol/l TM; and combination treatment, treated with both TM and 3-MA.
Hoechst 33258 staining
Hoechst 33258 assay was performed to measure apoptosis. The breast cancer cells were grown in 24 microwell plates and treated as described above. Apoptotic cells were detected by Hoechst 33258 staining following the manufacturer’s instructions (Apoptosis Hoechst staining kit; Beyotime Biotechnology, Jiangsu, China). Briefly, the cells were first fixed in 0.5 ml methanol for 30 min and then rinsed with PBS twice; 1 mg/ml Hoechst 33258 reagent was used to stain the apoptotic cells in the dark at room temperature for 5 min, after which the cells were again washed with PBS twice. The stained cells were examined and immediately photographed under a fluorescence microscope (Olympus, Tokyo, Japan) at an excitation wavelength of 330–380 nm. Apoptotic cells were identified on the basis of morphological changes in their nuclear assembly by observing chromatin condensation and fragmentation by Hoechst 33258 staining.
Colony formation assay
For this assay, the cells were seeded at 500 cells/well in 6-well plates and allowed to attach overnight. The cells were then treated with TM with or without 3-MA under standard cell culture conditions at 37°C and 5% CO2 in a humidified environment. After 5 days, the dishes were washed in PBS twice, fixed with methanol, stained with crystal violet (Fisher Scientific, Pittsburgh, PA, USA), washed with water and air dried. The number of colonies was determined by imaging with a Multimage™ Cabinet (Alpha Innotech Corp., San Leandro, CA, USA) and AlphaEase Fc software. Plating efficiency (PE, %) = number of colonies formed/number of cells plated ×100.
Western blot analysis
The concentrations of the samples were determined. They were then loaded onto a 10–15% sodium dodecyl sulfate polyacrylamide gel for electrophoresis (SDS-PAGE) and resolved at 70 V and then 120 V. The methanol-pre-treated polyvinylidene difluoride (PVDF) membrane was then soaked in transfer buffer (pH 8.3, 25 mmol/l Tris-HCl, 192 mmol/l glycine, 20% methanol) for 1 min. Proteins on the SDS-PAGE gel were then transferred to the PVDF membrane under 100 V for 120 min. The membrane was blocked overnight with 5% FBS/PBS at 4°C. Primary antibodies were diluted at 1:500 and incubated with a membrane for 2 h at room temperature. The membrane was then washed 3 times for 10 min each with PBS containing 0.05% Tween-20. Goat anti-mouse immunoglobulin G secondary antibody (1:2,000) was added and incubated with the membrane for 3 h at room temperature. The membrane was then washed 3 times using the same washing solution. The membrane was then developed for 1 min using an enhanced chemiluminescence kit with equal volumes of A and B solution, as previously described (23). After imaging, Image J version 1.44 software (Bio-Rad Laboratories, Hercules, CA, USA) was used to analyze the average density values.
Statistical analysis
All experiments described were performed at least in triplicate. Data are expressed as the means ± SEM. All statistical analyses were performed using a two-tailed paired Student’s t-test. P-values <0.05 were considered to indicate statistically significant differences.
Results
Human breast cancer cells show no apparent sensitivity to TM
The effects of TM on the viability of the breast cancer cell lines, MCF-7 and MDA-MB-231, were examined. The proliferation of breast cancer cells was found to be inhibited by relatively low concentrations of TM (Fig. 1). This inhibition became more pronounced with the increasing duration of exposure duration of exposure and the increasing TM concentrations. Following treatment with 12 μmol/l of TM for 24, 48 and 72 h, the survival rate of the MCF-7 cells dropped to 85.63, 83.59 and 79.39%, while the survival rate of the MDA-MB-231 cells was 85.12, 83.13 and 75.46%, respectively (Fig. 1), which was not statistically significant compared to the negative control (untreated) group. Following treatment with 12 mmol/l of TM for 24, 48 and 72 h, the survival rate of the MCF-7 cells dropped to 89.83, 88.50 and 80.43%, and the survival rate of the MDA-MB-231 cells dropped to 91.83, 83.96 and 82.48%, respectively (Fig. 1), which was not statistically significant compared to the negative control group.
Autophagy is induced by ER stress in breast cancer cells
TM inhibits N glycosylation and brefeldin A (BFA), which blocks protein transportation to the Golgi. Thus, TM induces ER stress (23). We wished to determine whether TM induces autophagy. To monitor the induction of ER stress, the expression of GRP78 and IRE1 was measured in the breast cancer cells following treatment with TM by western blot analysis (Fig. 2A). GRP78 is an indicator of ER stress and IRE1 is one of the 3 transmembrane ER stress sensors which are kept in an inactive state by binding to the ER chaperone, GRP78, thus preventing their oligomerization-induced activation. To distinguish the specific inhibition of mTOR-mediated cell proliferation from autophagy, the expression of the proteins associated with autophagy, LC3, beclin-1 and p62, was measured following treatment with TM. As shown in Fig. 2B, the expression of LC3 in the breast cancer cells was activated by TM treatment, and western blot analysis was used to detect the protein levels of LC3-I and LC3-II. The results revealed that the levels of LC3, particularly those of LC3-II, increased, leading to an increased ratio of LC3-II / LC3-I following treatment with TM. The expression of beclin-1 increased, whereas the expression of p62 increased at during the early stages of treatment, but then subsequently decreased.
3-MA inhibits autophagy induced by TM in breast cancer cells
3-MA is a common inhibitor of autophagy. We wished to determine whether 3-MA inhibits TM-induced autophagy. Beclin-1, p62 and LC3-II are markers of autophagy. As shown in Fig. 3, the expression of LC3-II, p62 and beclin-1 in the breast cancer cells was increased following treatment with TM; the results from western blot analysis confirmed that 3-MA suppressed the levels of LC3, p62 and beclin-1 induced by TM; in particular, the levels of LC3-II showed the greatest decrease, leading to a decreased ratio of LC3-II/LC3-I following treatment with TM.
3-MA promotes TM-induced cell death in breast cancer cells
The sensitivity of human breast cancer cells to the TM-induced inhibition of proliferation was not apparent. We therefore wished to determine whether 3-MA affects the viability of breast cancer cells. The results from MTT assay revealed that 3-MA induced cell death; however, these effects were not particularly prominent (Fig. 4A). In order to demonstrate that autophagy plays a decisive role in the promotion of cell death under ER stress conditions, MTT assay and colony formation assay were performed to determine whether 3-MA affects the viability of breast cancer cells treated with TM. In the MCF-7 and MDA-MB-231 cells, TM (0–12 μmol/l) produced a dose- and time-dependent reduction in cell growth. In the MCF-7 cells, the survival rate dropped to 83.59% after 48 h of treatment with 12 μmol/l TM (Fig. 1) and 90.09% with 2 mmol/l 3-MA treatment for 48 h (Fig. 4A). In the MDA-MB-231 cells, the survival rated dropped to 83.13% after 48 h of treatment with 12 μmol/l TM (Fig. 1) and 92.01% after 48 h of treatment with 2 mmol/l 3-MA (Fig. 4A). Subsequently, changes in MCF-7 and MDA-MB-231 cell viability were examined using TM alone or in combination with 3-MA. In the group treated with both TM and 3-MA, the viability of the breast cancer cells decreased more rapidly compared with the TM group. In the MCF-7 cells, combination treatment induced 57% cell death, i.e., a 31.21% increase over the rate of cell death in the TM group. In the MDA-MB-231 cells, combination treatment resulted in a cell death of 52.23%, i.e., an increase of approximately 30.42% compared with the TM group (Fig. 4A). Although the cell viability in the 3-MA group also decreased in both cell lines, the effects were not significant. These results suggest that 3-MA increases the rate of TM-induced cell death in breast cancer cells. Colony formation assays were also used to confirm that 3-MA enhances the sensitivity of human breast cancer cells to TM. Both cell lines formed fewer colonies when treated with 3-MA in combination with TM compared to treatment with TM alone (Fig. 4B).
Inhibition of autophagy facilitates TM-induced apoptosis
TM at 3 μmol/l was found to induce both autophagy and apoptosis simultaneously. 3-MA, a specific inhibitor of autophagy, potently suppressed TM-induced autophagy (Fig. 4C). When autophagy was suppressed by 3-MA in the TM-treated breast cancer cells, the cells were collected for staining with PI. Sub-G1 cell populations were counted using FACS. The results revealed that the rate of apoptosis in the MCF-7 cells that had been treated with 2 mmol/l of 3-MA, 3 μmol/l of TM, or 3 μmol/l of TM combined with 2 mmol/l of 3-MA for 48 h reached 12.4% (Fig. 4C-b), 16.3% (Fig. 4C-c) and 45.7% (Fig. 4C-d), respectively. The rate of apoptosis of the MDA-MB-231 cells treated with 2 mmol/l 3-MA, 3 μmol/l TM, or 3 μmol/l TM combined with 2 mmol/l 3-MA for 48 h reached 13.3% (Fig. 4C-b), 18.5% (Fig. 4C-c) and 43.9% (Fig. 4C-d), respectively, wheras the rate of apoptosis among the untreated breast cancer cells (MCF-7 and MDA-MB-231) was only 1.6% (Fig. 4C-a) and 1.8% (Fig. 4C-a), respectively. In this way, the rate of apoptosis among the breast cancer cells (MCF-7 and MDA-MB-231) treated with TM in combination with 3-MA was found to be significantly higher than among the cells treated with TM alone (Fig. 4D). To confirm that the inhibition of autophagy facilitates the apoptosis induced by TM, Hoechst 33258 (Fig. 4E) was used to observe nuclear fragmentation, one of the morphological hallmarks of apoptosis, as well as typical morphological changes associated with apoptosis, such as chromatin condensation. As shown in Fig. 4E, the cells treated with TM did not show appreciably higher levels of apoptosis than the other cells; however, the formation of apoptotic bodies and DNA fragmentation were apparent in the cells treated with 3-MA in combination with TM. By contrast, the cells in the control group did not show any abnormal morphology.
Role of the activation of the IRE1/JNK/beclin-1 pathway in the TM-induced apoptosis of breast cancer cells
The activation of ERK1/2 and c-Jun N-terminal kinase (JNK) was determined by western blot analysis. The cells were treated with 3 μmol/l of TM for different periods of time (0–6 h); the levels of phosphorylated and total protein of each MAPK were then detected (Fig. 5A). The levels of ERK1/2 increased at 1 h and reached maximal levels at 6 h. The phosphorylation of ERK1/2 was detected in the lysate of untreated breast cancer cells. The amount of phosphorylated ERK1/2 protein increased after 2 h and declined thereafter. We performed time-kinetic experiments in which phosphorylated and total JNK were analyzed by western blot analysis using specific antibodies. We then detected the levels of phospho-JNK and total JNK by western blot analysis using specific antibodies(Fig. 5A). The levels of phosphorylated JNK protein were detected in the lysate of untreated breast cancer cells. The levels of phosphorylated JNK protein increased after 1 h and declined thereafter. When probed with antibodies against total JNK, in comparison with that observed in the lysate of the untreated cells, the band showed change at the investigated time points of ER stress, which increased after 3 h, and then declined after 6 h. Thus, ER stressors upregulate and activate JNK proteins in breast cancer cells. However, as shown in Fig. 5B, 2 mmol/l of 3-MA reversed this effect. These results indicate that the activation of each MAPK is differentially regulated by TM and 3-MA.
Inhibition of autophagy by ER-localized Bcl-2
Bcl-2 family proteins are known to be closely related to apoptosis (24). Increasing evidence indicates that Bcl-2 family proteins are also related to autophagy (25). In this study, western blot analysis indicated that, in both types of breast cancer cells, the levels of the pro-apoptotic proteins, Bax and Bad, were higher in the cells treated with 3 μmol/l of TM for 24 h (Fig. 6) than in the untreated cells. More importantly, 2 mmol/l of 3-MA was found to enhance this effect. No significant changes in the expression of Bax and Bad were observed in the cells treated with 2 mmol/l of 3-MA for 24 h compared to the control (untreated) group. The levels of the anti-apoptotic proteins, Bcl-xL and Bcl-2, were higher in the cells treated with 3 μmol/l of TM for 24 h than in the untreated cells. 3-MA diminished this TM-induced increase in the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xl. Following treatment with 3-MA, the levels of Bcl-2/Bcl-xl in the MCF-7 cells and in the MDA-MB-231 cells decreased (Fig. 6A). Beclin-1 (also known as Atg6) was the first mammalian autophagy gene product to be identified (26). Beclin-1 is a haploinsufficient tumor suppressor. It was originally isolated as a Bcl-2-interacting protein. Bcl-2 has been shown to negatively regulate beclin-1-dependent autophagy through direct binding (15). Phosphorylation by JNK in turn activates Bim, while inhibiting Bcl-2 functions (27). In our study, western blot analysis revealed that, in both types of breast cancer cells, the levels of the anti-apoptotic proteins, Bcl-2/Bcl-xl, as well as those of IRE1, were increased following treament with TM. 3-MA diminished the increase in the levels of beclin-1 that had been induced by TM, but not those of IRE1 (Fig. 6B). The protein levels of beclin-1 increased in the cells treated with 3 μmol/l TM for 24 h. The results from western blot analysis revealed that LC3 was converted from LC3-I to LC3-II in the cells treated with 3 μmol/l TM for 24 h (Fig. 6B). More importantly, treatment with 2 mmol/l 3-MA reversed this effect.
Discussion
ER stress and autophagy are closely linked; both processes are characterized by opposing mechanisms, in that they either protect cells from death or they can trigger cell death. Under certain stress conditions, these processes play a protective role, such as the maintenance of a steady state of biosynthesis and the normal function of cells. However, when the damage is severe, these processes play a positive role by promoting apoptosis and initiating cell death (28).
In mammalian cells, UPR signaling is more complex than yeast (29). The UPR in mammalian cells is governed by 3 transmembrane ER stress sensors, namely PERK, IRE1 and ATF6. In addition to the UPR, ER stress releases Ca2+ from the ER, and the concentration of free Ca2+ in the cytoplasm increases. Depending on the state of the cell and the type of ER stress encountered, the outcome can be an increase in the capacity of the ER folding machinery, a reduction in the amount of protein centered in the ER, and the enhanced clearance of proteins from the ER, apoptosis or autophagy (30).
Therefore, in this study, by treating breast cancer cells with TM, we examined the role of the ER stress and autophagy in cell death induced by TM. We new insight into the mechanisms of action of autophagy and ER stress. We observed that both types of breast cancer cells were insensitive to TM at relatively low concentrations. We detected the expression of signature proteins of ER stress and autophagy in the breast cancer cells by western blot analysis; the results revealed that, in both types of breast cancer cells, TM induced the expression of LC3-II, beclin-1, p62, GRP78 and IRE1. Therefore, TM induced autophagy in both types of breast cancer cells; this protected the cell, making them insensitive to TM. 3-MA is a common inhibitor of autophagy. The results from western blot analysis confirmed that 3-MA suppressed the TM-induced levels of LC3, p62 and beclin-1, particularly those of LC3-II, leading to a decreased ratio of LC3-II / LC3-I following treatment with TM. Further experiments (staining with PI and Hoechst 33258 and colony formation) revealed that the inhibition of autophagy facilitated TM-induced apoptosis.
In mammalian cells, Ca2+ in the cytoplasm participates in the regulation of autophagy as a coordinator, which is induced by ER stress (31). As previously demonstrated, the regulation of autophagy signaling pathways induced by ER stress, hunger and viral infection is dependent on eIF2 alpha. However, the mechanism through which eIF2 regulates autophagy remains unknown, although ATF4-induced Atg12 expression seems to be involved in this process (32). On the contrary, Imaizumi (33) found that IRE1, rather than PERK, connects UPR to autophagy. It has been shown that in IRE1a- or ATF6-deficient mouse embryonic fibroblasts (MEFs) and PERK-deficient embryonic stem cells, the accumulation of LC3-positive vesicles triggered by TM or thapsigargin (an inhibitor of ER Ca2+ ATPase) fully depends on IRE1, rather than on PERK or ATF6 (34). As shown in a previous study, the thapsigargin-induced accumulation of LC3-positive vesicles was completely inhibited in MEFs deficient in TRAF, a cytosolic adaptor molecule that links active IRE1 to the activation of c-Jun N-terminal kinase (JNK) (35). Finally, a pharmacological inhibitor of JNK effectively inhibited the LC3 translocation in this model system, suggesting that the IRE1-TRAF2-JNK pathway is essential for the induction of autophagy in MEFs challenged with ER stressors. Of note, JNK has also been suggested as a mediator of autophagy induced by caspase inhibition and growth factor deprivation in fibrosarcoma cells and CD4+ T cells, respectively (35). This special mechanism (autophagy), which mainly uses the products from decomposed cells to produce energy, a process of self digestion, increases cell survival in order to protect cells if they do not have a sufficient supply of nutrients and oxygen (36). In the process of autophagy, the number of organelles decreases, which affects cell shrinkage. It has been found that, when autophagy reaches a certain threshold and nutrients are once again supplied to the cells, the cells are able to fully recover (37). Therefore, insufficient autophagy supports cells survival. On the other hand, if autophagy is out of balance, it will reach an irreversible point, and cell death will be triggered. This is a hotspot for current research as the process of the regulatory mechanism has not been fully elucidated.
In the present study, we demonstrated that both types of breast cancer cell lines (MCF-7 and MDA-MB-231) were insensitive to TM at relatively low concentrations. Western blot analysis showed that, in both types of breast cancer cells, TM induced the expression of IRE1, p-JNK, JNK, ERK1/2 and p-ERK1/2. However, 3-MA did not inhibit the expression of IRE1, whereas the expression of p-JNK, JNK, ERK1/2 and p-ERK1/2 was inhibited.
ERK-dependent autophagy plays an important role in neuronal cell death. By contrast, ERK activation causes disturbance in the fusion between autophagosomes and lysosomes and ultimately results in the inhibition of cell death by autophagy (38). The regulation of autophagy by ERK was further confirmed with the use of the PI3K inhibitor, 3-MA. 3-MA upregulated LC3-II accompanied by ERK activation, indicating that ERK may be involved in autophagy. However, we did not determine at which stage in the autophagy process the ERK signaling pathway is activated. Our findings indicate that the effect was associated with a high sensitivity to ER stress-mediated apoptosis and autophagy in breast cancer cells.
Of note, it has been suggested that JNK regulates autophagy through Bcl-2 phosphorylation, which prevents this protein from interacting with (and inhibiting) the essential regulator of autophagy, beclin-1 (39). In addition, JNK has been shown to control beclin-1 expression and thus regulate ceramide-induced autophagy (40). It is therefore possible that the activation of the IRE1/TRAF2/JNK arm of ER stress regulates autophagy through the modulation of beclin-1 function and expression.
Beclin-1 (also known as Atg6) was the first mammalian autophagy gene product to be identified. Beclin-1 is a haploinsufficient tumor suppressor that was originally isolated as a Bcl-2-interacting protein (41). Bcl-2 has been shown to negatively regulate beclin-1-dependent autophagy through direct binding (41). Beclin-1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type beclin-1. This regulatory activity of Bcl-2 in autophagy was found to be specifically attributable to the expression of this protein at the ER membrane (42), suggesting that signaling events originating from the ER may play an important role in the process.
In a previous study, Wei et al found that, upon nutrient withdrawal, JNK1 was activated and induced phosphorylation at multiple residues (Thr69, Ser70 and Ser87) in the non-structured loop of Bcl-2, located between the BH4 and BH3 domains (43). Autophagy and apoptosis are fundamental cellular pathways and both are regulated by JNK-mediated Bcl-2 phosphorylation (27). Wei et al found that, during nutrient starvation in HeLa cells, rapid Bcl-2 phosphorylation may initially occur to promote cell survival by disrupting the Bcl-2-beclin-1 complex, thus inducing autophagy (4 h) (43). After 16 h, when autophagy was no longer able to keep the cells alive, Bcl-2 phosphorylation disrupted the Bcl-2-Bax complex and activate the caspase 3-dependent pathway. This model can be used to understand the interrelationship between autophagy and apoptosis through JNK1-mediated Bcl-2 phosphorylation (44). Thus, Bcl-2 phosphorylation may not only be a mechanism for regulating autophagy and apoptosis, but also a mechanism for regulating the switch between the two pathways.
In this study, the results from MTT and colony formation assays indicated that cell proliferation was inhibited by exposure to TM. 3-MA combined with TM reduced cell viability more profoundly than either agent alone. These data were further supported by the results of colony formation assay. PI staining assay revealed that 3-MA promoted apoptosis in the TM-treated breast cancer cells. These data were further supported by Hoechst 33258 staining.
Subsequently, the molecular changes that follow TM treatment were observed using an immunoblot assay. Beclin-1 protein levels increased following treatment with TM. The results from western blot analysis revealed that LC3 was converted from LC3-I to LC3-II following treatment with TM. More importantly, treatment with 3-MA reversed these effects. Western blot analysis showed that, in both types of breast cancer cells, the level of the pro-apoptotic proteins, Bax and Bad, were higher in the cells treated with TM. 3-MA enhanced this effect. No significant differences in Bax and Bad expression were observed between the cells treated with 3-MA and the control group. The levels of the anti-apoptotic proteins, Bcl-2/Bcl-xL, increased in the cells treated with TM, but 3-MA diminished this increase. Following treatment with 3-MA alone, the levels of Bcl-2/Bcl-xL decreased.
These results suggest that autophagy is a protective response to apoptosis induced by TM. TM-induced apoptosis in breast cancer cells was found to be enhanced by 3-MA, an inhibitor of autophagy, through the downregulation of Bcl-2/Bcl-xL protein expresion. Our results indicate that the differential responses to TM are caused by the extent of the UPR and autophagy, both of which are regulated by the level of JNK and ERK activation.