γ-Tocotrienol-induced endoplasmic reticulum stress and autophagy act concurrently to promote breast cancer cell death.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 1

ARTICLE

Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 04/30/15 For personal use only.

␥-Tocotrienol-induced endoplasmic reticulum stress and autophagy act concurrently to promote breast cancer cell death Roshan V. Tiwari, Parash Parajuli, and Paul W. Sylvester

Abstract: The anticancer effects of ␥-tocotrienol are associated with the induction of autophagy and endoplasmic reticulum (ER) stress-mediated apoptosis, but a direct relationship between these events has not been established. Treatment with 40 ␮mol/L of ␥-tocotrienol caused a time-dependent decrease in cancer cell viability that corresponds to a concurrent increase in autophagic and endoplasmic reticulum (ER) stress markers in MCF-7 and MDA-MB-231 human breast cancer cells. ␥-Tocotrienol treatment was found to cause a time-dependent increase in early phase (Beclin-1, LC3B-II) and late phase (LAMP-1 and cathepsin-D) autophagy markers, and pretreatment with autophagy inhibitors Beclin-1 siRNA, 3-MA or Baf1 blocked these effects. Furthermore, blockage of ␥-tocotrienol-induced autophagy with Beclin-1 siRNA, 3-MA, or Baf1 induced a modest, but significant, reduction in ␥-tocotrienol-induced cytotoxicity. ␥-Tocotrienol treatment was also found to cause a decrease in mitogenic Erk1/2 signaling, an increase in stress-dependent p38 and JNK1/2 signaling, as well as an increase in ER stress apoptotic markers, including phospho-PERK, phospho-eIF2␣, Bip, IRE1␣, ATF-4, CHOP, and TRB3. In summary, these finding demonstrate that ␥-tocotrienol-induced ER stress and autophagy occur concurrently, and together act to promote human breast cancer cell death. Key words: ␥-tocotrienol, autophagy, breast cancer, endoplasmic reticulum stress, apoptosis. Résumé : Les effets anticancéreux du ␥-tocotriénol sont associés a` l’induction de l’autophagie et de l’apoptose médiée par le stress du réticulum endoplasmique (RE), mais le lien direct entre ces événements n’a pas été établi. Un traitement avec 40 ␮M de ␥-tocotriénol provoquait une diminution de la viabilité des cellules cancéreuses en fonction du temps qui correspond a` un accroissement concurrent des marqueurs de l’autophagie et du stress du réticulum endoplasmique (RE) chez les cellules humaines de cancer du sein MCF-7 et MDA-MB-231. Le traitement ␥-tocotriénol s’avérait provoquer un accroissement, en fonction du temps, des marqueurs précoces (Beclin-1, LC3B-II) et tardifs (LAMP-1 et cathepsine-d) de l’autophagie, et un prétraitement avec des inhibiteurs de l’autophagie comme un ARNic de Beclin-1, le 3-MA ou Baf-1 bloquait ces effets. De plus, le blocage de l’autophagie induite par le ␥-tocotriénol a` l’aide d’un ARNic de Beclin-1, de 3-MA ou de Baf-1 induisait une réduction modeste mais significative de la cytotoxicité induite par le ␥-tocotriénol. Le traitement au ␥-tocotriénol s’avérait aussi provoquer une diminution de la signalisation mitogène de Erk1/2, un accroissement de la signalisation dépendante au stress de p38 et JNK1/2, de même qu’un accroissement des marqueurs du stress apoptotique du RE incluant les phospho-PERK, phospho-eIF2␣, Bip, IRE1␣, ATF-4, CHOP et TRB3. En résumé, ces données démontrent que le stress du RE et l’autophagie induits par le ␥-tocotriénol surviennent de manière concurrente et agissent ensemble pour favoriser la mort des cellules humaines du cancer du sein. [Traduit par la Rédaction] Mots-clés : ␥-tocotriénol, autophagie, cancer du sein, stress du réticulum endoplasmique, apoptose.

Introduction Autophagy is commonly referred to as intracellular “selfeating” by which damaged cytoplasmic organelles or protein aggregates are packaged into membrane-bound autophagosomes that fuse with lysosomes to form autolysosomes, which then act to degrade and recycle their content (Chen et al. 2010; Klionsky 2005; Mathew et al. 2007; Rabinowitz and White 2010; Rikiishi 2012). However, excessive formation of autophagic vesicles can interfere with normal cellular function and lead to autophagy promotion of programmed cell death (Chen et al. 2010; Klionsky 2005). The exact role of autophagy in the etiology of cancer is complex and not completely understood. Experimental evidence has shown that autophagy can play a role in both the promotion of cancer cell survival and conversely, the promotion of cancer cell death (Shintani and Klionsky 2004; White and DiPaola 2009). The initiation of autophagy and the formation of autophagosomes is mediated by a group of autophagy-related proteins that

include Atg family member proteins, Beclin-1, class III phosphatidylinositol 3-kinase, LC3B-I, and LC3B-II (Klionsky et al. 2012; Rabinowitz and White 2010; Tassa et al. 2003). Elevations in the cellular expression of Beclin-1, the Atg5-Atg12 complex, and the conversion of LC3B-I to LC3B-II are hallmarks of autophagy (Baehrecke 2005; Ghavami et al. 2012; Kabeya et al. 2000; Klionsky et al. 2012; Tanida et al. 2004). The endoplasmic reticulum (ER) is an intracellular organelle that is involved in protein synthesis, but during times of stress the ER plays an important role in programmed cell death (Wali et al. 2009). ER stress-mediated apoptosis is associated with an increase expression of several proteins including protein kinase-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF-6), and inositol requiring kinase 1 (IRE1) (Han et al. 2013; Schroder and Kaufman 2005; Yoshida 2007). In addition, other proteins play a role in the initiation of ER stress-mediated apoptosis, including phosphorylated eukaryotic translational initiation

Received 4 September 2014. Revision received 22 February 2015. Accepted 6 March 2015. R.V. Tiwari, P. Parajuli, and P.W. Sylvester. School of Pharmacy, University of Louisiana at Monroe, 700 University Avenue, Monroe LA 71209, USA. Corresponding author: Paul W. Sylvester (e-mail: [email protected]). Biochem. Cell Biol. 93: 1–15 (2015) dx.doi.org/10.1139/bcb-2014-0123

Published at www.nrcresearchpress.com/bcb on 12 March 2015.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)


2

factor 2 (eIF2␣), C/EBP homology protein (CHOP), tribbles 3 (TRB3), and ATF-4, and increased expression of these proteins are the hallmarks of ER stress-mediated apoptosis (Lu et al. 2004; Ohoka et al. 2005; Siu et al. 2002; Su and Kilberg 2008; Wali et al. 2009; Xu et al. 2005). Previous studies have demonstrated that tocotrienols, members of the vitamin E family of compounds, display potent anticancer activity at treatment doses that have little or no effects on normal cell function or viability (McIntyre et al. 2000a). Studies have also shown that the anticancer effects of tocotrienols are associated with the induction of autophagy and ER stressmediated apoptosis (Jiang et al. 2012; Robison et al. 1998; Singh et al. 2012; Tiwari et al. 2014; Wali et al. 2009). However, a direct causal relationship between tocotrienol-induced autophagy and ER stress has not been established. Therefore, studies were conducted to characterize the interrelationship between ␥-tocotrienolinduced cytotoxicity, autophagy, and ER stress-mediated apoptosis in human MCF-7 and MDA-MB-231 breast cancer cells.

Materials and methods Reagents and antibodies All reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA), unless otherwise stated. Purified ␥-tocotrienol (>98% purity) was generously provided by First Tech International Ltd. (Hong Kong). The pan caspase inhibitor, zVADfmk, was purchased from R&D Systems (Minneapolis, Minnesota, USA). Antibodies for LC3BI/II, Beclin-1, Bcl-2, Bax, cleaved caspase-3, cleavedPARP, Atg12, p-p38, p-Erk1/2, p-JNK1/2, Bip, p-PERK, and p-eIF2␣ were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Erk-1, Erk-2, JNK, p38, LAMP-1, IRE1␣, TRB3, Scrambled siRNA, BECN1, siRNA and transfecting media were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). Antibodies for cathepsin-D, CHOP, and ATF4 were purchased from GeneTex, Inc. (Irvine, California, USA). Antibody for ␣-tubulin was purchased from Calbiochem (San Diego, California, USA). Goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from PerkinElmer Biosciences (Boston, Massachusetts, USA). Cell lines and culture conditions The estrogen-receptor negative MDA-MB-231, estrogen-receptor positive MCF-7 breast carcinoma cell lines, and the immortalized “normal” human MCF-10A mammary epithelial cell line were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA). MDA-MB-231 and MCF-7 breast cancer cells were cultured in modified Dulbecco’s Modified Eagle Medium (DMEM)/ F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 ␮g/mL insulin. The MCF10A cells were maintained in DMEM/F12 supplemented with 5% horse serum, 0.5 ␮g/mL hydrocortisone, 20 ng/mL EGF, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 ␮g/mL insulin. All cells were maintained at 37 °C in an environment of 95% air and 5% CO2 in a humidified incubator. For subculturing, cells were rinsed twice with sterile Ca2+- and Mg2+-free phosphate-buffered saline (PBS) and incubated in 0.05% trypsin containing 0.025% EDTA in PBS for 5 min at 37 °C. The released cells were centrifuged, resuspended in serum containing media, and counted using a hemocytometer. Experimental treatment To dissolve the highly lipophilic ␥-tocotrienol in aqueous culture media, a stock solution of ␥-tocotrienol was prepared by suspending it in a solution of sterile 10% BSA as described previously (McIntyre et al. 2000b; Tiwari et al. 2014; Wali et al. 2009). A stock solution of 3-methyladenine (3-MA) and bafilomycin A1 (Baf1) was prepared in DMSO, and was used to prepare various concentrations of treatment media. As required for individual experiments,

Biochem. Cell Biol. Vol. 93, 2015

ethanol and (or) DMSO was added to all treatment groups, such that the final concentration of these solvents never exceeded 0.1%. Measurement of viable cell number For cell viability studies, MCF-7, MDA-MB-231, and MCF-10A cells were initially seeded at a density of 1 × 104 cells/well in 96-well culture plates (6 wells/group) and maintained in control medium. After a 3 day culture period (approximately 70% confluency), cells were then divided into different treatment groups (6 wells/group) and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. Afterwards, viable cell number was determined using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, as described previously (Han et al. 2013; Wali et al. 2009). The optical density of each sample was measured at 570 nm on a microplate reader (SpectraCount, Packard BioScience Company, Meriden, Connecticut, USA) zeroed against a blank prepared from cell-free medium. Numbers of cells/well were calculated against a standard curve prepared by plating known cell densities, as determined by hemocytometer, in triplicate at the beginning of each experiment. Photomicrographs of cellular morphology MCF-7 and MDA-MB-231 human breast cancer cells were plated at an initial density of 7 × 105 cells/well in 6-well culture plates and maintained on control media for 3 days (approximately 70% confluency). Afterwards, cells were divided into vehicle-treated control and 40 ␮mol/L ␥-tocotrienol-treated groups, and returned to the incubator for a 24 h culture period. Afterwards, morphological changes in the different treatment groups was examined using a Nikon Eclipse TE2000-U phase-contrast microscope (Nikon Instruments Inc., Melville, NY, USA) at 100× magnification. Following brightfield examination, cells were then fixed with 100% methanol and stained with Giemsa for 10 min, washed under tap water, and then again examined with the phase-contrast microscope for the detection of autophagic vacuoles in the cells at 200× magnification. Detection of autophagic vacuoles with monodansylcadaverine It is well-established that the accumulation of autofluorescent monodansylcadaverine (MDC) in mature autophagic vacuoles (autolysosomes) is a specific marker for autophagy (Biederbick et al. 1995; Munafo and Colombo 2001). However, recent studies indicate that MDC is also a marker of earlier autophagic compartments, and the accumulation of MDC-positive vesicles is directly related to autophagy induction, both in cultured cells and in animal models (Iwai-Kanai et al. 2008; Munafo and Colombo 2001). MCF-7, MDA-MB-231, and MCF-10A cells initially plated at a density of 7 × 105 cells/well in 6-well culture plates and maintained on control media for 3 days (approximately 70% confluency). Cells were then divided into different treatment groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. The next day, all cells were treated with 0.05 mmol/L MDC and 0.2 ␮mol/L ethidium bromide at 37 °C for 15 min (Biederbick et al. 1995). Cells were then washed 5 times with PBS and lysed in 500 ␮L 10 mmol/L Tris-HCl buffer, pH 8, containing 0.1% triton X-100 (Munafo and Colombo 2001). Intracellular MDC accumulation in each well was measured using a fluorescence plate reader (BioTek FLx800, Winooski, Vermont, USA) with excitation wavelength of 360 nm and 525 nm emission filter. Total DNA content per well was determined with 530 nm excitation wavelength and 590 nm emission filter. Normalization of MDC fluorescence intensity was performed by dividing MDC fluorescence by total DNA for each well. MDC fluorescence/DNA was expressed as specific activity (arbitrary units)/well. Published by NRC Research Press



Tiwari et al.

Visualization of MDC-labeled vacuoles MCF-7 and MDA-MB-231 cells were initially plated at a density of 5 × 104 cells/chamber in 8-chamber glass culture slides (BD Falcon, USA) and maintained on control media for a 48 h culture period (approximately 70% confluency). Cells were then divided into vehicle-treated or 40 ␮mol/L ␥-tocotrienol treated groups and returned to the incubator for a 24 h culture period. The next day, cells were treated with 0.05 mmol/L MDC in PBS at 37 °C for 15 min. Cells were then washed 5 times with PBS and mounted with Vectashield medium containing DAPI (4=,6-diamidino-2phenylindole) (Vector Laboratories Inc., Burlingame, California, USA). The fluorescence images were captured using LSM Pascal confocal microscope (Carl Zeiss Microimaging Inc., Thornwood, NY, USA).

3

Fig. 1. ␥-Tocotrienol effects on human neoplastic MCF-7 and MDA-MB231, and MCF10A normal mammary epithelial cells. All cells were plated at density of 1 × 104 cells/well (6 replicates/group) in 96-well plates and maintained on media for a 3 day culture period (approximately 70% confluence). Afterwards, cells were divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. Viable cell number was determined using the MTT colorimetric assay. Vertical bars indicate mean cell number ± SEM in each treatment group. *P < 0.05 compared to cells in their respective untreated control group (0 h).

Fluorescent labeling for LC3B MCF-7 and MDA-MB-231 cells were initially plated at a density of 5 × 104 cells/chamber in 8-chamber glass culture slides and maintained with control media for a 48 h culture period. Cells were then divided into vehicle-treated and 40 ␮mol/L ␥-tocotrienol treated groups and returned to the incubator of a 24 h culture period. The next day, cells were washed 5 times with ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 6 min, and then permeabilized with 0.2% triton X-100 in PBS for 2 min. Fixed cells were washed 3 times in PBS and then blocked with 5% goat serum in PBS for 1 h at room temperature. Cells were stained with LC3B (1:400) primary antibody in 5% goat serum in PBS and incubated at 4 °C overnight. The next day, the cells were washed 5 times with ice-cold PBS followed by incubation with Alexa flour-488 conjugated secondary antibody (1:5000) in 5% goat serum in PBS for 1 h at room temperature. Afterwards, cells were washed 3 times with ice-cold PBS and then mounted with Vectashield medium containing DAPI (Vector Laboratories Inc.). The fluorescence images were captured using LSM Pascal confocal microscope (Carl Zeiss Microimaging Inc., Thornwood, NY, USA). Quantification of acidic vesicular organelles (AVO) with acridine orange staining MCF-7 and MDA-MB-231 cells were plated at an initial density of 1 × 106 cells/100 mm culture dish and maintained in control media for a 3 day culture period. Cell were then divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. The next day, cells were then stained with acridine orange (1 ␮g/mL) for 15 min in culture (Kanzawa et al. 2004). Afterwards, the cells were isolated with trypsin and washed 5 times with PBS. The cell pellets were then resuspended in 1.0 mL of PBS and then analyzed using a BD FACSCalibur flow cytometer and BD CellQuest Pro program (BD Bioscience, San Jose, California, USA). Western blot analysis MCF-7, MDA-MB-231, and MCF-10A cells were initially plated at a density of 1 × 106 cells/100 mm culture dish and maintained in control media for 3 days. Cells were then divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. Afterwards, cells were isolated with trypsin, washed with PBS, then whole cell lysates were prepared in Laemmli buffer, as described previously (Malaviya and Sylvester 2014; Shirode and Sylvester 2011). Protein concentration in each sample was determined using Bio-Rad’s protein assay kit (Bio Rad, Hercules, California, USA). Equal amounts of protein (50 ␮g/lane) from each sample were then subjected to electrophoresis through 15%– 20% SDS-polyacrylamide minigels. Proteins from minigels were transblotted at 30 V for 12–16 h at 4 °C onto a single 8 inch × 6.5 inch polyvinylidene fluoride (PVDF) membrane (PerkinElmer Life Sciences) in a Trans-Blot Cell apparatus (Bio-Rad) according to the methods described by Towbin et al. (1979). The PVDF membranes were then blocked with 2% BSA in 10 mmol/L Tris-HCl containing 50 mmol/L NaCl and 0.1% Tween 20, pH 7.4 (TBST). The Published by NRC Research Press



4


Fig. 2. Effects of ␥-tocotrienol on autofluorescent autophagy marker intensity in human neoplastic MCF-7 and MDA-MB-231, and normal immortalized MCF-10A mammary epithelial cells. All cells were initially plated at a density of 7 × 105 cells/well in 6-well culture plates and maintained on control media for 3 days (approximately 70% confluence). Afterwards, cells were divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h exposure period. Cells were then treated with 0.05 mmol/L MDC and 0.2 ␮mol/L ethidium bromide for 15 min at 37 °C. Cells were then washed, lysed, and MDC (wavelength 360 nm and emission filter 525 nm) and ethidium bromide (excitation wavelength 530 nm and emission filter 590) fluorescence was measure in each well. Normalization of treatment effects in each well was determined by dividing MDC fluorescence by ethidium bromide fluorescence (total DNA). Vertical bars indicate normalized mean MDC fluorescence ± SEM expressed as specific activity (arbitrary units) for each treatment group. *P < 0.05 compared to cells in their respective untreated control group (0 h).

blocked membranes were then incubated with specific primary antibodies diluted 1:1000 to 1:5000 in 2% BSA in TBST overnight at 4 °C, or against ␣-tubulin, diluted 1:5000 in 2% BSA in TBST for 2 h at room temperature. Membranes were then washed 5 times in TBST and incubated with respective horseradish peroxidaseconjugated secondary antibody diluted 1:5000 in 2% BSA in TBST for 1 h at room temperature and then washed 5 times in TBST. Blots were then visualized by chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, Illinois, USA). Images of protein bands from all treatment groups within a given experiment were acquired using Kodak Gel Logic 1500 Imaging System (Carestream Health Inc., New Haven, Connecticut, USA). Visualization of ␣-tubulin was used to ensure equal sample loading in each lane. Image of protein bands were acquired and scanning densitometric analysis was performed with Kodak molecular imaging software version 4.5 (Carestream Health Inc.). All experiments were repeated at least 3 times, and representative western blot images from each experiment are shown in the figures. Transient Transfection MCF-7 and MDA-MB-231cells were initially plated at a density of 7 × 105 cells/well (3 replicates per group) in 6-well culture plates in 2 mL antibiotic free media and allowed to adhere overnight. Transfections were performed using 5 ␮L lipofectamine 2000 (Invitrogen/Life Technologies, Grand Island, NY, USA) according to the manufacturer’s protocol. Briefly, cells were transfected with 100 pmol of scrambled or Beclin-1 siRNAs diluted in 2 mL of media and returned to the incubator. After a 6 h incubation period, the medium was replaced with fresh growth media containing 10% FBS, and cells were maintained in the incubator for 3 days. Cells were then treated with 0–40 ␮mol/L ␥-tocotrienol for a period of 24 h, then isolated with trypsin and prepared for subsequent western blot analysis. Statistical analysis Differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. A difference of P < 0.05 was considered statistically significant, compared with the vehicle-treated control group or as defined in the Figure legends.

Results ␥-Tocotrienol effects on malignant and normal mammary epithelial cell viability A cytotoxic treatment dose of 40 ␮mol/L ␥-tocotrienol was selected for use in these studies, based on results obtained in previous dose–response experiments using these cell lines (Tiwari et al. 2014). Treatment with 40 ␮mol/L ␥-tocotrienol induced a time-

dependent decrease in viable MCF-7 and MDA-MB-231 human breast cancer cell number but had no effect on immortalized normal MCF-10A human mammary epithelial cell number during the 24 h exposure period (Fig. 1). ␥-Tocotrienol effects on fluorescent autophagy marker intensity Following treatment with 40 ␮mol/L ␥-tocotrienol, MDC autofluorescent intensity significantly increased in a time-dependent manner in MCF-7 and MDA-MB-231 breast cancer cells throughout Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) Tiwari et al.

5


Fig. 3. Effects of ␥-tocotrienol on acidic vesicular organelle (AVO) levels as determined by flow cytometry in human neoplastic MCF-7 and MDA-MB-231, normal immortalized MCF-10A mammary epithelial cells. All cells were initially plated at a density of 7 × 105 cells/well in 6-well culture plates and maintained on control media for 3 days (approximately 70% confluence). Cells were then divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h exposure period. Afterwards, cells were incubated with 1 ␮g/mL acridine orange for 15 min at 37 °C and then subjected to flow cytometry analysis. Dot plots were generated using CellQuest Software. Cells displaying positive acridine orange staining appear in the upper right quadrant of each dot plot and represents the presence of AVO, a cellular marker for autophagy. The numbers appearing in the upper right quadrant of each dot plot represent the mean percentage ± SEM of cells with AVO in their respective treatment group. *P < 0.05 compared to cells in their respective untreated control group.

the 24 h treatment period (Fig. 2). In contrast, similar treatment had no effect on MDC autofluorescent intensity in normal immortalized MCF-10A human mammary epithelial cells (Fig. 2). Flow cytometric analysis of ␥-tocotrienol effects on the appearance of acidic vesicular organelle (AVO) Flow cytometric analysis was performed to quantify ␥-tocotrienolinduced autophagy in human neoplastic MCF-7 and MDA-MB-231, and normal MCF-10A mammary epithelial cells, as indicated by positive acridine orange staining, a direct indicator of intracellular AVO levels (Kanzawa et al. 2004). As shown in Fig. 3, green (x-axis, 510–530 nm, FL1-H channel) and red (y-axis, >650 nm, FL3-H channel) fluorescence emission with blue (488 nm) excitation produced from 1 × 104 cells/group was measured in dot plots. An increase in the intensity of bright red fluorescence (top right quadrant) is reflective of a corresponding increase in AVO and autophagy. Treatment with 40 ␮mol/L ␥-tocotrienol induced a time-dependent increase in AVO levels in neoplastic MCF-and MDA-MB-231 cells, but had little or no effect on normal MCF-10A mammary epithelial cells throughout the 24 h exposure period (Fig. 3). Since treatment with 40 ␮mol/L ␥-tocotrienol had no effect on cell growth, viability, or the induction of autophagy in MCF-

10A cells, treatment effects on these noncancerous cells were not evaluated in subsequent experiments. ␥-Tocotrienol effects on breast cancer cell morphology, autophagic vacuole levels, and MDC and LC3B fluorescence staining ␥-Tocotrienol treatment caused an increase in the appearance of damaged and (or) dying MCF-7 and MDA-MB-231 cancer cell number, compared to their corresponding vehicle-treated control groups (Fig. 4A). Treatment with 40 ␮mol/L ␥-tocotrienol for 24 h resulted in a relatively large increase in the appearance of large vacuoles in MCF-7 and MDA-MB-231 breast cancer cells, compared to cells in their vehicle-treated control groups (Fig. 4B). Figure 4C shows that treatment with 40 ␮mol/L ␥-tocotrienol for 24 h caused a relatively large increase in positive MDC fluorescent staining, a positive marker for autophagic vacuole formation (Chen et al. 2010; Tanida et al. 2004) in MCF-7 and MDA-MB-231 breast cancer cells, compared to cells in their respective vehicle-treated control groups. Positive MDC staining is visualized as red fluorescent cytoplasmic staining. Figure 4D shows that treatment with 40 ␮mol/L ␥-tocotrienol for 24 h caused a relatively large increase in positive LC3B fluorescent staining, a marker for autophagosomes Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 6



Fig. 4. MCF-7 and MDA-MB-231 breast cancer cell were treated with 0–40 ␮mol/L ␥-tocotrienol (␥-T3) for a 24 h culture period. Size bars in all photomicrographs represent 5 ␮m in length. (A) Treatment effects on tumor cell gross morphology as viewed in phase contrast photomicrographs (magnification 100×). (B) Treatment effects on the appearance of autophagic vacuoles, as visualized by Giemsa staining (200× magnification). Arrows indicate the presence of autophagic vacuoles in these cells. (C) Treatment effects on the appearance of large vacuoles, as visualized by MDC red fluorescent staining in confocal photomicrographs (200× magnification). Blue staining indicates counter staining of cell nuclei with DAPI. (D) Treatment effects on the appearance of LC3B, a marker for the presence of autophagosomes, as visualized by green fluorescent staining in confocal photomicrographs (200× magnification). Blue staining indicates counter staining of cell nuclei with DAPI. Size bars in photomicrographs represent 5 ␮m in length.

(Chen et al. 2010; Tanida et al. 2004) in MCF-7 and MDA-MB-231 breast cancer cells, compared to cells in their respective vehicletreated control groups. LC3B staining is visualized as green fluorescent diffuse cytoplasmic pools or punctate structures (puncta) in these breast cancer cell lines (Fig. 4D).

untreated control groups (Fig. 5B). Figure 5C shows that ␥-tocotrienol treatment caused a significant time-dependent increase in the ratio of LC3B-II/LC3B-I, a cellular marker of autophagy in both MCF-7 and MDA-MB-231 mammary cancer cell lines, compared to cells in the untreated control groups.

␥-Tocotrienol effects on cellular levels of LC3B-I, LC3B-II, Beclin-1, and Atg5-Atg12 Western blot analysis shows that exposure to 40 ␮mol/L ␥-tocotrienol had little or no effect on the relative levels and (or) conversion of LC3B-I to LC3B-II in immortalized normal human MCF-10A mammary epithelial cells throughout a 24 h treatment period (Fig. 5A). In contrast, similar treatment resulted in a timedependent increase in the conversion of LC3B-I (cytosolic form) to LC3B-II, the phosphatidylethanolamine-conjugated form that is associated with autophagosomes, and a corresponding increase in the relative levels of Beclin-1 and Atg5-Atg12 in MCF-7 and MDAMB-231 breast cancer cells, compared to cells in their respective

Beclin-1 siRNA blocks ␥-tocotrienol-induced elevations in Beclin-1 and LCB-II/LC3B-I To establish a direct relationship between ␥-tocotrienol-induced cytotoxicity and autophagy, small interfering RNA (siRNA) targeting Beclin-1 or scrambled RNA (negative control) was transfected into the MCF-7 and MDA-MB-231 breast cancer cell lines prior to a 24 h exposure to 40 ␮mol/L ␥-tocotrienol. Cells transfected with Beclin-1 siRNA or scrambled siRNA alone displayed no differences in viability (Fig. 6A). Cells transfected with scrambled siRNA and exposed to ␥-tocotrienol treatment displayed a significant decrease in cell viability, compared to cells in their respective vehicle-treated control groups (Fig. 6A). However, cells transfected with Beclin-1 Published by NRC Research Press


7


Fig. 5. Western blot analysis of ␥-tocotrienol effects on the relative protein levels that serve as markers for autophagy in (A) immortalized normal human (MCF-10A) mammary epithelial cells and (B) human (MCF-7 and MDA-MB-231) mammary cancer cells. All cells were initially plated at a density of 1 × 106 cells/100 mm culture plate and maintained on control media for 3 days (approximately 70% confluence). Cells were then divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h exposure period. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 ␮g/lane) followed by western blot analysis for LC3-I and LC3-II (20% gel), and Beclin-1, Atg5-Atg12, and ␣-tubulin (15% gel). (C) Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding ␣-tubulin. Vertical bars indicate the LC3-II/LC3-I ratio in optical density in each treatment group ± SEM. *P < 0.05 compared to their respective vehicletreated control group.

siRNA and treated with ␥-tocotrienol display a modest, but significant reduction in ␥-tocotrienol-induced cytotoxicity, compared to cells transfected with scrambled siRNA and treated with ␥-tocotrienol (Fig. 6A). Western blot analysis shows that cell transfected with scrambled siRNA displayed a relatively large increase in Beclin-1 levels and the LC3B-II/LC3B-I ratio following a 24 h exposure to 40 ␮mol/L ␥-tocotrienol, and this response to ␥-tocotrienol treatment was greatly attenuated in cells transfected with Beclin-1 siRNA (Fig. 6B). ␥-Tocotrienol treatment was also found to significantly increase the LC3B-II/LC3B-I ratio in cells transfected with scrambled siRNA, and this effect was significantly attenuated in cells transfected with Beclin-1 siRNA (Fig. 6C). ␥-Tocotrienol and autophagy inhibitor 3-MA effects on LC3B-I, LC3B-II, and Beclin-1 levels 3-MA is an inhibitor of autophagy that blocks the conversion of LC3B-I to LC3B-II (Petiot et al. 2000). MCF-7 and MDA-MB-231 breast cancer cells were pretreated with 0–10 mmol/L 3-MA for 1 h prior to a 24 h exposure to 0–40 ␮mol/L ␥-tocotrienol. Treatment with 3-MA alone had no effect on cell viability, while cells treated with ␥-tocotrienol alone displayed a significant decrease in cell viability, compared to cells in their respective vehicle-treated control groups (Fig. 7A). However, pretreatment with 3-MA caused a modest, but significant blockade of ␥-tocotrienol-induced cytotoxicity, compared to cells treated with ␥-tocotrienol alone (Fig. 7A). Western blot analysis showed that pretreatment with 3-MA alone had little or no effect, whereas treatment with ␥-tocotrienol alone induced a large increase in the relative levels of LC3B-I, LC3B-II, and Beclin-1 levels in both MCF-7 and MDA-MB-231 breast cancer

cells, compared to cells in their respective vehicle-treated control groups (Fig. 7B). However, pretreatment with 3-MA greatly reduced ␥-tocotrienol-induced elevations in LC3B-I, LC3B-II, and Beclin-1 levels in both breast cancer cell lines (Fig. 7B). In addition, treatment with ␥-tocotrienol alone was found to increase the LC3B-II/LC3B-I ratio, while pretreatment with 3-MA caused a significant attenuation of this effect of ␥-tocotrienol in both MCF-7 and MDA-MB-231 breast cancer cell lines (Fig. 7C). ␥-Tocotrienol and bafilomycin A1 (Baf1) effects on LAMP-1, cathepsin-D, LC3B-I, LC3B-II, and Beclin-1 levels Baf1 is an established inhibitor of late phase autophagy that acts to prevent maturation of autophagic vacuoles by inhibiting the fusion of autophagosomes with lysosomes (Yamamoto et al. 1998). LAMP-1 is a membrane glycoprotein lysosomal marker protein, and cathepsin D is a lysosomal aspartic protease that plays a key role in regulating autophagosome/lysosome fusion (Glick et al. 2010; Glunde et al. 2003). Western blot analysis shows that treatment with 40 ␮mol/L ␥-tocotrienol caused a time-dependent increase in LAMP-1 and cathepsin D levels in MCF-7 and MDA-MB-231 breast cancer cells throughout the 24 h treatment period (Fig. 8A). Cell viability studies show that treatment with 100 nmol/L Baf1 alone had no effect, whereas treatment with 40 ␮mol/L ␥-tocotrienol alone significantly reduced MCF-7 or MDA-MB-231 breast cancer cell viability, compared to cells in their respective vehicle-treated control groups (Fig. 8B). However, pretreatment with 100 nmol/L Baf1 1 h prior to a 24 h exposure period to 40 ␮mol/L ␥-tocotrienol Published by NRC Research Press



8


Fig. 6. (A) Cytotoxic effects of 0–40 ␮mol/L ␥-tocotrienol on MCF-7 and MDA-MB-231 cells transfected with scrambled siRNA or Beclin-1 siRNA. Transfections were performed using 5 ␮L lipofectamine 2000 according to the manufacturer’s protocol. All cells were plated at a density of 1 × 104 cells/well (six replicates/group) in 96-well plates and maintained on media for a 3 day culture period (approximately 70% confluence). Afterwards, cells were divided into different groups and treated with 0–40 ␮mol/L ␥-tocotrienol (␥-T3) for a 24 h culture period. Viable cell number was determined using the MTT colorimetric assay. Vertical bars indicate mean cell number ± SEM in each treatment group. *P < 0.05 compared to cells in their respective vehicle-treated control group. #P < 0.05 compared to cells transfected with scrambled siRNA and exposed to 40 ␮mol/L ␥-tocotrienol. (B) Western blot analysis of ␥-tocotrienol effects on the relative protein levels of LC3B-I, LC3B-II and Beclin-1 in MCF-7 and MDA-MB-231 cells. Cells were initially plated at a density of 7 × 105 cells/well (3 replicates per group) in 6-well culture plates in 2 mL antibiotic free media and allowed to adhere overnight. Transfections were performed using 5 ␮L lipofectamine 2000 according to the manufacturer’s protocol. Cells were then treated with 0–40 ␮mol/L ␥-tocotrienol (␥-T3) for a 24 h exposure period. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 ␮g/lane, 20% gel) followed by western blot analysis for LC3-I, LC3-II, Beclin-1, and ␣-tubulin. (C) Scanning densitometric analysis was performed on all blots done in triplicate, and the integrated optical density of each band was normalized with corresponding ␣-tubulin. Vertical bars indicate the LC3-II/LC3-I ratio in optical density in each treatment group ± SEM. *P < 0.05 compared to their respective vehicle-treated control group, #P < 0.05 compared to cells transfected with scrambled siRNA and exposed to 40 ␮mol/L ␥-tocotrienol (␥-T3).

caused modest, but significant attenuated response to ␥-tocotrienolinduced cytotoxicity in these breast cancer cell lines (Fig. 8B). Additional studies confirmed that treatment with 40 ␮mol/L ␥-tocotrienol caused a time-dependent increase in LAMP-1 and cathepsin D levels in MCF-7 and MDA-MB-231 breast cancer cells, while treatment with 100 nmol/L Baf1 alone caused a slight reduction in LAMP-1 and cathepsin-D (Fig. 8C). Pretreatment with Baf1 was found to block ␥-tocotrienol-induced elevations in LAMP-1 and cathepsin-D levels in these breast cancer cells (Fig. 8C). Results also showed that treatment ␥-tocotrienol alone significantly increased the conversion of LC3B-I to LC3B-II, and pretreatment with Baf1 significantly enhanced this effect in both breast cancer cell lines, compared to their corresponding control cells treated with ␥-tocotrienol alone (Figs. 8C and 8D).

Effects of ␥-tocotrienol on Erk1/2, JNK1/2, and p38 activation Western blot analysis showed that treatment with 40 ␮mol/L ␥-tocotrienol resulted in a time-dependent increase in the relative levels of the cellular stress markers phosphorylated-p38 (activated) and phosphorylated-JNK1/2 (activated), and a corresponding decrease in the mitogenic/survival marker phosphorylated-Erk1/2 (activated) in MCF-7 and MDA-MB-231 breast cancer cells (Fig. 9). Effects of ␥-tocotrienol on Bcl-2, Bax, and cleaved caspases-3 and PARP levels Western blot analysis showed that treatment with 40 ␮mol/L ␥-tocotrienol caused a time-dependent decrease in Bcl-2 (antiapoptotic), and a corresponding increase in Bax (pro-apoptotic), as well as, an increase in cleaved caspase-3 (activated) and cleaved-PARP Published by NRC Research Press



Tiwari et al.

9

Fig. 7. (A) Cytotoxic effects of 0–40 ␮mol/L ␥-tocotrienol on MCF-7 and MDA-MB-231 cells pretreated with 0–10 mmol/L 3-MA. All cells were plated at a density of 1 × 104 cells/well (six replicates/group) in 96-well plates and maintained on control media for a 3 day culture period (approximately 70% confluence). Afterwards, cells were divided into different groups and pretreated for 1 h with 0–10 mmol/L 3-MA prior to a 24 h exposure to 0–40 ␮mol/L ␥-tocotrienol (␥-T3). Viable cell number was determined using the MTT colorimetric assay. Vertical bars indicate mean cell number ± SEM in each treatment group. *P < 0.05 compared to cells in their respective vehicle-treated control group. #P < 0.05 compared to cells 40 ␮mol/L ␥-tocotrienol alone. (B) Western blot analysis of ␥-tocotrienol effects on the relative protein levels of LC3B-I, LC3B-II and Beclin-1 in MCF-7 and MDA-MB-231 cells pretreated with 0–10 mmol/L 3-MA. Cells were initially plated at a density of 1 × 106 cells/100 mm culture plate and maintained on control media for 3 days (approximately 70% confluency). Cells were then divided into different groups and pretreated with 0–10 mmol/L 3-MA for 1 h prior to a 24 h exposure period to 0–40 ␮mol/L ␥-tocotrienol. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 ␮g/lane, 20% gel) followed by western blot analysis for LC3-I, LC3-II, Beclin-1, and ␣-tubulin. (C) Scanning densitometric analysis was performed in triplicate and integrated optical density of each band was normalized with corresponding ␣-tubulin. Vertical bars indicate the LC3-II/LC3-I ratio in optical density in each treatment group ± SEM. *P < 0.05 compared to their respective vehicle-treated control group, #P < 0.05 compared to cells treated with 40 ␮mol/L ␥-tocotrienol alone.

(activated) protein levels in MCF-7 and MDA-MB-231 breast cancer cells (Fig. 10A). Figure 10B shows that ␥-tocotrienol treatment significantly increased the ratio of Bax/Bcl-2 in these breast cancer cell lines. Effects of combined pan caspase inhibitor and ␥-tocotrienol treatment on mammary tumor cell viability and ␥-tocotrienol effects on ER stress related protein levels Treatment for a 24 h culture period with 40 ␮mol/L ␥-tocotrienol alone significantly reduced, whereas treatment with 20 ␮mol/L zVADfmk alone (a pan caspase inhibitor) had no effect on MCF-7 or MDA-MB-231 cell viability (Fig. 11A). However, a combined treatment of zVADfmk was found to induce a complete blockade of the cytotoxic effects of ␥-tocotrienol on both breast cancer cell lines (Fig. 11A). Western blot analysis showed that treatment with

40 ␮mol/L ␥-tocotrienol induced a time-dependent increase in the relative levels of ER stress markers including Bip, IRE1, phosphorylated PERK (activated), phosphorylated eIF2␣ (inactivated), ATF-4, CHOP, and TRB3 in MCF-7 and MDA-MB-231 breast cancer cells (Fig. 11B).

Discussion The results demonstrate that ␥-tocotrienol-induced cytotoxicity in human MCF-7 and MDA-MB-231 breast cancer cells is associated with the concurrent induction of autophagy and ER stressmediated apoptosis. Treatment with a cytotoxic dose (40 ␮mol/L) of ␥-tocotrienol caused a time-dependent decrease in cancer cell viability that was directly correlated to an increase in autophagy and ER stress markers. Specifically, exposure to ␥-tocotrienol Published by NRC Research Press



10


Fig. 8. (A) Western blot analysis of ␥-tocotrienol effects on relative protein level of LAMP-1 and cathepsin-D. Cells were initially plated at a density of 1 × 106 cells/100 mm culture plate and maintained in control media for 3 days (approximately 70% confluence). Cell were then divided into different groups and treated with 0–40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. (B) Cytotoxic effects of 0–40 ␮mol/L ␥-tocotrienol (␥T3) on MCF-7 and MDA-MB-231 cells pretreated with 0–100 nM Baf1. All cells were plated at a density of 1 × 104 cells/well (six replicates/group) in 96-well plates and maintained on control media for a 3 day culture period (approximately 70% confluence). Afterwards, cells were divided into different groups and pretreated for 1 h with 0–100 nmol/L Baf1 prior to a 24 h exposure period to 0–40 ␮mol/L ␥-tocotrienol. Viable cell number was determined using the MTT colorimetric assay. Vertical bars indicate mean cell number ± SEM in each treatment group. *P < 0.05 compared to cells in their respective vehicle-treated control group. #P < 0.05 compared to cells 40 ␮mol/L ␥-tocotrienol alone. (C) Western blot analysis of ␥-tocotrienol (␥T3) and Baf1 treatment on LC3B-I, LC3B-II, LAMP-1 and cathepsin-D levels MCF-7 and MDA-MB-231 cells. Cells were initially plated at a density of 1 × 106 cells/100 mm culture plate and maintained on control media for 3 days (approximately 70% confluence). Cells were then divided into different groups and pretreated with 0–100 nmol/L Baf1 for 1 h prior to a 24 h exposure period to 0–40 ␮mol/L ␥-tocotrienol. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 ␮g/lane, 20% gel) followed by western blot analysis for LC3-I, LC3-II, LAMP-1, cathepsin-D, and ␣-tubulin. (D) Scanning densitometric analysis was performed in triplicate, and the integrated optical density of each band was normalized with corresponding ␣-tubulin. Vertical bars indicate the LC3-II/LC3-I optical density ratio in each treatment group ± SEM. *P < 0.05 compared to cells in their respective vehicle-treated control group. #P < 0.05 compared to cells treated with 40 ␮mol/L ␥-tocotrienol alone.

caused a progressive time-dependent increase in early phase (Beclin-1, LC3B-II) and late phase (LAMP-1 and cathepsin-D) autophagy markers, and pretreatment with autophagy inhibitors Beclin-1 siRNA, 3-MA, or Baf1 blocked these effects. During this same time period, ␥-tocotrienol caused a time-dependent decrease in mitogenic Erk signaling, a corresponding increase in stress-dependent p38 and JNK activation, and an increase in the expression of ER

stress apoptotic markers. Furthermore, pretreatment with autophagy inhibitors 3-MA or Baf1 caused a modest, but significant reduction in ␥-tocotrienol-induced cytotoxicity in these breast cancer cells. Although the role of autophagy in cancer etiology remains controversial, and studies have shown that autophagy can play a role in promoting either tumor cell survival or death (Baehrecke 2005; White and DiPaola 2009), the present findings Published by NRC Research Press


11


Fig. 9. Western blot analysis of ␥-tocotrienol effects on mitogenic/survival and stress signaling protein levels in MCF-7 and MDA-MB-231 breast cancer cells. All cells were initially plated at a density of 1 × 106 cells/100 mm culture plate and maintained in control media for 3 days (approximately 70% confluence). Cells were then divided into different groups and treated with 0–40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 ␮g /lane, 15% gel) followed by western blot analysis for phosphorylated-JNK1/2 (p-JNK1/2), JNK1/2, phosphorylated p38 (p-p38), p38, phosphorylated Erk1/2 (p-Erk1/2), total Erk1 and Erk2, and ␣-tubulin. The visualization of ␣-tubulin was used to ensure equal sample loading in each lane. All experiments were repeated at least 3 times.

Fig. 10. (A) Western blot analysis of ␥-tocotrienol effects on the relative levels of apoptotic protein markers in MCF-7 and MDA-MB-231 breast cancer cells. All cells were initially plated at 1 × 106 cells/100 mm culture plate and maintained in control media for 3 days (approximately 70% confluence). Cell were then divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (40 ␮g /lane, 15% gel) followed by western blot analysis for Bcl-2, Bax, cleaved PARP, and cleaved caspase-3. (B) Scanning densitometric analysis was performed in triplicate and the integrated optical density of each band was normalized with corresponding ␣tubulin. Vertical bars represent the Bax/Bcl-2 optical density ratio in each treatment group ± SEM. *P < 0.05 compared to cell in their respective untreated control group.

provide strong evidence to suggest that ␥-tocotrienol-induced ER stress and autophagy occurs concurrently and together act to promote the self-destruction of human breast cancer cells. Previous studies have reported that the cytotoxic effects of tocotrienols against cancer cells are mediated by several mechanisms, including apoptosis and (or) autophagy (Wali et al. 2009; Tiwari et al. 2014; Jiang et al. 2012). However, a clear understand-

ing of the exact interrelationship between autophagy and ER stress is not presently understood. ␥-Tocotrienol-induced ER stress has been shown to be involved in activating the PERK/eIF2␣/ ATF-4 pathway in breast cancer cells (Wali et al. 2009; Patacsil et al. 2012; Park et al. 2010). The PERK/eIF2␣/ATF-4 pathway has also been shown to play an important role in the conjugation of LC3B I to Atg5-Atg12 and in the modulation of autophagy-related Published by NRC Research Press



12


Fig. 11. (A) Effects of pan caspase inhibitor zVADfmk on ␥-tocotrienol-induced cytotoxicity. All cells were plated at a density of 1 × 104 cells/ well (6 replicates/group) in 96-well plates and maintained on media for a 3 day culture period (approximately 70% confluence). Afterwards, cells were divided into different groups and treated with 0–40 ␮mol/L ␥-tocotrienol and (or) 0–20 ␮mol/L zVADfmk for a 0–24 h culture period. Viable cell number was determined using the MTT colorimetric assay. Vertical bars indicate mean cell number ± SEM in each treatment group. *P < 0.05 compared to cells in their respective untreated control group. (B) Western blot analysis of ␥-tocotrienol effects on ER stress signaling protein levels in MCF-7 and MDA-MB-231 breast cancer cell lines. All cells were initially plated at 1 × 106 cells/100 mm culture plate and maintained in control media for 3 days (approximately 70% confluence). Cell were then divided into different groups and treated with 40 ␮mol/L ␥-tocotrienol for a 0–24 h culture period. Afterwards, whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 ␮g /lane, 15% gel) followed by western blot analysis for Bip, IRE1␣, phosphorylated PERK (p-PERK), phosphorylated eIF2␣ (p-eIF2␣), ATF-4, CHOP, TRB3, and ␣-tubulin. The visualization of ␣-tubulin was used to ensure equal sample loading in each lane. All experiments were repeated at least 3 times.

gene expression (Kouroku et al. 2007; Cheng and Yang 2011; Ghavami et al. 2012). Since ␥-tocotrienol stimulates the activation of the PERK/eIF2␣/ATF-4 pathway, LC3B-I conjugation to Atg5-Atg12, and the conversion of LC3B-I from its cytosolic soluble form to its lipidated and autophagosome bound form LC3B-II in MCF-7 and MDA-MB-231 breast cancer cells, these data provide new evidence suggesting that ␥-tocotrienol induced autophagy appears to be trigged by ER stress activation of the PERK/eIF2␣/ATF-4 pathway. Studies are currently underway to determine the exact intracellular targets that are directly involved in mediating ␥-tocotrienolinduced ER stress and autophagy in breast cancer cells. It is well-established that phosphorylation eIF2␣ by PERK leads to a decrease in the efficient translation of most of mRNAs and prevents further protein synthesis (Dong et al. 2008; Fels and Koumenis 2006; Ron and Walter 2007; Wali et al. 2009). During ER stress, only selected mRNAs, such as ATF-4, are translated (Lu et al. 2004; Vattem and Wek 2004). The target genes of ATF-4 are asparagine synthetase (ASNS) and CHOP, and the expression of CHOP is elevated in response to severe or prolonged ER stress (Siu et al. 2002; Su and Kilberg 2008). CHOP directly regulates target genes, such as Bcl-2, (Han et al. 2013; Szegezdi et al. 2006) and promotes programmed cell death through the induction of TRB3, which increases cellular responsiveness to death receptor mediated ap-

optosis (Ohoka et al. 2005; Wali et al. 2009; Ding et al. 2007; Oh and Lim 2009; Urano et al. 2000; Verfaillie et al. 2010). Experimental findings show that ␥-tocotrienol-induced activation of eIF2␣ was associated with an increase in AFT-4, CHOP, and their downstream target TRB3 in MCF-7 and MDA-MB-231 breast cancer cells. Previous studies showed that elevations in TRB3 is involved in ␥-tocotrienol-induced ER stress mediated apoptosis in the highly malignant mouse +SA cells mammary tumor cells (Wali et al. 2009). Furthermore, combined treatment of ␥-tocotrienol with the pan caspase inhibitor, zVADfmk, was found to completely block the cytotoxic effects of ␥-tocotrienol. The present findings provide further evidence demonstrating the activation of the PERK/eIF2␣/ATF-4 pathway during ␥-tocotrienol-induced ER stress mediated apoptosis. ␥-Tocotrienol-induced autophagy was previously shown to be associated with an increase in expression of cellular markers LC3B-II and Beclin-1 that appear during the early stages of autophagy, (Jiang et al. 2012; Tiwari et al. 2014). However the effects of ␥-tocotrienol on the expressing of cellular markers LAMP-1 and cathepsin D that appear during the late stages of autophagy had not previously been investigated. Acute exposure to ␥-tocotrienol was found to cause a time-dependent increase in LAMP-1 and cathepsin D. During the late stages of autophagy, LAMP-1 is required Published by NRC Research Press


13


Fig. 12. Schematic representation of the proposed molecular mechanism mediating ␥-tocotrienol concurrent induction of autophagy and ER stress-mediated apoptosis in breast cancer cells.

for fusion of autophagosomes to the lysosomes in the degradation pathway, whereas cathepsin D is a lysosomal protease that participates in the degradation of autophagosomal content (Glick et al. 2010; Glunde et al. 2003). Furthermore, pretreatment with Baf1, a late phase inhibitor of autophagy that acts to inhibit the vacuolar H+ ATPase proton pump and ultimately prevent the maturation of autophagy vacuoles by inhibiting autophagosomelysosome fusion (Yamamoto et al. 1998), blocked these ␥-tocotrienoldependent effects. These findings provide novel information indicating that ␥-tocotrienol plays a role in promoting the formation of autolysosomes during the later stages of autophagy in malignant breast cancer cells. ER stress also leads to the release of calcium ions from ER lumen into the cytosol, where it acts as a second messenger to initiate the activation of pathways involved in apoptosis and autophagy (Hoyer-Hansen and Jaattela 2007; Xu et al. 2014). Specifically, ER stress activation of IRE1␣ stimulates JNK and p38 signaling, and JNK and p38 act to inactivate Bcl-2 and prevent Bcl-2 negative regulation of Beclin-1 (Ding et al. 2007; Hoyer-Hansen and Jaattela 2007; Oh and Lim 2009; Pattingre et al. 2005; Urano et al. 2000; Verfaillie et al. 2010; Xu et al. 2014). It is also well-established that mitogen-dependent activation of Erk1/2 and Akt signaling promotes cell survival by increasing anti-apoptotic Bcl-2 family protein levels (Lee et al. 2012; Lu and Xu 2006; Sun et al. 2008). ␥-Tocotrienol treatment was found to decrease Erk1/2 and Akt mitogenic signaling, while at the same time increase stressdependent JNK and p38 signaling in MCF-7 and MDA-MB-231 breast cancer cells. Interaction between the anti-apoptotic protein Bcl-2 and the autophagy protein Beclin-1 has been shown to be a critical interface point between autophagy and apoptosis (Pattingre et al. 2005). Beclin-1 is localized within the trans-Golgi network, where it participates in the formation of autophago-

somes, and Bcl-2 binds to and inhibits Beclin-1 activity (Kihara et al. 2001). ␥-Tocotrienol-induced elevations in Beclin-1 levels was also found to be directly associated with a corresponding decrease in Bcl-2 and a corresponding increase in pro-apoptotic Bax levels in MCF-7 and MDA-MB-231 breast cancer cells. ␥-Tocotrienol treatment increases the Bax/Bcl-2 ratio, which has previously been shown to promote autophagy (Raisova et al. 2001; Tiwari et al. 2014) and at the same time increase expression of the apoptotic markers cleaved caspases-3 and cleaved PARP (Bachawal et al. 2010). The present findings further demonstrate that ␥-tocotrienolinduced ER stress is associated with a suppression in mitogenic Erk1/2 signaling, an increase in stress-dependent p38 and JNK signaling, and a subsequent increase in the Bax/Bcl-2 ratio and apoptosis. Acute exposure for 24 h to 40 ␮mol/L ␥-tocotrienol was found to be extremely cytotoxic to MCF-7 and MDA-MB-231 breast cancer, but not the noncancerous MCF-10A mammary epithelial cells. These findings indicate that malignant cells display a selective sensitivity to anticancer effects of ␥-tocotrienol and are in agreement with previous findings that show cells with the greatest degree of tumor progression or malignancy were found to be the most sensitive to the cytotoxic effects of tocotrienols, followed by noninvasive neoplastic, preneoplastic, and normal mammary epithelial cells, respectively, (McIntyre et al. 2000a, 2000b; Sylvester and Theriault 2003). The exact reason why malignant cells are more sensitive to the cytotoxic effects of ␥-tocotrienol than noncancerous cells is presently unknown. However, ␥-tocotrienolinduced cytotoxicity in estrogen-receptor positive MCF-7 and estrogen-receptor negative MDA-MB-231 breast cancer cells was found to be occur independently of estrogen receptor status (Nesaretnam et al. 1998; Yu et al. 1999). The possibility exists that noncancerous cells possess specific compensatory mechanisms Published by NRC Research Press



14

that provide protection against ␥-tocotrienol-induced ER stressmediated apoptosis and autophagy, and these self-survival mechanisms may be dysfunctional in malignant cells. Additional studies are required to determine if this hypothesis is correct. In summary, results from this study demonstrate that the cytotoxic effects of ␥-tocotrienol are associated with the concurrent induction of autophagy and ER stress-mediated apoptosis. Since blockade of ␥-tocotrienol-induced autophagy results in a modest attenuation of ␥-tocotrienol-induced cytotoxicity, these findings provide evidence that ␥-tocotrienol-induced ER stress and autophagy acts together to promote cell death in human breast cancer cells. A schematic representation of the interrelationship and intracellular mechanisms involved in mediating ␥-tocotrienolinduced autophagy and ER stress mediated apoptosis in promotion cytotoxicity are summarized in Fig. 12.

Acknowledgements The authors would like to thank First Tech International Ltd. for generously providing ␥-tocotrienol for use in these studies. This work was performed at the School of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana, USA, and supported in part by grants from First Tec International Ltd. (Hong Kong), the Malaysian Palm Oil Council (MPOC), the Louisiana Cancer Foundation, and the Louisiana Campuses Research Initiative (LACRI). The authors would also like to thank Dr. Karen P Briski for her assistance in studies with the confocal microscope.

References Bachawal, S.V., Wali, V.B., and Sylvester, P.W. 2010. Enhanced antiproliferative and apoptotic response to combined treatment of gamma-tocotrienol with erlotinib or gefitinib in mammary tumor cells. BMC Cancer, 10: 84. doi:10. 1186/1471-2407-10-84. PMID:20211018. Baehrecke, E.H. 2005. Autophagy: dual roles in life and death? Nat. Rev. Mol. Cell Biol. 6(6): 505–510. doi:10.1038/nrm1666, 10.1038/nrn1720. PMID:15928714. Biederbick, A., Kern, H.F., and Elsasser, H.P. 1995. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur. J. Cell Biol. 66(1): 3–14. PMID:7750517. Chen, Y., Azad, M.B., and Gibson, S.B. 2010. Methods for detecting autophagy and determining autophagy-induced cell death. Can. J. Physiol. Pharmacol. 88(3): 285–295. doi:10.1139/Y10-010. PMID:20393593. Cheng, Y., and Yang, J.M. 2011. Survival and death of endoplasmic-reticulumstressed cells: Role of autophagy. World J. Biol. Chem. 2(10): 226–231. Ding, W.X., Ni, H.M., Gao, W., Yoshimori, T., Stolz, D.B., Ron, D., and Yin, X.M. 2007. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am. J. Pathol. 171(2): 513–524. doi:10.2353/ajpath.2007.070188. PMID:17620365. Dong, D., Ni, M., Li, J., Xiong, S., Ye, W., Virrey, J.J., et al. 2008. Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development. Cancer Res. 68(2): 498–505. doi:10.1158/0008-5472.CAN-07-2950. PMID:18199545. Fels, D.R., and Koumenis, C. 2006. The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biol. Ther. 5(7): 723–728. doi:10.4161/cbt.5.7.2967. PMID:16861899. Ghavami, S., Yeganeh, B., Stelmack, G.L., Kashani, H.H., Sharma, P., Cunnington, R., et al. 2012. Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts. Cell Death Dis. 3: e330. doi:10.1038/cddis.2012.61. PMID:22717585. Glick, D., Barth, S., and Macleod, K.F. 2010. Autophagy: cellular and molecular mechanisms. J. Pathol. 221(1): 3–12. doi:10.1002/path.2697. PMID:20225336. Glunde, K., Guggino, S.E., Solaiyappan, M., Pathak, A.P., Ichikawa, Y., and Bhujwalla, Z.M. 2003. Extracellular acidification alters lysosomal trafficking in human breast cancer cells. Neoplasia, 5(6): 533–545. doi:10.1016/S14765586(03)80037-4. PMID:14965446. Han, J., Back, S.H., Hur, J., Lin, Y.H., Gildersleeve, R., Shan, J., et al. 2013. ER-stressinduced transcriptional regulation increases protein synthesis leading to cell death. Nat. Cell Biol. 15(5): 481–490. doi:10.1038/ncb2738. PMID:23624402. Hoyer-Hansen, M., and Jaattela, M. 2007. Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell Death Differ. 14(9): 1576–1582. doi:10.1038/sj.cdd.4402200. PMID:17612585. Iwai-Kanai, E., Yuan, H., Huang, C., Sayen, M.R., Perry-Garza, C.N., Kim, L., and Gottlieb, R.A. 2008. A method to measure cardiac autophagic flux in vivo. Autophagy, 4(3): 322–329. doi:10.4161/auto.5603. PMID:18216495. Jiang, Q., Rao, X., Kim, C.Y., Freiser, H., Zhang, Q., Jiang, Z., and Li, G. 2012. Gamma-tocotrienol induces apoptosis and autophagy in prostate cancer cells by increasing intracellular dihydrosphingosine and dihydroceramide. Int. J. Cancer, 130(3): 685–693. doi:10.1002/ijc.26054. PMID:21400505. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., et al.


2000. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19(21): 5720–5728. doi:10.1093/ emboj/19.21.5720. PMID:11060023. Kanzawa, T., Germano, I.M., Komata, T., Ito, H., Kondo, Y., and Kondo, S. 2004. Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ. 11(4): 448–457. doi:10.1038/sj.cdd.4401359. PMID: 14713959. Kihara, A., Kabeya, Y., Ohsumi, Y., and Yoshimori, T. 2001. Beclinphosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep. 2(4): 330–335. doi:10.1093/embo-reports/kve061. PMID:11306555. Klionsky, D.J. 2005. The molecular machinery of autophagy: unanswered questions. J. Cell Sci. 118(1): 7–18. doi:10.1242/jcs.01620. PMID:15615779. Klionsky, D.J., Abdalla, F.C., Abeliovich, H., Abraham, R.T., Acevedo-Arozena, A., Adeli, K., et al. 2012. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 8(4): 445–544. doi:10.4161/auto.19496. PMID:22966490. Kouroku, Y., Fujita, E., Tanida, I., Ueno, T., Isoai, A., Kumagai, H., and Momoi, T. 2007. ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamineinduced LC3 conversion, an essential step for autophagy formation. Cell Death Differ. 14(2): 230–239. Lee, W.K., Chakraborty, P.K., Roussa, E., Wolff, N.A., and Thevenod, F. 2012. ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP. Biochim. Biophys. Acta, 1823(10): 1864–1876. doi:10.1016/j.bbamcr.2012.06.003. PMID:22705154. Lu, P.D., Harding, H.P., and Ron, D. 2004. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J. Cell Biol. 167(1): 27–33. doi:10.1083/jcb.200408003. PMID:15479734. Lu, Z., and Xu, S. 2006. ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life, 58(11): 621–631. doi:10.1080/15216540600957438. PMID:17085381. Malaviya, A., and Sylvester, P.W. 2014. Synergistic Antiproliferative Effects of Combined gamma -Tocotrienol and PPAR gamma Antagonist Treatment Are Mediated through PPAR gamma -Independent Mechanisms in Breast Cancer Cells. PPAR Res. 2014: 439146. doi:10.1155/2014/439146. PMID:24729783. Mathew, R., Karantza-Wadsworth, V., and White, E. 2007. Role of autophagy in cancer. Nat. Rev. Cancer, 7(12): 961–967. doi:10.1038/nrc2254. PMID:17972889. McIntyre, B.S., Briski, K.P., Gapor, A., and Sylvester, P.W. 2000a. Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. Proc. Soc. Exp. Biol. Med. 224(4): 292–301. doi:10.1046/j.1525-1373.2000.22434.x. PMID:10964265. McIntyre, B.S., Briski, K.P., Tirmenstein, M.A., Fariss, M.W., Gapor, A., and Sylvester, P.W. 2000b. Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells. Lipids, 35(2): 171–180. doi:10.1007/BF02664767. PMID:10757548. Munafo, D.B., and Colombo, M.I. 2001. A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J. Cell Sci. 114(20): 3619–3629. PMID:11707514. Nesaretnam, K., Stephen, R., Dils, R., and Darbre, P. 1998. Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status. Lipids, 33(5): 461–469. doi:10.1007/s11745-998-0229-3. PMID:9625593. Oh, S.H., and Lim, S.C. 2009. Endoplasmic reticulum stress-mediated autophagy/ apoptosis induced by capsaicin (8-methyl-N-vanillyl-6-nonenamide) and dihydrocapsaicin is regulated by the extent of c-Jun NH2-terminal kinase/ extracellular signal-regulated kinase activation in WI38 lung epithelial fibroblast cells. J. Pharmacol. Exp. Ther. 329(1): 112–122. doi:10.1124/jpet.108.144113. PMID:19139269. Ohoka, N., Yoshii, S., Hattori, T., Onozaki, K., and Hayashi, H. 2005. TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J. 24(6): 1243–1255. doi:10.1038/sj.emboj.7600596. PMID: 15775988. Park, S.K., Sanders, B.G., and Kline, K. 2010. Tocotrienols induce apoptosis in breast cancer cell lines via an endoplasmic reticulum stress-dependent increase in extrinsic death receptor signaling. Breast Cancer Res. Treat. 124(2): 361–375. Patacsil, D., Tran, A.T., Cho, Y.S., Suy, S., Saenz, F., Malyukova, I., Ressom, H., Collins, S.P., Clarke, R., and Kumar, D. 2012. Gamma-tocotrienol induced apoptosis is associated with unfolded protein response in human breast cancer cells. J. Nutr. Biochem. 23: 93–100. Pattingre, S., Tassa, A., Qu, X., Garuti, R., Liang, X.H., Mizushima, N., et al. 2005. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell, 122(6): 927–939. doi:10.1016/j.cell.2005.07.002. PMID:16179260. Petiot, A., Ogier-Denis, E., Blommaart, E.F., Meijer, A.J., and Codogno, P. 2000. Distinct classes of phosphatidylinositol 3=-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 275(2): 992–998. doi:10.1074/jbc.275.2.992. PMID:10625637. Rabinowitz, J.D., and White, E. 2010. Autophagy and metabolism. Science, 330(6009): 1344–1348. doi:10.1126/science.1193497. PMID:21127245. Raisova, M., Hossini, A.M., Eberle, J., Riebeling, C., Wieder, T., Sturm, I., et al. 2001. The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J. Invest. Dermatol. 117(2): 333–340. doi:10.1046/j.0022-202x.2001.01409.x. PMID:11511312. Rikiishi, H. 2012. Novel Insights into the Interplay between Apoptosis and Autophagy. Int. J. Cell Biol. 2012: 317645. doi:10.1155/2012/317645. PMID: 22496691. Published by NRC Research Press



Tiwari et al.

Robison, L.M., Sylvester, P.W., Birkenfeld, P., Lang, J.P., and Bull, R.J. 1998. Comparison of the effects of iodine and iodide on thyroid function in humans. J. Toxicol. Environ. Health A, 55(2): 93–106. doi:10.1080/009841098158539. PMID:9761130. Ron, D., and Walter, P. 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol. 8(7): 519–529. doi:10.1038/ nrm2199. PMID:17565364. Schroder, M., and Kaufman, R.J. 2005. ER stress and the unfolded protein response. Mutat. Res. 569(1–2): 29–63. doi:10.1016/j.mrfmmm.2004.06.056. PMID:15603751. Shintani, T., and Klionsky, D.J. 2004. Autophagy in health and disease: a doubleedged sword. Science, 306(5698): 990–995. doi:10.1126/science.1099993. PMID: 15528435. Shirode, A.B., and Sylvester, P.W. 2011. Mechanisms Mediating the Synergistic Anticancer Effects of Combined gamma-Tocotrienol and Celecoxib Treatment. J. Bioanal. Biomed. 3: 1–7. PMID:22140606. Singh, B.N., Kumar, D., Shankar, S., and Srivastava, R.K. 2012. Rottlerin induces autophagy which leads to apoptotic cell death through inhibition of PI3K/ Akt/mTOR pathway in human pancreatic cancer stem cells. Biochem. Pharmacol. 84(9): 1154–1163. doi:10.1016/j.bcp.2012.08.007. PMID:22902833. Siu, F., Bain, P.J., LeBlanc-Chaffin, R., Chen, H., and Kilberg, M.S. 2002. ATF4 is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthetase gene. J. Biol. Chem. 277(27): 24120–24127. doi:10.1074/ jbc.M201959200. PMID:11960987. Su, N., and Kilberg, M.S. 2008. C/EBP homology protein (CHOP) interacts with activating transcription factor 4 (ATF4) and negatively regulates the stressdependent induction of the asparagine synthetase gene. J. Biol. Chem. 283(50): 35106–35117. doi:10.1074/jbc.M806874200. PMID:18940792. Sun, W., Wang, Q., Chen, B., Liu, J., Liu, H., and Xu, W. 2008. Gamma-tocotrienolinduced apoptosis in human gastric cancer SGC-7901 cells is associated with a suppression in mitogen-activated protein kinase signalling. Br. J. Nutr. 99(6): 1247–1254. doi:10.1017/S0007114507879128. PMID:18081943. Sylvester, P.W., and Theriault, A. 2003. Role of tocotrienols in the prevention of cardiovascular disease and breast cancer. Curr Topics Nutraceut. Res. 1(2): 121–136. Szegezdi, E., Logue, S.E., Gorman, A.M., and Samali, A. 2006. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 7(9): 880–885. doi:10. 1038/sj.embor.7400779. PMID:16953201. Tanida, I., Ueno, T., and Kominami, E. 2004. LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell Biol. 36(12): 2503–2518. doi:10.1016/j.biocel. 2004.05.009. PMID:15325588. Tassa, A., Roux, M.P., Attaix, D., and Bechet, D.M. 2003. Class III phosphoinositide 3-kinase-Beclin1 complex mediates the amino acid-dependent

15

regulation of autophagy in C2C12 myotubes. Biochem. J. 376(3): 577–586. doi:10.1042/BJ20030826. PMID:12967324. Tiwari, R.V., Parajuli, P., and Sylvester, P.W. 2014. gamma-Tocotrienol-induced autophagy in malignant mammary cancer cells. Exp. Biol. Med. (Maywood), 239(1): 33–44. doi:10.1177/1535370213511022. PMID:24231340. Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76(9): 4350–4354. doi:10.1073/pnas. 76.9.4350. PMID:388439. Urano, F., Wang, X., Bertolotti, A., Zhang, Y., Chung, P., Harding, H.P., and Ron, D. 2000. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science, 287(5453): 664–666. doi:10. 1126/science.287.5453.664. PMID:10650002. Vattem, K.M., and Wek, R.C. 2004. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 101(31): 11269–11274. doi:10.1073/pnas.0400541101. PMID:15277680. Verfaillie, T., Salazar, M., Velasco, G., and Agostinis, P. 2010. Linking ER Stress to Autophagy: Potential Implications for Cancer Therapy. Int. J. Cell Biol. 2010: 930509. doi:10.1155/2010/930509. PMID:20145727. Wali, V.B., Bachawal, S.V., and Sylvester, P.W. 2009. Endoplasmic reticulum stress mediates gamma-tocotrienol-induced apoptosis in mammary tumor cells. Apoptosis, 14(11): 1366–1377. doi:10.1007/s10495-009-0406-y. PMID: 19771520. White, E., and DiPaola, R.S. 2009. The double-edged sword of autophagy modulation in cancer. Clin. Cancer Res. 15(17): 5308–5316. doi:10.1158/1078-0432. CCR-07-5023. PMID:19706824. Xu, C., Bailly-Maitre, B., and Reed, J.C. 2005. Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 115(10): 2656–2664. doi:10.1172/ JCI26373. PMID:16200199. Xu, Y., Wang, C., and Li, Z. 2014. A new strategy of promoting cisplatin chemotherapeutic efficiency by targeting endoplasmic reticulum stress. Mol. Clin. Oncol. 2(1): 3–7. doi:10.3892/mco.2013.202. PMID:24649299. Yamamoto, A., Tagawa, Y., Yoshimori, T., Moriyama, Y., Masaki, R., and Tashiro, Y. 1998. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 23(1): 33–42. doi:10.1247/csf. 23.33. PMID:9639028. Yoshida, H. 2007. ER stress and diseases. FEBS J. 274(3): 630–658. doi:10.1111/j. 1742-4658.2007.05639.x. PMID:17288551. Yu, W., Simmons-Menchaca, M., Gapor, A., Sanders, B.G., and Kline, K. 1999. Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Nutr. Cancer, 33(1): 26–32. doi:10.1080/01635589909514744. PMID:10227040.

Published by NRC Research Press

beclin-1 in breast cancer cells.

Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: a protective role.

Fluoxetine induces cytotoxic endoplasmic reticulum stress and autophagy in triple negative breast cancer.

Targeting BIP to induce Endoplasmic Reticulum stress and cancer cell death.

Endoplasmic reticulum stress and cancer.

Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage.

BIP to induce endoplasmic reticulum stress, autophagy and apoptosis.

Endoplasmic Reticulum Stress, Unfolded Protein Response, and Cancer Cell Fate.

Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders.

Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells.

PERK induces resistance to cell death elicited by endoplasmic reticulum stress and chemotherapy.

Cancer Microenvironment and Endoplasmic Reticulum Stress Response.

Endoplasmic reticulum stress, genome damage, and cancer.

Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation.

Endoplasmic reticulum stress in endometrial cancer.

Ampelopsin-induced autophagy protects breast cancer cells from apoptosis through Akt-mTOR pathway via endoplasmic reticulum stress.

Methylglyoxal induces cell death through endoplasmic reticulum stress-associated ROS production and mitochondrial dysfunction.

autophagy pathway is involved in cholesterol-induced pancreatic β-cell injury.

XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death.

Endoplasmic reticulum and autophagy in rat hepatocytes.

Carfilzomib Triggers Cell Death in Chronic Lymphocytic Leukemia by Inducing Proapoptotic and Endoplasmic Reticulum Stress Responses.

Long and short (timeframe) of endoplasmic reticulum stress-induced cell death.

Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells.