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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Oleandrin induces apoptosis via activating endoplasmic reticulum stress inbreast cancer cells

Xiao-xi Lia, Da-qing Wangb, Cheng-guang Suia, Fan-dong Menga, Shu-lan Sunc, Jian Zhenga,You-hong Jianga,*aMolecular Oncology Laboratory of Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, PR Chinab The People’s Hospital of Liaoning Province, No.33 Wenyi Road, Shenhe District, Shenyang, Liaoning, PR Chinac Central Laboratory, Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute, No. 44 Xiaoheyan Road, Dadong District, Shenyang, Liaoning, PRChina

A R T I C L E I N F O

Keywords:OleandrinBreast cancerApoptosisEndoplasmic reticulum stress

A B S T R A C T

Background: Breast cancer is the most common malignant tumor in women. Due to limited treatment outcomeand high rate of metastasis, the prognosis is especially poor for triple-negative breast cancer. It is urgent todiscover and develop novel agents for treatment of breast cancer. Herein, we investigated the potential me-chanisms of Oleandrin’s (a cardiac glycoside) cytotoxic activity against breast cancer cells.Methods: Cell proliferation was assessed by xCELLigence Real-Time Cell Analyzer (RTCA)-MP system. Apoptoticcells were detected by using Annexin V/PI staining and nuclear fragments observation. The effect of oleandrin onATP1B3 expression and markers of ER stress were determined by western blot. A primary cell sensitivity assaywas performed via a collagen gel droplet-embedded culture drug sensitivity method (CD-DST).Results: Oleandrin suppressed cell proliferation and colony formation in the three breast cancer cell lines but didnot affect normal mammary epithelial cells. Additionally, the expression of ATP1B3 was higher in the threebreast cancer cell lines compared to MCF10A cells. Treatment with oleandrin increased the number of apoptoticcells and led to nuclear pyknosis, fragmentation, and apoptotic body formation in breast cancer cells.Furthermore, oleandrin treatment increased expression of Bax and Bim but decreased that of Bcl-2. Treatmentwith oleandrin also upregulated the expression of endoplasmic reticulum stress associated proteins, includingeIF2α, ATF4, and CHOP, but not PERK. oleandrin treatment also induced the phosphorylation of PERK andeIF2α. Of note, oleandrin exhibited antitumor effects on patient-derived breast cancer cells under three-di-mensional culture conditions.Conclusions: Taken together, our results suggest that oleandrin induces mitochondrial-mediated apoptosis byactivating endoplasmic reticulum stress in breast cancer. Moreover, oleandrin may be an effective strategy forthe treatment of breast cancer.

1. Introduction

Breast cancer is the most common malignant tumor in women withapproximately 2.89 million new cases per year and 626,000 deathsworldwide in 2018 [1,2]. Currently, the treatment of breast cancerrelies on surgery, chemotherapy, radiotherapy, endocrine therapy andantibody therapy [3]. The prognosis is especially poor for triple-nega-tive breast cancer, which accounts for 15 % of breast carcinomas, due tolimited treatment options and high rate of metastasis [4,5]. Althoughthe standard treatment and novel drug delivery system has improvedsurvival, further improvements must be made to increase survival

[6–9]. Therefore, it is crucial to discover new and more effective drugs.Cardiac glycosides (CGs), derived from Digitalis, such as digoxin,

digitoxin, and ouabain, have been used to treat congestive heart failureand several types of arrhythmias [10]. In myocytes, CGs increase theNa+ influx and decrease the K+ influx through inhibition of Na+/K+-ATPase [11]. The increased intracellular Na+ concentration activatesthe exchange of Na+ and Ca2+, and the resulting increased the in-tracellular Ca2+ concentration increases cardiomyocyte contractility.Recently, studies have discovered the antitumor activity of CGs [12,13].UNBS1450, which belongs to the CG family, induces tumor apoptosis byregulating the cleavage of caspase 8, 9, and 3/7 via the NF-κB pathway

https://doi.org/10.1016/j.biopha.2020.109852Received 25 November 2019; Received in revised form 27 December 2019; Accepted 10 January 2020

⁎ Corresponding author at: Molecular Oncology Laboratory of Cancer Research Institute, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, 110001, PR China.

E-mail address: jiangyouhong2000@aliyun.com (Y.-h. Jiang).

Biomedicine & Pharmacotherapy 124 (2020) 109852

0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

T

[14]. Frese demonstrated that CGs upregulates DR4 and DR5, de-creasing the resistance of lung cancer cells to Apo2/TRAIL-inducedapoptosis [15]. Previous studies have shown that treatment with the CGdigitalis effectively reduces the mortality rate of patients with breastcancer, demonstrating the efficacy of CGs in breast cancer treatment[16]. However, the therapeutic use of CGs is limited due to their car-diovascular toxicity. Some studies have shown that treatment with CGsat nanomolar concentrations exhibits anticancer effects without af-fecting healthy cells [17–19].

Oleandrin, a type of CG, induces mitochondrial apoptosis in humancolorectal cancer cells [20]. Moreover, oleandrin suppresses cell pro-liferation, colony formation, and invasion in triple negative breastcancer cells via inhibition of the STAT-3 pathway [21]. However,whether oleandrin induces breast cancer cell apoptosis is not wellknown. Herein, we show that oleandrin suppresses cell proliferation inthree human breast cancer cell lines at nanomolar concentrationswithout affecting mammary epithelial cells. Furthermore, oleandrininduces cell apoptosis through endoplasmic reticulum stress.

2. Materials and methods

2.1. Cell lines and cell culture

The mammary epithelial cell line MCF10A was purchased from theAmerican Type Culture Collection (ATCC). Human breast cancer celllines: MDA-MB-231, MCF7, and SK-BR-3 were obtained from the CellBank of the Chinese Academy of Sciences (Shanghai, China). MCF10Acells were cultured in MEGM (Lonza, Morristown, NJ, USA) with100 ng/ml cholera toxin. MDA-MB-231 cells were cultured in LeibovitzL-15 (Hyclone, GE Healthcare Life Sciences, Logan, UT, USA). MCF7cells were cultured in minimum Eagle’s medium (Hyclone, GEHealthcare Life Sciences, Logan, UT, USA). SK-BR-3 cells were culturedin high-glucose Dulbecco’s Modified Eagle’s medium (Hyclone, GEHealthcare Life Sciences, Logan, UT, USA). All medium contained 10 %fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Inc.,Waltham, MA, USA). The cells were cultured at 37 °C in a 5 % CO2

incubator (Thermo Fisher Scientific, Inc.).

2.2. Patients

This study was approved by the ethics committee of the LiaoningCancer Hospital Ethics Review Approval No. 20181235. A total of 20female participants with pathologically diagnosed breast cancerwithout neoadjuvant chemotherapy was enrolled in the present study.Informed consents were provided to all the participants. The clinicalfeatures of 20 participants were shown in Table 1.

2.3. Compounds and antibodies

Oleandrin was purchased from MedChemExpress (MonmouthJunction, NJ, USA) and its chemical structure was shown in Fig. 1.Oleandrin was dissolved in sterile dimethyl sulfoxide (DMSO), and thefinal concentration of DMSO is less than 0.1 % in all experiment. Theinformation of primary antibodies as follow: anti-Bax#2774, anti-Bim#60434 and anti-β-actin#3700were purchased from Cell SignalingTechnology, Inc. Danvers, MA, USA. The anti-Bcl-2(33-6100) and Su-perSignal West Pico PLUS Chemiluminescent Substrate were obtainedfrom Thermo Fisher Scientific, Inc. Anti-PERK(ab79483), anti-eI-F2α(ab5369), anti-elF2α (phospho S52)(ab227593), anti-ATF4(ab23760), anti-CHOP(ab11419), goat anti-mouse IgG-horse-radish peroxidase (HRP)(ab205719), goat anti-Rabbit IgG-HRP(ab6721), anti-PERK (phospho T982)(ab192591) and anti-ATP1B3 an-tibody (ab137055) were purchased from Abcam (Cambridge, MA,USA).

2.4. Cell viability assay

Before we performed xCELLigence Real-Time Cell Analyzer (RTCA)-MP system (ACEA Biosciences, Inc., San Diego, CA, USA) experiment,the IC50 of Oleandrin in MCF-7, SK-BR-3 and MB-MDA-231 cells wasdetermined by MTT assay. The IC50 of Oleandrin ranged from 1 to30 nM. Base on this result, the Real-Time Cell Analyzer experimentwere designed. Cell concentration was adjusted to 1× 105 cells/ml incomplete medium. Next, 100 μl of cell suspension were seeded into E-16plates, which contained a biocompatible microelectrode array. The E-16 plates and the device were placed in an incubator. Electrical im-pedance, which reflects cell viability, was monitored in every 5min andpresented as the cell index. The cell index was normalized to the timepoint of agent treatment.

2.5. Clonogenic assay

A total of 2×103 cells were seeded into a 6-well plate in 2mlcomplete medium. When the cells were attached to the plate after 18 h,oleandrin was added into each well at the half-maximal inhibitoryconcentration (IC50) concentration. After 24 h treatment, cells werethen rinsed twice with PBS and cultured in fresh medium withoutoleandrin for 14 days. Subsequently, the colonies were fixed with for-malin and stained with crystal violet.

Table 1Clinical characteristics of the participants.

NO. Age cTNMa ER PR C-erbB-2 Ki67 Moleculer subtypes

1 62 T2N1M0 90 % 40 % ++ 8 % lumina A2 54 T3N1M0 70 % 80% – 10 % lumina A3 56 T2N2bM0 60 % 30 % + 10 % lumina A4 47 T1cN0M0 90 % 30 % + 10 % lumina A5 61 T2N0M0 90 % 95 % + 12 % lumina A6 67 T2N1M0 10 % 8 % +++ 10 % lumina B7 72 T3N2bM0 90 % 70 % ++ 20 % lumina B8 61 T2N0M0 95 % 90 % +++ 10 % lumina B9 58 T2N0M0 90 % 2 % +++ 50 % lumina B10 54 T2N1M0 90 % 90 % ++ 35 % lumina B11 63 T3N1M0 – – +++ 60 % HER-2+

12 62 T3N0M0 – – +++ 70 % HER-2+

13 46 T2N1M0 – – +++ 60 % HER-2+

14 53 T2N0M0 – – +++ 8 % HER-2+

15 57 T2N1M0 – – +++ 35 % HER-2+

16 48 T3N1M0 – – + 40 % TNBC17 72 T3N0M0 – – ++ 30 % TNBC18 64 T2N0M0 – – – 60 % TNBC19 67 T3N2bM0 – – ++ 70 % TNBC20 62 T2N2bM0 – – ++ 50 % TNBC

Abbreviation: ER, estrogen receptor. PR, progesterone receptor. C-erbB-2, erb-b2 receptor tyrosine kinase 2.

a TNM staging is carried out according to the American Joint Committee onCancer (AJCC) eighth edition standard.

Fig. 1. The chemical structure of Oleandrin.

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2.6. Apoptosis analysis

The 5×105 cells harvested in each group were stained with 5 μlAnnexin V-fluorescein isothiocyanate (FITC) and 5 μl propidium iodide(PI) in 500 μl buffer. After 10min, cells were rinsed twice with PBS.DMSO-treated cells served as the untreated control. Apoptosis wasanalyzed by flow cytometry (BD Accuri™ C6, BD Biosciences, FranklinLakes, NJ, USA).

2.7. Nuclear morphology characterization

The cells were treated with oleandrin for 24 h. Cells were thenrinsed twice with PBS and stained with Hoechst 33342 Solarbio,Beijing, China at a concentration of 10 ug/ml at 37 °C. After 30min,cells were washed with PBS 3 times and observed under a DMi8fluorescent microscope Leica, Wetzlar, Germany.

2.8. Western blotting analysis

The treated and control cells were harvested and protein was ex-tracted for western blotting. Primary antibodies were diluted accordingto the product description and incubated at 4 °C overnight. The sec-ondary antibodies (goat anti-mouse IgG-HRP (1:10,000) or goat anti-rabbit IgG-HRP (1:15,000) against the primary antibodies were applied.Immunodetection was performed using a Bio-Rad GelDoc XR+ system(Bio-Rad Laboratories, Inc., Hercules, CA, USA).

2.9. Primary breast cancer cell sensitivity assay

A primary cell sensitivity assay was performed with collagen geldroplet-embedded culture drug sensitivity method (CD-DST; Kurabo,Osaka, Japan) as previously described [22]. Briefly, breast cancerspecimens were obtained from patients undergoing surgical resection.Tissues were isolated and digested with EZ enzyme (Nitta Gelatin Inc.,Osaka, Japan). Disassociated primary breast cancer cells were culturedin a collagen gel-coated flask (Nitta Gelatin Inc.) for 24 h. Adherentcells were then harvested and seeded in three-dimensional (3D) col-lagen droplets, and then treatment with 20 nM or 40 nM oleandrin.After 24 h of treatment, the medium was removed and replaced withprepared culture media-2 (PCM-2; Kurabo) without FBS for 7 days.Droplets were stained with neutral red and fixed with formalin. Cellviability relative to optical density was analyzed using the PrimageSystem (Solution Systems, Tokyo, Japan).

2.10. Statistical analysis

The results were analyzed via one-way analysis of variance by usingthe SPSS software (version 21, IBM Corp., Armonk, NY, USA). Multiplecomparisons between the control and treatment groups were evaluatedusing Dunnett’s Least Significant Difference test. Data is presented asmean ± SD. P < 0.05 was considered to be statistically significant.

3. Results

3.1. Oleandrin suppresses the proliferation and colony formation of breastcancer cells, but not mammary epithelial cells

To investigate the inhibitory effects of oleandrin on breast cancercell viability, MCF7 (luminal A subtype), SK-BR-3 (HER-2+ subtype),and MDA-MB-231 (triple negative breast cancer, TNBC) cells weretreated with oleandrin, then cell proliferation was monitored using theRTCA-MP system every 5min for 72 h. The control group was treatedwith DMSO. As shown in Fig. 2A, oleandrin significantly inhibits breastcancer cell growth (MCF-7, SK-BR-3 and MDA-MB-231) in a dose- andtime-dependent manner. Of note, oleandrin did not affect MCF10Aproliferation, even at higher concentrations (Fig. 2A). The IC50 of

oleandrin at 24 h and 48 h for MCF7 was 14.5 and 6.07 nM, respec-tively; for SK-BR-3 cells, 6.13 and 1.42 nM, respectively; and for MDA-MB-231 cells, 24.62 and 11.47 nM, respectively. Proliferation of each ofthe three molecular subtypes of breast cancer cells was inhibited byoleandrin at nanomolar level. To further demonstrate this result, breastcancer cells were treated with oleandrin and colony formation wasanalyzed (Fig. 2B). Oleandrin significantly suppressed colony formationof the three breast cancer cell lines. Previous studies have suggestedthat the Na+/K+-ATPase α3 subunit (ATP1B3) may be the target ofoleandrin [23]. Herein, the protein expression level of ATP1B3 in 3breast cancer cells (MCF7, SK-BR-3, MDA-MB-231) and MCF10A weredetermined by western-blot analysis. As result shown in Fig. 2C, theexpression level of ATP1B3 was lower in MCF10A than MCF7, SK-BR-3,and MDA-MB-231 cells. This result suggests that the cytotoxic activityof oleandrin against breast cancer cells may be associated with theexpression level of ATP1B3.

3.2. Effect of oleandrin on inducing apoptosis of breast cancer cells

Given that oleandrin exhibited potent cytotoxic effect in breastcancer cells, we further investigate the effects of oleandrin on apoptosisof breast cancer cells, including non-triple negative breast cancer cellline MCF7 and TNBC cell line MDA-MB-231. After treated with olean-drin at IC50 concentration for 24 h, apoptotic cells were analyzed bystaining with Annexin V-FITC and PI. DMSO-treated cells served as thecontrol. Apoptotic cells were defined as either Annexin V-FITC+ PI−

(early apoptosis) or Annexin V-FITC+ PI+ (late apoptosis). Comparedto the DMSO-treated control groups, the number of apoptotic cells in-creased in the breast cancer cells after oleandrin treatment (Fig. 3A).Additionally, oleandrin-induced apoptosis was further assessed withnuclear staining and observation by fluorescence microscopy. As seenin Fig. 3B, nuclear pyknosis, fragmentation, and apoptotic bodies wereobserved in oleandrin-treated breast cancer cells, whereas the controlcells displayed normal round nuclei. These results suggest that olean-drin induces breast cancer cell apoptosis.

3.3. Oleandrin initiates mitochondrial apoptosis signaling pathway

Endogenous activation of the mitochondrial-signaling pathway isinvolved in apoptosis regulation. The Bcl-2 family of proteins, includingthe anti-apoptotic protein Bcl-2 and pro-apoptotic proteins Bax andBim, serve important roles in the mitochondrial apoptotic pathway[24]. In the present study, Bcl-2, Bax, and Bim were detected by wes-tern blot. As presented in Fig. 3C, compared to the control group, theexpression levels of Bax and Bim were upregulated and Bcl-2 wasdownregulated following treatment with oleandrin. Overall, these re-sults demonstrate that oleandrin-induced apoptosis is associated withthe mitochondrial apoptosis signaling pathway.

3.4. Oleandrin activates ER stress in breast cancer cells

ER stress plays an important role in the regulation of the mi-tochondrial apoptosis pathway [25]. In the present study, the key ERstress proteins were examined after 12 h of oleandrin treatment. Ex-pression of PERK, a protein located on the ER that detects ER stress, didnot significantly differ between the control and oleandrin-treatedgroups. However, phospho-PERK increased after treatment witholeandrin in both MCF7 and MDA-MB-231 cells. Phospho-PERK phos-phorylates eIF2α, phospho-eIF2α activates ATF4, and ATF4 furtherupregulates CHOP [26]. As shown in Fig. 4, increased expression andphosphorylation of eIF2α correlates with increased expression of ATF4and CHOP. These results suggest that the ER stress pathway is activatedin oleandrin-treated MCF7 and MDA-MB-231 cells.

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3.5. Effect of oleandrin on the proliferation of primary breast cancer cells

To investigate whether oleandrin may be a potential therapeuticdrug for breast cancer, the primary breast cancer cells were treated witholeandrin via a CD-DST assay. DMSO treated cells served as the control.

Cells with high viability were prominently stained with neutral red(Fig. 5). Compared with the control group, the cell viabilities werereduced by oleandrin in Lumina A subtype, Lumina B subtype, HER-2+

subtype and TNBC.

Fig. 2. Oleandrin inhibits the cell growth and colony formation in human breast cancer cells, but does not affect growth of normal mammary epithelial cells. (A) Atotal of 1× 104 human mammary epithelial cells (MCF10A) and human breast cancer cells (MCF7, SK-BR-3, and MDA-MB-231) were treated with oleandrin.MCF10A was treated with concentrations ranging from 200 nM to 3.13 nM. MCF7 and SK-BR-3 cells were treated with concentrations ranging from 50 nM to0.78 nM. MDA-MB-231 was treated with concentrations ranging from 100 nM to 1.56 nM. DMSO-treated cells were used as control. The cell index was normalized tothe starting point of oleandrin treatment, and 100 % was set as the normalized cell index from control group. The relative cell viability of oleandrin treatment groupwas analyzed, and IC50 was calculated. (B) A total of 1000 cells of MCF7, SK-BR-3 and MDA-MB-231 were seeded into 6-well plate and treated with oleandrin at theconcentration of IC50 or 2× IC50 for 24 h. Cells were cultured for 14 days and the number of colonies was counted. (C) The expression of ATP1B3 in MCF10A, MCF7,SK-BR-3, and MDA-MB-231 cells was determined by using western blot. β-actin was used as a loading control. Data are presented as the mean ± SD of the mean fromthree independent experiments **P < 0.01 vs. control.

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Fig. 3. Oleandrin induces apoptosis in human breast cancer.(A) A total of 1× 105 MCF7 and MDA-MB-231 cells were treated with oleandrin or DMSO for 24 h, and then stained with Annexin V-FITC and PI. Cell apoptosis wasdetected using flow cytometry. The Annexin V-FITC+PI−/+ cells were compared to control groups. **P < 0.01 vs. control. B Control and oleandrin-treated cellswere stained with Hoechst 33342, and observed by fluorescence microscopy. C MCF7 and MDA-MB-231 cells were treated with oleandrin or DMSO for 24 h. Theexpression of Bcl-2, Bim, and Bax was detected by western blot using β-actin as a loading control.

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4. Discussion

Oleandrin, isolated from the leaves of Nerium oleander, is cytotoxicto a variety of tumors. Compared to other CGs (including gitoxigenin,convallatoxin, strophanthidin, and neriifolin), oleandrin selectively andmore strongly suppresses colon cancer cell growth without reducing theviability of normal human colonic epithelial cells. Na+/K+-ATPase α3subunit maybe the target of oleandrin, and this may explain the se-lectivity [23,27]. In the present study, the expression of the Na+/ K+-ATPase α3 subunit in mammary epithelial cell line MCF10A and breastcancer cell lines MDA-MB-231, MCF7, and SK-BR-3 were comparedusing western blot. The result showed that protein expression of theNa+/K+-ATPase α3 subunit was lower in MCF10A compared to thebreast cancer cell lines. We observed that oleandrin significantly in-hibited proliferation in MDA-MB-231, MCF7, and SK-BR-3 cells, but notin MCF10A cells. In addition, the genotype background of 3-breastcancer cells as follow: MCF-7 (Lumina A), SK-BR-3 (HER2+), MDA-MB-231 (TNBC). The IC50 of Oleandrin in MCF7, SK-BR-3 and MDA-MB-231is 14.5, 6.12 and 24.62 nM, respectively. SK-BR-3 exhibits more sensi-tive to Oleandrin than MCF7 and MDA-MB-231 cells, this result may beassociated with high-level expression of ATP1B3 in SK-BR-3 cells. In thefuture, we will provide the evidences that Oleandrin could directlytarget ATP13B in vitro and in vivo breast cancer model [28,29].

Based on the expression level of human epidermal growth factorreceptor 2 (HER2), hormone receptor (HR) including estrogen (ER) andprogesterone (PR), breast cancers can be classified into 5 subtypes:Luminal A (HR+/HER2-), Luminal B (HR+/HER2+), HER2-enriched(HR-/HER2+), basal-like (about 75 % of this type of breast cancers

belongs to triple-negative) and normal breast-like tumors [4]. HR- orHER2-targeted therapy have successfully used for the treatment of Lu-minal A, Luminal B and HER2-enriched breast cancers[]. Due to loss ofmolecular targets, no therapeutic responses were observed in TNBC,and cytotoxic chemotherapy is the only available treatment option forTNBCs [30,31]. Though cardiac glycosides exhibit potent cytotoxicactivity in breast cancer at low-nM level [27,32,33]. Of note, moreevidences demonstrated that cardiac glycosides bind with ER and canincrease risk of breast and uterus cancer incidences (risk ratios:1.3-1.5)[34]. Thus, we will consider the side effect and therapeutic efficacy ofoleandrin in breast cancers in the future, especially in ER+breastcancer.

Apoptosis plays a key role in cancer cell death after therapy [35].Previous studies have shown that the NF-κB pathway, the mitochon-drial apoptosis pathway, and DR4 and DR5 are involved in CG-inducedapoptosis. However, in the present study, we showed that ER stress wasinvolved in oleandrin-induced apoptosis in breast cancer cells. The ERis important for the regulation of intracellular Ca2+ homeostasis, pro-tein folding, and protein maturation [36,37]. Several studies havefound that ER stress is associated with apoptosis [38]. Hypoxia, Ca2+

concentration, and oxidative injury affect ER homeostasis and hinderprotein folding [39–41]. The result of unfolded protein accumulation inthe ER is ER stress. ER stress triggers a stress response via the unfoldedprotein response (UPR) pathway. The three major sensors on the ERthat regulate ER stress are inositol-requiring protein 1 alpha (IRE1-α),activating transcription factor 6 (ATF6), and protein kinase RNA-likeendoplasmic reticulum kinase (PERK) [42]. Activated PERK increasesphosphorylation of Ser at position 51 in eukaryotic initiation factor 2

Fig. 4. Oleandrin induces apoptosis via activating ER stress.MCF7 and MDA-MB-231 cells were treated with oleandrin for 24 h. The expression of PERK, p-PERK, elF2α, p-elF2α, ATF-4 and CHOP was detected by using westernblot. β-actin was used as loading control.

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alpha (eIF2α) which inhibits global protein synthesis. The phosphory-lated eIF2α promotes expression of activating transcription factor 4(ATF4) and C/EBP homologous protein (CHOP). CHOP is a critical pro-apoptosis transcription factor that regulates the expression of Bcl-2family proteins [43,44]. In the present study, we observed that olean-drin induced MDA-MB-231 and MCF7 cell apoptosis after 24 h, whichwas represented by Annexin V-FITC+ PI−/+ staining and Hoechst33342 staining. Furthermore, oleandrin treatment increased the ex-pression of Bim and Bax and decreased the expression of pro-survivalprotein Bcl-2. The Bcl-2 family of proteins regulates the mitochondrialapoptosis pathway. Our results are consistent with a previous studyshowing that oleandrin induces apoptosis in human colon cancer cellsvia the mitochondrial pathway. To further investigate the possiblemechanism involved in oleandrin-induced mitochondrial apoptosis, theexpression of UPR downstream signaling proteins such as PERK wereanalyzed. After 6 h of oleandrin treatment, PERK, p-PERK and p-eIF2αwere upregulated in MDA-MB-231 and MCF7 cells, but eIF2α was notaffected. Moreover, ATF4 and CHOP, downstream targets of the PERK

pathway, were upregulated. The activation of PERK signaling pathwayprecedes apoptosis.

5. Conclusions

Taken together, our results suggest that oleandrin induces mi-tochondrial-mediated apoptosis by activating endoplasmic reticulumstress in breast cancer. Moreover, oleandrin may be an effectivestrategy for the treatment of breast cancer.

Funding

This work was supported by Natural Science Foundation of LiaoningProvince (20180550618 to Shu-lan Sun) and Science and TechnologyPlan Project of Liaoning Province(NO.2013225585).

Fig. 5. Oleandrin decreases the viability of primary breast cancer cells.Primary breast cancer cells were obtained from lumina A, lumina B, HER-2+ and TNBC patients. Oleandrin sensitivity was evaluated by a collagen gel droplet-embedded culture drug sensitivity assay. Cell viabilities were quantified by staining with neutral red and detected by image analysis. The results presented wererepresentative of each subtype patients. The cell viability of control group was set as 100 %. Data are presented as the mean ± SD from three droplets. **P < 0.01vs. control.

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Authors' contributions

X Li and D Wang mainly performed the experiments and drafted themanuscript. X Li, D Wang, C Sui, F Meng, S Sun, J Zheng and Y Jiangplayed an important role in coordinating the study and performing theanalysis with constructive discussions. X Li, D Wang, C Sui, F Mengprepared the samples and performed the experiments; X Li and Y Jiangconceived the study and revised the manuscript. All authors discussedthe results and contributed to the writing and editing of the manuscript.

Declaration of Competing Interest

The authors have no conflict of interest.

Acknowledgement

Not applicable.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.biopha.2020.109852.

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