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CD24- phenotype breast cancer stem cells
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Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells Babak Namia,* , Huseyin Donmezb , Nadir Kocakb a b
Department of Medical Genetics and Women and Children’s Health Research Institute (WCHRI), University of Alberta, Edmonton, Canada Department of Medical Genetics, Selcuk University Medical Faculty, Konya, Turkey
A R T I C L E I N F O
Article history: Received 21 April 2016 Accepted 13 June 2016 Keywords: Tunicamycin Endoplasmic reticulum stress Breast cancer Cancer stem cells CD44 CD24
A B S T R A C T
Tunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis has role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. In this study we show effect of tunicamycin on subpopulation and invasion of CD44+/CD24- MCF7 breast cancer stem cells. CD44+/CD24- cells were isolated from MCF7 cell line by fluorescence activated cell sorting (FACS) and treated with tunicamycin. ER stress was monitored by evaluation of X-box binding protein 1(XBP-1) mRNA splicing, cleaved activating transcription factor 6 (ATF6) nuclear translocation and CCAAT/ enhancer-binding protein homologous protein (CHOP) expression. CD44+/CD24- subpopulation was analyzed using flow cytometry. Invasion was investigated by scratch assay, trypan blue staining, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation and in vitro migration assays. Increased level of spliced XBP-1, ATF6 nuclear translocation and CHOP protein expression were detected in CD44+/CD24- and original MCF7 cells treated with tunicamycin. Also, a significant decline in CD44+/CD24- cell subpopulation was determined in the cells treated with tunicamycin. The results also showed inhibited invasion, increased cell death, suppressed proliferation and reduced migration in the CD44+/CD24- and CD44+/CD24- rich MCF7 cell culture, under effect of tunicamycin. Our results indicate that CD44+/CD24- phenotype MCF7 cells are susceptible to tunicamycin. The results showed that tunicamycin-induced ER stress suppresses CD44+/CD24- phenotype cell subpopulation and in vitro invasion and accelerates tumorosphore formation. These results suggest that tunicamycininduced ER stress inhibits CD44+/CD24- phenotype MCF7 breast cancer stem cells. We conclude that using ER-targeting chemicals like tunicamycin is an interesting approach to target breast cancer stem cells inside tumor. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction Emerging evidence has suggested that the capability of a tumor to grow and its resistance against therapies is dependent on a small subset of cells within a tumor, called cancer stem cells (CSCs). Cancer stem cells have been reported to express stem cell surface markers and possess self-renewal potency. It has been proposed that these cells are only cells with the ability to regenerate malignant cells and force the growth of the cancer (Lobo et al.,
* Corresponding author. E-mail addresses: [email protected] (B. Nami), [email protected] (H. Donmez), [email protected] (N. Kocak).
2007). Breast CSCs have been characterized to have the cell surface phenotype CD44+/CD24 (Al-Hajj et al., 2003). CD44 is a celladhesion molecule involved in binding of cells to hyaluronic acid (Peach et al., 1993), whereas CD24 is a regulator of chemokine (CX-C motif) receptor 4 (CXCR4) that involved in breast tumor formation in low level expression (Schabath et al., 2006), Generally, CD44+/CD24- phenotype cells represent averagely 1–4% of all cells in a breast tumor and their population enhancement exhibits increased tumorigenicity and invasion (Al-Hajj et al., 2003; Sun et al., 2013). Although data have been provided to support resistance of CSCs against death inducing mechanisms (Sun et al., 2013). researchers believe CSCs could be Achilles' heel of cancer in CSC-targeted therapies (Wicha, 2014).
http://dx.doi.org/10.1016/j.etp.2016.06.004 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: B. Nami, et al., Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.004
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In this study we investigated cytotoxic effect of tunicamycin in CD44+/CD24- phenotype breast cancer stem cells. Tunicamycin is an antibiotic that inhibits glycoprotein processing by blocking UDP-HexNAc: polyprenol-P HexNAc-1-P family of enzymes in human the enzyme GlcNAc phosphotransferase (GPT) (Duksin and Bornstein, 1977; Heifetz et al., 1979). Tm inhibits N-linked glycosylation of proteins and causes blocking of DNA synthesis and cell cycle arrest in G1 phase (Ettlinger et al., 1986; Vai et al., 1987). N-linked glycosylation is essential for protein folding and inhibition by Tm impede proteins folding correctly eventually causes accumulation unfolded proteins in the endoplasmic reticulum (ER) lumen (Parodi, 2000; Ware et al., 1995). Accumulation of unfolded protein in ER lumen is a toxic phenomenon that leads unfolded protein response (UPR). UPR is characterized by endoplasmic reticulum (ER) stress a cellular mechanism that sets up in response to unfolded protein accumulation in the ER lumen (van den Berg et al., 2000; Chakrabarti et al., 2011). This mechanism is elucidated with activation of three different pathways parallel to each other which is promoted by the ER transmembrane proteins inositol-Requiring Enzyme 1a (IRE1a), PRKR-Like Endoplasmic Reticulum Kinase (PERK) and activating transcription factor 6 (ATF6) (Chakrabarti et al., 2011; Schroder and Kaufman, 2006). The signaling leads to regulation of asset of downstream genes through X-box binding protein 1(XBP-1), activating transcription factor 4 (ATF4) and cleaved activating transcription factor 6 (ATF6) transcription factors (Chakrabarti et al., 2011; Schroder and Kaufman, 2006). Essentially, ER stress pursues two primary aims including maintain normal cellular function by halting protein translation and synthesis (Brostrom and Brostrom, 1998) and activation of the signaling pathways leading to up-regulation of the molecular chaperones involved in protein folding and degradation (Shenkman et al., 2007). The failure to achieve these two objectives, the ER stress leads cell towards death via apoptosis (Tabas and Ron, 2011) and autophagy (Yorimitsu et al., 2006). ER stress is in charge of guarding the intracellular homeostasis by haltering cell survival and death mechanisms. It has been demonstrated that ER stress-induced cell death is regulated by Ca2 homeostasis and the pro-apoptotic protein CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) (Xu et al., 2005). ER stress has also a key role in development of inflammation (Hotamisligil, 2010), obesity and diabetes (Nakatani et al., 2005), neurodegenerative diseases (Cnop et al., 2012; Hetz and Mollereau, 2014), heart diseases (Yamaguchi et al., 2003) and cancer (Schonthal, 2013). The role of ER stress in promotion of calcium—induced apoptosis through CHOP protein has already been demonstrated (Han et al., 2013; Pyrko et al., 2007). Thus, ER stress has been suggested as a promising target for tumor cell death and cancer therapy. Effectiveness of tunicamycin as an anti-cancer chemical has been demonstrated in various human cancer models (de Freitas et al., 2011; Hou et al., 2013; Morin and Bernacki, 1983; Shiraishi et al., 2005). Therefore, in this study, we aimed to investigate the effect of tunicamycin-induced ER stress on breast CSC subpopulation and invasion in vitro. 2. Materials and methods 2.1. Chemical and antibodies Tunicamycin was purchased from Sigma-Aldrich (St. Louis, USA). It was dissolved in dimethyl sulfoxide (DMSO) and stored at +4 C. Optimum concentration of tunicamycin was determined by testing ranges 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg of tunicamycin in per milliliter of culture medium regarding cytotoxicity (tested by morphological observation and trypan blue staining) and ER stress activity (tested by evaluating XBP-1mRNA splicing) potentials (data not shown).
Rabbit anti human CHOP polyclonal IgG (sc-200), rabbit anti ATF6 (N-terminus) polyclonal IgG (sc-22799), rabbit anti human GAPDH polyclonal IgG (sc-25778), HRP conjugated goat anti rabbit polyclonal IgG (sc-2004) and FITC-conjugated goat anti rabit polyclonal IgG (sc-2012) antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, USA). FITC-conjugated mouse anti human CD44 monoclonal (MA1-12127) and PE-conjugated mouse anti human CD24 monoclonal (MA1-10154), FITC-conjugated Mouse IgG isotype control (MA1-10413) and PE-conjugated mouse IgG isotype control (MA1-10415) antibodies were from Thermo Scientific (Rockford, USA). 2.2. Cell culture MCF7 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) that was completed with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin (100 unit/ml) and 1% streptomycin (10 mg/ml) with incubation at 37 C in a 5% CO2 humidified incubator. After the cell culture reached 80% confluency, the cells were trypsinized with 0.25% trypsin and harvested. In order to perform the experiments, the cells were seeded as 2 104, 5 104, 105 and 5 105 cells in 96, 24, 12 and 6-well plates respectively in triplicate for each one and cultured at the condition maintained above for 24 h before start to treatment. 2.3. RNA isolation and XBP-1 mRNA splicing assay Total RNA was isolated using TRIzol reagent (Invitrogen, Waltham, USA) according to protocol described previously (Rio et al., 2010). Synthesis of cDNA from the total RNA was carried out using Transcriptor High Fidelity cDNA Synthesis1 (Roche, Basel, Switzerland) kit by applying the oligo (dT)18 primer pairs following the manufacturer's instructions. cDNA of XBP-1 was amplified by PCR using forward 50 -TTACGAGAGAAAACTCATGGCC30 and reverse primer 50 -GGGTCCAAGTTGTCCAGAATGC-30 . Primers sequences were obtained from literature (Samali et al., 2010) and were synthesized by Biomers Inc. (Ulm, Germeny). After the PCR cycling, the product was ran in 15% polyacrylamide gel by vertical electrophoresis (Bio-Rad Laboratories, Berkeley, USA) at 40 mA electric current for an hour. After the electrophoresis, the gel was stained with ethidium bromide solution and the spliced 263 bp and unspliced 289 bp XBP-1 bands were imaged in a gel documentation system (Bio-Rad Laboratories, Berkeley, USA). 2.4. Western blotting Total protein was extracted by adding proper volume of RIPA buffer (Sigma-Aldrich, St. Louis, USA) to cells and spining down. The protein samples were prepraed by adding a same volume of 4 x lammeli buffer and then incubation at 95 C for 5 min. Amount 40 mg of total protein was loaded onto each well of the % 4–15 gradiyant gel and was ran at 40 mA electric current for an hour. After the electrophoresis, the proteins transferred on the PVDF membrane (Bio-Rad Laboratories, Berkeley, USA) using a transBlot1 TurboTM Transfer system (Bio-Rad Laboratories, Berkeley, USA) at 1.3 A electric current for 7 min. Blocking of membrane was done with 5% non-fat milk solution for an hours in 50 rpm agitation. The immunoblotting was accomplished by incubation of the membrane with 1 mg/ml of primary antibody solution and then 0.1 mg/ml of secondary antibody both for 2 h at room temperature. At the end of the blotting, the membrane was incubated in Immunstar WesternC Chemiluminescent (Bio-Rad Laboratories, Berkeley, USA) substrate solution for 10 min then imaged by a gel documentation system.
Please cite this article in press as: B. Nami, et al., Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.004
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2.5. Immunofluorescence assay Cells were cultured on the immunofluorescence slides 48 h before treatment starts. After treatment period, the slides were rinsed in PBS and the cells were fixed by cold methanol for 5 min. Blocking was done with incubation of slides in 1% BSA solution for an hour. After the blocking, the slides were incubated in 1 mg/ml solution of the primary antibody for an hour. Afterwards, the slides were rinsed in PBS tree times each for 5 min. Thereafter, the slides were incubated in 1 mg/ml solution of secondary antibody for an hour in darkness then were washed completely in PBS and mounted by 40 ,6-diamidino-2-phenylindole (DAPI). The slides were observed under a confocal fluorescence microscope (Nikon, Tokyo, Japan). 2.6. Flow cytometry and FACS analysis Flow cytometry and fluorescence-activated cell sorting (FACS) analysis were performed as described previously (Nami et al., 2015).
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2 mg/ml of tunicamycin or same volume of PBS. After the period, cells were washed two times with PBS, fixed by 4% formaldehyde and then stained with acidified hematoxylin dye. At the end, the stain intensity of the migrated cell colonies in the threshold subsequent areas was measured by ImageJ software (NIH, Maryland, USA). 2.9. Trypan blue staining and MTT assays Trypan blue staining and MTT assays was done as described previously (Acar et al., 2015). 2.10. Statistical analysis The statistical significances were considered applying twotailed student’s t-test and analysis of variance (ANOVA) using the GraphPad Prism1 V.5.00 software. Also, the image base data were analyzed using ImageJ software (NIH, Maryland, USA), p < 0.050 was considered as statistically significant. 3. Results
2.7. Scratch assay 3.1. Tunicamycin stimulates ER stress in CD44+/CD24- MCF7 cells The cells were seeded in 6-well plates and cultured in the standard condition. After the culture reached about 80% confluency, scratches as plus sign with 4 mm in width were created on the middle of the plates. The cell culture was continued up to 24 h in presence of 2 mg/ml of tunicamycin or same volume of PBS. After this period, the cells were rinsed three time in PBS and fixed by 4% formaldehyde and then were stained with acidified hematoxylin dye (Sigma-Aldrich, St. Louis, USA). 2.8. In vitro migration assay Same amount of CD44+/CD24- enriched cells were seeded in corner of 100 mm plate and cultured for 48 h. After the cells attached, a threshold adjusted on 2 cm distance from the cell enclosure and the culture was continued up to 24 h in presence of
In result of dose determination, the optimum concentration of tunicamycin was determined as 2 mg/ml (data is not shown). To investigate CD44+/CD24- cell response to ER stress inducer, the cells we sorted from original MCF7 cell culture and treated with 2 mg/ml of tunicamycin for 24 h and then ER stress was monitored. Monitoring of ER stress was done by assessment of XBP-1 mRNA splicing, ATF6 protein translocation and CHOP protein expression. A result, enhancement in XBP-1 mRNA splicing rate was determined in both original MCF7 culture and CD44+/CD24MCF7 cells both treated with tunicamycin in comparison with related negative controls (Fig. 1A). The XBP-1 splicing is a marker to recognize activated ER stress through IREa sensor. Another sensor of ER stress is ATF6 transmembrane that migrates from ER to nucleus during ER stress. Nuclear translocation of ATF6 under
Fig. 1. Tunicamycin stimulates ER stress in CD44+/CD24- MCF7 breast cancer stem cells. The original MCF7 and FACS sorted CD44+/CD24- MCF7 cells were cultured with 2 mg/ ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 h. (A) Quantitative PCR result of 289 bp unspliced XBP-1 (uXBP-1) and 263 bp spliced XBP-1 (sXBP-1) showed a significant XBP-1 mRNA splicing enhancement in original MCF7 (p < 0.001) and CD44+/CD24- MCF7 cells (p < 0.001) due to treatment with tunicamycin for 24 h. (B) Confocal fluorescence image of FITC immunofluorescense staining of cleaved ATF6. Nuclear translocation of cleaved ATF6 was observed in original and CD44+/CD24- MCF7 cells due to 24 h’ treatment with tunicamycin. Scale of the bars is 10 mm. (C) Increased expression of CHOP protein in original and CD44+/CD24MCF7 cells due to 24 h’ treatment with tunicamycin that was evaluated with western blotting.
Please cite this article in press as: B. Nami, et al., Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.004
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effect of tunicamycin was also observed in CD44+/CD24- cells as well as original MCF7 cells compared to negative controls (Fig. 1B). Moreover, increased expression of CHOP protein, a downstream gene of ER stress, was determined in the both cell types treated with tunicamycin in comparison with negative controls (Fig. 1C). These result indicates high level activity of ER stress in the cells under effect of tunicamycin. To confirm this, we treated the cells with 2 mM dithiothreitol (DTT) as another ER stress stimulator agent for 24 h and monitored ER stress performing same methods. Results of DTT treated cells were as same as tunicamycin (data not shown).
3.2. Tunicamycin accelerates CD44+/CD24- MCF7 spheroid formation At the consequence of 24 h’ culture of original MCF7 cells in presence of 2 mg/ml of tunicamycin, a defined dismorpholgy related to stress was observed (Fig. 2A). Also, spheroids were detected in the CD44+/CD24- cells cultured in serum free medium after 4 days’ treatment with 2 mg/ml of tunicamycin in 2D culture environment (Fig. 2B).
3.3. Tunicamycin suppresses CD44+/CD24- MCF7 cell subpopulation In order to determine subpopulation of CD44+/CD24- cells under effect of tunicamycin-induced ER stress, we cultured MCF7 cells in presence of tunicamycin for 24 h and analyzed subpopulation of CD44+/CD24- cells in the same population of original MCF7 culture. According to the results shown in Fig. 5, the mean percentage of tunicamycin treated CD44+/CD24- cell subpopulation in the culture was 0.15%, while this value was 1.15% in untreated negative control cells (p < 0.010; Fig. 3). This result shows a declined subpopulation of CD44+/CD24- cells in the original MCF7 cell culture due to tunicamycin in comparison with negative controls.
3.4. Tunicamycin suppresses CD44+/CD24- cell invasion We established CD44+/CD24- enriched cell culture by adding FACS sorted CD44+/CD24- phenotype cells to the original MCF7 culture until its subpopulation reaches 10% in order to enhance involvement of CD44+/CD24- cell with tunicamycin treatments and exam conditions. Invasion was assessed by scratch, trypan blue viability, MTT and in vitro migration assays. The cells left to be cultured in presence of 2 mg/ml of tunicamycin or same volume of PBS for 24 h. Scratch assay was performed to investigate invasion of CD44+/CD24- rich cells. As result width of the wound in the cultures treated with tunicamycin was significantly greater than negative controls (p < 0.001; Fig. 4A). Trypan blue cell survival assay revealed that percentage of viable cells in tunicamycin treated cultures was 71.82%, whereas this value was 87.53% in the negative control cultures (p < 0.001; Fig. 4B). This result shows a dramatic death rate of CD24+/CD24- cells because of tunicamycin treatment. Moreover, significant reduction in the proliferation of both original MCF7 and CD24+/CD24- cells were determined in tunicamycin treated cells (p < 0.001; Fig. 4C). In order to validate these observation, we examined in vitro migration of CD44+/CD24rich cells under treatment with Tm. The results revealed that the number and distance from threshold of secondary colonies in tunicmycin treated cell cultures were significantly lower compared to negative controls (p < 0.001; Fig. 4D). This finding suggests that tuncamycin-induced ER stress could inhibit the cells migration in culture model. By and large, these results show that ER stress suppressed invasion of CD44+/CD24- MCF7 cells. Altogether these results revealed that tunicamycin inhibites the invasion of CD44 +/CD24- cells and cause significant regression (p < 0.010; Fig. 4E). 4. Discussion Tunicamycin cause UPR and ER stress by blocking N-linked glycosylation process in ER lumen. The cytotoxicity of tunicamycin in breast tumor cells has been demonstrated through it effect in ER
Fig. 2. ER stress causes MCF7 cell dysmorphology and accelerates tumorsphere formation. Attached original MCF7 and CD44+/CD24- MCF7 cells were treated with 2 mg/ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 h respectively in FCS containing and serum free culture medium. (A) dysmorphological evidence in attached original MCF7 cells due to activated ER stress. (B) CSCs spheroids were observed in the cell culture treated with tunicamycin for 4 days but not in negative controls. Scale of the bars is 100 mm.
Please cite this article in press as: B. Nami, et al., Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.004
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Fig. 3. ER stress decreases CD44+/CD24- MCF7 breast cancer stem cell subpopulation. MCF7 cells were cultured with 2 mg/ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 h and then the CD44+/CD24- cells population analyzed by flow cytometry. (A) Flow cytometry analysis plate of MCF7 cells in terms of CD44 and CD24 expression. (B) Decrease in the subpopulation of cells with CD44+/CD24- phenotype (P2 plate; p < 0.010) and increase in the subpopulation of else phenotypes cells due to activated ER stress (P1-P2; p < 0.010).
stress-mediate apoptosis and autophagy (Clarke et al., 2012; Han et al., 2013). ER stress is an essential mechanism for regulation of cellular homeostasis when the protein synthesis is impaired. It has been demonstrated that dysfunction of ER stress in many cancer cells can lead tumor cell to survival and growth (Schonthal, 2013). ER stress is identified with up-regulation of chaperon proteins, endoplasmic reticulum associated degradation (ERAD) genes, autophagy factors and also their positive regulators like XBP-1, CHOP, ATF4 and ATF6 that aid the cell in a struggle to survive. The other feature of ER stress is promotion of apoptotic and autophagic cell death via up-regulating CHOP and calcium influx (Xu et al., 2005). Consequently, this phenomenon acts as a double-edged sword its cutting edges have been used to target cancer. So far, the critical role of ER stress in regulation of breast cancer has been extensively explained (Clarke et al., 2012). Most recent reports have demonstrated that some ER stress factors especially XBP-1 and ER chaperon protein GRP78 have a key role in regulation of CSCs though insufficient supporting information (Chen and Zhou, 2005; Li et al., 2013). These results suggest that ER stress might have a couple of function in CSCs. We here showed that XBP-1 mRNA splicing, CHOP protein expression and ATF6 nuclear translocation is up-regulated in breast CSCs with CD44+/CD24phenotype when the cells are exposed to ER stress inducer agent tunicamycin (Fig. 5). Our data suggest that breast CSCs are susceptible to tunicamycin-induced ER stress and targeting of protein synthesis
machinery. Our results also support the notion that stimulation of ER stress in the cells causes reduction in the size of CD44+/CD24subpopulation. So that, the percentage of CSCs in the cell culture under ER stress condition was about eightfold lower than negative control, indicating that the reduction could be attributed to activated ER stress by tunicamycin. Apoptosis and autophagy are two canonical consequence of ER stress when CHOP protein is upregulated. Our results showed an up-regulation of CHOP protein in CSCs under the effect of tunicamycin. CHOP is a pro-apoptotic factor that is able to promote programmed cell death via apoptosis and autophagy pathways (Pyrko et al., 2007; Tabas and Ron, 2011; Yorimitsu et al., 2006). On the other hand, we recently reported that autophagic condition suppresses CD44+/CD24- cell subpopulation in MCF7 culture model (Nami et al., 2015). The considerable fact is that autophagy is a consequence event of ER stress per se. Reduced subpopulation of CSCs might due to CD44+/CD24- cells loss through ER stress-associated cell death. But the challenging point here is that CD44+/CD24- and other phenotypic cells were equally exposed to tunicamycin therefore showed same level upregulation of ER stress elements. With regard to identical response of all phenotype MCF7 cells to ER stress, it is expected equal decrease in subpopulation of all phenotypes and not just of CD44 +/CD24- cells. Unfortunately, it is not clear whether cancer stem cells arise by conversion of normal stem cells or tumor cell reverse differentiation. Furthermore, many studies have demonstrated that autophagy plays a prevail role in cellular differentiation during
Please cite this article in press as: B. Nami, et al., Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.004
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Fig. 4. ER stress suppresses CD44+/CD24- MCF7 breast cancer stem cell invasion. The original MCF7, CD44+/CD24- enriched MCF7 cells and CD44+/CD24- MCF7 cells were treated with 2 mg/ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 and then invasions were evaluated using different tests. (A) Scratch assay result of CD44+/CD24- enriched MCF7 cells showed an inhibited invasion in result of tunicamycin (p < 0.001). Scale of the bars is 1 mm. (B) Trypan blue staining results of CD44+/CD24- cells revealed a decreased viable (p < 0.001) and increased death cell due to tunicamycin (p < 0.001). (C) MTT assay elucidated a decrease in proliferation of both original MCF7 (p < 0.001) and CD44+/CD24- MCF7 cells (p < 0.001) in the result of tunicamycin. The letter A indicates absorbance. (D) Migrated cell colonies of CD44+/CD24- enriched MCF7 cells with activated ER stress at four different distance from the threshold were significantly lower in comparison with the negative controls (p < 0.001). (E) Fold change reduction in invasion of both original MCF7 and CD44+/CD24- MCF7 cells due to tunicamycin that obtained with ANOVA analysis of tests which described above. Considering to p values there is elicited that retreat of CD44+/CD24- MCF7 cells was greater than original MCF7 (p < 0.010).
development. Therefore, changes in self-renewal and differentiation potency of the cells might be due to ER stress-induced autophagic condition. Moreover, Salemi et al. (Salemi et al., 2012) reported that autophagy acts as a necessary factor in regulation of stem cell self-renewal and differentiation; however, functional properties of CSCs highly diverse in normal stem cells. Therefore, decrease in CSC subpopulation might also due to effect of ER stress on the CSC self-renewal and differentiation properties. Although, we did not investigate here the self-renewal and differentiation of CD44+/CD24- cells in this study. We showed a suppressed invasion of CD44+/CD24- MCF7 cells including reduced proliferation, survival and migration under
tunicamycin treatment. It is previously reported that breast CSCs with CD44+/CD24- phenotype are the core element for tumor growth, invasion and metastasis as well as its resistance against radio- and chemotherapies and relapse. Basically CSC mass inside a tumor is associated with enhanced invasive properties and elevated expression of genes involved in malignancy of the tumor (Sun et al., 2013; Wicha, 2014). Similarly, it has been revealed that CD44+/CD24- subpopulation in primary tumors has a direct linkage with tumor regrowth, malignancy and invasion and metastasis owing to its high capacity for DNA repair and resistance against chemotherapies (Al-Hajj et al., 2003; Sun et al., 2013; Wicha, 2014). A number of recent publications reported that CSCs
Please cite this article in press as: B. Nami, et al., Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.004
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subpopulation of CSCs inside original bulk cell culture. We suggest that studding ER stress in CSCs is a requisite in order to understand the biology of CSC inside tumors and also to develop ER stress inducing therapies. Although, ER targeted chemotherapeutics on cancer has not been clinically examined yet; evaluation of this type of treatment is suggested with focus on CSCs. Thus we conclude that using ER-targeting agents like tunicamycin is an effective choice to target breast cancer stem cells inside tumor.
Conflict of interest The authors declare no conflict of interest. Acknowledgments
Fig. 5. Model summarizing the effect of tunicamycin-induced ER stress in CD44 +/CD24- MCF7 breast cancer stem cell. Tunicamycin causes accumulation of unfolded proteins in ER lumen inducing unfolded protein response in the cell. In this condition mRNA of XBP-1 is spliced by endoribonuclease domain of ER transmembrane protein IRE1 and XBP-1 protein is synthesized. ATF6 is releases from ER membrane and then is localized in nucleus. Both XBP-1 and ATF6 are bzipper transcription factors which induces expression of CHOP protein. CHOP protein is a pro-apoptotic factor which can provoke cell death via different pathways.
may be inhibited by upregulation of ER stress factors. Given these, we suppose that tunicamycin-induced ER stress inhibited the cell invasion through suppression of CSCs in the culture, however further studies are needed to clarify. Recently, it was shown that breast cancer cell lines were also capable of generating non-adherent spheres termed tumorspheres. These CSC spheroids develop in the result of CSC selfrenewal activity that has morphologically been characterized previously (Al-Hajj et al., 2003). Formation of tumorspheres commonly requires specific culturing conditions such as celloptimized media with defined supplements to retain the cells undifferentiated. Therefore, culturing tumor cells in serumcontaining medium can lead cells to be differentiated with markedly distinct morphologies and growth characteristics. Despite the essential culture factors are needed for tumorshphere formation, it also requires a long culture period in some cases weeks to be formed. In contrast, our result revealed that tumorshphere could be formed in just 4 days under effect of tunicamycin without additional supplements. This shows that ER stress promotes a set of unknown mechanisms and accelerates tumorspheres formation in regular cell culture condition. Given that the exact reason of stem cell spheroid formation still remains unclear, we suggest investigation of possible function of ER stress in the formation of tumorspheres. Numerous reports have pointed out that CSCs can survive from the effects of chemotherapy and subsequently support the regrowth of the tumor, but the clinically relevant aspect is that this property can be utilized as a target for cancer therapy. Therefore, many researchers propagandize that CSCs could be Achilles' heel of cancer. So far, targeting of ER and protein processing pathways to promote ER stress in tumor cell have been demonstrated promising outcomes. 5. Conclusion Evidently the present study shows that breast CSCs are susceptible to tunicamycin effect. Our result revealed that tunicamycin treatment suppresses breast CSCs invasion though inducing ER stress in the cells that consequences decline in
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Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells Babak Namia,* , Huseyin Donmezb , Nadir Kocakb a b
Department of Medical Genetics and Women and Children’s Health Research Institute (WCHRI), University of Alberta, Edmonton, Canada Department of Medical Genetics, Selcuk University Medical Faculty, Konya, Turkey
A R T I C L E I N F O
Article history: Received 21 April 2016 Accepted 13 June 2016 Keywords: Tunicamycin Endoplasmic reticulum stress Breast cancer Cancer stem cells CD44 CD24
A B S T R A C T
Tunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis has role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. In this study we show effect of tunicamycin on subpopulation and invasion of CD44+/CD24- MCF7 breast cancer stem cells. CD44+/CD24- cells were isolated from MCF7 cell line by fluorescence activated cell sorting (FACS) and treated with tunicamycin. ER stress was monitored by evaluation of X-box binding protein 1(XBP-1) mRNA splicing, cleaved activating transcription factor 6 (ATF6) nuclear translocation and CCAAT/ enhancer-binding protein homologous protein (CHOP) expression. CD44+/CD24- subpopulation was analyzed using flow cytometry. Invasion was investigated by scratch assay, trypan blue staining, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation and in vitro migration assays. Increased level of spliced XBP-1, ATF6 nuclear translocation and CHOP protein expression were detected in CD44+/CD24- and original MCF7 cells treated with tunicamycin. Also, a significant decline in CD44+/CD24- cell subpopulation was determined in the cells treated with tunicamycin. The results also showed inhibited invasion, increased cell death, suppressed proliferation and reduced migration in the CD44+/CD24- and CD44+/CD24- rich MCF7 cell culture, under effect of tunicamycin. Our results indicate that CD44+/CD24- phenotype MCF7 cells are susceptible to tunicamycin. The results showed that tunicamycin-induced ER stress suppresses CD44+/CD24- phenotype cell subpopulation and in vitro invasion and accelerates tumorosphore formation. These results suggest that tunicamycininduced ER stress inhibits CD44+/CD24- phenotype MCF7 breast cancer stem cells. We conclude that using ER-targeting chemicals like tunicamycin is an interesting approach to target breast cancer stem cells inside tumor. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction Emerging evidence has suggested that the capability of a tumor to grow and its resistance against therapies is dependent on a small subset of cells within a tumor, called cancer stem cells (CSCs). Cancer stem cells have been reported to express stem cell surface markers and possess self-renewal potency. It has been proposed that these cells are only cells with the ability to regenerate malignant cells and force the growth of the cancer (Lobo et al.,
* Corresponding author. E-mail addresses: [email protected] (B. Nami), [email protected] (H. Donmez), [email protected] (N. Kocak).
2007). Breast CSCs have been characterized to have the cell surface phenotype CD44+/CD24 (Al-Hajj et al., 2003). CD44 is a celladhesion molecule involved in binding of cells to hyaluronic acid (Peach et al., 1993), whereas CD24 is a regulator of chemokine (CX-C motif) receptor 4 (CXCR4) that involved in breast tumor formation in low level expression (Schabath et al., 2006), Generally, CD44+/CD24- phenotype cells represent averagely 1–4% of all cells in a breast tumor and their population enhancement exhibits increased tumorigenicity and invasion (Al-Hajj et al., 2003; Sun et al., 2013). Although data have been provided to support resistance of CSCs against death inducing mechanisms (Sun et al., 2013). researchers believe CSCs could be Achilles' heel of cancer in CSC-targeted therapies (Wicha, 2014).
http://dx.doi.org/10.1016/j.etp.2016.06.004 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: B. Nami, et al., Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.004
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In this study we investigated cytotoxic effect of tunicamycin in CD44+/CD24- phenotype breast cancer stem cells. Tunicamycin is an antibiotic that inhibits glycoprotein processing by blocking UDP-HexNAc: polyprenol-P HexNAc-1-P family of enzymes in human the enzyme GlcNAc phosphotransferase (GPT) (Duksin and Bornstein, 1977; Heifetz et al., 1979). Tm inhibits N-linked glycosylation of proteins and causes blocking of DNA synthesis and cell cycle arrest in G1 phase (Ettlinger et al., 1986; Vai et al., 1987). N-linked glycosylation is essential for protein folding and inhibition by Tm impede proteins folding correctly eventually causes accumulation unfolded proteins in the endoplasmic reticulum (ER) lumen (Parodi, 2000; Ware et al., 1995). Accumulation of unfolded protein in ER lumen is a toxic phenomenon that leads unfolded protein response (UPR). UPR is characterized by endoplasmic reticulum (ER) stress a cellular mechanism that sets up in response to unfolded protein accumulation in the ER lumen (van den Berg et al., 2000; Chakrabarti et al., 2011). This mechanism is elucidated with activation of three different pathways parallel to each other which is promoted by the ER transmembrane proteins inositol-Requiring Enzyme 1a (IRE1a), PRKR-Like Endoplasmic Reticulum Kinase (PERK) and activating transcription factor 6 (ATF6) (Chakrabarti et al., 2011; Schroder and Kaufman, 2006). The signaling leads to regulation of asset of downstream genes through X-box binding protein 1(XBP-1), activating transcription factor 4 (ATF4) and cleaved activating transcription factor 6 (ATF6) transcription factors (Chakrabarti et al., 2011; Schroder and Kaufman, 2006). Essentially, ER stress pursues two primary aims including maintain normal cellular function by halting protein translation and synthesis (Brostrom and Brostrom, 1998) and activation of the signaling pathways leading to up-regulation of the molecular chaperones involved in protein folding and degradation (Shenkman et al., 2007). The failure to achieve these two objectives, the ER stress leads cell towards death via apoptosis (Tabas and Ron, 2011) and autophagy (Yorimitsu et al., 2006). ER stress is in charge of guarding the intracellular homeostasis by haltering cell survival and death mechanisms. It has been demonstrated that ER stress-induced cell death is regulated by Ca2 homeostasis and the pro-apoptotic protein CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) (Xu et al., 2005). ER stress has also a key role in development of inflammation (Hotamisligil, 2010), obesity and diabetes (Nakatani et al., 2005), neurodegenerative diseases (Cnop et al., 2012; Hetz and Mollereau, 2014), heart diseases (Yamaguchi et al., 2003) and cancer (Schonthal, 2013). The role of ER stress in promotion of calcium—induced apoptosis through CHOP protein has already been demonstrated (Han et al., 2013; Pyrko et al., 2007). Thus, ER stress has been suggested as a promising target for tumor cell death and cancer therapy. Effectiveness of tunicamycin as an anti-cancer chemical has been demonstrated in various human cancer models (de Freitas et al., 2011; Hou et al., 2013; Morin and Bernacki, 1983; Shiraishi et al., 2005). Therefore, in this study, we aimed to investigate the effect of tunicamycin-induced ER stress on breast CSC subpopulation and invasion in vitro. 2. Materials and methods 2.1. Chemical and antibodies Tunicamycin was purchased from Sigma-Aldrich (St. Louis, USA). It was dissolved in dimethyl sulfoxide (DMSO) and stored at +4 C. Optimum concentration of tunicamycin was determined by testing ranges 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg of tunicamycin in per milliliter of culture medium regarding cytotoxicity (tested by morphological observation and trypan blue staining) and ER stress activity (tested by evaluating XBP-1mRNA splicing) potentials (data not shown).
Rabbit anti human CHOP polyclonal IgG (sc-200), rabbit anti ATF6 (N-terminus) polyclonal IgG (sc-22799), rabbit anti human GAPDH polyclonal IgG (sc-25778), HRP conjugated goat anti rabbit polyclonal IgG (sc-2004) and FITC-conjugated goat anti rabit polyclonal IgG (sc-2012) antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, USA). FITC-conjugated mouse anti human CD44 monoclonal (MA1-12127) and PE-conjugated mouse anti human CD24 monoclonal (MA1-10154), FITC-conjugated Mouse IgG isotype control (MA1-10413) and PE-conjugated mouse IgG isotype control (MA1-10415) antibodies were from Thermo Scientific (Rockford, USA). 2.2. Cell culture MCF7 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) that was completed with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin (100 unit/ml) and 1% streptomycin (10 mg/ml) with incubation at 37 C in a 5% CO2 humidified incubator. After the cell culture reached 80% confluency, the cells were trypsinized with 0.25% trypsin and harvested. In order to perform the experiments, the cells were seeded as 2 104, 5 104, 105 and 5 105 cells in 96, 24, 12 and 6-well plates respectively in triplicate for each one and cultured at the condition maintained above for 24 h before start to treatment. 2.3. RNA isolation and XBP-1 mRNA splicing assay Total RNA was isolated using TRIzol reagent (Invitrogen, Waltham, USA) according to protocol described previously (Rio et al., 2010). Synthesis of cDNA from the total RNA was carried out using Transcriptor High Fidelity cDNA Synthesis1 (Roche, Basel, Switzerland) kit by applying the oligo (dT)18 primer pairs following the manufacturer's instructions. cDNA of XBP-1 was amplified by PCR using forward 50 -TTACGAGAGAAAACTCATGGCC30 and reverse primer 50 -GGGTCCAAGTTGTCCAGAATGC-30 . Primers sequences were obtained from literature (Samali et al., 2010) and were synthesized by Biomers Inc. (Ulm, Germeny). After the PCR cycling, the product was ran in 15% polyacrylamide gel by vertical electrophoresis (Bio-Rad Laboratories, Berkeley, USA) at 40 mA electric current for an hour. After the electrophoresis, the gel was stained with ethidium bromide solution and the spliced 263 bp and unspliced 289 bp XBP-1 bands were imaged in a gel documentation system (Bio-Rad Laboratories, Berkeley, USA). 2.4. Western blotting Total protein was extracted by adding proper volume of RIPA buffer (Sigma-Aldrich, St. Louis, USA) to cells and spining down. The protein samples were prepraed by adding a same volume of 4 x lammeli buffer and then incubation at 95 C for 5 min. Amount 40 mg of total protein was loaded onto each well of the % 4–15 gradiyant gel and was ran at 40 mA electric current for an hour. After the electrophoresis, the proteins transferred on the PVDF membrane (Bio-Rad Laboratories, Berkeley, USA) using a transBlot1 TurboTM Transfer system (Bio-Rad Laboratories, Berkeley, USA) at 1.3 A electric current for 7 min. Blocking of membrane was done with 5% non-fat milk solution for an hours in 50 rpm agitation. The immunoblotting was accomplished by incubation of the membrane with 1 mg/ml of primary antibody solution and then 0.1 mg/ml of secondary antibody both for 2 h at room temperature. At the end of the blotting, the membrane was incubated in Immunstar WesternC Chemiluminescent (Bio-Rad Laboratories, Berkeley, USA) substrate solution for 10 min then imaged by a gel documentation system.
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2.5. Immunofluorescence assay Cells were cultured on the immunofluorescence slides 48 h before treatment starts. After treatment period, the slides were rinsed in PBS and the cells were fixed by cold methanol for 5 min. Blocking was done with incubation of slides in 1% BSA solution for an hour. After the blocking, the slides were incubated in 1 mg/ml solution of the primary antibody for an hour. Afterwards, the slides were rinsed in PBS tree times each for 5 min. Thereafter, the slides were incubated in 1 mg/ml solution of secondary antibody for an hour in darkness then were washed completely in PBS and mounted by 40 ,6-diamidino-2-phenylindole (DAPI). The slides were observed under a confocal fluorescence microscope (Nikon, Tokyo, Japan). 2.6. Flow cytometry and FACS analysis Flow cytometry and fluorescence-activated cell sorting (FACS) analysis were performed as described previously (Nami et al., 2015).
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2 mg/ml of tunicamycin or same volume of PBS. After the period, cells were washed two times with PBS, fixed by 4% formaldehyde and then stained with acidified hematoxylin dye. At the end, the stain intensity of the migrated cell colonies in the threshold subsequent areas was measured by ImageJ software (NIH, Maryland, USA). 2.9. Trypan blue staining and MTT assays Trypan blue staining and MTT assays was done as described previously (Acar et al., 2015). 2.10. Statistical analysis The statistical significances were considered applying twotailed student’s t-test and analysis of variance (ANOVA) using the GraphPad Prism1 V.5.00 software. Also, the image base data were analyzed using ImageJ software (NIH, Maryland, USA), p < 0.050 was considered as statistically significant. 3. Results
2.7. Scratch assay 3.1. Tunicamycin stimulates ER stress in CD44+/CD24- MCF7 cells The cells were seeded in 6-well plates and cultured in the standard condition. After the culture reached about 80% confluency, scratches as plus sign with 4 mm in width were created on the middle of the plates. The cell culture was continued up to 24 h in presence of 2 mg/ml of tunicamycin or same volume of PBS. After this period, the cells were rinsed three time in PBS and fixed by 4% formaldehyde and then were stained with acidified hematoxylin dye (Sigma-Aldrich, St. Louis, USA). 2.8. In vitro migration assay Same amount of CD44+/CD24- enriched cells were seeded in corner of 100 mm plate and cultured for 48 h. After the cells attached, a threshold adjusted on 2 cm distance from the cell enclosure and the culture was continued up to 24 h in presence of
In result of dose determination, the optimum concentration of tunicamycin was determined as 2 mg/ml (data is not shown). To investigate CD44+/CD24- cell response to ER stress inducer, the cells we sorted from original MCF7 cell culture and treated with 2 mg/ml of tunicamycin for 24 h and then ER stress was monitored. Monitoring of ER stress was done by assessment of XBP-1 mRNA splicing, ATF6 protein translocation and CHOP protein expression. A result, enhancement in XBP-1 mRNA splicing rate was determined in both original MCF7 culture and CD44+/CD24MCF7 cells both treated with tunicamycin in comparison with related negative controls (Fig. 1A). The XBP-1 splicing is a marker to recognize activated ER stress through IREa sensor. Another sensor of ER stress is ATF6 transmembrane that migrates from ER to nucleus during ER stress. Nuclear translocation of ATF6 under
Fig. 1. Tunicamycin stimulates ER stress in CD44+/CD24- MCF7 breast cancer stem cells. The original MCF7 and FACS sorted CD44+/CD24- MCF7 cells were cultured with 2 mg/ ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 h. (A) Quantitative PCR result of 289 bp unspliced XBP-1 (uXBP-1) and 263 bp spliced XBP-1 (sXBP-1) showed a significant XBP-1 mRNA splicing enhancement in original MCF7 (p < 0.001) and CD44+/CD24- MCF7 cells (p < 0.001) due to treatment with tunicamycin for 24 h. (B) Confocal fluorescence image of FITC immunofluorescense staining of cleaved ATF6. Nuclear translocation of cleaved ATF6 was observed in original and CD44+/CD24- MCF7 cells due to 24 h’ treatment with tunicamycin. Scale of the bars is 10 mm. (C) Increased expression of CHOP protein in original and CD44+/CD24MCF7 cells due to 24 h’ treatment with tunicamycin that was evaluated with western blotting.
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effect of tunicamycin was also observed in CD44+/CD24- cells as well as original MCF7 cells compared to negative controls (Fig. 1B). Moreover, increased expression of CHOP protein, a downstream gene of ER stress, was determined in the both cell types treated with tunicamycin in comparison with negative controls (Fig. 1C). These result indicates high level activity of ER stress in the cells under effect of tunicamycin. To confirm this, we treated the cells with 2 mM dithiothreitol (DTT) as another ER stress stimulator agent for 24 h and monitored ER stress performing same methods. Results of DTT treated cells were as same as tunicamycin (data not shown).
3.2. Tunicamycin accelerates CD44+/CD24- MCF7 spheroid formation At the consequence of 24 h’ culture of original MCF7 cells in presence of 2 mg/ml of tunicamycin, a defined dismorpholgy related to stress was observed (Fig. 2A). Also, spheroids were detected in the CD44+/CD24- cells cultured in serum free medium after 4 days’ treatment with 2 mg/ml of tunicamycin in 2D culture environment (Fig. 2B).
3.3. Tunicamycin suppresses CD44+/CD24- MCF7 cell subpopulation In order to determine subpopulation of CD44+/CD24- cells under effect of tunicamycin-induced ER stress, we cultured MCF7 cells in presence of tunicamycin for 24 h and analyzed subpopulation of CD44+/CD24- cells in the same population of original MCF7 culture. According to the results shown in Fig. 5, the mean percentage of tunicamycin treated CD44+/CD24- cell subpopulation in the culture was 0.15%, while this value was 1.15% in untreated negative control cells (p < 0.010; Fig. 3). This result shows a declined subpopulation of CD44+/CD24- cells in the original MCF7 cell culture due to tunicamycin in comparison with negative controls.
3.4. Tunicamycin suppresses CD44+/CD24- cell invasion We established CD44+/CD24- enriched cell culture by adding FACS sorted CD44+/CD24- phenotype cells to the original MCF7 culture until its subpopulation reaches 10% in order to enhance involvement of CD44+/CD24- cell with tunicamycin treatments and exam conditions. Invasion was assessed by scratch, trypan blue viability, MTT and in vitro migration assays. The cells left to be cultured in presence of 2 mg/ml of tunicamycin or same volume of PBS for 24 h. Scratch assay was performed to investigate invasion of CD44+/CD24- rich cells. As result width of the wound in the cultures treated with tunicamycin was significantly greater than negative controls (p < 0.001; Fig. 4A). Trypan blue cell survival assay revealed that percentage of viable cells in tunicamycin treated cultures was 71.82%, whereas this value was 87.53% in the negative control cultures (p < 0.001; Fig. 4B). This result shows a dramatic death rate of CD24+/CD24- cells because of tunicamycin treatment. Moreover, significant reduction in the proliferation of both original MCF7 and CD24+/CD24- cells were determined in tunicamycin treated cells (p < 0.001; Fig. 4C). In order to validate these observation, we examined in vitro migration of CD44+/CD24rich cells under treatment with Tm. The results revealed that the number and distance from threshold of secondary colonies in tunicmycin treated cell cultures were significantly lower compared to negative controls (p < 0.001; Fig. 4D). This finding suggests that tuncamycin-induced ER stress could inhibit the cells migration in culture model. By and large, these results show that ER stress suppressed invasion of CD44+/CD24- MCF7 cells. Altogether these results revealed that tunicamycin inhibites the invasion of CD44 +/CD24- cells and cause significant regression (p < 0.010; Fig. 4E). 4. Discussion Tunicamycin cause UPR and ER stress by blocking N-linked glycosylation process in ER lumen. The cytotoxicity of tunicamycin in breast tumor cells has been demonstrated through it effect in ER
Fig. 2. ER stress causes MCF7 cell dysmorphology and accelerates tumorsphere formation. Attached original MCF7 and CD44+/CD24- MCF7 cells were treated with 2 mg/ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 h respectively in FCS containing and serum free culture medium. (A) dysmorphological evidence in attached original MCF7 cells due to activated ER stress. (B) CSCs spheroids were observed in the cell culture treated with tunicamycin for 4 days but not in negative controls. Scale of the bars is 100 mm.
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Fig. 3. ER stress decreases CD44+/CD24- MCF7 breast cancer stem cell subpopulation. MCF7 cells were cultured with 2 mg/ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 h and then the CD44+/CD24- cells population analyzed by flow cytometry. (A) Flow cytometry analysis plate of MCF7 cells in terms of CD44 and CD24 expression. (B) Decrease in the subpopulation of cells with CD44+/CD24- phenotype (P2 plate; p < 0.010) and increase in the subpopulation of else phenotypes cells due to activated ER stress (P1-P2; p < 0.010).
stress-mediate apoptosis and autophagy (Clarke et al., 2012; Han et al., 2013). ER stress is an essential mechanism for regulation of cellular homeostasis when the protein synthesis is impaired. It has been demonstrated that dysfunction of ER stress in many cancer cells can lead tumor cell to survival and growth (Schonthal, 2013). ER stress is identified with up-regulation of chaperon proteins, endoplasmic reticulum associated degradation (ERAD) genes, autophagy factors and also their positive regulators like XBP-1, CHOP, ATF4 and ATF6 that aid the cell in a struggle to survive. The other feature of ER stress is promotion of apoptotic and autophagic cell death via up-regulating CHOP and calcium influx (Xu et al., 2005). Consequently, this phenomenon acts as a double-edged sword its cutting edges have been used to target cancer. So far, the critical role of ER stress in regulation of breast cancer has been extensively explained (Clarke et al., 2012). Most recent reports have demonstrated that some ER stress factors especially XBP-1 and ER chaperon protein GRP78 have a key role in regulation of CSCs though insufficient supporting information (Chen and Zhou, 2005; Li et al., 2013). These results suggest that ER stress might have a couple of function in CSCs. We here showed that XBP-1 mRNA splicing, CHOP protein expression and ATF6 nuclear translocation is up-regulated in breast CSCs with CD44+/CD24phenotype when the cells are exposed to ER stress inducer agent tunicamycin (Fig. 5). Our data suggest that breast CSCs are susceptible to tunicamycin-induced ER stress and targeting of protein synthesis
machinery. Our results also support the notion that stimulation of ER stress in the cells causes reduction in the size of CD44+/CD24subpopulation. So that, the percentage of CSCs in the cell culture under ER stress condition was about eightfold lower than negative control, indicating that the reduction could be attributed to activated ER stress by tunicamycin. Apoptosis and autophagy are two canonical consequence of ER stress when CHOP protein is upregulated. Our results showed an up-regulation of CHOP protein in CSCs under the effect of tunicamycin. CHOP is a pro-apoptotic factor that is able to promote programmed cell death via apoptosis and autophagy pathways (Pyrko et al., 2007; Tabas and Ron, 2011; Yorimitsu et al., 2006). On the other hand, we recently reported that autophagic condition suppresses CD44+/CD24- cell subpopulation in MCF7 culture model (Nami et al., 2015). The considerable fact is that autophagy is a consequence event of ER stress per se. Reduced subpopulation of CSCs might due to CD44+/CD24- cells loss through ER stress-associated cell death. But the challenging point here is that CD44+/CD24- and other phenotypic cells were equally exposed to tunicamycin therefore showed same level upregulation of ER stress elements. With regard to identical response of all phenotype MCF7 cells to ER stress, it is expected equal decrease in subpopulation of all phenotypes and not just of CD44 +/CD24- cells. Unfortunately, it is not clear whether cancer stem cells arise by conversion of normal stem cells or tumor cell reverse differentiation. Furthermore, many studies have demonstrated that autophagy plays a prevail role in cellular differentiation during
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Fig. 4. ER stress suppresses CD44+/CD24- MCF7 breast cancer stem cell invasion. The original MCF7, CD44+/CD24- enriched MCF7 cells and CD44+/CD24- MCF7 cells were treated with 2 mg/ml of tunicamycin (Tm) as ER stress stimulator, or 2 ml/ml of PBS as negative control for 24 and then invasions were evaluated using different tests. (A) Scratch assay result of CD44+/CD24- enriched MCF7 cells showed an inhibited invasion in result of tunicamycin (p < 0.001). Scale of the bars is 1 mm. (B) Trypan blue staining results of CD44+/CD24- cells revealed a decreased viable (p < 0.001) and increased death cell due to tunicamycin (p < 0.001). (C) MTT assay elucidated a decrease in proliferation of both original MCF7 (p < 0.001) and CD44+/CD24- MCF7 cells (p < 0.001) in the result of tunicamycin. The letter A indicates absorbance. (D) Migrated cell colonies of CD44+/CD24- enriched MCF7 cells with activated ER stress at four different distance from the threshold were significantly lower in comparison with the negative controls (p < 0.001). (E) Fold change reduction in invasion of both original MCF7 and CD44+/CD24- MCF7 cells due to tunicamycin that obtained with ANOVA analysis of tests which described above. Considering to p values there is elicited that retreat of CD44+/CD24- MCF7 cells was greater than original MCF7 (p < 0.010).
development. Therefore, changes in self-renewal and differentiation potency of the cells might be due to ER stress-induced autophagic condition. Moreover, Salemi et al. (Salemi et al., 2012) reported that autophagy acts as a necessary factor in regulation of stem cell self-renewal and differentiation; however, functional properties of CSCs highly diverse in normal stem cells. Therefore, decrease in CSC subpopulation might also due to effect of ER stress on the CSC self-renewal and differentiation properties. Although, we did not investigate here the self-renewal and differentiation of CD44+/CD24- cells in this study. We showed a suppressed invasion of CD44+/CD24- MCF7 cells including reduced proliferation, survival and migration under
tunicamycin treatment. It is previously reported that breast CSCs with CD44+/CD24- phenotype are the core element for tumor growth, invasion and metastasis as well as its resistance against radio- and chemotherapies and relapse. Basically CSC mass inside a tumor is associated with enhanced invasive properties and elevated expression of genes involved in malignancy of the tumor (Sun et al., 2013; Wicha, 2014). Similarly, it has been revealed that CD44+/CD24- subpopulation in primary tumors has a direct linkage with tumor regrowth, malignancy and invasion and metastasis owing to its high capacity for DNA repair and resistance against chemotherapies (Al-Hajj et al., 2003; Sun et al., 2013; Wicha, 2014). A number of recent publications reported that CSCs
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subpopulation of CSCs inside original bulk cell culture. We suggest that studding ER stress in CSCs is a requisite in order to understand the biology of CSC inside tumors and also to develop ER stress inducing therapies. Although, ER targeted chemotherapeutics on cancer has not been clinically examined yet; evaluation of this type of treatment is suggested with focus on CSCs. Thus we conclude that using ER-targeting agents like tunicamycin is an effective choice to target breast cancer stem cells inside tumor.
Conflict of interest The authors declare no conflict of interest. Acknowledgments
Fig. 5. Model summarizing the effect of tunicamycin-induced ER stress in CD44 +/CD24- MCF7 breast cancer stem cell. Tunicamycin causes accumulation of unfolded proteins in ER lumen inducing unfolded protein response in the cell. In this condition mRNA of XBP-1 is spliced by endoribonuclease domain of ER transmembrane protein IRE1 and XBP-1 protein is synthesized. ATF6 is releases from ER membrane and then is localized in nucleus. Both XBP-1 and ATF6 are bzipper transcription factors which induces expression of CHOP protein. CHOP protein is a pro-apoptotic factor which can provoke cell death via different pathways.
may be inhibited by upregulation of ER stress factors. Given these, we suppose that tunicamycin-induced ER stress inhibited the cell invasion through suppression of CSCs in the culture, however further studies are needed to clarify. Recently, it was shown that breast cancer cell lines were also capable of generating non-adherent spheres termed tumorspheres. These CSC spheroids develop in the result of CSC selfrenewal activity that has morphologically been characterized previously (Al-Hajj et al., 2003). Formation of tumorspheres commonly requires specific culturing conditions such as celloptimized media with defined supplements to retain the cells undifferentiated. Therefore, culturing tumor cells in serumcontaining medium can lead cells to be differentiated with markedly distinct morphologies and growth characteristics. Despite the essential culture factors are needed for tumorshphere formation, it also requires a long culture period in some cases weeks to be formed. In contrast, our result revealed that tumorshphere could be formed in just 4 days under effect of tunicamycin without additional supplements. This shows that ER stress promotes a set of unknown mechanisms and accelerates tumorspheres formation in regular cell culture condition. Given that the exact reason of stem cell spheroid formation still remains unclear, we suggest investigation of possible function of ER stress in the formation of tumorspheres. Numerous reports have pointed out that CSCs can survive from the effects of chemotherapy and subsequently support the regrowth of the tumor, but the clinically relevant aspect is that this property can be utilized as a target for cancer therapy. Therefore, many researchers propagandize that CSCs could be Achilles' heel of cancer. So far, targeting of ER and protein processing pathways to promote ER stress in tumor cell have been demonstrated promising outcomes. 5. Conclusion Evidently the present study shows that breast CSCs are susceptible to tunicamycin effect. Our result revealed that tunicamycin treatment suppresses breast CSCs invasion though inducing ER stress in the cells that consequences decline in
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