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NOTE The effect of calcitriol on endoplasmic reticulum stress response Biochem. Cell Biol. Downloaded from by San Francisco (UCSF) on 04/29/15 For personal use only.

Ela Haddur, Ali Burak Ozkaya, Handan Ak, and Hikmet Hakan Aydin

Abstract: Calcitriol, the active form of vitamin D, is known for its anticancer properties including induction of apoptosis, inhibition of angiogenesis, and metastasis. Calcitriol also increases intracellular calcium triggering apoptosis in a calpaindependent manner. Since the main storage unit for cellular calcium is endoplasmic reticulum (ER) and a decrease in ER calcium levels might induce ER stress associated cell death, we hypothesized that the cellular actions of calcitriol occur via ER stress. We have evaluated induction of ER stress by assessing BIP expression and XBP-1 splicing in breast cancer cell lines (MCF-7 and MDA-MB-231) and mammary epithelial cell line MCF10A. Our results suggest that cytotoxic concentrations of calcitriol induce an ER stress related response indicated as increased BIP levels and XBP-1 splicing not only in breast cancer cells but also in mammary epithelial cell line. However, vehicle treatment also induced a similar response de-emphasizing the importance of such effect. Calcitriol also failed to activate calpains, further weakening the idea of ER stress as the main mechanism for apoptotic effects of calcitriol. Taken together our results suggest an association between ER stress and vitamin D signaling. However present data indicates that ER stress by itself is not sufficient to explain anticancer properties of calcitriol. Key words: vitamin D, cancer, ER stress, unfolded protein response. Résumé : Le calcitriol, la forme active de la vitamine D, est connu pour ses propriétés anti-cancéreuses incluant l'induction de l'apoptose et l'inhibition de l'angiogenèse et de la métastase. Le calcitriol accroît aussi l'apoptose déclenchée par le calcium intracellulaire de manière dépendante de la calpaïne. Puisque la principale unité d'entreposage du calcium cellulaire est le réticulum endoplasmique (RE) et qu'une diminution des niveaux de calcium du RE peut induire la mort cellulaire associée a` un stress du RE, les auteurs ont émis l'hypothèse que les actions cellulaires du calcitriol s'exercent par l'intermédiaire du stress du RE. Ils ont évalué l'induction du stress du RE en mesurant l'expression de BIP et l'épissage de XBP-1 dans des lignées de cancer du sein (MCF-7 et MDA-MB-231) et dans la lignée épithéliale mammaire MCF10A. Leurs résultats suggèrent que les concentrations cytotoxiques de calcitriol induisent une réponse reliée au stress du RE comme le montrait l'augmentation des niveaux de BIP et de l'épissage de XBP-1, non seulement dans les lignées de cancer du sein mais aussi dans la lignée épithéliale mammaire. Cependant, le traitement au véhicule induisait une réponse similaire, ce qui désaccentuait l'importance d'un tel effet. Le calcitriol ne parvenait pas a` activer les calpaïnes, affaiblissant davantage l'idée que le stress du RE soit le principal mécanisme responsable des effets apoptotiques du calcitriol. Dans l'ensemble, les résultats des auteurs suggèrent qu'il existe une association entre le stress du RE et la signalisation de la vitamine D. Cependant, les données actuelles indiquent que le stress du RE en soi n'est pas suffisant pour expliquer les propriétés anti-cancéreuses du calcitriol. [Traduit par la Rédaction] Mots-clés : vitamine D, cancer, stress du RE, réponse aux protéines dépliées.

Introduction Calcitriol (1,25-dihydroxyvitamin D3), the active form of vitamin D, is a secosteroid hormone responsible for regulation of calcium and phosphate metabolism (Adams and Hewison 2010). Calcitriol has potent anticancer properties such as inhibition of tumor growth, angiogenesis and metastasis, and induction of apoptosis shown in various types of cancer both in vitro and in vivo (Krishnan and Feldman 2011). As a regulator of calcium, calcitriol also acts on cellular calcium homeostasis to increase intracellular calcium concentrations, which in return activates calcium-dependent cysteine proteases called calpains (Mathiasen et al. 2002). Elevation of cytoplasmic calcium may play a central role in pro-apoptotic effects of calcitriol, since calpains are known inducers of apoptosis (Squier et al. 1994). Endoplasmic reticulum (ER) is a membrane-bound organelle responsible for several cellular functions including post-translational folding of proteins and calcium storage (Sitia and Braakman

2003). Alterations in ER calcium levels disrupt the folding process because of the need for molecular chaperons for calcium to function (Ron and Walter 2007). Accumulation of unfolded or misfolded proteins due to dysfunctional folding mechanisms is known as ER stress (Ron and Walter 2007). Cellular response to ER stress is referred to as unfolded protein response (UPR), in which translation is slowed down, protein degradation is induced, and folding capacity of ER is increased to maintain ER homeostasis (Ron and Walter 2007). BIP (Binding immunoglobulin protein), a molecular chaperone protein located in the lumen of the ER, binds to hydrophobic regions of unfolded proteins to prevent their escape from ER during ER stress (Shen et al. 2002). Expression of BIP correlates with the load of unfolded proteins, and therefore may be used as a marker for ER stress (Ron and Walter 2007). Dissociation of BIP enables release of ER stress initiators into the cytoplasm and induces translational alterations via ER stress related transcription factors (Shen et al. 2002). Among these transcription factors, XBP-1 is the most commonly used marker

Received 24 November 2014. Revision received 6 March 2015. Accepted 17 March 2015. E. Haddur, A.B. Ozkaya, H. Ak, and H.H. Aydin. Ege University, School of Medicine, Department of Medical Biochemistry, Bornova, Izmir 35100, Turkey. Corresponding author: H.H. Aydin (e-mails: [email protected]; [email protected]). Biochem. Cell Biol. 93: 1–4 (2015)

Published at on 6 April 2015.

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for ER stress due to its UPR specific upregulation induced by ATF6 and splicing induced by IRE1 (Lee et al. 2003; Yoshida et al. 2001). UPR may act both as a survival pathway or apoptotic pathway, depending on the severity of ER stress (Szegezdi et al. 2006; Tsang et al. 2010). There is evidence in the literature suggesting an association between the effects of calcitriol and ER stress. Cytotoxic doses of calcitriol are known to induce apoptotic cell death, which is one of the possible outcomes of ER stress response (Deeb et al. 2007). It was previously reported that calcitriol induces calcium release from endoplasmic reticulum, increasing cytoplasmic calcium and triggering an apoptosis-like cell death (Mathiasen et al. 2002). Such a decrease in ER calcium levels would lead to ER stress and UPR. We have concluded that the effects of calcitriol may be explained by induction of ER stress and hypothesized that calcitriol induces ER stress, and its cellular effects can be explained by UPR.

Materials and methods Cell culture All studied cell lines were kindly provided by Ege University Medical School Department of Medical Oncology. Breast cancer cell lines MCF-7 and MDA-MB-231 were cultured in RPMI medium (Lonza) with 10% fetal bovine serum (Lonza) and 1% penicillinstreptomycin (Sigma-Aldrich) supplement. Immortalized mammary epithelial cell line MCF-10A was cultured in DMEM medium (Lonza) with 10% fetal bovine serum (Lonza), 1% penicillin-streptomycin (Sigma-Aldrich), 0.4% EGF (epidermal growth factor, New England Biolabs), 0.5% hydrocortisone (Biochrom), 0.1% insulin (SigmaAldrich) supplement. All cell lines were maintained in a CO2 incubator with standard incubation conditions. Cells (104 cells per well) were seeded onto 96-well plates for viability experiments, and 20 × 104 cells per flask were seeded onto 25 cm2 for other experiments. Cells were treated with different concentrations of calcitriol (Cayman), reconstituted in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at 20 mmol/L and diluted with proper media for viability experiments. For all other experimental procedures, a cytotoxic concentration of calcitriol and an appropriate concentration of vehicle (DMSO) were used. Cell viability assay Cell Counting Kit-8 (Sigma-Aldrich), a colorimetric assay that measures mitochondrial succinate dehydrogenase activity as an indicator of cell viability, was used to determine the effect of calcitriol on cell viability. Cells were treated with different concentrations of calcitriol and incubated for 24 h. Cell viability was determined according to the manufacturer’s instructions. Mean absorbance (450 nm) of nontreated samples was considered as 100%, and viability of other samples calculated accordingly. Each sample measurement was repeated at least 6 times to calculate mean absorbance values. Calculated IC50 values were used for other experiments. Western blot Cells were left nontreated (NT), treated with calcitriol, or DMSO for 24 h. Cells were collected and protein isolation was carried out using modified RIPA buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% NP40, 0.1% SDS, 1% protease inhibitor cocktail, and 5 mmol/L EDTA, Sigma-Aldrich). Protein concentration of each sample was determined by BCA assay (Sigma-Aldrich). Proteins (50 ␮g) were separated on 10% SDS-polyacrylamide gel and transferred to PVDF membranes via wet-blotting overnight. Antibodies used for immunoblotting (anti-BIP and proper HRP-tagged secondary antibodies) were purchased from Cell Signaling and (anti-␤-actin) Sigma-Aldrich. Visualization of the membranes was carried out with ECL plus kit (Pierce) in a dark room. Densitometric analysis was carried out with Vision Works LS (UVP) image acquisition and analysis software.

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Real-time PCR Cells were left NT, treated with calcitriol, or DMSO for 24 h. Cells were collected and total RNA was isolated with RNAeasy Mini Kit (Qiagen). cDNA synthesis was carried out with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and qPCR Probe Master Mix (RTA) was used for PCR in Applied Biosystems 7900 fast device. All protocols were carried out according to the manufacturer’s instructions. The following primers were used for detection of XBP-1 (specific to both spliced and unspliced isoforms): forward 5=-AAG CCA AGG GGA ATG AAG T-3= and reverse 5=-CCAGAATGCCCAACAGGATA-3= (Lee et al. 2003). The following probes were used for specific detection of spliced and unspliced isoforms of XBP-1: spliced FAM-GCT GAG TCC GCA GCA GGT GCA G-MGB and unspliced VIC-AGC ACT CAG ACT ACG TGC ACC T-MGB (Davies et al. 2008). All PCR reactions include both primers and probes, and each sample measurement was repeated at least 3 times. The ratio of spliced XBP-1 to unspliced XBP-1 was calculated for each sample according to the ⌬CT method. Briefly, CT values of unspliced form of XBP-1 were subtracted from CT values of spliced form for each sample to obtain ⌬CT values. Fold changes were calculated from ⌬CT values according to the formula: 2–⌬CT. Calpain activity Cells were left NT, treated with calcitriol, or DMSO for 24 h. Calpain activity was measured with Calpain Activity Fluorometric Assay Kit (Biovision) according to the manufacturer’s instructions, with at least two replicates for each sample and positive control. The method is based on the detection of the cleavage of calpain substrate Ac-LLY-AFC, which emits yellow-green light (␭max = 505 nm) upon cleavage by calpain. Activity was determined for each sample containing 55 ␮g of protein, determined by BCA assay, and for 1 ␮g of active calpain (positive control) by measuring fluorescence emission values at 505 nm (excitation = 400 nm). Statistical analyses The effect of calcitriol on XBP-1 splicing and calpain activity was statistically evaluated using Student's t-test by comparing calcitriol treatment to vehicle (DMSO) treatment. P values less than 0.05 were considered significant. GraphPad Prism 5.3 was used for statistical analysis and to draw figures.

Results Calcitriol induces growth inhibition To determine cytotoxic concentrations of calcitriol we first performed a cell viability assay using Cell Counting Kit-8. Calcitriol inhibited growth of breast cancer cell lines (MCF-7 and MDAMB-231) and mammary epithelial cell line (MCF-10A) in a similar dose-dependent manner (Fig. 1). IC50 was determined to be approximately 40 ␮mol/L for MCF-7 (Fig. 1A) and 50 ␮mol/L for MDA-MB-231 and MCF-10A (Figs. 1B and 1C) cell lines. All other experiments were conducted by using these concentrations. Calcitriol alters BIP expression and XBP-1 splicing After determining cytotoxic concentrations of calcitriol, we investigated the effects of calcitriol on BIP expression and XBP-1 splicing. Treatment with calcitriol for 24 h induced a minor elevation in expression of BIP compared to vehicle treatment in all cell lines (Figs. 2A and 2B). Expression of BIP in NT cells was dramatically lower in MCF-7 and MCF10A compared to MDA-MB-231 cells. This high basal expression of BIP in estrogen receptor negative breast cancer was previously reported and thought to play a role in the aggressiveness of such tumors (Fernandez et al. 2000). Surprisingly DMSO treatment (0.20% for MDA-MB-231 and MCF10A and 0.25% for MCF-7) also induced BIP expression in MCF-7 and MCF10A cells, emphasizing that stress-inducing effect of DMSO occurs even in low concentrations. Calcitriol treatment also induced splicing of XBP-,1 as shown in Fig. 2C. Basal spliced/ Published by NRC Research Press

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Fig. 1. Calcitriol inhibits cell viability. MCF-7, MDA-MB-231, and MCF10A cells were treated with increasing concentrations of calcitriol for 24 h, and viability was assessed with Cell Counting Kit-8. Each value represents mean of at least 6 replicates. Viability of non-treated cells was considereds 100%, and inhibition of viability in calcitriol treated cells was calculated accordingly. Calculated mean viability values are presented as bars, and error bars represent standard error. IC50 was determined to be approximately 40 ␮mol/L for MCF-7 (A) and 50 ␮mol/L for MDA-MB-231 (B) and MCF-10A (C) cells.

Fig. 2. Calcitriol effect on BIP expression and XBP-1 splicing. Cells were treated with IC50 concentrations of calcitriol (CAL), corresponding concentrations of DMSO, or left non-treated for 24 h. Alterations in BIP expression was determined by western blot (A) and evaluated by densitometric analysis (B). (C) Ratio of spliced/unpsliced isoform of XBP-1 was determined by PCR and evaluated by ⌬CT method. At least 3 replicates were studied for each sample. Statistical significance was evaluated with Student's t-test and P values less than 0.05 were marked with (*). (D) Calpain activity was measured with Calpain Activity Fluorometric Assay Kit (Biovision). Each sample was studied in duplicate. PC stands for positive control (active calpain). All error bars represent standard error.

unspliced XBP-1 ratio was similar (≈2 fold) in all tested cell lines. Spliced isoform was further induced with calcitriol treatment at different levels in all cell lines, when compared to NT cells. However, DMSO treatment induced XBP-1 splicing more effectively than calcitriol treatment in MCF-7 cells. Calcitriol-based induction of XBP-1 was significant only in MDA-MB-231 cell line with a P value of 0.0256. Calpain activity was also determined in cells treated with calcitriol to evaluate calcitriol-mediated apoptotic response via decreased cytoplasmic calcium levels. Calcitriol treatment did not alter calpain activity in the studied cell lines (Fig. 2D). Therefore, apoptotic effects of calcitriol cannot be explained by ER stress associated low cytoplasmic calcium levels.

Discussion The growth inhibitory (James et al. 1996), anti-angiogenic (Mantell et al. 2000), and anti-metastatic (Flanagan et al. 2003) functions of calcitriol on breast cancer cell lines has been previously reported. However the role of calcitriol in ER stress is relatively unknown. In this study we evaluated the effects of calcitriol on ER stress. Our results showed a slight elevation in expression of BIP, a chaperon protein that is increased during ER stress to induce UPR signal by releasing PERK, ATF6, and IRE1 from ER (Shen et al. 2002), following calcitriol treatment in all cell lines in a similar manner (Fig. 2A). However DMSO also induced BIP expres-

sion, even more dominantly than calcitriol, de-emphasizing this effect of calcitriol. Calcitriol also altered splicing of XBP-1, an ER stress transcription factor spliced by IRE1 (Yoshida et al. 2001). XBP-1 spliced isoform was the dominant form in all tested samples, even in NT samples, with 2:1 ratio indicating basal stress (Fig. 2C). However, calcitriol treatment further increased this ratio, but only in MDAMB-231 and MCF-10A cell lines (Fig. 2C). It is important to note that DMSO treatment also increased XBP-1 splicing, especially in MCF-7 cells. Although these results might indicate induction of ER stress associated response by calcitriol treatment, its importance for cellular actions of calcitriol is arguable. Inability of calcitriol to activate calpains, even though the opposite was previously suggested by Mathiasen et al., weakens the idea of ER stress (and low cytoplasmic calcium) associated apoptosis as the main mechanism for anti-proliferative effects of calcitriol. However in the mentioned study, MCF-7 cells were treated with calcitriol for longer periods of time (4 days), and in vitro induction data was not as significant as in vivo. It is important to point out that growth inhibitory and ER stress inducing effects of calcitriol is not limited to cancer cells and calcitriol affects normal mammary epithelial cells in a similar manner, making utilization of this process for cancer treatment partially insignificant. Published by NRC Research Press

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At this point significance of the connection between calcitriol and ER stress is open to dispute, as the induction of both ER stress markers are also affected by DMSO and as calcitriol fails to activate calpains. On the other hand, DMSO is known to act in cell differentiation in a manner similar to calcitriol (Brackman et al. 1995), which might explain its effects on XBP-1 splicing and BIP expression. Also, calcitriol further induced these markers, suggesting a role for calcitriol in ER stress independent of the actions of DMSO. Since there is no activation of calpains induced by calcitriol, it is logical to assume that the induction of ER stress by calcitriol occurs via other pathways such as accumulation of unfolded proteins or disruption of the redox state (Tsang et al. 2010). Even though ER stress is not sufficient by itself to explain cellular functions of calcitriol and obtained results do not confirm our original hypothesis, we believe this report might be a starting point for further studies aiming to elucidate the role of ER stress in vitamin D signaling.

Acknowledgements This project is supported by Ege University Scientific Research Project Grant No. 2012-TIP-072.

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The effect of calcitriol on endoplasmic reticulum stress response.

Calcitriol, the active form of vitamin D, is known for its anticancer properties including induction of apoptosis, inhibition of angiogenesis, and met...
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