Calcium signaling affects migration and proliferation differently in individual cancer cells due to nifedipine treatment

Journal Pre-proofs Calcium signaling affects migration and proliferation differently in individual cancer cells due to nifedipine treatment Barbora Chovancova, Veronika Liskova, Svetlana Miklikova, Sona Hudecova, Petr Babula, Adela Penesova, Angelika Sevcikova, Erika Durinikova, Marie Novakova, Miroslava Matuskova, Olga Krizanova PII: DOI: Reference:

S0006-2952(19)30394-6 https://doi.org/10.1016/j.bcp.2019.113695 BCP 113695

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

12 September 2019 5 November 2019

Please cite this article as: B. Chovancova, V. Liskova, S. Miklikova, S. Hudecova, P. Babula, A. Penesova, A. Sevcikova, E. Durinikova, M. Novakova, M. Matuskova, O. Krizanova, Calcium signaling affects migration and proliferation differently in individual cancer cells due to nifedipine treatment, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113695

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Calcium signaling affects migration and proliferation differently in individual cancer cells due to nifedipine treatment

Barbora Chovancovaa, Veronika Liskovaa, Svetlana Miklikovab, Sona Hudecovaa, Petr Babulac,d, Adela Penesovaa, Angelika Sevcikovaa,e, Erika Durinikovab, Marie Novakovac,d, Miroslava Matuskovab, Olga Krizanovaa,c

aInstitute

of Clinical and Translational Research, Biomedical Research Center SAS,

Bratislava, Slovak Republic; bCancer Research Institute, Biomedical Research Center SAS, Bratislava, Slovak Republic; cDepartment of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; dInternational Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic;eDepartment of Chemistry, Faculty of Natural Sciences, University of Ss. Cyril and Methodius, Trnava, Slovakia

Corresponding author: Prof. Olga Krizanova, Ph.D., D.Sc. Institute of Clinical and Translational Research Biomedical Research Center, Slovak Academy of Sciences Dubravska cesta 9, 84505 Bratislava, Slovakia e-mail: [email protected] Telephone: (+421)232295312 Category: Toxicology

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Abstract Several papers have reported that calcium channel blocking drugs were associated with increased breast cancer risk and worsened prognosis. One of the most common signs of breast tumors is the presence of small deposits of calcium, known as microcalcifications. Therefore, we studied the effect of dihydropyridine nifedipine on selected calcium transport systems in MDA-MB-231 cells, originating from triple negative breast tumor and JIMT1 cells that represent a model of HER2-positive breast cancer, which possesses amplification of HER2 receptor, but cells do not response to HER2 inhibition treatment with trastuzumab. Also, we compared the effect of nifedipine on colorectal DLD1 and ovarian A2780 cancer cells. Both, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and type 1 sodium calcium exchanger (NCX1) were upregulated due to nifedipine in DLD1 and A2780 cells, but not in breast cancer MDA-MB-231 and JIMT1 cells. On contrary to MDA-MB-231 and JIMT1 cells, in DLD1 and A2780 cells nifedipine induced apoptosis in a concentration-dependent manner. After NCX1 silencing and subsequent treatment with nifedipine, proliferation was decreased in MDA-MB-231, increased in DLD1 cells, and not changed in JIMT1 cells. Silencing of IP3R1 revealed increase in proliferation in DLD1 and JIMT1 cells, but caused decrease in proliferation in MDA-MB-231 cell line after nifedipine treatment. Interestingly, after nifedipine treatment migration was not significantly affected in any of tested cell lines after NCX1 silencing. Due to IP3R1 silencing, significant decrease in migration occurred in MDAMB-231 cells after nifedipine treatment, but not in other tested cells. These results support different function of the NCX1 and IP3R1 in the invasiveness of various cancer cells due to nifedipine treatment.

Key words: Breast cancer; Sodium calcium exchanger 1; Inositol 1,4,5-trisphosphate receptor; Apoptosis; Migration

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1. Introduction Calcium signaling is directly or indirectly involved in tumor´s fate. In tumor cells, calcium signaling is remodeled or deregulated and plays an important role in initiation, progression and metastasis formation. Altered calcium signaling pathways might help tumor cells to overcome the anti-tumor protective mechanisms [1, 2]. On the other hand, selectively increased calcium can help to induce apoptosis through modulation and/or increased expression of some calcium transport systems [3-6]. For example, melatonin performs its anticancer effect by inducing apoptosis in tumors through different targeting of calcium transport systems [6]. Sulphoraphane induces apoptosis through inositol 1,4,5-trisphosphate receptors in ovarian carcinoma [4]. In pheochromocytoma, triptolide induces apoptosis through upregulation of calcium transport system SERCA type 3 [7]. Besides apoptosis, disrupted calcium homeostasis might also trigger programmed immunogenic necrosis in cancer [8]. The important role of calcium in tumor progression and metastasis represents opportunity for targeting altered calcium signaling during tumorigenesis. L-type voltage calcium channels (CaV1.1 – CaV1.4) are localized on plasma membrane, and allow entry of calcium ions into the cell. In tumor cells, change in protein levels and expression of voltage dependent calcium channels was observed, for example in human colorectal cancer [9-11], pancreatic adenocarcinoma [12], skin squamous cell carcinoma [13], and prostate carcinoma [14]. Dihydropyridines are L-type voltage-dependent calcium channel blockers widely used in the treatment of cardiovascular diseases, such as hypertension and angina pectoris [15], but antiproliferative effect of these compounds was described as well [16]. Dihydropyridines are divided into three generations - the most frequently used dihydropyridines are nifedipine and nicardipine (first generation), nimodipine (second generation) and amlodipine (third generation) [17, 18]. Dihydropyridines inhibit calcium entry into the cell by interaction with α1 subunit of L-type voltage-dependent calcium channel 3

[17]. Lee and colleagues reported that dihydropyridines, such as nifedipine, inhibit proliferation of human brain tumor cells [19]. Other study showed that nifedipine increased the migration of colon cancer cell line HCT116 [20]. Also, nifedipine was shown to stimulate proliferation and migration of breast cancer cells and promoted formation of metastasis [21, 22]. However, increased risk of breast cancer by long-term use of calcium channel blockers was not observed in the study of Wilson and colleagues [23]. Verapamil, other calcium channel blocker, has no effect on tumor growth, proliferation and migration of breast cancer cells [21, 22]. Pahor and colleagues [24] have shown that use of calcium-channel blocker was associated with significantly increased risk ratio for cancers of the uterus and adnexa uteri, and lymphatic and hemopoietic organs. In their work the hazard ratio was increased, but did not reach significance for stomach, colon, rectum, breast, prostate, and urinary tract cancers. Also, these authors reported that risk is significantly dependent on dose-response gradient of calcium-channel blockers [24]. Long-term use of calcium channel blockers (for example nifedipine and diltiazem) was reported to be associated with higher risk of breast [25-27] and prostate cancers [28, 29]. Nevertheless, some other papers claimed that use of antihypertensive drugs, such as calcium channel blockers, is not associated with higher risk of breast cancer [30-32]. Differences in signaling pathways, by which dihydropyridines promote their effects in breast cancer compared to other types of cancers, are still not fully understood, mainly because breast cancer is a heterogeneous group of diseases. According to the expression of hormone receptors, oncogenes, and tumor suppressor genes there are three basic therapeutic groups of breast cancer: 1) with the expression of estrogen and progesterone receptors (ER+, PR+, HER2-), 2) with HER2 oncogene amplification (HER2- amplified) and 3) tumors without estrogen and progesterone receptors and HER2 positivity, called triple-negative carcinomas (ER-, PR-, HER2-, triple-negative or basal-like). Thus, current knowledge about the effect of 4

dihydropyridines on cell’s proliferation and migration in different types of breast cancer and the mechanism of their action is still unclear. Since dihydropyridines are known as blockers of calcium channels, this work was built on previous observations from different laboratories that nifedipine might have different effects on various malignities, e.g. increased proliferation/migration vs. no effect in breast cancers, or beneficial effects in glioblastoma. These effects are probably realized through potentiation of pro- or antiapoptotic effects. Therefore, we focused our interest downstream from dihydropyridine calcium channels. Since mitochondrial pathway of apoptosis is induced by higher release of calcium from ER, we hypothesize that type 1 IP3 receptors are modulated by altered nifedipine effects in various cell lines. Also, another goal of our work was to connect calcium transport in nifedipinetreated cells with possible changes in proliferation and/or migration in breast cancer cells vs. other cancer cells. We used breast MDA-MB-231 (representing a model of triple negative breast cancer) and JIMT1 cancer cell line (that is an unique model of HER2-positive breast cancer, which possesses amplification of HER2 receptor, but cells do not response to HER2 inhibition treatment with trastuzumab), ovarian A2780 cancer cell line (since it is a part of reproductive system) and colorectal DLD1 cancer cell line (a typical representative of hypoxic tumors). 2. Materials and methods 2.1. Cell cultivation, treatment and proliferation Experiments were performed on human colon adenocarcinoma cell line DLD1 (CCL-221, ATCC, USA), human ovarian cancer cell line A2780 (93112519, Sigma-Aldrich, USA), human breast cancer cell line MDA-MB-231 (HTB-26, ATCC, USA), and human breast cancer cell line JIMT1 (ACC 589, DSMZ, Germany). Cell lines were cultured in RPMI medium (Sigma-Aldrich, USA) or Dulbecco Minimal Essential Medium (DMEM; Sigma5

Aldrich, USA) with a high glucose (4.5g/L) and L-glutamine (300μg/mL), supplemented with 10% fetal bovine serum (Sigma-Aldrich, USA), penicillin (Calbiochem, USA; 100U/mL) and streptomycin (Calbiochem, USA; 100μg/mL). Cells were cultured in humidified atmosphere at 37°C and 5% CO2. After plating, cells were treated with nifedipine (Sigma-Aldrich, USA) in a final concentration 1, 10 and 100µM for 24h. Proliferation of DLD1/MDA-MB231/JIMT1 cells was determined in IncuCyte ZOOMTM kinetic imaging system (Essen Bioscience) on 96-well plate using 3000/2000/4000 cells per well. The effect of nifedipine on proliferation of tumor cells was followed for the period of 5 days. Cell proliferation was evaluated by IncuCyte ZOOM™ 2016A software (Essen BioScience, UK) and expressed as means of octaplicates ± SEM. All experiments were performed on cell passages 4-10. 2.2. Determination of cell cycle Changes in cell cycle were determined by flow cytometric measurement of DNA content of nuclei labeled with propidium iodide (PI; Roche Diagnostics, USA). Briefly, cells (3 x 105) were collected using cell scrapper, washed twice with cold PBS and incubated in 300μl PBS with 0.05% Triton X-100 (Sigma-Aldrich, USA) and 15µl RNAse A (10mg/ml; Roche Diagnostics, USA) for 20 min at 37 °C. Afterwards, cells were cooled on ice for at least 10 min before PI (50µg/ml) was added. Finally, the stained cells were analyzed using a FACS Aria II flow cytometer (Becton Dickinson) equipped with 488nm excitation laser and filters for cell cycle analysis: log 675/40 - sub-G0; lin 585/42 - DNA cell cycle histogram; (585/42 peak area vs. peak height for doublets discrimination). Forward/side light scatter characteristic was used to exclude the cell debris from the analysis. 2.3. Cell migration assay Sixty thousand DLD1 cells, 35000 MDA-MB-231 cells, and 50000 JIMT1 cells per well were plated on ImageLock 96-well plates (Essen BioScience, UK), and let to adhere for 24h. Forty6

eight thousand DLD1 cells, 28000 MDA-MB-231 cells, and 35000 JIMT1 cells per well were plated on ImageLock 96-well plates (Essen BioScience, UK), and let to adhere for 24h after silencing of either IP3R1 or NCX1. Confluent monolayers were then wounded with wound making tool (IncuCyte WoundMaker; Essen BioScience), washed twice and supplemented with fresh culture medium. Images were taken every 2h for the next 26h in the IncuCyte ZOOM™ kinetic imaging system (Essen BioScience, UK). Cell migration was evaluated by IncuCyte ZOOM™ 2016A software (Essen BioScience, UK) based on the relative wound density measurements and expressed as means of octaplicates ± SEM. 2.4. Gene silencing Cells were grown in 24-well or 96-well plates in RPMI or DMEM medium with 10% FBS. Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously in [33]. ON-TARGET plus SMART pool human ITPR1 (L-006207-00-0005, Dharmacon, Thermo Scientific, USA) and NCX1 (L-007620-00-0005, Dharmacon, Thermo Scientific, USA) siRNAs were applied to the final concentration of 50pmol (96-well plate) or 100pmol (24-well plate) per well for 48h. The same procedure was performed with ON-TARGET plus Non-targeting Pool (Dharmacon, Thermo Scientific, USA), which serves for the determination of baseline cellular responses in RNAi experiments. After the first 24h of silencing, nifedipine (10µM and/or 100µM) was applied for additional 24h. All groups of cells were then harvested and used in further experiments. 2.5. Cytosolic [Ca2+]i staining by Fura2-AM fluorescent dye Cells were plated on a 24-well plate at the density of 5 x 105. After the treatment, cells were washed with 1ml of serum-free medium and loaded with 5µM FURA-2 AM; (Sigma-Aldrich, USA) in the presence of 0.5% pluronate (Sigma-Aldrich,USA) and 0.1nM 7

ionomycin (Sigma-Aldrich, USA) in serum-free medium for 60 min at 37°C in the dark. The cells were then washed three times with 500µl of phosphate saline buffer (PBS). Fluorescence was measured on the fluorescence scanner Synergy II (BioTek, Germany) at λex 340/380nm and λem 516nm. The results were calculated as ratio between 340nm and 380nm and expressed as relative fluorescence units (RFU). 2.6. Detection of apoptosis with Annexin-V-FLUOS After the nifedipine treatment, DLD1, A2780, MDA-MB-231, and JIMT1 cells were gently scraped and pelleted at 1000 rpm for 5 min. Cells were then washed with 1ml of PBS, cell pellet was resuspended in 200μl of Annexin-V-FLUOS/ propidium iodide labeling solution (Roche Diagnostics, USA) and incubated at room temperature in dark for 20 min according to the manufacturer´s protocol. After the incubation, samples were diluted with 400µl PBS, placed on ice and measured on BD FACSCanto II flow cytometer (Becton Dickinson, Ann Arbor, USA). Results were evaluated with a Flowing software version 2.5.1. 2.7. Isolation of RNA and real-time PCR Total RNA was isolated using TRI Reagent (Sigma-Aldrich, USA). The purity and integrity of isolated RNAs were checked on GeneQuant Pro spectrophotometer (Amersham Biosciences, UK). Reverse transcription was performed using 1.5µg of total RNAs and Ready-To-Go You-Prime First-Strand Beads with the pd(N6) primer (GE Healthcare Life Sciences, UK). The real-time PCR amplification and detection was carried out on the Applied Biosystems StepOneTM RealTime PCR Systems (Applied Biosystems, USA) as described in [6]. The expression of target genes sodium-calcium exchanger type 1 (NCX1), voltagedependent calcium channel L-type, alpha 1C subunit (CaV1.2), voltage-dependent calcium channel L-type, alpha 1D subunit (CaV1.3), inositol trisphosphate receptor type 1 (IP3R1), inositol trisphosphate receptor type 2 (IP3R2), and inositol trisphosphate receptor type 3 8

(IP3R3) was normalized to the expression of housekeeping gene β-actin. For detection of the β-actin, NCX1, CaV1.2, CaV1.3, IP3R1, IP3R2, and IP3R3 following primers were designed: human β-actin: 5’- ACA TCT GCT GGA AGG TGG AC- 3’ (forward); 5’- TCC TCC CTG GAG AAG AGC TA - 3’ (reverse); human NCX1: 5’- TCC CAT CTG TGT TGT GTT CGC - 3’ (forward); 5’- TCA TCT TGG TCC CTC TCA TC - 3’ (reverse); human CaV1.2: 5’CAT CAC CAA CTT CGA CAA CTT C - 3' (forward); 5'- CAG GTA CGCC TTT GAG ATC TTC TTC - 3' (reverse) [34]; human CaV1.3: 5’- GCA AAC TAT GCA AGA GGC ACC AG - 3' (forward); 5'- GGG AGA GAG ATC CTA CAG GTG G - 3' (reverse) [35]; human IP3R1: 5'- TCT ATG AGC AGG GGT GAG ATG AG - 3' (forward); 5'- GGA ACA CTC GGT CAC TGG AT - 3' (reverse) [36]; human IP3R2: 5'- ATG CGT GTG TCC TTG GAT GC - 3' (forward); 5'- GTA GCA GAA GTA GCT GAT TG - 3' (reverse) [37]; human IP3R3: 5'- AGT GAG AAG CAG AAG AAG G - 3' (forward); 5'- CAT CCG GGG GAA CCA GTC - 3' (reverse) [38]. 2.8. Western blot analysis Cells were scraped and resuspended in 10mM Tris–HCl pH 7.5, 1mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, USA) and subjected to centrifugation for 10 min at 10,000×g at 4°C. The pellet was resuspended in Tris buffer containing the 50µM 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS; Sigma-Aldrich, USA), and incubated for 20 min at 4°C. The lysate was centrifuged for 10 min at 10,000×g at 4°C. Protein concentration in supernatants was determined by Modified Lowry Protein Assay Kit (Thermo Scientific, USA). Protein extract from each sample was separated by electrophoresis on gradient SDS polyacrylamide gels and proteins were transferred to Hybond PVDF blotting membrane (GE Healthcare, Life Sciences, USA) using semidry blotting (Owl, Inc.). The membranes were blocked in 5% non-fat dry milk in TBS-T overnight at 4°C and then incubated with primary mouse monoclonal antibodies: IP3R1 (SAB5200080, Sigma9

Aldrich, USA; 300kDa), NCX1 (ab2869, Abcam, Cambridge, UK; 120kDa), CaV1.2 (ab84814, Abcam, Cambridge, UK; 240kDa), CaV1.3 (ab84811, Abcam, Cambridge, UK; 245kDa) and β-actin (ab6276, Abcam, Cambridge, UK; 42kDa). Horseradish peroxidaselinked secondary goat anti-mouse antibody (ab6789, Abcam, Cambridge, UK) and chemiluminescence detection system (LuminataTM Crescendo Western HRP Substrate, Millipore) were used for visualization. Each membrane was digitally captured using an imaging system (C-DiGit, LI-COR). 2.9. Immunofluorescence Cells grown on glass coverslips were fixed in ice-cold methanol. Non-specific binding was blocked by incubation with PBS containing 3% bovine serum albumin (BSA, Sigma-Aldrich, USA) for 60 min at a room temperature. Cells were then incubated with primary antibodies diluted in PBS with 1% BSA (PBS-BSA) for 1h at 37°C. In these experiments, the anti-NCX1 rabbit polyclonal antibody (1:200 dilution, π11-13, Swant, Switzerland) against the full length canine cardiac NCX1 was used. This antibody recognizes all splice variants of NCX1 and does not cross react with other NCX isoforms. Afterwards, cells were washed three times with PBS with 0.02% TWEEN (Sigma-Aldrich, USA) for 10 min, incubated with Alexa Fluor-488 donkey anti-rabbit IgG (1:1000 dilution, Thermo Fisher Scientific, USA) in PBS-BSA for 1h at 37°C, and washed as described previously. Also, to determine CaV1.3 mouse monoclonal antibody (1:250 dilution, ab84811, Abcam, Cambridge, UK) was used, and to determine CaV1.2 mouse monoclonal antibody (1:100 dilution, ab84814, Abcam, Cambridge, UK) was used. Cells were washed four times with PBS with 0.02% TWEEN (Sigma-Aldrich, USA) for 10 min, incubated with Alexa Fluor-594 goat anti-mouse IgG (1:1000 dilution, Thermo Fisher Scientific, USA) in PBS-BSA for 1h at 37°C and washed as previously. Finally, cover-slips were mounted onto slides in mounting medium with DAPI (Sigma-Aldrich, USA). Images of all samples were acquired with the same microscope setup. Cells were visualized by 10

epifluorescence microscopy using Nikon Eclipse Ti-S/L100 (Nikon, Japan); NIS elements software (Nikon, Japan) was used to process images and to evaluate the resultant pictures. 2.10. Statistical analysis The results are presented as mean ± S.E.M. Each value represents an average of at least 3 wells from at least three independent cultivations of DLD1 and/or A2780 and/or MDA-MB-231 cells and/or JIMT1 cells. Significant differences among groups were determined by ANOVA or Graphpad, verified by Wilcoxon-Mann-Whitney test as described in [39]. For multiple comparisons, an adjusted t-test with p values corrected by the Bonferroni method was used (Instat, GraphPad Software). 3. Results 3.1. Nifedipine affects differently proliferation and migration in DLD1 and MDA-MB-231 cells Treatment with 10µM or 100µM nifedipine for 24h caused significant decrease in proliferation rate in DLD1 cells (Figure 1A) and no changes in cell migration (Figure 1B). In these cells small, but significant difference in S phase of cell cycle was detected in nifedipine treated cells compared to untreated cells (Figure 1A, inset). In MDA-MB-231 cells, nifedipine treatment at the concentration of 10µM did not cause any significant changes in proliferation (Figure 1C) and no change in cell cycle (Figure 1C, inset), but at the concentration of 100µM significant decrease in proliferation was detected. Also, partial suppression in migration of MDA-MB-231 cells (Figure 1D) was seen at the nifedipine concentration 100µM, while no significant changes were determined in MDA-MB-231 cells at 10µM nifedipine (Figure 1D). 3.2. Nifedipine affects levels of intracellular calcium and apoptosis induction differently in breast cancer cell lines compared to other cancer cell lines 11

Since nifedipine is a calcium channel blocker, we proposed that changes in proliferation and migration after nifedipine treatment might be affected by altered calcium signaling. In ovarian A2780 tumor cells and DLD1 colorectal carcinoma cells, nifedipine treatment for 24h increased the cytosolic calcium concentration (Figure 2A,B). The most pronounced increase was observed at 100µM nifedipine. Nifedipine did not cause any significant changes in cytosolic calcium in MDA-MB-231 cells (Figure 2C) and also in JIMT1 cells (Figure 2D). Further, we measured apoptosis induction in A2780, DLD1, MDA-MB-231, and JIMT1 cells after nifedipine treatment (Figure 2E-H). We observed increase in apoptosis induction after nifedipine treatment for 24h in a concentration dependent manner in A2780 (Figure 2E) and DLD1 (Figure 2F) cell lines, but not in MDA-MB-231 (Figure 2G) or JIMT1 cells (Figure 2H). Apoptosis/necrosis was determined in all above-mentioned cells after 10µM nifedipine treatment in a time dependent manner (24h, 48h, 72h). In A2780 and DLD1 cells, nifedipine (10µM) increased apoptosis in a time dependent manner (Figure 2I,J). On the other hand, nifedipine (10µM) decreased late apoptosis after 48h and 72h in MDA-MB-231 cells (Figure 2K), while no significant changes were observed in JIMT1 cells (Figure 2L). 3.3. Expression of the selected calcium transport systems differs in breast cancer vs other cancer cell lines Since nifedipine is the L-type calcium channel blocker, we measured expression of the CaV1.2 and CaV1.3 after nifedipine treatment (10µM). We observed significant elevation in the expression of CaV1.2 and CaV1.3 mRNA and protein after nifedipine treatment in DLD1 (Figure 3A,B,C,D) and A2780 tumor cells (Figure 3E,F,G,H). In breast cancer cells MDAMB-231 and JIMT1 we did not identify CaV1.2 mRNA (Figure 3I,M), but we detected CaV1.2 protein (Figure 3J,N). The expression of CaV1.3 was not changed due to nifedipine treatment, both on mRNA and protein levels in MDA-MB-231 and JIMT1 cells (Figure 3K,L,O,P). In DLD1 and A2780 cells, mRNA levels (Figure 4A,C) and also protein levels (Figure 4B,D) of 12

the NCX1 were elevated after nifedipine treatment. In MDA-MB-231 and JIMT1 breast cancer cells no changes in NCX1 mRNA (Figure 4E,G) were detected. NCX1 protein was not changed in MDA-MB 231 cells (Figure 4F), but decrease was observed in JIMT1 cells after nifedipine treatment for 24h (Figure 4H). Immunofluorescence performed on DLD1, A2780, MDA-MB-231 and JIMT1 cells using CaV1.2 antibody showed increased signal in DLD1, A2780 and JIMT cells, but not in MDA-MB-231 cells (Figure 4I). This result corresponds to western blot analysis and point to possible differences among MDA-MB-231 and other cell lines. Immunofluorescence revealed partial co-localization of the NCX1 (green signal) and CaV1.3 (red signal) in untreated and also in nifedipine-treated DLD1, A2780, JIMT1 and MDA-MB-231 cells (Figure 5). Interestingly, intensity of NCX signal (green) was decreased in JIMT1 cells after nifedipine treatment, but not in MDA-MB-231 cells (Figure 5). The IP3R1 mRNA and protein levels were significantly increased in A2780 and DLD1 cells (Figure 6A,D,E,H), but not in MDA-MB-231 and JIMT1 cells after nifedipine treatment (Figure 6I,L,M,P). Nifedipine did not cause any changes in mRNA levels of the type 2 and 3 IP3 receptor in all studied cell lines (Figure 6B,C,F,G,J,K,N,O). 3.4. Proliferation and migration is modulated differently by nifedipine in DLD1 vs breast cancer cell lines Further, we tested impact of IP3R1 (IP1) and NCX1 silencing on proliferation in DLD1 cells (Figure 7). Group of DLD1 cells, where scrambled siRNA was added, was used as a control for DLD1 cells with silenced NCX1 or IP3R1, since scrambled siRNA alone affected proliferation of DLD1 cells (Figure 7B). When NCX1 was silenced in DLD1 cells, proliferation was increased in group of cells with/without nifedipine (Figure 7D,G), although in nifedipine-treated cells increase in proliferation was much more pronounced (Figure 7G; p=0.0029) compared to groups treated with scrambled siRNA (scr). Similar results were

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observed, when IP3R1 (Figure 7E,H), or combination of NCX1 and IP3R1 was silenced (Figure 7F,I). Also, we tested effect of IP3R1 and NCX1 silencing on proliferation in breast cancer JIMT1 cells (Figure 8). On contrary to DLD1 cells, silencing of the NCX1 did not have effect on proliferation in group of JIMT1 cells with/without nifedipine treatment (Figure 8D,G) compared to scrambled siRNA (scr). Group of JIMT1 cells, where scrambled siRNA was added (scr), was used as a control for JIMT1 cells with silenced NCX1 or IP3R1. Interestingly, silencing of the IP3R1 (Figure 8E) and also combination of silenced NCX1 and IP3R1 in JIMT1 cells revealed increase in proliferation (Figure 8E,F) in untreated cells. After the treatment with nifedipine, increase in JIMT1 cells with silenced IP3R1 was still visible (Figure 8H), although it was not as large as in untreated cells. Contrary to untreated cells, combined silencing of NCX1 and IP3R1 did not show differences in proliferation in nifedipine treated cells (Figure 8I). Further we tested the effect of silencing of IP3R1 and NCX1 on proliferation of breast cancer MDA-MB-231 cells (Figure 9). Silencing of NCX1, IP3R1 and combination of NCX1 and IP3R1 did not have effect on proliferation of MDA-MB-231 cells without nifedipine treatment (Figure 9D,E,F) compared to scrambled siRNA (scr). Group of MDA-MB-231 cells, where scrambled siRNA was added (scr), was used as a control for MDA-MB-231 cells with silenced NCX1 or IP3R1, since scrambled siRNA alone affected proliferation of MDA-MB231 cells. Silencing of NCX1, IP3R1 significantly decreased proliferation in groups with nifedipine treatment (Figure 9G,H). Migration of DLD1 cells was not affected by nifedipine (Figure 10A). Neither silencing of the NCX1 (Figure 10D,G), nor silencing of the IP3R1 (IP1; Figure 10E,H) revealed changes in migration of the DLD1 cells. Also, when both, NCX1 and IP3R1 were silenced, no significant 14

change compared to controls treated with scrambled siRNA (scr) was observed (Figure 10F,I). Treatment of JIMT1 cells with nifedipine resulted in decreased migration (Figure 11A). In JIMT1 cells, silencing of the NCX1 (Figure 11D,G) resulted in decreased migration in group without nifedipine treatment, but not in nifedipine-treated group. Silencing of the IP3R1 (IP1; Figure 11E,H) did not have any effect of migration of JIMT1 cells. Combined silencing of the NCX1 and IP3R1 did not have effect on JIMT1 migration both in control group (Figure 11F), and also in nifedipine-treated group (Figure 11I). Interestingly, in another breast cancer cell line - in MDA-MB-231 cells, silencing of the NCX1 (Figure 12D,G) did not show any changes in migration in groups with/without nifedipine treatment, while silencing of the IP3R1 (IP1; Figure 12E,H) significantly decreased cell migration in the presence of nifedipine compared to scrambled control and did not have any effect in migration of untreated MDA-MB-231 cells. Combined silencing of the NCX1 and IP3R1 revealed decrease in MDA-MB-231 migration in nifedipine-treated group (Figure 12I), but not in untreated group (Figure12F). 3.5. Nifedipine affects calcium transport through NCX1 and IP3R1 in DLD1 and A2780 cells, but not in breast cancer MDA-MB-231 and JIMT1 cells In order to verify that silencing of the NCX1 and/or IP3R1 affects calcium transport, we determined levels of the cytosolic calcium in controlled and individually silenced cells (Figure 13). Silencing of the NCX1 decreased levels of cytosolic calcium in DLD1 (Figure13A) and A2780 cells (Figure 13B), but not in MDA-MB-231 (Figure 13C) and JIMT1 cells (Figure 13D). The same result was observed, when IP3R1 was silenced. When both these transporters were silenced, additional decrease in levels of cytosolic calcium occured in A2780 and DLD1 cells, but not in MDA-MB-231 and JIMT1 cells (Figure 13).

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4. Discussion Nifedipine as a potent antihypertensive drug is commonly used for a treatment of hypertension, or some other cardiovascular complications. However, in women suffering from breast cancer it might represent a potential danger, although results are still controversial. It was already suggested that nifedipine increased proliferation rate and migration of breast cancer cells [21, 22], although Devore and co-workers [31] postulated that antihypertensive medication use was largely not associated with the risk of invasive breast cancer among women. Interestingly, in other types of cancer, the effect of nifedipine might be beneficial [40, 41], e.g. nifedipine was shown to potentiate pro-apoptotic effect of cisplatin in glioblastoma cells [40]. Therefore, initially we compared proliferation and migration of DLD1 cells derived from colorectal carcinoma and breast adenocarcinoma cell line MDA-MB-231 after exposure to nifedipine. We observed significant decrease in proliferation of DLD1 cells, but no significant changes in proliferation of MDA-MB-231 cells after 10µM nifedipine treatment. Migration of DLD1 cells was not affected by nifedipine, but was significantly slower than migration of the MDA-MB-231 cells. Intracellular signaling pathways that underlie individual subtypes of breast cancer are different and thus chemotherapeutics targeting only one pathway will be effective only in some of the breast cancer subtypes [42]. The NeoALTTO study compared trastuzumab, lapatinib, and both inhibitors, in combination with paclitaxel for 12 weeks prior to breast surgery. The combination arm exhibited a higher rate of pathologic complete response (51.3%) than either the trastuzumab (29.5%) or lapatinib (24.7%) arms [43]. Therefore, in order to understand properly mechanism of calcium signaling in breast cancer, in further experiments we used two different breast cancer cell lines, MDA-MB-231 and JIMT1. MDAMB-231 represents a highly aggressive, invasive and poorly differentiated triple-negative breast cancer cell line, since it lacks estrogen and progesterone receptor expression, and also 16

human epidermal growth factor receptor 2 (HER2). JIMT1 is a unique model of HER2positive breast cancer, which possesses amplification of HER2 receptor, but these cells do not response to HER2 inhibition treatment with trastuzumab [44-46]. The proper therapy for this HER2 positive cancer does not exist, since the cells are resistant to trastuzumab and the prognosis of this group of patients is poor. The proper third-line therapy is still under development. Nifedipine as a calcium channel blocker affects the intracellular calcium concentration. We have shown that nifedipine increased levels of cytosolic calcium in DLD1 and A2780 cells in a concentration-dependent manner, but not in any of studied breast cancer cells - MDA-MB231 or JIMT1 cells. Increase in cytosolic calcium might be from the extracellular space, or due to release of calcium from the endoplasmic reticulum (ER). The sustained high cytoplasm Ca2+ is toxic for cells by activating cell death signaling [47]. A sharp fall in the [Ca2+]ER or sustained decrease in [Ca2+]ER, accompanied with release of ER-Ca2+ into the cytoplasm, can induce apoptotic cell death [48]. We observed increase in apoptosis induction in a concentration and a time-dependent manner in DLD1 and A2780 cells, but not in MDA-MB231 and JIMT1 cells. In MDA-MB-231 cells, we have seen significant time-dependent decrease in a number of apoptotic cells, which might suggest that nifedipine acts protectively on these type of breast cancer cells. Expression of the CaV1.2 and CaV1.3 was increased in DLD1 and A2780 cells, while MDAMB-231 and JIMT1 lack the expression of CaV1.2 and have not changed the expression of CaV1.3. Guo et al. [21] also reported that insignificant increase in [Ca2+]i was observed by nifedipine in MDA-MB-231 cells, thus suggesting that effect of nifedipine on the proliferation and migration of MDA-MB-231 cells was not related with calcium channel and cellular Ca2+. These authors also concluded that MDA-MB-231 cells did not express the CaV1.2 and CaV1.3 subtype. The bioinformatics analysis confirmed that CaV1.2 exhibited low expressions in the 17

majority of types of cancer, including brain, lymphoma, ovarian, bladder, prostate, renal, salivary gland, cervix, and colorectal cancers, as compared to normal tissue [49]. These authors suggested that alterations in CaV1.2 expression may have an adverse effect on tissue homeostasis, which may result in tumorigenesis. Expression of CaV1.3 subtype was observed in all tested cancer cell lines. While in DLD1 and A2780 cells expression of this channel subtype was upregulated by nifedipine treatment, in MDA-MB-231 and JIMT1 cells it was not modulated by nifedipine. Ca2+ protein α1D of CaV1.3 channel was shown to be overexpressed in colorectal cancer biopsies compared to normal tissues [20]. Gene silencing experiments targeting α1D reduced the migration and the basal cytosolic Ca2+ concentration of HCT116 colon cancer cell line and modified the cytosolic Ca2+ oscillations induced by the sodium/calcium exchanger NCX1/3 working in its reverse mode [20]. NCX1 is a plasma membrane transporter transporting calcium out and sodium into the cell in majority of cells under physiological conditions. Nevertheless, in some cancer cells NCX1 operates in so called “reverse mode”, thus transporting calcium into the cells and sodium out of the cell [50, 51]. Reverse mode NCX1 is involved in proton maintenance in hypoxic tumors, when it couples to sodium/proton exchanger 1 (NHE1) and this complex extrudes protons from the cell [51]. Due to the nifedipine treatment, NCX1 was increased in DLD1 and A2780 cells. In MDA-MB-231 cells, nifedipine has no effect on NCX1 expression and surprisingly, in JIMT1 cells nifedipine did not change mRNA levels of the NCX1, but decreased its protein levels, as determined by Western blot analysis and also by immunofluorescence. Silencing of the NCX1 resulted in increase in the proliferation compared to group treated with scrambled siRNA in DLD1 cells, and this increase was even more pronounced in the presence of nifedipine. Based on these results we concluded that NCX1 might suppress proliferation in DLD1 cells. These experiments cannot be performed on A2780 cells, since these cells do not form a monolayer. Nevertheless, silencing of the 18

NCX1 did not have any effect on proliferation of JIMT1 and MDA-MB-231 cells in untreated conditions, but after nifedipine treatment there was a significant decrease of proliferation rate in NCX1 silenced MDA-MB-231 cells. This result would point to opposite effect of the NCX1 in nifedipine-treated MDA-MB-231 cells compared to DLD1 cells. Moreover, NCX1 might function differently in JIMT1 and MDA-MB-231 cells. NCX1 silencing did not have effect on migration of DLD1 and MDA-MB-231 cells in normal cells, or after nifedipine treatment. In JIMT1 cells, migration of cells with silenced NCX1 was decreased in untreated cells, but unaffected in nifedipine-treated cells, probably due to lower expression of the NCX1. Since it was already published that release of calcium from the ER through IP3R1 can induce apoptosis [4, 52], we focused also on this type of receptor. We observed that IP3R1 is upregulated by nifedipine treatment in DLD1 and A2780 cell, but not in MDA-MB-231 and JIMT1 cells, which corresponded to the apoptotic status already discussed above. In DLD1 cells, silencing of the IP3R1 increased proliferation and nifedipine treatment even pronounced this outcome. Similar effect was observed in JIMT1 cells, although due to nifedipine treatment, increase in proliferation was not so extensive. Proliferation of the JIMT1 cells was slower than proliferation of the MDA-MB-231 cells. Silencing of the IP3R1 in MDA-MB-231 cells did not have any effect on proliferation in untreated conditions, but nifedipine treatment caused decreased proliferation in group of cells with silenced IP3R1. From these results it is apparent that IP3R1 acts differently in JIMT1 and MDA-MB-231 cells. In DLD1 and JIMT1 cells, migration was not affected by IP3R1 silencing neither in normal, nor in nifedipine treated cells. Nevertheless, in MDA-MB-231 cells, silencing of the IP3R1 significantly decreased migration in nifedipine treated cells. From these results it is apparent that IP3R1 plays a different role in MDA-MB-231 cells compared to JIMT1 and DLD1 cells.

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Triple-negative breast cancer is an aggressive form of the disease with usually high histologic grade [53]. According to the American Cancer Society the 5-year survival rates for breast cancer are near to 100% in stage 0 to 1, about 93 % in stage 2, 72% in stage 3, and 22% in stage 4 (metastatic). The distant recurrence rate in the first year after the breast cancer diagnosis is 3 times higher in triple-negative cancer than in non-triple negative cancers [53]. In our experiments we observed different involvement of NCX1 and IP3R1 in migration of the triple-negative MDA-MB-231cells and JIMT1 breast cancer cells. Differences in calcium signaling might account for, or at least contribute to invasiveness of different types of breast cancer cells. Mechanism, how can nifedipine modulate expression is not clear. Two papers are reporting the effect of calcium channel blockers (CCBs) on responsive elements. Sugiura et al. [54] proposed a possible mechanism responsible for beneficial effects of these blockers on renal protection. CCBs inhibited extracellular matrix production by suppression of TGFß expression which was, at least in part, mediated by suppression of AP-1. The suppression of AP-1 was effect also thought to contribute to the inhibition of cell proliferation. Other paper by Hayashi et al. [55] showed the effect of CCBs on NFkB activation in the mesangial cells. Taken together, mechanism of dihydropyridine action is very complicated and may differ in various cancer cells. Therefore, further studies will be need to carify this issue. We have shown that nifedipine affects proliferation and migration differently in diverse cancer cell lines. Silencing of the NCX1 and IP3R1 in the presence/absence of nifedipine affects differently proliferation and migration in JIMT1 and MDA-MB-231 breast cancer cells, compared to colorectal carcinoma DLD1 cells. Moreover, marked differences in the proliferation and migration due to NCX1/IP3R1 were observed in JIMT1 and MDA-MB-231 cells. These results might point to different role of nifedipine in different cancer cells.

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Acknowledgement This work was supported by grants APVV-16-0246 and VEGA 2/0038/19, also by the project no. LQ1605 from the National Program of Sustainability II (MEYS CR) and MUNI/A/1255/2018. The kinetic measurement by IncuCyte ZOOM Imaging system was enabled by financial support from the Slovak Cancer Research Foundation. Authors wish to thank Mrs. Marta Sirova for the help with cell cultures, Mgr. Michal Zuzcak for the help with Western blots and Bc. Katarina Kovacova for the help with RNA determinations. Abbreviations CaV1.1 - CaV1.4: isoforms of the L-type voltage calcium channel IP3R: inositol 1,4,5-trisphosphate receptor NCX1: sodium/calcium exchanger type 1 NHE1: sodium/proton exchanger type 1 SERCA: sarco/endoplasmic reticulum Ca2+-ATPase Conflict of interest The authors declare that they have no competing interests. References [1] J. Parkash, K. Asotra, Calcium wave signalling in cancer cells, Life Sci, 87 (2010), pp. 587-595. doi: 10.1016/j.lfs.2010.09.013 [2] N. Prevarskaya, R. Skryma, Y. Shuba, Targeting Ca2+ transport in cancer: close reality or long perspective? Expert Opin Ther Targets, 17 (2013), pp. 225-241. doi: 10.1517/14728222.2013.741594

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[48] T. Capiod, Y. Shuba, R. Skryma, N. Prevarskaya, Calcium signalling and cancer cell growth, Subcell Biochem, 45 (2007), pp. 405–427. [49] N.N. Phan, Ch.Y. Wang, Ch.F. Chen, Z. Sun, M.D. Lai, Y.Ch. Lin, Voltage-gated calcium channels: Novel targets for cancer therapy. Oncol Lett, 14 (2017), pp. 2059–2074. doi: 10.3892/ol.2017.6457 [50] S.R. Sennoune, J. M. Santos, F. Hussain, R. Martínez-Zaguilán, Sodium calcium exchanger operates in the reverse mode in metastatic human melanoma cells, Cell Mol Biol, 61 (2015), pp. 40-49. [51] I. Szadvari, S. Hudecova, B. Chovancova, M. Matuskova, D. Cholujova, L. Lencesova, D. Valerian, K. Ondrias, P. Babula, O. Krizanova, Sodium/calcium exchanger is involved in apoptosis induced by H2S in tumor cells through decreased levels of intracellular pH, Nitrci Oxide, 87 (2019), pp. 1-9. doi: 10.1016/j.niox.2019.02.011 [52] I. Rezuchova, S. Hudecova, A. Soltysova, M. Matuskova, E. Durinikova, B. Chovancova, M. Zuzcak, M. Cihova, M. Burikova, A. Penesova, L. Lencesova, J. Breza, O. Krizanova, Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells, Cell Death Dis, 10 (2019), pp. 186. doi: 10.1038/s41419019-1433-4 [53] W.D. Foulkes, I.E. Smith, J.S. Reis-Filho, Triple-negative breast cancer, N Engl J Med, 363 (2010), pp. 1938-1948. doi: 10.1056/NEJMra1001389 [54] T. Sugiura, E. Imai, T. Moriyama, M. Horio, M. Hori, Calcium channel blockers inhibit proliferation and matrix production in rat mesangial cells: possible mechanism of suppression of AP-1 and CREB activities. Nephron, 85 (2000), pp. 71-80.

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Figure legends Figure 1. Proliferation (A, C) and migration (B, D) of DLD1 (A, B) and MDA-MB-231 (C, D) cells after nifedipine (NIF) treatment. Nifedipine was used at concentrations 10μM and 100μM. In DLD1 cells, reduced proliferation (A; NIF 10μM - p=0.009; NIF 100μM p=0.002) and no change in cell migration (B; NIF 10μM - p=0.123; NIF 100μM - p=0.061) was observed after nifedipine treatment. In MDA-MB-231 cells, 10µM nifedipine did not cause any changes in proliferation, but 100µM nifedipine caused significant decrease in proliferation (C; NIF 10μM - p=0.060; NIF 100μM - p=0.0001) and also in migration (D; NIF 10μM - p=0.847; NIF 100μM - p=0.0010). In DLD1 cells, S phase was significantly decreased due to nifedipine treatment (A, inset), but in MDA-MB-231 cells no changes in cell cycle were determined after treatment with nifedipine (C, inset). Results are displayed as mean ±SEM, n=8. Statistical significance was calculated by Graphpad, verified by WilcoxonMann-Whitney test, * - p<0.05. Figure 2. Determination of apoptosis (E-H), apoptosis/necrosis (I-L) and changes in levels of the cytosolic calcium (A-D) in A2780 (A,E,I), DLD1 (B,F,J), MDA-MB-231 (C,G,K), and JIMT1 (D,H,L) cells after nifedipine treatment. In tumor cell lines A2780 (A) and DLD1 (B), 24h treatment of nifedipine (final concentration 1µM (NIF1), 10µM (NIF10) and 100µM (NIF100)) increased levels of cytosolic calcium. In tumor cell lines MDA-MB-231 (C) and JIMT1 (D), nifedipine did not significantly change levels of the cytosolic calcium. Nifedipine treatment increased early apoptosis in A2780 (E) and DLD1 (F) cell lines in a concentration30

dependent manner, but no significant changes were observed in apoptosis induction in MDAMB-231 (G) and JIMT1 (H) cell line after 24h of nifedipine treatment. Also, apoptosis/necrosis was increased by nifedipine in a time dependent manner in A2780 (I) and DLD1 (J) cell lines, but in MDA-MB-231 (K) cell line decrease in apoptosis/necrosis occurred after 48h and 72h of treatment and in JIMT1 cells no significant change was observed during 24-72h of nifedipine treatment. cont – untreated control, 24NIF10 – nifedipine treatment for 24h (final concentration 10µM), 48NIF10 - nifedipine treatment for 48h (final concentration 10µM), 72NIF10 - nifedipine treatment for 72h (final concentration 10µM). Results are displayed as mean ±S.E.M. Each column represents an average of at least 6 samples from two independent cultivations. Statistical significance * represents p˂0.05, ** p˂0.01 and *** p˂0.001 compared to untreated control. Figure 3. Changes in expression of the CaV1.2 (A,B,E,F,I,J,M,N) and CaV1.3 (C,D,G,H,K,L,O,P) in DLD1 (A-D), A2780 (E-H), MDA-MB-231 (I-L), and JIMT1 (M-P) due to the nifedipine treatment (NIF, 10µM, 24h). In DLD1 and A2780 cell lines, mRNA levels of CaV1.2 (A,E) and CaV1.3 (C, G) were increased due to NIF treatment. In MDA-MB231 and JIMT1 cell lines, no CaV1.2 mRNA was detected (I,M), although clear signal was observed by western blot analysis (J,N). The mRNA and protein levels of CaV1.3 (K,O,L,P) were not increased after nifedipine treatment. cont – untreated control. Results are displayed as mean ±S.E.M. Each column represents an average of 6-9 samples from three independent cultivations. Statistical significance * represents p˂0.05 and ** p˂0.001. Figure 4. Changes in expression of the NCX1 (A-H) and CaV1.2 immunofluorescence (I) in DLD1 (A,B,I), A2780 (C,D,I), MDA-MB-231 (E,F,I), and JIMT1 (G,H,I) due to the nifedipine treatment (NIF, 10µM, 24h). In DLD1 and A2780 cell lines, nifedipine significantly increased the NCX1 on mRNA (A, C) and protein levels (B, D). Protein expression of the NCX1 was not changed in MDA-MB-231 cells (F), but decreased in JIMT1 31

cells (H). cont – untreated control. Results are displayed as mean ±S.E.M. Each column represents an average of 6-9 samples from three independent cultivations. Statistical significance * represents p˂0.05 and ** p˂0.001. Immunofluorescence of CaV1.2. (I) in the control (cont) and nifedipine (NIF) treated group in DLD1, A2780, MDA-MB-231 and JIMT1 cells. Insets show the negative controls without primary antibody for each type of cells. Scale bar represents 10 m. Figure 5. Immunofluorescence of CaV1.3 (red signal) and NCX1 (green signal) in DLD1, JIMT1, A2780 and MDA-MB-231 tumor cell lines in control conditions and in nifedipine (10µM NIF) treated groups. Nuclei were counterstained with DAPI (blue signal). Colocalization of these two transporters was observed in all types of cells (yellow signal; merged). Scale bar represents 10μm. Figure 6. Changes in expression of the IP3R1 (A,E,I,M,D,H,L,P), IP3R2 (B,F,J,N) and IP3R3 (C,G,K,O) in DLD1 (A-D), A2780 (E-H), MDA-MB-231 (I-L), and JIMT1 (M-P) cells after 24h nifedipine treatment (NIF; 10µM). In tumor cell lines DLD1 and A2780, nifedipine increased IP3R1 mRNA (A, E) and protein (D, H) levels. Nifedipine did not cause any changes in IP3R2 (B, F) and IP3R3 (C, G) mRNA expression neither in DLD1 cells, nor in A2780 cells. No changes in IP3R1 (I, M), IP3R2 (J, N) and IP3R3 (K, O) mRNA were observed in MDA-MB-231 and JIMT1 breast tumor cell lines. cont – untreated control. Results are displayed as mean ±S.E.M. Each column represents an average of 6-9 samples from three independent cultivations. Statistical significance *** represents p˂0.001. Figure 7. Proliferation of DLD1 cells in control (D, E, F) and nifedipine-treated group (G, H, I; NIF; 10µM) after silencing of NCX1 (D, G), IP3R1 (IP1; E, H), or combination of both (NCX1+IP1; F, I). Proliferation of cells with silenced NCX1, IP1, or NCX1+IP1 was compared with cells, where scrambled siRNA (scr) was used, since scr decreased rate of 32

proliferation (B; p=0.0008). Significantly increased proliferation was observed in control, scrambled siRNA treated DLD1 cells with silenced NCX1 (p=0.0163), IP1 (p=0.0041), or in group of cells with double silencing of NCX1+IP1 (p=0.0100). Even more pronounced changes were observed in nifedipine-treated cells after silencing NCX1 (p=0.0029), IP1 (p=0.0018), or NCX1+IP1 (p=0.0034). Results are displayed as mean ±SEM, n=8. Statistical differences were determined by Graphpad, verified by Wilcoxon-Mann-Whitney test. Figure 8. Proliferation of JIMT1 cells in control and nifedipine-treated group (NIF; 10µM) after silencing of the NCX1 (D, G), IP3R1 (IP1; E, H), or combination of both (NCX1+IP1; F, I). Proliferation of cells with silenced NCX1, IP1, or NCX1+IP1 was compared with cells, where scrambled siRNA (scr) was used, since scr decreased rate of proliferation (p=0.0001). No change in proliferation was observed in control, untreated JIMT1 cells with silenced NCX1 (p=0.0679). Nevertheless, significant increase was observed in untreated JIMT1 cells, where IP1 (p=0.0001) or combination of NCX1 and IP1 was silenced (p=0.0005). In the similarly silenced groups of JIMT1 cells treated with nifedipine, no increase in silenced groups was observed. Results are displayed as mean ±SEM, n=8. Statistical differences were determined by Graphpad, verified by Wilcoxon-Mann-Whitney test. Figure 9. Proliferation of MDA-MB-231 cells in control and nifedipine-treated group (NIF; 10µM) after silencing of the NCX1 (D, G), IP3R1 (IP1; E, H), or combination of both (NCX1+IP1; F, I). Proliferation of cells with silenced NCX1, IP1, or NCX1+IP1 was compared with cells, where scrambled siRNA (scr) was used, since scr decreased rate of proliferation (p=0,0134). No changes in proliferation was observed in untreated cells with silenced NCX1, IP1 or combination of both. However, significant decrease was determined in groups with silenced NCX1, IP1 after nifedipine treatment. Results are displayed as mean ±SEM, n=8. Statistical differences were determined by Graphpad, verified by WilcoxonMann-Whitney test. 33

Figure 10. Migration of DLD1 cells in control and nifedipine-treated group (NIF; 10µM, 24h) after silencing of NCX1 (D, G), IP3R1 (IP1; E, H), or combination of both (NCX1+IP1; F, I). Migration of cells with silenced NCX1, IP1, or NCX1+IP1 was compared with cells, where scrambled siRNA (scr) was used. No significant change was observed in any tested group with/without nifedipine and silenced NCX1, IP1, or combination of both. Results are displayed as mean ±SEM, n=8. Statistical differences were evaluated by Graphpad, verified by Wilcoxon-Mann-Whitney test. Figure 11. Migration of JIMT1 cells in control and nifedipine-treated group (NIF; 10µM, 24h) after silencing of NCX1 (D, G), IP3R1 (IP1; E, H), or combination of both (NCX1+IP1; F, I). Migration of cells with silenced NCX1, IP1, or NCX1+IP1 was compared with cells, where scrambled siRNA (scr) was used, since silencing affects cell migration (B). Significant change was observed only in group without nifedipine treatment and silenced NCX1 (D, p=0.0445), but not in the group with silenced NCX1 and treated with nifedipine (G; p=0.1488). Results are displayed as mean ±SEM, n=8. Statistical differences were evaluated by Graphpad, verified by Wilcoxon-Mann-Whitney test. Figure 12. Migration of MDA-MB-231 cells in control and nifedipine-treated group (NIF; 10µM, 24h) after silencing of NCX1 (D,G), IP3R1 (IP1; E, H), or combination of both (NCX1+IP1; F, I). Migration of cells with silenced NCX1, IP1, or NCX1+IP1 was compared with cells, where scrambled siRNA (scr) was used. Significant change was observed in tested groups treated with nifedipine and silenced either IP1 (H; p=0.0039) alone, or in combination with NCX1 (I; p=0.0281). Results are displayed as mean ±SEM, n=8. Statistical differences were evaluated by Graphpad, verified by Wilcoxon-Mann-Whitney test. Figure 13. Determination of levels of cytosolic calcium in DLD1 (A), A2780 (B), MDA-MB231 (C) and JIMT1 (D) cells in control and nifedipine treated cells after silencing NCX1,

34

IP3R1, or combination of both. Results are displayed as mean ±SEM, n=8. Statistical significance was evaluated by one-way ANOVA. Statistical significance * represents p˂0.05, ** p˂0.01 and *** p˂0.001 compared to untreated control. Statistical significance # represents p˂0.05, ## p˂0.01 and ### p˂0.001 compared to nifedipine treated group.

35

percentage of DLD1 cells

Proliferation DLD1

C

control NIF10

80

60

20

20

15

15

*

10

B

control NIF10

80

60

10

5 0

Proliferation MDA-MB-231 percentage of MDA-MB-231 cells

Figure 1 A

G0/G1

S

G2

Migration DLD1

D

5 0

G0/G1

S

G2

Migration MDA-MB-231

Figure 2

NIF10 NIF100

14 12

***

10

***

8 6 4 2 0

I

cont

NIF1

NIF10

35

NIF100

***

30 25

***

20

** *

1.6

1.5

1.5 cont

F

NIF1

NIF10 NIF100

***

14 12

6 4

2 cont

NIF1

NIF10

5

cont

24NIF1048NIF1072NIF10

NIF10 NIF100

14

NIF100

J 25

***

20

** *

** *

5 0

cont

24NIF10 48NIF10 72NIF10

1.2 1.0 0.8

H

cont

NIF1

cont

NIF1

NIF10 NIF100

12 10

6 4 2 0

1.4

14

8

K

10

10

NIF1

10

8

0

cont

G

JIMT1, 24h

0.6

12

10

15

15

0

1.7

1.6

Annexin V-FLUOS positive cells (%)

NIF1

1.6

1.8

1.7

Anexin V-FLUOS/PI positive cells (%)

Annexin V-FLUOS positive cells (%)

cont

2.0 1.9

1.8

1.5

1.8

RFU/μg protein

1.6

2.1

Annexin V-FLUOS positive cells (%)

1.7

*

1.9

2.0

MDA-MB-231, 24h

RFU/μg protein

2.0

RFU/μg protein

1.8

E

***

2.1

1.9

Anexin V-FLUOS/PI positive cells (%)

RFU/μg protein

2.2

*

2.0

2.2

DLD1, 24h

***

D

Annexin V-FLUOS positive cells (%)

A2780, 24h

2.1

C

cont

NIF1

NIF10 NIF100

15

8 6 4 2 0

L Anexin V-FLUOS/PI positive cells (%)

B

Anexin V-FLUOS/PI positive cells (%)

A

NIF10 NIF100

15 12

10

* *

5

0

cont

*

24NIF1048NIF1072NIF10

9 6 3 0

cont

24NIF1048NIF1072NIF10

10

A2780

actin protein (a.u.)

**

12

NIF

9

Cav1.2/

6

3

NIF

Cav1.2 240 kDa -actin 42 kDa

8 6

**

4 2 0 cont

0 NIF

J

20

6 actin protein (a.u.)

cont

MDA-MB-231 15

10

cont

NIF

NIF

5 4

3

-actin 42 kDa

NIF

2

Cav1.2/

5

0 cont

1 0

NIF

cont

N

cont 6

20 actin protein (a.u.)

JIMT1 15

Cav1.2/

10

5

5 4

NIF

Cav1.2 240 kDa -actin 42 kDa

*

3 2 1 0

0 cont

NIF

cont

NIF

actin protein (a.u.) Cav1.3/

cont

H

cont

A2780

6

3

-actin 42 kDa

4 3

1 0

cont

9

5

Cav1.3 245 kDa

2

0

3

NIF

NIF

6

3

9

O

*

9

K Cav1.2 240 kDa

*

0

NIF

G

3

1

0

cont

4

2

actin protein (a.u.)

cont

cont

3

Cav1.3 245 kDa -actin 42 kDa

5

Cav1.3/

0

6

NIF

cont

L

MDA-MB-231 6

10 actin protein (a.u.)

NIF

**

9

Cav1.3/

cont

2

NIF

6

0 cont

P

NIF

JIMT 1 6

cont

NIF

NIF

8

Cav1.3 245 kDa -actin 42 kDa

6 4 2 0 cont 10

actin protein (a.u.)

0

M

actin mRNA (a.u.)

4

cont

DLD1

Cav1.3/

3

*

Cav1.3 mRNA/ actin mRNA (a.u.)

6

6

D

Cav1.3

12

Cav1.3 mRNA/ actin mRNA (a.u.)

9

-actin 42 kDa

8

C

actin mRNA (a.u.)

actin protein (a.u.)

12

Cav1.2 240 kDa

Cav 1.3 mRNA/

**

NIF

10

F

Cav1.2 mRNA/ actin mRNA (a.u.) Cav1.2 mRNA/

actin mRNA (a.u.)

I

cont

B

actin mRNA (a.u.)

DLD1

Cav 1.3 mRNA/

Cav1.2

Cav1.2/

Cav1.2 mRNA/ actin mRNA (a.u.)

15

E

Cav1.2 mRNA/

Figure 3

A

cont

8

NIF

NIF

Cav1.3 245 kDa -actin 42 kDa

6 4 2 0

0 cont

NIF

cont

NIF

B

*

7

12 10 8 6

NCX1/

2

1

A2780 actin protein (a.u.)

6

NCX1/

6

2

4 2

cont

NIF NCX1 120 kDa -actin 42 kDa

5

***

4 3

1

NIF

cont

MDA-MB-231

F

7

8 6

actin protein (a.u.)

10

6

NCX1/

12

2

4 2

MDA-MB-231

NIF

cont

NIF NCX1 120 kDa

5

-actin 42 kDa

4 3

MDA-MB-231

cont

1 0

cont

NIF

H

20

JIMT1 15

10

5

cont

NIF

cont 7

JIMT1

NIF

cont

NIF NCX1 120 kDa

6 5

-actin 42 kDa

4 3

***

2 1 0 cont

NIF

JIMT1

0

NCX1/ actin protein (a.u.)

NCX1 mRNA/

NIF

0

0

actin mRNA (a.u.)

**

7

**

8

NCX1 mRNA/

actin mRNA (a.u.)

10

E actin mRNA (a.u.)

3

D A2780

Cont

-actin 42 kDa

4

cont

12

Cav1.2

NCX1 120 kDa

5

NIF

C

NCX1 mRNA/

NIF

A2780

cont

Figure 4

I

0

0

0

cont

6

2

4

G

DLD1

DLD1

14

DLD1

actin protein (a.u.)

NCX1

16

NCX1 mRNA/

actin mRNA (a.u.)

A

NIF

Figure 5

NC

DLD1

JIMT1

A2780

MDA-MB-231

cont Cav1.3

cont NCX1

cont merged

NIF Cav1.3

NIF NCX1

NIF merged

cont

8 6

16 14 12 10 8 6 4 2 0 NIF

14

10 8 6

18

6

4

2 0 cont

NIF

actin protein (a.u.) IP3R1/

7

16 14 12 10

12 9

3 0 NIF

14

10 8 6 4

2 0 cont

NIF

4 2

NIF

IP3R1 300 kDa

A2780

-actin 42 kDa

**

5 4 3 0

cont

NIF

18

L7

MDA-MB-231

15 12 9 6 3

cont

MDA-MB-231

NIF

cont

NIF IP3R1 300 kDa

6 5

-actin 42kDa

4 3 2 1 0

0

O JIMT1

12

6

K

MDA-MB-231

cont

8

NIF

cont

6

0

NIF

15

N JIMT1

12

cont

1

H

A2780

18

0

J

MDA-MB-231

cont

2

2

cont

actin protein (a.u.)

18

NIF

4

**

3

actin protein (a.u.)

10

-actin 42 kDa

4

IP3R1/

12

IP3R2 mRNA/ cont

IP3R3 mRNA/ actin mRNA (a.u.)

14

Figure 6 IP3R1 300 kDa

5

20

16

NIF

6

NIF

G

A2780

DLD1

cont

0 cont

IP3R3 mRNA/

***

NIF

F

actin mRNA (a.u.)

actin mRNA (a.u.) IP3R1 mRNA/ actin mRNA (a.u.) IP3R1 mRNA/

A2780

IP3R2 mRNA/

IP3R1 mRNA/

actin mRNA (a.u.)

E 20 18 16 14 12 10 8 6 4 2 0

0

NIF

actin mRNA (a.u.)

cont

2

DLD1

IP3R1/

0

4

20 18 16 14 12 10 8 6 4 2 0

D7

cont

NIF

14

P7 JIMT1

actin protein (a.u.)

2

M

6

IP3R3

12 10 8 6

6

cont

JIMT1

NIF

cont

2

-actin 42 kDa

4 3

1 0

0 cont

NIF

NIF IP3R1 300 kDa

5

2

4

IP3R1/

4

I

8

IP3R2 mRNA/

6

10

IP3R2 mRNA/

IP3R1 mRNA/

8

actin mRNA (a.u.)

10

12

IP3R3 mRNA/

12

14

C

DLD1

actin mRNA (a.u.)

14

IP3R2

actin mRNA (a.u.)

***

B

actin mRNA (a.u.)

actin mRNA (a.u.)

16

DLD1

IP3R2 mRNA/

IP3R1

actin mRNA (a.u.)

A

cont

NIF

Figure 7

Proliferation - DLD1

A

B p=0.0257

D

p=0.0163

G

C

E

p=0.0041

H p=0.0029

p=0.0005

p=0.0008

F

p=0.01

I p=0.0018

p=0.0034

Figure 8

Proliferation – JIMT1

A

B p=0.3715

D

p=0.0679

G

C

E

p=0.0001

H p=0.6630

p=0.0137

p=0.0001

F

p=0.0005

I p=0.0196

p=0.3853

Figure 9

Proliferation -MDA-MB-231

A

B p=0.3949

D

p=0.3493

G

C p=0.0134

E

p=0.1369

H p=0.0029

p=0.0002

F

p=0.4270

I p=0.0056

p=0.1302

Figure 10

Migration - DLD1

A

B p=0.1966

D

p=0.1731

G

C p=0.3104

E

p=0.2024

H p=0.2156

p=0.9019

F

p=0.4012

I p=0.3279

p=0.1227

Figure 11

Migration - JIMT1

A

B p=0.0102

D

C p=0.0001

E p=0.0445

G

F p=0.6498

H p=0.1488

p=0.0005

p=0.1026

I p=0.0774

p=0.5523

Figure 12

Migration-MDA-MB-231

A

B p=0.1813

D

C p=0.0004

E p=0.0669

G

F p=0.6843

H p=0.9438

p=0.0086

p=0.3952

I p=0.0039

p=0.0281

Figure 13

* * *** ***

1.8 1.6

## ###

1.4 1.2

***

1.0

0.8

***

0.6

###

***

0.4

0.2

cont

silNCX

sil IP1 silNCXIP1

0.0

scr

MDA-MB-231

cont

silNCX

sil IP1 silNCXIP1 scr

cont

silNCX sil IP1 silNCXIP1 scr

JIMT1

D cytosolic calcium (a.u.)

2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

A2780

2.2 2.0

C cytosolic calcium (a.u.)

B

DLD1

cytosolic calcium (a.u.)

cytosolic calcium (a.u.)

2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

#

A

2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

cont

silNCX sil IP1 silNCXIP1 scr

cont NIF10