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Original Article

The combined effects of all‑trans‑retinoic acid and docosahexaenoic acid on the Mina Abdolahi1, induction of apoptosis in human breast Fazel Shokri2, Mostafa Hosseini3, Maryam cancer MCF‑7 cells Shadanian1, Ali‑Akbar Saboor‑Yaraghi1,4 ABSTRACT Introduction: Breast cancer is one of the most women’s cancers in the worldwide. In vivo and in vitro studies showed that 1Department of all‑trans‑retinoic acid (ATRA) and docosahexaenoic acid (DHA) can modulate differentiation and apoptosis in both cancer and immune Cellular and Molecular Nutrition, Faculty of cells. Nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) activation in the presence of their ligands, plays a critical Nutritional Sciences role in the proliferation, differentiation, and apoptosis of normal cells. and Dietetics, Tehran Aim of Study: We hypothesized that ATRA and DHA, as ligands of RARs and RXRs respectively, may have synergistic effects on University of Medical the induction of apoptosis in MCF‑7 human mammary carcinoma cell lines. Sciences, Departments of 2Immunology and Materials and Methods: MCF‑7 cells were seeded in a 24‑well plate at 3 × 105 cells per well. The cells were treated with 5 μM 3Epidemiology and ATRA, 30 μM DHA, and various combinations of them over a 3‑day trial. Apoptosis was measured by Annexin V‑FITC kit and flow Biostatistics, School cytometery. of Public Health, Tehran University of Results: Our results showed that the combination treatment of ATRA and DHA (5 µM and 30 µM and half dose at 2.5 µM and Medical Sciences, 15 µM, respectively) in a dose‑dependent manner induced apoptosis rate in MCF‑7 cells significantly more than single treatment 4Food Microbiology of ATRA or DHA, as compared to control group (P < 0.05). Research Center, Tehran University of Conclusion: We conclude that the combination of ATRA and DHA at the well‑balanced proportion may be effective in cancer cell Medical Sciences, apoptosis. Further studies provide details about the potential synergistically effects of combination treatment of ATRA and DHA in Tehran, Iran growth inhibition and differentiation of human mammary cancer cells. For correspondence: Dr. Ali‑Akbar KEY WORDS: All‑trans‑retinoic acid, apoptosis, breast cancer, docosahexaenoic acid, MCF‑7 cell Saboor‑Yaraghi, Department of Cellular and Molecular Nutrition, Faculty of INTRODUCTION cancer.[8] Early studies have shown the protective Nutritional Sciences effects of omega‑3 fatty acids and retinoids in and Dietetics, Tehran [9,10] University of Medical Breast cancer is one of the most women’s cancers breast cancer. Possible mechanisms include Sciences, Tehran, Iran. in the worldwide and leading cause of death cell abnormal growth suppression, neoplastic Food Microbiology from cancer is fifth leading cause of death from suppression, induction of differentiation, and Research Center, cancer.[1] Breast cancer incidence rate and its apoptosis.[11,12] These components in contrast to Tehran University of mortality are increased rapidly in developing their effects in cancer cells do not induce apoptosis Medical Sciences, [2] Tehran, Iran. countries over the last 20 years. Some breast significantly in the normal and noncancerous cells E‑mail: asaboor@ [13‑15] cancer risk factors are nonmodifiable including including breast and colon cancer cells. tums.ac.ir family history, obesity, lactation, menstrual and reproductive history.[3] The critical role of nutrition Epidemiological studies have shown fish oil may and diet as a modifiable risk factor in cancer reduce breast cancer. Fish oil is a rich source of long Access this article online etiology derived from antioxidant benefits and chain n‑3 fatty acids, especially docosahexaenoic Website: www.cancerjournal.net anti‑cancer properties of selected nutrients.[4] In acid (DHA).[16] Enhanced apoptotic effects of DHA DOI: 10.4103/0973-1482.154071 cancer, balance between cell division and death is were shown in MCF‑7 human breast carcinoma PMID: *** disrupted.[5] Apoptosis or programmed cell death cells in vitro and in vivo in animal models.[17,18] DHA Quick Response Code: is an important control mechanism following inhibits breast tumors incidence, progression, DNA damage.[6] Therefore, reduced apoptosis or metastasis, and reactive oxygen species production its resistance can modulate cancer cell growth.[7] lead to cell death.[19] Numerous studies have Current strategies for cancer therapy is focused indicated that all‑trans‑retinoic acid (ATRA), the on apoptosis as a desirable target of cell control in active metabolite of Vitamin A, has anti‑cancer

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Abdolahi, et al.: The combined effects of ATRA and DHA on the apoptosis of breast cancer cells effects and considered a potent inhibitor of malignant For cell treatments, cells were seeded in 24‑well plates with a transformation.[20] density of 3 × 105 cells per well. The cells were incubated for 24 h at above‑mentioned condition until the cells reached 80% All‑trans‑retinoic acid regulates cell cycle progression[21] and confluence. Then media was removed and replaced with fresh enhances sensitivity of cells to the anti‑cancer agents in some FBS‑free media containing ATRA and DHA alone at different type of cancers.[22] ATRA can reduce MCF‑7 breast carcinoma concentrations of 1, 5, and 10 µM for ATRA and 15, 30, and cell proliferation and induce apoptosis in vitro.[23] An even 40 µM for DHA; Both at 24, 48, 72 and 96 h as pilot. At the next greater reduction of cancer cell progression is observed when step on the based on pilot results, the cells treated with 5 µM retinoids were treated in combination with omega‑3 fatty of ATRA, 30 µM of DHA, and combination of them including acids.[24] Heterodimerization of retinoic acid receptors (RARs) ATRA 5 µM + DHA 30 µM and ATRA 2.5 µM + DHA 15 µM and retinoid X receptors (RXRs) and binding to their ligands, for an incubation period of 72 h. The last treatments run in DHA and ATRA, is responsible for synergistic effects of retinoids two independent experiments each performed in triplicates. and fatty acids on carcinogenesis. The RXR‑RAR heterodimers bind to DNA and activate gene expression, cell proliferation, Cell apoptosis assay and cell differentiation.[25] According to previous studies, there Apoptosis were analyzed using an Annexin V‑FITC/PI is a potential for combined synergistic effects of ATRA and DHA apoptosis detection kit (BioVision, K101‑25, Milpitas, CA, on induction of apoptosis in breast cancer cells. The purpose USA) according to the manufacture’s protocol. Briefly, of this study was to investigate the effects of a combination MCF‑7 cells incubated with different proportion of treatment treatment of ATAR and DHA on the induction of apoptosis in solutions for different incubation times, as mentioned MCF‑7 human mammary carcinoma cell lines. above. Control wells treated with (0.1% DMSO). Cells were trypsinized, and washed with 500 µl of binding buffer, MATERIALS AND METHODS and incubated with 5 µl conjugate Annexin V‑FITC and 5 µl propidium iodide (PI) for 15 min in the dark and room Cell culture temperature and analyzed by flow cytometry (FACS Calibur, MCF‑7 human mammary carcinoma cells were obtained BD Biosciences, USA) method. from National Cell Bank of Iran (NCBI Code: C135), which is affiliated to Pasteur Institute of Iran. Cells Statistical analysis were cultured in RPMI‑1640 (GIBCO, USA) containing Student’s paired t‑test and ANOVA test were used to determine 10% fetal bovine serum (FBS, GIBCO, USA) and 1% a statistically significant difference between treatment

penicillin‑streptomycin (GIBCO, USA) at 37°C with 5% CO2. and control groups. A P < 0.05 was considered statistically The cells cultured in 1 × 75 cm2 cell culture flasks until reach significant. 80% confluence. Then MCF‑7 cells were washed with trypsin solution and centrifuged for 5 min at 1800 RPM. The cells RESULTS were resuspended by gently vortexing in 1 mL fresh media after discarding the supernatant. Cells were counted using The results of the pilot experiment have shown in Figures 1 and 2. the trypan blue method on a hemocytometer to count the Different concentrations of ATRA and DHA induced apoptosis number of viable cells in 1 mL. in MCF‑7 cells compared to the control except for the first 24 h (data not shown). As shown in figures, the highest For cell counting, once 80% of confluence is reached, apoptosis rate were found in ATRA at 5 µM and DHA at 30 µM MCF‑7 cells were washed with trypsin solution and centrifuged concentrations that obtained statically significant following for 5 min at 1800 RPM. The cells were resuspended by light 72 h incubation (P < 0.05). gently vortexing in 1 mL fresh media after discarding the supernatant. Cells were counted using the trypan blue method The results of next step of experiment showed that the on a hemocytometer to count the number of viable cells in single treatments of ATRA at 5 µM and DHA at 30 µM and 1 mL. combination treatment of them (ATRA plus DHA at dosage of 2.5 µM + 15 µM or 5 µM + 30 µM, respectively) after 72 h Preparation all‑trans‑retinoic acid and docosahexaenoic acid significantly induced apoptosis in MCF‑7 cells compared to the and cell treatments control (7.12%, 9.23%, 9.82%, and 12.49% increasing apoptosis All‑trans‑retinoic acid and DHA were purchased from rate respectively) (P < 0.05) [Figure 3]. Sigma–Aldrich (St. Louis, MO, USA). They were dissolved in 100% dimethyl sulfoxide (DMSO) (MP Biomedicals, The In addition, in combination groups significant induction Netherlands) under dim‑light condition and were stored of apoptosis was observed when compared to ATRA group at −80°C until use. The compounds were diluted from the stock alone (in ATRA 2.5 + DHA 15 µM group 2.7% and in ATRA solutions to a final concentration of ATRA at 1, 5, and 10 µM 5 + DHA 30 µM group 5.37% increasing). The promotion of and DHA at 15, 30, and 40 µM prior to being incubated with apoptosis between DHA 30 µM and combined treatment at the cells. Negative control cells were treated with 0.1% DMSO. half dose of single treatment (ATRA 2.5 µM + DHA 15 µM) did

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Abdolahi, et al.: The combined effects of ATRA and DHA on the apoptosis of breast cancer cells

ATRA It is necessary to mention that FITC Annexin V positive cells are in early apoptosis, whereas FITC Annexin V and PI positive Control 60 cells are in late apoptosis. Results are expressed as a percentage of positive cells. 1 umol 40 DISCUSSION 5 umol % Apoptosis 20 In the present study, the combination treatment of ATRA, the 10 umol most active metabolite of Vitamin A, and DHA, an omega‑3 0 fatty acid, ligands of RAR and RXR, respectively, enhanced a 48 72 96 synergistic and dose‑time dependent effect on apoptosis in Time (hours) breast cancer MCF‑7 cells. For the best of our knowledge, this is a novel effect that not demonstrated before. Figure 1: Percentage of apoptosis in MCF-7 cell line treated with ATRA at different concentrations and different incubation times Retinoic acid plays an important role in the gene transcription and as a necessary factor in the growth of epithelial cells.[26] DHA RA exerts its effects through interaction with the nuclear receptors, RARs and RXR. These receptors are ligand‑dependent 80 transcription factors. ATRA especially binds to RAR and activate Control (0.1% it, while 9‑cis RA binds to both RARs and RXRs and activate [27,28] s 60 DMSO) them.

15 umol 40 Docosahexaenoic acid, a member of the omega‑3 30 umol polyunsaturated fatty acids, has antineoplastic properties.[29] % Apoptosi It has been observed that DHA causes cancer cell death by 20 40 umol induction of cytotoxicity in tumor cells[30] and increasing uptake of anticancer drugs, and enhancing pro‑oxidant and [10] 0 pro‑apoptic effects of chemotherapeutic agents. Binding of 48 72 96 ATRA to RARs, facilitates binding of DHA to RXRs and active Time (hours) RAR/RXR heterodimers are formed. When ATRA binds to RAR, facilitates binding of DHA to RXR and leads to RAR‑RXR Figure 2: Percentage of apoptosis in MCF-7 cell line treated with DHA heterodimers. RAR‑RXR heterodimers bind to RA response at different concentrations and different incubation times elements and modulate the expression of the target genes involved in normal cell proliferation, differentiation, and 25 apoptosis.[17,25] * 20 * * The results of the present study showed that DHA increased [14] * MCF‑7 apoptosis which is in consistent with the literature. 15 Previous studies demonstrated that ATRA is a ligand for RARs and may amplify the effect of RXRs ligand, that is, DHA in % Apoptosis 10 MCF‑7 cells.[24] It is suggested that ATRA and DHA can target enteric and extrinsic apoptosis pathways through their effects 5 on key proteins and enzymes which are involved in apoptosis pathways. This combination can increase the expression of 0 control ATRA 5 umol DHA ATRA 2.5 umol + ATRA 5 umol + pro‑apoptotic genes such as Bax and Bak while reduce gene 30 umol DHA 15 umol DHA 30 umol expression of anti‑apoptotic proteins including Bcl‑2 and [31,32] Figure 3: Comparison of apoptosis rate in different single and Bcl‑XL. ATRA and DHA have also enhancing effects on combination treatments of ATRA and DHA on MCF-7 cells after 72 activity and gene expression of caspases, such as caspase 8, hours. Data are expressed as mean±SE (2 independed experiments 9, and 3 as a dose‑depended manner.[33,34] performed in triplicate). *P < 0.05 vs. Control group Our finding is parallel with the study of Crowe and not differ statistically but excessive apoptosis rate was seen Chandraratna, which demonstrated that AGN194204, the in half dose combined group (0.59% increasing). In addition, synthetic RXR‑selective ligand, and linolenic acid which binds apoptosis rate in combined treatment of ATRA 5 µM + DHA to peroxisome proliferator‑activated receptors (PPARs) can 30 µM compared to a single treatment of DHA, showed a enhance the effect of apoptosis through heterodimerization significant difference (3.26% increasing) (P < 0.05). of RXRs/PPARs in breast cancer cell lines.[35] It has been

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Abdolahi, et al.: The combined effects of ATRA and DHA on the apoptosis of breast cancer cells shown PPARs‑γ have anti‑carcinogenesis effects on breast for adenomas/tumors. Cancer Lett 2003;193:17‑24. cancer cells as shown combination of PPAR‑RXR dimers 14. Wang Q, Yang W, Uytingco MS, Christakos S, Wieder R. ligands enhance activity of responsive promoter in breast 1,25‑Dihydroxyvitamin D3 and all‑trans‑retinoic acid sensitize [36,37] breast cancer cells to chemotherapy‑induced cell death. Cancer Res cancer cells. 2000;60:2040‑8. 15. Seegers JC, de Kock M, Lottering ML, Grobler CJ, van Papendorp DH, In general, there are no many literatures on the field of Shou Y, et al. Effects of gamma‑linolenic acid and arachidonic efficacy of combination therapy trough nuclear receptors acid on cell cycle progression and apoptosis induction in normal ligands on breast cancer prevention or treatment. Previous and transformed cells. 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