Mahdizadeh et al. DARU Journal of Pharmaceutical Sciences (2016) 24:17 DOI 10.1186/s40199-016-0155-8 RESEARCH ARTICLE Open Access Crocin suppresses multidrug resistance in MRP overexpressing ovarian cancer cell line Shadi Mahdizadeh1, Gholamreza Karimi2, Javad Behravan3,4, Sepideh Arabzadeh3, Hermann Lage5 and Fatemeh Kalalinia3,6* Abstract Background: Crocin, one of the main constituents of saffron extract, has numerous biological effects such as anti-cancer effects. Multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) are important elements in the failure of cancer chemotherapy. In this study we aimed to evaluate the effects of crocin on MRP1 and MRP2 expression and function in human ovarian cancer cell line A2780 and its cisplatin-resistant derivative A2780/RCIS cells. Methods: The cytotoxicity of crocin was assessed by the MTT assay. The effects of crocin on the MRP1 and MRP2 mRNA expression and function were assessed by real-time RT-PCR and MTT assays, respectively. Results: Our study indicated that crocin reduced cell proliferation in a dose-dependent manner in which the reduction in proliferation rate was more noticeable in the A2780 cell line compared to A2780/RCIS. Crocin reduced MRP1 and MRP2 gene expression at the mRNA level in A2780/RCIS cells. It increased doxorubicin cytotoxicity on the resistant A2780/RCIS cells in comparison with the drug-sensitive A2780 cells. Conclusion: Totally, these results indicated that crocin could suppress drug resistance via down regulation of MRP transporters in the human ovarian cancer resistant cell line. Keywords: Crocin, Multidrug resistance, MRP1, MRP2, A2780, A2780/RCIS Background (ABC family) [4, 5]. One important class of the ABC fam- Cancer is a leading cause of death in the world that ily is the human multidrug resistance-associated protein broadly affects more and less economically developed (MRP) family which contains seven members. Several countries [1]. Using different chemotherapy regimens members of the MRP family especially MRP1 and MRP2 is a common method in cancer treatment, but there is are involved in the detoxification and protection of the some limitation on the effectiveness of chemotherapy host against xenobiotic materials. They are also assumed that leads to poor therapeutic results [2]. One of the to cause drug resistance through their ability in transport- most important reasons of treatment failure during ing a wide range of anticancer drugs out of the cells and chemotherapy is the multidrug resistance (MDR) their presence in many different types of tumors [6]. phenomenon. The MDR tumors are resistant to che- In the last decades, different research explored new motherapeutic agents which are structurally and func- botanical candidates with potential anti-cancer effects tionally different from the initial anticancer drug. that has opened a window to developing safer and more Typical MDR occurs through overexpression of the effective anti-cancer therapies [7, 8]. Crocus sativus is a membrane efflux proteins that pump anticancer drugs out plant of the Iridaceae family. Stigmas of Crocus sativus of the cells [3]. These pharmaceutical transporter proteins flowers (saffron) contain various chemical substances belong to the ATP-binding cassette transporters family [9]. Crocin is a major glycosylated carotenoid found in saffron [10] that has various pharmacological effects * Correspondence: [email protected] like protecting the myocardial cell against hypoxia 3Biotechnology Research Center, School of Pharmacy, Mashhad University of damage [11], antioxidant [12, 13], anti-atherosclerosis Medical Sciences, P. O. Box 91775-1365, Mashhad, Iran 6Medical Genetic Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mahdizadeh et al. DARU Journal of Pharmaceutical Sciences (2016) 24:17 Page 2 of 8 [14, 15], antidepressant [16] and anti-inflammatory ef- 40, 60, 80 and 100 μM) in culture medium prior to the fects [17, 18]. In addition, different studies have shown start of each experiment. anticancer activities of crocin against human leukemia, breast, colorectal, and bladder cancer cell lines [19–23]. Cell culture and treatment Based on these facts, it is expected that crocin could Cells were cultured in RPMI-1640 contained FBS potentially be used clinically for the prevention and 10 % (v/v), penicillin (100 U/mL), and streptomycin treatment of cancer in the near future. (100 μg/mL) at 37 °C in humidified air containing It has shown that crocin inhibits Lipopolysaccharides CO2 5 %. For MTT and real-time PCR studies, ovar- (LPS)-induced nitric oxide (NO) release from brain ian cancer cells were incubated for 4–72hwithcro- microglial cells and reduces the LPS-stimulated pro- cin (0–100 μM). For MRP activity analysis, all cell ductions of tumor necrosis factor-alpha, interleukin-1 lines were co-treated with different concentrations of beta, and intracellular reactive oxygen species, which ef- crocin (0–100 μM) and doxorubicin (0–500 nM) for fectively cause decreased NF-kappa B activation [17, 24]. 4–72 h. This study was obtained the approval of the On the other hand, it has been previously showed that Research Ethics Committee of Mashhad University of sulindac, the nonsteroidal anti-inflammatory drug, gen- Medical Sciences (code No: IR.MUMS.REC.1390.301). erates oxidative stress via induction of reactive oxygen species (ROS) production, which finally leads to the MTT cytotoxicity assay higher expression of MRP1 and MRP3 in human colo- Drug sensitivity of the A2780 cell line and drug-resistant rectal cancer cell lines [25]. These evidences suggest cell line A2780/RCIS were confirmed by MTT assay. that crocin might affect the protein expression of MDR Cells were seeded at an initial density of 104 cells/well in proteins. In the present study, we aimed to evaluate the 96-well plates. The plates were incubated at 37 °C in a effects of crocin on the expression and function of 5 % CO2-supplemented atmosphere for 24 h. Subconflu- MRP1 and MRP2 in the human ovarian carcinoma cell ent cells were treated with different concentrations of lines A2780 and its cisplatin-resistant derivative A2780/ crocin and doxorubicin in a final volume of 100 μlof RCIS cells (MRP2-overexpressing cell line). standard growth medium in each well. The control wells had DMSO in the growth medium at equal volumes to those used for the test compounds. Cell viability was mea- Methods sured after 4–72 h, using 3-(4, 5-dimethylthiazol-2-yl)-2, Materials 5-diphenyl tetrazolium bromide (MTT). The reduced Fetal bovine serum (FBS) and RPMI 1640 with L-glutamine MTT dye was solubilized with DMSO (100 μl/well) and were purchased from Gibco (USA) and Biosera (UK), absorbance was determined on an ELISA plate reader respectively.MTT,DMSO,trypanblue,doxorubicin (BioTek, Bad Friedrichshall, Germany) with a test wave- and penicillin G/streptomycin were obtained from length of 550 nm and a reference wavelength of Sigma-Aldrich (Germany). Crocin was generously pro- 630 nm. Each experiment was performed in triplicate vided by Dr. Seyed Ahmad Mohajeri (Pharmaceutical and was repeated at least three times. The percentage Research Center, Mashhad University of Medical Sciences, of cell proliferation was calculated using the ratio of Iran). RNA tripure isolation kit was obtained from Roche ODtest/ODcontrol. Applied Science, Germany and Real-time EXPRESS One-Step SYBR GreenER™ Kit was purchased from Real-Time RT-PCR Invitrogen, USA. The MRP-overexpressing, cisplatin- Total cellular RNA was extracted using tripure isolation resistant ovarian cancer cell line, A2780/RCIS and its reagent. The total amount of RNA was measured using parental cisplatin sensitive cell line A2780 were gener- a NanoDrop 1000 spectrophotometer (Thermo Fisher ously provided by Professor Herman Lage (Molecular Scientific, Wilmington, DE) and the acceptable purity Pathology Department, Charite Campus Mitte, Berlin, was in the range of 1.8-2.2 for the A260/A230 and Germany). A260/A280 ratios. Real-time RT-PCR was performed to measure the expression levels of MRP1 and MRP2 in ovarian cancer cell lines using the EXPRESS One-Step Preparation of the crocin solution SYBR GreenER™ Kit and real-time cycler Mx3000P™ Total crocin was extracted and crystallized from saf- Stratagen (Stratagen, USA). The primers had the fol- fronstigmasanditspuritywastestedwithHPLCand lowing sequences: MRP1: 5´-GTGTTTCTGGTCAGCC wasmorethan96%[26].Crocinwasdissolvedin CAACT-3´ (forward) and 5´-TTGGATCTCAGGATGG DMSO (dimethyl sulfoxide) and PBS to a final concen- CAGG-3´ (reverse); MRP2, 5′-AGCAGCCATAGAGCT tration of 1024 mM and stored at −20 °C. The drug GGCCCTT-3′ (forward) and 5′-AGCAAAACCAGGA was freshly diluted to its final concentration (10, 20, GCCATGTGCC-3′ (reverse); β-actin: 5´-TCATGAAG Mahdizadeh et al. DARU Journal of Pharmaceutical Sciences (2016) 24:17 Page 3 of 8 TGTGACGTGGACATC-3´ (forward) and 5´-CAGGA show significant differences between the data and p GGAGCAATGATCTTGATCT-3´ (reverse). Reactions values < 0.05 were considered significant. were performed with an initial cDNA synthesis step at 50 °C for 5 min, followed by the denaturation step at 95 °C for 2 min and PCR amplification cycles Results (40 cycles at 95 °C for 15 s, 60 °C for 1 min). Rela- Effect of crocin on the proliferation rate of A2780 cancer tive expression levels for MRP1 or MRP2 were nor- cell lines malized to the β-actin by the MxPro-Mx3000P To investigate the effects of crocin on the cell survival of system.