Mirmalek et al. Cancer Cell Int (2016) 16:3 DOI 10.1186/s12935-016-0279-4 Cancer Cell International PRIMARY RESEARCH Open Access Cytotoxic and apoptogenic effect of hypericin, the bioactive component of Hypericum perforatum on the MCF‑7 human breast cancer cell line Seyed Abbas Mirmalek1, Mohammad Amin Azizi2, Ehsan Jangholi3, Soheila Yadollah‑Damavandi3, Mohammad Amin Javidi4, Yekta Parsa3,7*, Tina Parsa3, Seyed Alireza Salimi‑Tabatabaee2, Hossein Ghasemzadeh kolagar5 and Reza Alizadeh‑Navaei6 Abstract Background: Breast cancer is the most prevalent malignancies among the women that have a high mortality. Previ‑ ous studies demonstrated that hypericin, a bioactive component of Hypericum perforatum have a cytotoxic effect on the malignant cell lines. However, an anti-carcinogenic activity of hypericin on MCF-7 is uncertain. To investi‑ gate the cytotoxic effect of hypericin on MCF-7 cells, a human breast adenocarcinoma cell-line, that resistance to chemotherapy. Methods: The MCF-7 and fibroblast (as normal cell line) were treated with various concentrations of hypericin, and Cisplatin as a positive control for 24 and 48 h. Cytotoxicity activity was measured and confirmed by MTT assay and Trypan blue staining, respectively. In addition, Apoptosis were determined by Annexin V/Propidium Iodide assay. Immunocytochemistry (ICC) analysis for bcl2 and p53 proteins performed to further investigate different expression of these genes in different samples. Results: Both cisplatin and the hypericin exhibited a dose-dependent cytotoxic effect in the MCF-7 cell line. Although the LD50 of the hypericin was significantly lower when compared to cispaltin (5 vs. 20 μg/ml), it continued to decrease the growth rate of the MCF-7 cells when tested at higher concentration than LD50. In contrast, cisplatine, at higher concentration than LD50, completely inhibited the growth of the MCF-7 in 48 h. Regarding Annexin V/ Propidium results, treatment of MCF-7 cells with LD50 concentration of cisplatin and hypericin showed 60 and 52 % apoptosis in 24 h, respectively. ICC analysis for bcl2 and p53 also confirmed our results; in treated samples for the dose of LD50 in 24 and 48 h of cisplatin and hypercin, more cells expressed p53 (guardian of cells in front of tumor forma‑ tion/progression) and less expressed bcl2 (which has anti apoptotic activity) compared to untreated samples. Conclusions: Considering that hypericin showed to be cytotoxic, it seems to be a chemopreventive agent and a good candidate for antineoplastic drug development. Keywords: MCF-7, Breast Cancer, Apoptosis, Hypericum perforatum, Hypericin, Cytotoxic *Correspondence: [email protected] 7 Medical Research Center, Azad University, Tehran Medical Branch, Attarimoqaddam Ave, Haqani Ave, Dr. Shariati St, Tehran, P. O. BOX : 19395‑1495, Iran Full list of author information is available at the end of the article © 2016 Mirmalek et al. 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. Mirmalek et al. Cancer Cell Int (2016) 16:3 Page 2 of 9 Background studies of H. perforatum revealed that the main chemical Breast cancer is one of the most common human malig- components are flavonoids, phloroglucinols and naph- nancies, accounting for 22 % of all cancers diagnosed in thodiathrones, of these, the content of flavonoids are the women and among the most frequent causes of cancer richest [13]. mortality in women worldwide [1, 2]. Metastatic breast Hypericin is one of the main constitute of H. perfora- cancer is considered incurable with median survival esti- tum, which in addition to its well-known anti-depres- mates of around 2–3 years, nevertheless treatments with sant activity has been proven to exhibit potent cytotoxic endocrine, cytotoxic, or targeted therapies can improve and pro-apoptotic effects against tumor cell lines in or preserve the quality of life and extend endurance. low micro molar concentrations, and to inhibit tumor- Breast cancer represents a complex and heterogeneous induced angiogenesis [8, 14]. disease comprising distinct pathologies with specific his- Although, extensive research has been carried out on tological features, therapeutic responses, metastatic dis- anti-carcinogenic activity of H. perforatum, no single semination patterns, and patient outcomes [3]. study exists which in MCF-7 cells [15–17]. Hence, this For the first time, Soule et al. [4] introduced the MCF-7 study was aimed to determine the antineoplastic activity cell line, which was derived from a patient with meta- of hypericin on human breast cell line MCF-7. static breast cancer. Despite many studies were estab- lished MCF-7 cells as the first hormone-responsive breast Results cancer cell line [5]. The benefit of the MCF-7 cell line as MTT assay an investigative tool led to its adoption in laboratories To determine cytotoxicity effect of hypericin on the worldwide [5, 6]. Various evidences include the ability MCF-7 cell viability MTT assay performed. As it is to undergo DNA fragmentation, differential sensitivi- shown in Fig. 1, the LD50 for 24 h for hypericin evalu- ties to estrogens and anti-estrogens, differential expres- ated to be 5 (μg/ml); and that of cisplatin was 20 (μg/ml). sion of estrogen receptors (ER), ER-mRNA, progesterone Treatment of fibroblasts (as a normal cell line) with the receptors and differences in tumorigenicity and prolifera- concentrations up to 30 (μg/ml) of hypericin and/or cis- tive rates. It has been demonstrated that the MCF-7 cell platin have not significant effect on the cells survival in line is a novel tool for the subject area of breast cancer 24 h (P = 0.062 and P = 0.054, respectively). resistance to chemotherapy, because it seems to mirror LD50 for 48 h for hypericin evaluated to be 0.5 (μg/ml) the heterogeneity of tumor cells in vivo [7]. The human (Fig. 2a); and that of cisplatin was 7.5 (μg/ml) (Fig. 2b). breast cancer cell line MCF-7 provides an unlimited Interestingly cells viability increased with the increase in source of homogenous self-replicating material, free of hypericin concentration. Treatment of fibroblasts with contaminating stromal cells, and can be easily cultured in the concentrations up to 10 (μg/ml) of hypericin and 15 simple standard media. Such a cell line is ideal to study (μg/ml) of cisplatin have not significant effect on the cells the interaction between a chemo-preventive drug and a survival in 48 h. cancer cell. The mechanism by which a chemo-preven- tive drug inhibits the proliferation of a cancer cell can be Flow cytometery better evaluated in vitro where the other physiological AnnexinV/PI flow cytometery was performed to quan- regulatory mechanisms, which are present in the in vivo tify cells that underwent apoptosis after cisplatin and system, are absent. hypericin treatment. As it is shown in Fig. 3, treatment Exploring the plant chemical diversity appears to be a of MCF-7 cells with LD50 concentration of cisplatin vital strategy for development of novel anticancer drugs and hypericin showed 60 and 52 % apoptosis in 24 h, characterized by alternative mode of action or lower respectively. toxicity. The intensive screening of plant-derived com- pounds for antineoplastic activity during the last several Trypan blue staining decades has contributed to the founding of various drug To further investigate the effect of hypericin on MCF-7 classes occupying a substantial portion of the anticancer cells death, trypan blue staining performed. After stain- drug market [8]. Recent evidence suggests that the mem- ing cells which had treated with 0.5 (μg/ml) concentra- bers of the Guttiferae family, e.g. diverse Hypericum, tion of hypericin or 7.5 (μg/ml) concentration of cisplatin Garcinia, Clusia, Cratoxylumspecies, appear to be a valu- for 48 h, cells became blue (Fig. 4a, b). Interestingly stain- able resource of cytotoxic compounds [8–10]. Hypericum ing cells which had treated with hypericin in higher con- perforatum, known as St. John’s wort, is the most stud- centration than LD50 [>0.5 (μg/ml)] did not became blue ied of Hypericum species and it is known for its phar- (Fig. 4c). However, cells which had treated with cisplatin macological antidepressant activities, its antiviral and in higher concentration of LD50 [>7.5 (μg/ml)], became antibacterial properties [11, 12]. Previous phytochemical blue after staining. Mirmalek et al. Cancer Cell Int (2016) 16:3 Page 3 of 9 Fig. 1 MTT assay results for cisplatin and hypericin in 24 h on MCF‑7 cell line. Treatment of MCF‑7 cells with 5 (μg/ml) concentration of hypericin showed 50 % cell death ( LD50 or IC50) (a, blue column), whereas LD50 of cisplatin in this time calculated to be 20 (μg/ml) (b, blue = column).*P < 0.05, **P < 0.01 Fig. 2 MTT assay results for cisplatin and hypericin in 48 h on MCF‑7 cell line. Treatment of MCF‑7 cells with 0.5 (μg/ml) concentration of hypericin showed 50 % cell death ( LD50 or IC50) (a, blue column), whereas LD50 of cisplatin in this time calculated to be 7.5 (μg/ml) (b, blue column). = *P < 0.05, **P < 0.01 Expression of bcl2 and p53 in different samples extracted from the H. perforatum or chemically synthe- To confirm that either treatment of cells with hypercin sized. It has been used for the photodynamic therapy of and/or cisplatin induce cell death via apoptosis and cancer and other conditions [19].