Cytotoxic effect of sanguiin H-6 on MCF-7 and MDA-MB-231 human breast carcinoma cells

Bioorganic & Medicinal Chemistry Letters 27 (2017) 4389–4392

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Cytotoxic effect of sanguiin H-6 on MCF-7 and MDA-MB-231 human breast carcinoma cells Eun-Ji Park a,f, Dahae Lee b,c,f, Seon-Eun Baek a, Ki Hyun Kim c, Ki Sung Kang b, Tae Su Jang d, Hye Lim Lee b, Ji Hoon Song e,⇑, Jeong-Eun Yoo a,⇑ a

Department of Obstetrics and Gynaecology, College of Korean Medicine, Daejeon University, Daejeon 35235, Republic of Korea College of Korean Medicine, Gacheon University, Seongnam 13120, Republic of Korea School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea d Institute of Green Bio Science & Technology, Seoul National University, PyeongChang 25354, Republic of Korea e Department of Medicine, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 23 June 2017 Revised 8 August 2017 Accepted 10 August 2017 Available online 12 August 2017 Keywords: Sanguiin H-6 Apoptosis Estrogen receptor MCF-7 cells MDA-MB-231 cells

a b s t r a c t Sanguiin H-6 is a dimer of casuarictin linked by a bond between the gallic acid residue and one of the hexahydroxydiphenic acid units. It is an effective compound extracted from Rubus coreanus. It has an anticancer effect against several human cancer cells; however, its effect on breast cancer cells has not been clearly demonstrated. Thus, we aimed to investigate the anticancer effect and mechanism of action of sanguiin H-6 against two human breast carcinoma cell lines (MCF-7 and MDA-MB-231). We found that sanguiin H-6 significantly reduced cell viability in a concentration-dependent manner. It also increased the rates at which MCF-7 and MDA-MB-231 cells underwent apoptosis. Furthermore, sanguiin H-6 induced the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, which resulted in apoptosis. However, cleavage of caspase-9 was only detectable in MCF-7 cells. In addition, sanguiin H-6 increased the ratio of Bax to Bcl-2 in both MCF-7 and MDA-MB-231 cells. These findings suggest that sanguiin H-6 is a potent therapeutic agent against breast cancer cells. In addition, it exerts its anticancer effect in an estrogen-receptor-independent manner. Ó 2017 Elsevier Ltd. All rights reserved.

Introduction The most common type of cancer in women is breast cancer, which is also the leading cause of death worldwide. Breast cancers are typically classified by the expression of estrogen receptors (ER), progesterone receptors, or human epidermal growth factor receptors.1 Treatment with tamoxifen or ER-modulating agents is a typical therapeutic approach for breast cancer. However, breast cancer cells become resistant to these types of chemotherapy, which are initially effective. It has been reported that many patients with breast cancer who receive these agents have an increased risk of relapse.2,3 Moreover, usage of ER antagonists is restricted by drug resistance, adverse effects, and ineffectiveness against ER-negative breast cancer.4 Thus, novel compounds that can suppress the growth of ER-positive and ER-negative breast cancer cells without causing serious side effects are desirable.

⇑ Corresponding authors. f

E-mail addresses: [email protected] (J.H. Song), [email protected] (J.-E. Yoo). These two authors contributed equally to the work described in this study.

http://dx.doi.org/10.1016/j.bmcl.2017.08.019 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

Most cancers develop from increased cell proliferation with failed removal of damaged cells.5 Many studies have suggested that effective strategies for cancer therapy are inhibition of growth and activation of apoptosis of cancer cells. It has been repeatedly reported that many natural products have a potent anticancer effect either in vitro or in vivo.6,7 For instance, in vitro and in vivo studies have shown that berry extract, which is a natural product, has a potential anticancer effect that is mediated via inhibition of proliferation and induction of apoptosis.7,8 Sanguiin H-6 is one of the major ellagitannins found in the extracts of berries, such as strawberries, cranberries, cloudberries, and red raspberries. It is also found in the crude drug Sanguisorbae radix (Fig. 1A).9,10 Sanguiin H-6 is a dimer of casuarictin linked by a bond between the gallic acid residue and one of the hexahydroxydiphenic acid units. It is a polyphenol compound that possesses strong antioxidant activity and prevents the production of nitric oxide.10,11 We recently reported that sanguiin H-6 effectively inhibits the proliferation of human ovarian carcinoma cells and lung cancer cells. It also induced apoptosis of the cells.12,13 However, the effect of sanguiin H-6 on breast cancer cells has not been clearly demonstrated.

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Fig. 1. Cytotoxic effect of sanguiin H-6 on MCF-7 and MDA-MB-231 cells. (A) Chemical structure of sanguiin H-6. (B) MCF-7 cells were treated with the indicated concentrations of sanguiin H-6 and analyzed for their viability. Sanguiin H-6 decreased cell viability in a dose-dependent manner. (C) MDA-MB-231 cells were treated with the indicated concentrations of sanguiin H-6 for 24 h. Sanguiin H-6 significantly decreased the viability of the cells.

In the present study, we examined the cytotoxic effect of sanguiin H-6 on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells, which are breast carcinoma cell lines. Each cell line was treated with different concentrations of sanguiin H-6 for 24 h and assessed for viability. Sanguiin H-6 at concentrations of up to 100 lM reduced the viabilities of both MCF-7 (Fig. 1B) and MDAMB-231 (Fig. 1C) in a concentration-dependent manner. The maximum effect of sanguiin H-6 was observed at a concentration of 100 lM, at which the viabilities of MCF-7 and MDA-MB-231 cells reduced to approximately 69% and 63%, respectively. These results suggest that sanguiin H-6 is cytotoxic to the human breast cancer cells. Inhibition of cell proliferation and induction of apoptosis are the most common strategies considered in the development of anticancer drugs. The reduction in cell viability caused by sanguiin H-6 could have resulted from inhibition of cell proliferation and induction of apoptosis. Therefore, we evaluated whether sanguiin H-6 induces apoptosis of MCF-7 and MDA-MB-231. The cells were exposed to different concentrations of sanguiin H-6 for 24 h and stained with Alexa Fluor 488 annexin V conjugate, which detects externalized phosphatidylserine by the loss of membrane integrity during the latter stage of cell death. Fluorescent images shown in Fig. 1A indicate that sanguiin H-6 induced apoptosis of both

MCF-7 and MDA-MB-231 cells. The bar graphs in Fig. 2B represent the percentages of apoptotic cells (annexin-V-positive cells) observed during total cell count examination. Our quantitative data showed that treatment with sanguiin H-6 at a concentration of 100 lM for MCF-7 and 50 lM for MDA-MB-231 significantly increased the rate of apoptosis by 33.7% and 40.7%, respectively (Fig. 2A and B). These data indicate that sanguiin H-6 mediates apoptosis in human breast carcinoma cells; however, the underlying mechanism needs to be identified. There are two major apoptotic pathways (intrinsic and extrinsic pathway) mediated by activation of series of caspases, which play an important role in apoptosis.14,15 It is well known that caspase-8 and caspase-9 are the upstream caspases in the intrinsic and extrinsic apoptotic pathways, respectively.16 Activation of caspase-8 and -9 induces activation of caspase-3 during the execution phase of apoptosis.17 To investigate the mechanism underlying the apoptosis induced by sanguiin H-6, we evaluated the activation of a series of caspases. The results showed that sanguiin H-6 induced the cleavage of caspase-8 in MCF-7 cells (Fig. 3A) and increased the cleavage of caspase-8 and -9 in MDA-MB-231 cells (Fig. 3B). However, the cleavage of caspase-3 was increased by sanguiin H-6 in both cell lines (Fig. 3A and B). Activated caspase-3 cleaves poly (ADP-ribose) polymerase (PARP) into an 85-kDa fragment, which

E.-J. Park et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4389–4392

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Fig. 2. Induction of apoptosis by sanguiin H-6. (A) MCF-7 cells were exposed to 50 or 100 lM sanguiin H-6 for 24 h. The percentage of apoptotic cells was increased by sanguiin H-6. (B) MDA-MB-231 cells were treated with 25 or 50 lM sanguiin H-6 for 24 h. The treatment increased the percentage of apoptotic cells.

Fig. 3. Mechanism underlying the apoptosis induced by sanguiin H-6. (A) MCF-7 cells were exposed to 50 or 100 lM sanguiin H-6 for 24 h. Sanguiin H-6 increased the cleavage of caspase-8, caspase-3, and PARP, but not that of caspase-9. (B) MDA-MB-231 cells were treated with 25 or 50 lM sanguiin H-6 for 24 h, after which western blot analysis was performed. Sanguiin H-6 increased the cleavage of caspase-8, -9, and -3, as well as that of PARP.

is a key characteristic feature of apoptosis.18 Therefore, we probed the cleavage of PARP by western blot analysis. Our results showed that sanguiin H-6 induced proteolytic cleavage of PARP in both the MCF-7 and MDA-MB-231 cells (Fig. 3A and B). Furthermore, the results indicated that sanguiin H-6 mediates apoptosis in human breast carcinoma cells by activating caspase-3 and cleaving PARP.

Taken together, the results indicate that the cytotoxic effect of sanguiin H-6 on human breast carcinoma cells results from the induction of apoptosis via activation of a series of caspases. Finally, evidence suggests that the Bcl-2 gene family, which includes anti- and pro-apoptotic proteins, is a major regulator of apoptosis. These genes are balanced by the formation of dimers

Fig. 4. Effect of sanguiin H-6 on the ratio of Bax to Bcl-2. (A) MCF-7 cells were exposed to 50 or 100 lM sanguiin H-6 for 24 h. Sanguiin H-6 increased Bax expression but not Bcl-2 expression. (B) MDA-MB-231 cells were treated with 25 or 50 lM sanguiin H-6 for 24 h. The treatment decreased Bcl-2 expression.

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between anti- and pro-apoptotic protein members.19 It has been reported that an increase in the ratio of Bax to Bcl-2 is a critical determinant of apoptosis.19,20 Therefore, we carried out a western blot analysis to determine whether the ratio of Bax to Bcl-2 was increased by sanguiin H-6 in MCF-7 and MDA-MB-231 cells. In MCF-7 cells, sanguiin H-6 increased Bax expression; however, it did not affect Bcl-2 expression (Fig. 4A). In contrast, in MDA-MB231 cells, Bcl-2 expression was decreased by sanguiin H-6, whereas Bax expression did not change (Fig. 4B). Although the two proteins were expressed differently in the two cell lines, the ratio of Bax to Bcl-2 was increased by sanguiin H-6 in both cell lines. This indicates that sanguiin H-6 induces apoptosis of human breast carcinoma cells by increasing the ratio of Bax to Bcl-2. Our results from the present study suggest that sanguiin H-6 prevents cell growth and induces apoptosis via activation of the caspase cascade. In addition, it increases the ratio of Bax to Bcl-2 independent of ER. Therefore, sanguiin H-6 may be a suitable therapeutic agent against both ER-positive and ER-negative breast cancer. Conflicts of interest The authors declare no competing financial interest. Acknowledgement This research was supported by the Daejeon University Fund (2016). A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08. 019. References 1. Tate CR, Rhodes LV, Segar HC, et al. Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat. Breast Cancer Res. 2012;14:R79.

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