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Different anticancer effects of Saxifragifolin A on estrogen receptor-positive and estrogen receptor-negative breast cancer cells.

ABSTRACT

Background: Breast cancer is the leading cause of cancer-related death among women worldwide. For treating breast cancer, numerous natural products have been considered as chemotherapeutic drugs.

Hypothesis/purpose: The present study aims to investigate the apoptotic effect of Saxifragifolin A (Saxi A) isolated from Androsace umbellate in two different human breast cancer cells which are ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells, and examine the molecular basis for its anticancer actions.

Study design: The inhibitory effects of Saxi Aon cell survival were examined in MCF-7 cells and MDA-MB-231 cells in vitro.

Methods: MTT assays, Annexin V/PI staining analysis, ROS production assay, Hoechst33342 staining and Western blot analysis were performed.

Results: Our results showed that MDA-MB-231 cells were more sensitive to Saxi A-induced apoptosis than MCF-7 cells. Saxi A induced apoptosis in MDA-MB-231 cells through ROS-mediated and caspase-dependent pathways, whereas treatment with Saxi A induced apoptosis in MCF-7 cells in a caspase-independent manner. In spite of Saxi A-induced activation of MAPKs in both breast cancer cell lines, only p38 MAPK and JNK mediated Saxi A-induced apoptosis. In addition, cell survival of shER[alpha]-transfected MCF-7 cells was decreased, while MDA-MB-231 cells that overexpress ER[alpha] remained viable.

Conclusion: Saxi A inhibits cell survival in MCF-7 cells and MDA-MB-231 cells through different regulatory pathway, and ER[alpha] status appears to be important for regulating Saxi A-induced apoptosis in breast cancer cells. Thus, Saxi A may have a potential therapeutic use for treating breast cancer.

Keywords:

Saxifragifolin A

MCF-7 cell

MDA-MB-231 cell

ROS

MAPK

ER [alpha]

Introduction

Breast cancer is currently one of the most prevalent cancers and the leading cause of cancer-related deaths in women worldwide (Jemal et al., 2010). It is reported that breast cancer accounts for 23% (1.38 million) of the total new cancer cases and approximately half a million people die from the disease every year (Jemal et al., 2011). Despite significant advances in understanding the biological and clinical nature of breast cancer, the incidence and mortality rates of the disease are still increasing.

Estrogen receptor [alpha] (ER[alpha]) is overexpressed in over half of all breast cancers and about 70% of the patients respond to anti-estrogen drugs such as tamoxifen (Ali and Coombes, 2000). Patients with estrogen receptor [alpha] expressing (ER-positive) tumors have a better prognosis and are inclined to respond to hormonal therapy whereas ER-negative tumors are more aggressive and unresponsive to anti-estrogens (Rochefort et al., 2003). Thus, therapeutic options for ER-negative breast cancer are restricted to conventional cytotoxic therapy. Moreover, hormone therapy and chemotherapy are not fully effective because of their non-specific mechanisms of action and the presence of resistant cancer cells (Gonzalez-Angulo et al., 2007). Therefore, novel therapies and new chemopreventive agents are urgently needed to treat breast cancer, especially for hormone-independent breast cancer.

Natural products have been suggested as potential sources of chemotherapeutic drugs in the treatment of breast cancer (Bishayee et al., 2011). Previous studies have shown that triterpenoids, a major group of natural substances widely distributed in plants, such as betulinic acid and ginsenosides, possess anticancer properties, and specifically targets breast cancer (Petronelli et al., 2009). Saxifragifolin A (Saxi A) is an oleanane triterpenoid isolated from Androsace umbellata (Lour.) Merr. (Primulaceae) which is used as an anticancer and antiphlogistic herb in the treatment of sore throat, traumatic injury, and solid tumors in traditional Chinese medicine (Wang et al., 2008). It is reported that triterpenoid saponins extracted from Androsace umbellata exhibit cytotoxic activity in several types of cancer cells, including HepG2, Hep3B, A549, HCT15, and Raw 264.7 cells (Park et al., 2010; Wang et al., 2008; Zhang et al., 2007). Moreover, Saxifragifolin D inhibits breast cancer cell survival through ROS-dependent endoplasmic reticulum stress (Shi et al., 2013). However, the cytotoxic effect of Saxi A on breast cancer cells and the underlying mechanisms remain unexploited.

In the present study, we examined the apoptotic effect of Saxi A in two breast cancer cell lines, MCF-7 and MDA-MB-231, and the significance of ER[alpha] status on Saxi A-induced cytotoxicity. Our findings suggest that Saxi A inhibits cell viability in MCF-7 cells through ROS and caspase-independent pathways, while the survival of MDA-MB231 cells can be reduced by Saxi A treatment in a ROS and caspase-dependent manner. In addition, we demonstrate that ER[alpha] may play an important role in Saxi A-induced apoptosis in breast cancer cells.

Materials and methods

Saxifragifolin A purification

The whole plants of Androsace umbellata (Lour.) Merr. were collected at the end of April 2009 in Changnyeong, Gyeongsangnam-do, Korea. A voucher specimen (SKKU-Ph-09-93) has been deposited at the School of Pharmacy, Sungkyunkwan University.

The dried whole plants of A. umbellata (300 g) were cut into small pieces and extracted with MeOH (15 l) three times at room temperature. After extraction, the total filtrate was concentrated under reduced pressure and the residue (70.1 g) was suspended in MeOH[H.sub.2]O (9:1, 700 ml). The resulting solution was extracted with hexane (700 ml x 3). The aqueous MeOH solution was adjusted to 30% [H.sub.2]O and partitioned with C[H.sub.2][Cl.sub.2] three times. The aqueous MeOH solution was evaporated in vacuo, and the residue was suspended in [H.sub.2]O (700 ml) and consecutively partitioned with EtOAc (700 ml) and n-BuOH (700 ml). Each of the organic fractions was evaporated under reduced pressure to give hexane (5.75 g), EtOAc (1.03 g), C[H.sub.2][Cl.sub.2] (3.37 g), n-BuOH (27.52 g), and [H.sub.2]O (26.88 g) fractions. A portion of the n-BuOH fraction was subjected to Sephadex LH-20 column chromatography (70% MeOH-[H.sub.2]O). Fractions were combined according to their TLC patterns to furnish five fractions (B1-B5). Fraction B2 was chromatographed on a RP-18 column and eluted with 60% MeOH in [H.sub.2]O to give 10 fractions designated as B2-1 to B2-10. Saxifragifolin A was afforded as white amorphous powder by reversed-phase [C.sub.18] HPLC (60% MeOH) and recycling HPLC with a JAIGEL-GS 310 column (MeOH only) from fractions B2-5. Saxifragifolin A was determined to be above 95% pure by HPLC analysis with RI detection.

Cell lines and reagents

The human breast cancer cell lines (MDA-MB-231 and MCF-7) were obtained from American Type Culture Collection (Manassas, Virginia, USA). MDA-MB-231 cells were propagated in DMEM supplement with 10% (v/v) fetal bovine serum and antibiotics (100 IU/ml of penicillin and 100 [micro]g/ml of streptomycin). MCF-7 cells were cultured in RPM1 1640 supplement with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 IU/ml of penicillin and 100 [micro]g/ml of streptomycin). Both human breast cancer cells were cultured at 37[degrees]C in a humidified atmosphere containing 5% C[O.sub.2]. FBS, DMEM, RPMI 1640, penicillin G, and streptomycin were obtained from Life Technologies Inc. (Carlsbad, CA, USA). Trypsin-EDTA and propidium iodide (PI) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Dihydroethidium 2'7'-dichlorodihydrofluorescein diacetate ([H.sub.2]DCFDA) was obtained from Molecular Probes (Eugene, OR, USA). ER[alpha], extra-cellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERKl/2, p38 MAPK, phospho-p38 MAPK, JNK, phospho-JNK, B[cl.sub.2], Bcl-xL, Bax, and A1F antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Cytochrome c, caspase-3, and caspase-8 antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Unless otherwise indicated, all the chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

MTT assay

Cell cytotoxicity was measured by quantitative colorimetric assay with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylterazoIium bromide (MTT) assay as described elsewhere (Nguyen et al., 2015). Breast cancer cells were seeded at a concentration of 1 x [10.sup.5] cells/well in 96-well plates and treated with Saxi A for 24 h. The cells were washed once with D-PBS and incubated with 25 mg/ml of MTT (Sigma, St. Louis, MO) for 4 h at 37[degrees]C and 5% C[O.sub.2]. The formazan blue crystals formed by the reduction of MTT were dissolved in 150 ml of DMSO. The amount of formazan was determined by measuring the absorbance at 540 nm using a Molecular Device microplate reader (Molecular Devices, Eugene, OR, USA).

Annexin V/PI staining analysis

Cells undergoing apoptosis were identified using the annexin V-FITC/propidium iodine (PI) apoptosis detection kit (BD Pharmingen, San Diego, CA, USA) following the manufacturer's protocol. Briefly, after treatment with indicated concentrations of Saxi A for 12 h, the cells were harvested by centrifugation at 300 x g, washed twice with ice-cold PBS, pelleted, and resuspended at concentration of 1 x [10.sup.6] cells/ml in 100 [micro]l of binding buffer plus 5 [micro]l of Annexin V-FITC conjugate and 10 [micro]l of PI solution. The cell solution was incubated in the dark for 15 min at room temperature, and 300 [micro]l of binding buffer was added to the solution. At least 10,000 cells were subjected to flow cytometry (Becton-Dickinson Biosciences, Drive Franklin Lakes, USA) to identify viable, apoptotic, and necrotic populations. They were quantified using CellQuest software according to the manufacturer's instructions.

ROS production assay

CM[H.sub.2]-DCFDA (5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; acetyl ester, Molecular Probes, Eugene, OR), a redox-sensitive fluorescent dye, was used to evaluate the intracellular ROS level by flow cytometry analysis. MCF-7 cells and MDA-MB-231 cells were treated with various concentrations of Saxi A for 12 h. The cells were stained for 30 min at 37[degrees]C with 10 [micro]M CM[H.sub.2]-DCFDA. After the cells were collected, the intracellular ROS level was measured by flow cytometry. At least 10,000 cells from each sample were analyzed. The changes in the concentration of endogenous ROS were expressed as a percentage of untreated control cells.

Hoechst33342 staining

Morphological detection of apoptotic cells was determined using Hoechst33342 staining. Briefly, breast cancer cells were grown on 22-mm diameter glass coverslips. The cells were then pretreated with 10 mM N-acetyl cysteine (NAC) for 2 h, and exposed to Saxi A (7 [micro]g/ml) for 12 h. NAC, a widely used thiol-containing antioxidant, was used to inhibit ROS production. After treatment with Saxi A, the cells were washed with PBS, fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, and washed again with PBS. They were permeabilized with 1% BSA/0.2% Triton X-100/PBS for 1 h before incubation with Hoechst33342-containing PBS solution for 5 min at room temperature. The cells were washed again with permeabilization buffer. The coverslips were mounted to glass slides and photographed with a fluorescence microscope (BX51-Olympus Optical).

Western blot analysis

MCF-7 and MDA-MB-231 cells were harvested and washed twice in PBS. After washing with PBS, the cells were suspended in 70 ml of Buffer A [10 mM HEPES (pH 7.9), 1.5 mM Mg[Cl.sub.2], 10 mM KC1, 0.5 mM DTT, 0.5 mM PMSF and Protease Inhibitor Cocktail] and incubated on ice for 15 min. Nonidet P-40 (0.5%) was added to lyse the cells that were vortexed for 10 s and centrifuged twice at 500 x g for 10 min at 4[degrees]C. After centrifugation, the nuclear pellet was obtained and the supernatants were further centrifuged at 10,000 x g for 30 min at 4[degrees]C. The resultant mitochondrial pellets were suspended in Buffer A, sonicated, and stored at -70[degrees]C until used. The supernatant from the previous step was further centrifuged at 100,000 x g for 1 h at 4[degrees]C to obtain the cytoplasmic fraction, which was stored at--70 [degrees]C until used. Protein concentration was determined by using the Bio-Rad protein assay (Bio-Rad Lab, Hercules, CA, USA) with BSA as the standard. Subcellular fractions were solubilized in Laemmli sample buffer and separated by SDS polyacrylamide gel. The fractionated proteins were electrophoretically transferred to an immobilon polyvinylidene difluoride membrane (Amersham, Arlington heights, IL, USA) and probed with the appropriate antibodies. The blots were developed using an enhanced chemiluminescence (ECL) kit (Amersham). In all immunoblotting experiments, the blots were reprobed with an anti-[beta]-actin antibody which served as a control for the protein loading.

[FIGURE 1 OMITTED]

Plasmid and shRNA

To generate HA-tagged ER[alpha], the coding region of ER[alpha] was amplified with forward primer 5'-CCGGAATTCGCCACCATGACCATGACCCTCCACACCAAAG-3' and reverse primer 5'-ATATCTCGAGTTAGCTCTGCAATGTTCC-3'. The amplified ER[alpha] was cloned between EcoRI and XhoI sites in the pcDNA3/HA-ER[alpha] vector. To knockdown ER[alpha] expression, an oligonucleotide of shER[alpha] 5'-GATCCCCTTCAGATAATCGACGCCAGTTCAAGAGACTGGCGTCGATTATCTGAATTTTTGGAAA-3' was designed. Oligonucleotides were annealed and cloned into the pSuper vector between BglII and HindIII sites (Oligoengine, Seattle, WA).

Statistical analysis

Results are reported as means [+ or -] SEM. One-way analysis of variance was used to determine significance among groups, after which a modified t-test with the Bonferroni correction was used for comparison between individual groups. Significant values (p < 0.05) are represented by an asterisk.

Results

Identification of Saxifragifolin A

The compound isolated from the n-BuOH fraction of Androsace umbellata MeOH extract was identified as Saxi A (Fig. 1) by comparing its spectral data (ESI-MS, [sup.1]H- and [sup.13]C NMR) (data not shown) with the values in the literature (Park et al., 2010; Waltho et al., 1986).

Differential suppressive effect of Saxi A on cell survival in MCF-7 and MDA-MB-231 cells

To investigate whether Saxi A inhibits cell viability in two different human breast cancer cell lines, we treated ER[alpha]-positive MCF-7 cells and ER[alpha]-negative MDA-MB-231 cells with 0.01, 0.1, 1, 10, 100 [micro]g/ml of Saxi A for 24 h and performed MTT cytotoxicity assay. As shown in Fig. 2A, the [IC.sub.50] value for MCF-7 cells was 7.82 [micro]g/ml compared to 3.88 [micro]g/ml in MDA-MB-231 cells. This result showed that MDA-MB-231 cells were more sensitive to Saxi A (p < 0.05) than MCF-7 cells. We also examined whether Saxi A induced apoptosis using flow cytometry analysis. Since the cell viability of two breast cancer cell lines was the most different between 5 and 10 [micro]g/ml (Fig. 2A), MCF-7 cells and MDA-MB-231 cells were stimulated with 5, 7, 10 [micro]g/ml of Saxi A for 12 h and stained with Annexin V/PI. Flow cytometry showed that stimulation with Saxi A induced apoptotic cell death in a concentration-dependent manner in both cell lines (Fig. 2B). Treatment with 10 [micro]g/ml of Saxi A significantly increased the population of necrotic, early apoptotic and late apoptotic cells to 4.46, 7.35 and 3.12, respectively, in MCF-7 cells. Flow cytometric analysis also showed an increase in the population of necrotic, early apoptotic and late apoptotic cells in MDA-MB-231 cells to 3.02, 24.98 and 27.12, respectively. These results suggest that ER[alpha]-positive MCF-7 cells are more resistant to Saxi A-induced apoptosis than ER[alpha]-negative MDA-MB-231 cells.

Effect of Saxi A on intracellular ROS generation

To determine whether Saxi A-induced apoptosis in both breast cancer cell lines was ROS dependent, we measured endogenous ROS production using flow cytometry analysis. MCF-7 and MDA-MB-231 cells were treated with 5, 7, 10 [micro]g/ml of Saxi A for 12 h and then stained with CM[H.sub.2]-DCFDA for 30 min. As shown in Fig. 3A, ROS levels significantly increased in MDA-MB-231 cells after treatment with Saxi A in a concentration-dependent manner. However, Saxi A treatment did not significantly generate ROS in MCF-7 cells. To further investigate the effect of ROS on Saxi A-induced cell morphological alterations, we stained both breast cancer cell lines with Hoechst 33342 and the cells were visualized with a fluorescence microscope. Treatment of MDA-MB-231 cells with 7 [micro]g/ml of Saxi A for 12 h caused an increase in nuclear fragmentation and condensation (Fig. 3B). However, nuclear morphological changes induced by Saxi A were reduced by pretreatment with the ROS scavenger, NAC for 2 h in MDA-MB-231 cells. Meanwhile, no obvious changes in nuclear morphology were found in Saxi A-treated MCF-7 cells. These results were further confirmed by flow cytometry analysis. As shown in Fig. 3C, treatment with 7 [micro]g/ml of Saxi A induced apoptosis in MDA-MB-231 cells, whereas the population of apoptotic cells was significantly reduced by pretreatment with NAC for 2 h. Meanwhile, NAC treatment had no protective effect on Saxi A-induced cell death in MCF-7 cells. These results suggest that Saxi A induces apoptosis in MDA-MB-231 cells via a ROS-dependent pathway.

Effect of Saxi A on the expression of apoptotic proteins and caspase activation

The Bcl-2 protein family is an important regulator in apoptotic process, and includes the anti-apoptotic proteins [Bcl.sub.2] and Bcl-xL and pro-apoptotic protein Bax. To evaluate the effect of Saxi A on the expression of Bcl-2 family proteins, the levels of Bcl-2, Bcl-xL and Bax were determined by Western blot analysis (Fig. 4A). Treatment of MCF-7 cells with Saxi A decreased Bcl-2 and Bcl-xL protein expression, and slightly increased Bax expression, whereas treatment of MDA-MB-231 cells caused a significant decrease in the levels of Bcl-2 and Bcl-xL and significantly increased Bax expression in a concentration-dependent manner. Since the release of cytochrome c from the mitochondrial intermembrane spaces to the cytosol is a major event in the mitochondria-dependent apoptotic pathway and the translocation of truncated apoptosis-inducing factor (AIF) from the inner mitochondrial membrane into the cytosol occurs in the caspase-independent apoptotic pathway, we examined whether Saxi A induces the release of cytochrome c and AIF from mitochondria into the cytosol. As shown in Fig 4B, Saxi A treatment in both cell lines decreased the levels of cytochrome c and AIF in the mitochondrial fraction in a concentration-dependent manner, and significantly increased in the cytosolic fraction. Additionally, we investigated whether caspases were activated in Saxi A-induced apoptosis in breast cancer cells. As shown in Fig. 4C, Saxi A cleaved caspases-3 and -8 in MDA-MB-231 cells. However, Saxi A did not significantly have an effect on the activation of caspase-8 in MCF-7 cells. Taken together, these results suggest that Saxi A induced apoptosis in MDA-MB-231 cells through a caspase-dependent pathway, while the induced apoptosis in MCF-7 cells in a caspase-independent manner.

[FIGURE 2 OMITTED]

Effect of Saxi A on MAPKs activity

Numerous previous studies have shown that mitogen-activated protein kinases (MAPKs), including ERK 1/2, p38 MAPK, and JNK induced apoptosis in various types of cells, particularly in cancer cells. To determine whether Saxi A-induced apoptosis is regulated by MAPKs signaling pathway in breast cancer cells, the activation of MAPKs was examined by Western blot analysis. As shown in Fig. 5A, Saxi A treatment in both cell lines increased ERK 1/2 and p38 MAPK phosphorylation in a time-dependent manner. The phosphorylation of JNK was significantly elevated following 30 min of exposure to Saxi A in both MCF-7 and MDA-MB-231 cells. To confirm the relevance of MAPKs in Saxi A-induced apoptosis, we treated both cell lines with an ERK 1/2 inhibitor (PD98059), p38 MAPK inhibitor (SB203580), and a JNK inhibitor (SP600125). Pretreatment of breast cancer cells with SP600125 for 2 h before Saxi A exposure significantly reduced apoptosis (Fig. 5B). In addition, SB203580 inhibited Saxi A-induced apoptosis in MDA-MB-231 cells. Pretreatment with PD98059 had no effect on Saxi A-induced apoptosis in either breast cancer cell line. Collectively, these findings suggest that Saxi A increased MAPK phosphorylation in both MCF-7 and MDA-MB-231 cells and it appears that all MAPKs, except ERK 1/2, play an important role in Saxi A-induced apoptosis in breast cancer cells.

The role of ER[alpha] in Saxi A-induced apoptosis

As shown in the results above, ER[alpha]-negative MDA-MB-231 cells were more sensitive to Saxi A-induced apoptosis compared to ER[alpha]-positive MCF-7 cells. To investigate if ER[alpha] plays a role in the sensitivity of MDA-MB-231 cells to Saxi A-induced apoptosis, MCF-7 cells were transfected with ER[alpha]-specific shRNA to downregulate ER[alpha] expression and MDA-MB-231 cells were transfected with HA-tagged ER[alpha] to overexpress ER[alpha] The expression levels of apoptotic proteins in both breast cancer cell lines were detected by Western blot analysis. As shown in Fig. 6A, Bcl-2 expression in MCF-7 cells was significantly decreased by Saxi A treatment, and treatment with shER[alpha] further reduced expression levels. However, shER[alpha] transfection drastically increased Bax protein expression levels in Saxi A-treated MCF-7 cells. Similarly, Saxi A treatment in MDA-MB-231 cells decreased Bcl-2 expression, whereas the overexpression of ER[alpha] significantly increased Bcl-2 expression. Saxi A increased Bax expression, but was decreased by shER[alpha] pretreatment. To further confirm the effect of ER[alpha] on Saxi A-induced apoptosis, breast cancer cells were stained with Annexin V/PI and examined by flow cytometry analysis. As expected, treatment with Saxi A for 12 h increased the apoptotic cell population in MCF-7 cells transfected with shER[alpha] (Fig. 6B). On the contrary, ER[alpha] overexpressed MDA-MB-231 cells were resistant to treatment by Saxi A (Fig. 6C). Taken together, these results demonstrate that ER[alpha] knockdown in MCF-7 cells accelerated apoptosis induced by Saxi A treatment, whereas ER[alpha] overexpression in MDA-MB-231 cells restored cell viability from Saxi A-induced apoptosis.

[FIGURE 3 OMITTED]

Discussion

Recent studies have shown that triterpenoid saponins from Androsace umbellata inhibit cell proliferation and induce apoptosis in human hepatoma (Wang et al., 2008; Zhang et al., 2007) and Raw 264.7 cells (Park et al., 2010). Especially, Saxifragifolin D was reported to stimulate ROS production, trigger endoplasmic reticulum stress, and finally induce autophagy and apoptosis in breast cancer cells (Shi et al., 2013). However, the cytotoxic effect of Saxi A remains unknown. Our study reveals that Saxi A inhibited cell proliferation and increased the apoptotic cell population in both breast cancer cell lines, but ER-negative MDA-MB-231 cells were more sensitive to Saxi A-induced cell death. The anticancer effects on both ER-positive and ER-negative breast cancer cells are supported by a recent report suggesting [alpha]-Santarol, a terpenoid isolated from sandalwood, also has anti-neoplastic effects on both breast cancer cell lines (Santha et al., 2013).

It has been suggested that ROS acts as redox messengers in intracellular signaling while unregulated ROS production may be involved in apoptosis (Circu and Aw, 2010). It is also widely accepted that the ROS generation is closely related with certain mechanisms in the early stages of apoptosis and mitochondrial dysfunction (Herrera et al., 2001). Our results show that the generation of ROS was elevated concentration-dependently in MDA-MB-231 cells, while MCF-7 cells were unresponsive to Saxi A treatment. Additionally, chromatin condensation, one of morphological features in apoptotic cells, in addition to cell shrinkage and membrane blebbing (Green and Reed, 1998), was observed in Saxi A-induced MDA-MB-231 cells, and was inhibited by NAC treatment. Further support for this conclusion comes from flow cytometry observation that treatment with Saxi A increased the apoptotic cell population of MDA-MB-231 cells and NAC treatment reduced the population of apoptotic cells. Our results are similar to the finding that Saxifragifolin D increased ROS production in MDA-MB-231 cells, which were inhibited by pretreatment with NAC (Shi et al., 2013). Thus, the present data suggest that Saxi A affects ROS generation in breast cancer cells differently, and only MDA-MB-231 cells undergo apoptosis in a ROS-dependent manner by Saxi A.

The mitogen-activated protein kinase (MAPK) family, which includes ERK 1/2, p38, MAPK, and JNK, plays an essential role in intracellular signaling networks and controls multiple cellular mechanisms such as proliferation, differentiation, stress response, and especially the apoptotic process (Stork and Schmitt, 2002). Previous studies have proved that MAPK signaling is involved in the cell survival of breast cancer cells (Mester and Redeuilh, 2008). Furthermore, it has been shown that natural products extracted from medical plants induced cell death in human breast cancer cells through the activation and phosphorylation of MAPKs (Chen et al., 2011; Li et al., 2014). Our study shows that Saxi A increased MAPKs phosphorylation in both MCF-7 and MDA-MB-231 cells, while JNK in MCF-7 cells and p38 and JNK in MDA-MB-231 cells were related to Saxi Ainduced apoptosis. Similar results have been obtained from several reports which demonstrate that JNK and p38 MAPKs are involved in the apoptotic process of breast cancer cells (Park et al., 2011; Rabi and Banerjee, 2008).

Generally, two major pathways are involved in apoptosis. They are the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. The extrinsic pathway is initiated by death receptors interacting with their respective ligands, leading to the cleavage of initiator caspase-8. The active caspase-8 then cleaves executioner caspase-3. In the intrinsic pathway, the death signals inside the cells initiate the process. The disruption of the mitochondria membrane potential results in the release of cytochrome c, which subsequently triggers the activation of caspases-9 and -3, and eventually promotes apoptosis (Elmore, 2007). Bcl-2 family proteins play important roles in regulating mitochondria permeability and caspase activation. Among these proteins, Bcl-2 and Bcl-xL proteins are thought to be anti-apoptotic effector proteins which bind to the outer membrane of the mitochondria and prevent the release of cytochrome c. Bax has been identified as pro-apoptotic protein and is responsible for permeabilizing the membrane because of damage due to cellular stress (Youle and Strasser, 2008). In this study, the data revealed that Saxi A had different inhibitory effects on cell growth in two breast cancer cell lines; ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells. Treatment with Saxi A reduced the cell viability of MCF-7 cells through the mitochondrial pathway, as supported by the results showing increased Bax expression, decreased Bcl-2 and Bcl-xL expression, and the release of cytochrome c and AIF from mitochondria into the cytosol. Saxi A-induced apoptosis in MDA-MB-231 cells was also associated with the expression of Bax, Bcl-2 and Bcl-xL, the same as listed above. However, the release of cytochrome c and AIF as well as the activation of caspase-8 and caspase-3 was related to Saxi A-triggered apoptoic cell death in MDA-MB-231 cells. Given that MCF-7 cells are deficient in caspase-3 (Janicke, 2009), a distinct difference between the two breast cancer cells in Saxi A-induced apoptotic process could be the existence of ERa.

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Up to date, the most common classification of breast cancer in choosing suitable chemotherapeutic reagents is based on the ER[alpha] status (Andre and Pusztai, 2006). ER is highly expressed and serves as a prognostic factor in most types of breast cancer cells which develop and proliferate in an estrogen-dependent manner (Ali and Coombes, 2000; Ijichi et al., 2011). To treat these tumors, various natural materials have been reported to inhibit breast cancer cell growth by down-regulating ER[alpha] expression (Jang et al., 2011; Li et al., 2011). Though ER-positive breast cancers can be cured by ER-targeted or anti-estrogen therapy, ER-negative breast cancers are unresponsive to treatment and more aggressive. Therefore, the development of new effective therapeutic agents is required for these more aggressive types of breast cancer. Our results show that suppressing ER[alpha] by shER[alpha] transfection decreased cell survival in Saxi A-stimulated MCF-7 cells, whereas the overexpression of ER[alpha] by HA-ER[alpha] inhibits the cytotoxic effects of Saxi A in MDA-MB-231 cells. That is to say, in contrast with other natural substances that target ER[alpha], Saxi A induces apoptosis more effectively in the absence of ER[alpha] in both breast cancer cell lines compared to non-transfected controls. Previous studies have shown that Wogonin (Chung et al., 2008), [alpha]-Santalol (Santha et al., 2013) and Anethole (Chen and deGraffenried, 2012) have apoptotic effects in both ER-positive and ER-negative human breast cancer cell lines. In addition, several natural compounds have anticancer effects in ER-negative MDA-MB-231 cells (He et al., 2014; Kim et al., 2013). Taken together, Saxi A induces breast cancer cell death more in ER-negative MDA-MB-231 cells than ER-positive MCF-7 cells because of the absence of ER[alpha].

Conclusion

In summary, our report elucidates the anticancer effects of Saxi A on two breast cancer cell lines, ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells, via different apoptotic processes. These findings also indicate that ER[alpha] could be an important regulator in Saxi A-induced apoptosis of breast cancer cells. Collectively, Saxi A has the potential to be used as a new chemotherapeutic agent in the treatment of different types of breast cancers. Further investigations including in vivo studies using animal models will contribute to elucidating the clinical use of Saxi A for breast cancer treatment.

ARTICLE INFO

Article history:

Received 16 January 2015

Revised 8 May 2015

Accepted 26 May 2015

Conflict of interest

The authors have no conflicts of interest to declare.

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Kyung-Ho Kim (1), Ji-Yun Kim (1) Jong-Hwan Kwak, Suhkneung Pyo *

School of Pharmacy, Sungkyunkwan University, Suwon, Kyunggi-do, 440-746, Republic of Korea.

* Corresponding author. Tel: +82 31 290 7713; fax: +82 31 290 7733.

E-mail address: snpyo@skku.edu (S. Pyo).

(1) These authors equally contributed to this work.

http://dx.doi.org/10.1016/j.phymed.2015.05.057
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Author:Kim, Kyung-Ho; Kim, Ji-Yun; Kwak, Jong-Hwan; Pyo, Suhkneung
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Aug 15, 2015
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