Rhein Induces Apoptosis in Human Breast Cancer Cells

Human breast cancers cells overexpressing HER2/neu are more aggressive tumors with poor prognosis, and resistance to chemotherapy. This study investigates antiproliferation effects of anthraquinone derivatives of rhubarb root on human breast cancer cells. Of 7 anthraquinone derivatives, only rhein showed antiproliferative and apoptotic effects on both HER2-overexpressing MCF-7 (MCF-7/HER2) and control vector MCF-7 (MCF-7/VEC) cells. Rhein induced dose- and time-dependent manners increase in caspase-9-mediated apoptosis correlating with activation of ROS-mediated activation of NF-κB- and p53-signaling pathways in both cell types. Therefore, this study highlighted rhein as processing anti-proliferative activity against HER2 overexpression or HER2-basal expression in breast cancer cells and playing important roles in apoptotic induction of human breast cancer cells.

Breast cancer is one of the most common cancers in women, resulting from gene amplification and/or overexpression of some oncogenes like HER2/neu (also known as ErbB2) and oestrogen receptors [10,11]. Overexpression of HER2/neu in nearly 30% of human breast cancers correlates with more aggressive tumors and poor prognosis [12]. HER-2-overexpressing cells appear to be resistant to some classes of chemotherapy agents [12], but sensitive to others [13]. Overexpression of HER2/neu was positively correlated with p53 nuclear accumulation and tumor metastasis, negatively with hormonal receptor status [14]. Tyrosine kinase activity of HER2/neu could phosphorylate HER3 and then activate phosphatidylinositol 3-kinase (PI3K) involved in malignance of tumors [15]. Trastuzumab (Herceptin) is widely used for treatment of HER2-positive breast cancer; resistance to trastuzumab occurs in some patients [16]. Therefore, HER2/neu is a potentially therapeutic target for breast cancer, influencing efficacy of chemotherapy.
Engineering human breast cancer MCF-7 cells that expresses basal level of HER2/neu for Overexpression of HER2/neu has been used to analyze biological properties of HER2 overexpression [15,17]. This study investigates apoptotic effects of anthraquinone derivatives of rhubarb root on both types of breast cancer cells, HER2-overexpressing MCF-7 cells (MCF-7/HER2) and control vector MCF-7 cells (MCF-7/VEC). Of 7 anthraquinone derivatives, only rhein showed antiproliferative and apoptotic effects on both MCF-7/HER2 and MCF-7/VEC cells. We also analyzed apoptotic mechanism of rhein on both human breast cancer cell lines with basal level and Overexpression of HER2.

Western Blotting Analysis.
Cell lysates that were harvested from MCF-7/VEC and MCF-7/HER2 cells with or without treatment of anthraquinone derivatives were dissolved in 2X SDS-PAGE sample buffer without 2-mercaptoethanol, and boiled for 10 min. Cell lysate proteins were resolved on 12% SDS-PAGE gels and transferred to nitrocellulose paper. Resultant blots were blocked with 5% skim milk and reacted with properly diluted monoclonal antibodies against HER2/c-neu (Ab-3,3B5), caspase 9, Apoptosis signalregulating kinase 1 (ASK1), P21, and β-actin (Cell Signaling Technology) for 3 h incubation. Immune complexes were detected by horseradish peroxidase-conjugated goat antimouse IgG antibodies, followed by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech).

MTT Cytotoxicity Test.
Aloe emodin, emodin, rhein, chrysophanol, sennoside A, sennoside B, and 9-, 10-anthraquinone were purchased from Sigma Chemical Company (St. Louis, MO, USA). In vitro antiproliferative effect of each compound on MCF-7/VEC and MCF-7/HER2 cells was examined using MTT assay. Cells were plated in 96well plates (5 × 10 4 cells/well) and then treated with serial dilution of each tested compound. After treatment for 48 h, 25 μL of a MTT solution at 5 mg/mL was added to each well and incubated at 37 • C in 5% CO 2 for 3 h. After subsequent washing three times with phosphate buffer saline, 100 μL DMSO was added into plates for dissolving formazan crystals. OD 570−630 in each well was measured with a micro-ELISA reader and survival rate used to indicate suppressive effects of each compound on MCF-7/VEC and MCF-7/HER2 cells. Survival rate (%) = ((A control − A experiment )/A control ) × 100%. Cytotoxic concentration giving 50% (CC50) was calculated by computer program (provided by John Spouge, NCBI, NIH).

Detection of Cell Cycle and Apoptosis by Flow Cytometry.
MCF-7/VEC and MCF-7/HER2 cells were harvested 24,48, and 72 h posttreatment with or without serial dilutions of rhein. Cells were fixed using 70% ethanol at 4 • C overnight, then re-suspended in PBS containing 50 μg/mL PI and 0.1 mg/mL RNase and 0.1% Triton X-100 in darkroom. After 30 min incubation at 37 • C, cells were tested by flow cytometry (Becton-Dickinson, San Jose, Calif, USA) equipped with an argon ion laser at 488 nm wavelength, then rates of sub G1 (apoptotic), G1, S, and G2 phase cells were determined. In addition, rhein-induced cytopathic effect (CPE) of MCF-7/VEC and MCF-7/HER2 cells was observed 48 h posttreatment using light microscopy.

In Vivo Signaling Transduction Pathway Assay with
Cis-Reporter Plasmids. Cis-reporter plasmids pAP1-Luc, pNF-κB-Luc, and p53-Luc were purchased from Stratagene Company. MCF-7/VEC and MCF-7/HER2 cells were cotransfected with cis-reporter plasmid and internal control reporter pRluc-C1 (BioSignal Packard) in 6-well plates, using the Arrest-In transfection reagent. After one-day incubation, transfected cells were seeded into 24-well plates using the MEM containing 10% FBS, then treated with or without 20 μg/mL rhein. After 4 h posttreatment, enzyme activity of firefly and Renilla luciferases in indicated cells was measured by dual Luciferase Reporter Assay System (Promega) and Luminometer TROPIX TR-717 (Applied Biosystems). Relative firefly luciferase activity of the cis-acting reporter was normalized by Renilla luciferase.

Rhein Influenced Cell Cycle Phase of MCF-7/VEC and MCF-7/HER2 Cells.
To examine the effect of rhein on cell cycle phases, both types were treated with or without rhein at concentrations of 10 μg/mL and 100 μg/mL, and then harvested 24, 48, and 72 hours posttreatment. Cells were stained with propidium iodide and analyzed for cell cycle fractions by flow cytometry (Figure 3). Rhein induced concentration-and time-dependent manner increase in sub-G1 (apoptosis) fractions of MCF-7/VEC and MCF-7/HER2 cells (P < 0.05, Figure 3(a)). The rate of G1 phase in MCF-7/VEC cells significantly increased 24 hours posttreatment with rhein at a concentration of 100 μg/mL (P < 0.05), but no effect of rhein on G1 phase in MCF-7/HER2 cells was detected (Figure 3(b)). Rhein at concentrations of 10 and 100 μg/mL caused a significant S phase arrest in MCF-7/HER2 cells 48 and 72 hours posttreatment (P < 0.05), but rhein at a concentration of 100 μg/mL decreased S phase . Moreover, rhein increased the rate of G1 phase, but decreased the fraction of S phase in MCF-7/VEC cells (Figures 3(b) and 3(c)). The difference of cell cycle distribution in response to rhein could be due to cell  [22,23]. Cell cycle analysis of breast cancer BT-474 cells and MCF-7 cells indicated 1alpha(OH)D5 inducing an S phase decrease in MCF-7 cells and a G2 phase decrease in BT-474 cells [22]. Moreover, resveratrol induced apoptosis of MCF-7 cells via S phase arrest, but not MDA-MB-231 cells [23]. Western blot analysis indicated activation of caspase 9 in both cell types (Figure 4), responsible for dose-dependent manners on rhein-induced apoptosis of breast cancer cells. Results demonstrate that S phase arrest of MCF-7/HER2 cells and increased G1 phase of MCF-7/VEC cells correlated with rhein-induced caspase-9-mediated apoptosis. Rheininduced S phase arrest of MCF-7/HER2 cells correlates with antiproliferative and apoptotic mechanisms of rhein on human hepatocellular and tongue carcinoma cells [19,24]. Also, G1 arrest of MCF-7/VEC cells induced by rhein proves consistent with rhein-induced apoptosis of human lung cancer A-549 cells [25] and cervical cancer Ca Ski cells [26].

Rhein Induced ROS-Mediated Activation of ASK1 in MCF-7/VEC and MCF-7/HER2
Cells. Intracellular ROS generation reportedly triggers activation of caspase 9 and ASK1 signaling in apoptotic responses [27][28][29]. Therefore, we assessed effects of rhein treatment on intracellular ROS and ASK1 levels in MCF-7/VEC and MCF-7/HER2 cells ( Figures 5 and 6). Cells were cultured with rhein at 37 • C for 48 hours. After being washing twice with PBS, cells were stained with DCFH-DA, then analyzed by flow cytometry. Rhein treatment caused concentration-dependent increase of intracellular ROS in MCF-7/VEC and MCF-7/HER2 cells (P < 0.05, Figure 5). Western blot with anti-ASK1 antibodies revealed rhein as concentration-dependently triggering ASK1 level in each type of cell ( Figure 6). Results indicate ROS-mediated ASK1 signaling in rhein-induced apoptosis of both types. The findings were in agreement with incremental level of intracellular ROS in rhein-induced apoptosis of human tongue cancer cells, promyelocytic leukemia cells, and nasopharyngeal carcinoma cells [24,30,31].

Rhein Activated p53/p21 Signaling in MCF-7/VEC and MCF-7/HER2 Cells.
To probe correlation between in vivo signaling pathways and rhein-induced growth arrest and apoptosis, cells in response to rhein were further characterized by dual reporter assays with internal control reporter pRluc-C1 and luciferase reporter plasmid containing direct repeat elements of AP1, NF-κB, and p53 ( Figure 7). The cells were harvested 4 h posttreatment, and then the relative expression of firefly luciferase driven from the indicated cis-reporter plasmid was normalized by Renilla luciferase.
Evidence-Based Complementary and Alternative Medicine  Relative intensity of firefly luciferase revealed that rhein significantly activated p53-and NF-κB promoters (P < 0.05), but induced no significant change of AP1-derived reporter activity in each type of cell ( Figure 7). Moreover, p53-or NF-κB-derived reporter activity increased more than 1.7-fold in rhein-treated cells than mock cells. Definite increase of rhein-induced NF-κB-and p53-derived reporter activity in both types of cells indicated rhein modulating NF-κB-and p53-signaling pathways in breast cancer cells. Our result was inconsistent with the function of NF-κB mediating the cell survival response by inhibiting p53-dependent apoptosis and upregulating anti-apoptotic members of the Bcl2 family [32]. However, recent evidences show pro-apoptotic property of NF-κB via p53-dependent apoptosis in breast cancer MCF7/ADR, melanoma M14, and pro-B cells [33][34][35][36]. Activation of NF-κB induces upregulation of pro-apoptotic death receptor 5 (DR5) in breast cancer MCF-7 and MDA-MB-231 cells, correlating with TNF-related apoptosis inducing    Figure 7: Effect of rhein on in vivo signal pathways in human breast cancer cells. MCF-7/VEC and MCF-7/HER2 cells were transiently co-transfected with cis-reporter plasmids (pAP1-Luc, pNF-κB-Luc, and p53-Luc) and an internal control reporter (pRluc-C1), and then treated with rhein for 24 h. Firefly and Renilla Luciferase enzymes were measured; the relative firefly luciferase activity was normalized by Renilla Luciferase. * P value < 0.05; * * P value < 0.01 compared with untreated cells. ligand-mediated apoptosis [37]. In addition, oxidative stress has demonstrated activation of both NF-κB and p53 [38,39]. Doxycycline-induced superoxide-mediated apoptosis indicated NF-κB as a proapoptotic factor by activating the p53signaling pathway [39]. Monoamine neurotoxin-induced apoptosis of peripheral blood lymphocytes is associated with activation of NF-κB, p53, and c-Jun transcription factors [40]. Thus, rhein-induced activation of both NF-κB-and p53-signaling pathways might correlate with ROS-mediated ASK1 signaling in breast cancer cells (Figures 5 and 6). Moreover, rhein has reported activation of p53/p21 signaling pathway with in rhein-induced apoptosis of human lung cancer cells, cervical cancer cells, and hepatoblastoma cells [25,26,41]. We also demonstrated rhein-inducing dosedependent increases of p21 protein in MCF-7/VEC and MCF-7/HER2 cells ( Figure 8). Therefore, rhein-induced apoptosis of breast cancer cells could be associated with ROSmediated activation of NF-κB-and p53-signaling pathways.
In conclusion, this study highlighted rhein versus six other anthraquinone derivatives, as processing antiproliferative activity against both types of breast cancer cells with HER2 overexpression or HER2-basal expression. Rhein showed micromolar IC50 values on growth inhibition of both types via caspase-9-mediated apoptosis. ROS-mediated activation of NF-κB-and p53-signaling pathways might play important roles in rhein-induced apoptosis of human breast cancer cells.