Acta Pharmacologica Sinica (2011) 32: 1402–1410 © 2011 CPS and SIMM All rights reserved 1671-4083/11 $32.00 www.nature.com/aps
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Original Article
Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo Qian-mei ZHOU#, Xiu-feng WANG#, Xin-jun LIU, Hui ZHANG, Yi-yu LU, Shi-bing SU* Research Center for Traditional Chinese Medicine Complexity System, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Aim: To investigate the efficacy of mitomycin C (MMC) in combination with curcumin in suppressing human breast cancer in vitro and in vivo. Methods: Human breast cancer MCF-7 cells were used. Cell viability was measured using MTT assay. The cell cycle phase was detected with flow cytometric analysis. Cell cycle-associated proteins were examined using Western blot analysis. MCF-7 breast cancer xenografts were established to monitor tumor growth and cell cycle-associated protein expression. Results: Curcumin inhibited MCF-7 breast cancer cell viability in a concentration-dependent manner (IC50 value=40 μmol/L). Similarly, MMC inhibited the cell viability with an IC50 value of 5 μmol/L. Combined treatment of MMC and curcumin showed a synergistic antiproliferative effect. In the presence of curcumin (40 μmol/L), the IC50 value of MMC was reduced to 5 μmol/L. In MCF-7 xenografts, combined administration of curcumin (100 mg/kg) and MMC (1-2 mg/kg) for 4 weeks produced significantly greater inhibition on tumor growth than either treatment alone. The combined treatment resulted in significantly greater G1 arrest than MMC or curcumin alone. Moreover, the cell cycle arrest was associated with inhibition of cyclin D1, cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2) and CDK4, along with the induction of the cell cycle inhibitor p21 and p27 both in MCF-7 cells and in MCF-7 xenografts. These proteins were regulated through p38 MAPK pathway. Conclusion: The results suggest that the combination of MMC and curcumin inhibits MCF-7 cell proliferation and cell cycle progression in vitro and in vivo via the p38 MAPK pathway. Keywords: curcumin; mitomycin C; human breast cancer MCF-7 cells; combined chemotherapy; cell cycle; xenografts; p38 MAPK; cyclin; cyclin-dependent kinases (CDKs); p21 Acta Pharmacologica Sinica (2011) 32: 1402–1410; doi: 10.1038/aps.2011.97; published online 10 Oct 2011
Introduction
Cell growth and proliferation are tightly regulated by various interactions between molecules[1]. Several major checkpoints in the cell cycle are controlled by multiple protein kinases[2, 3]. The cyclin-dependent kinases (CDKs) join with regulatory proteins called cyclins to drive the cell through the cell cycle[4]. Specific complexes regulate each step of the cycle. Cyclin D1/CDK2 and CDK4 drive progression through G1, cyclin E/ CDK2 controls entry into S phase, and cyclin A/CDK2 controls S-phase progression. CDK inhibitors (CDKIs) such as p21 and p27[5] block specific interactions. In many cell types, aberrant expression of cell cycle regulatory proteins can induce cell #
The first two authors contributed equally to this work. * To whom correspondence should be addressed. E-mail
[email protected] Received 2011-03-15 Accepted 2011-06-23
cycle progression[6, 7]. The mitogen-activated protein kinase (MAPK) family is activated in response to proliferative cues[8]. P38 MAPK is a major subfamily of mammalian MAPK. It has been reported that p38 MAPK activation is involved in the induction of a G1/S checkpoint[9] that results in the accumulation of p21[10, 11]. Moreover, p21 establishes a G1/S checkpoint by inactivating CDK2[10]. In addition, p38 MAPK can stabilize p21 in vivo[9] and can down-regulate the level of cyclin D1[12]. The cell cycle controls cell proliferation and cancer is a disease of inappropriate cell proliferation. The excess cells initiate a vicious cycle where cells are insensitive to the signals that are involved in adhesion, differentiation and death in normal cells[13]. Modulation of the cell cycle also contributes to oncotherapy. The majority of human breast cancers display deregulated overexpression of cyclin D1, whereas in normal cells its expression is tightly regulated by mitogenic signal-
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ing involving the p21 Ras pathway[14]. Research on cell cycle progression and cell cycle regulatory proteins in human breast cancer cell lines has been performed in recent years[15, 16]. Curcumin (diferuloylmethane), a yellow colored polyphenol and a natural plant phenolic food additive, is an active component of the perennial herb Curcuma longa (commonly known as turmeric)[17]. Curcumin exhibits anti-cancer activities both in vitro and in vivo through a variety of mechanisms. It inhibits proliferation and induces apoptosis in a wide array of cancer cell types in vitro, including cells from cancers of the bladder, breast, lung and other tissues[18]. It also controls the cell cycle and causes cell cycle arrest in various cancers. Curcumin inhibited cell cycle progression through the downregulation of cyclin D1 in vitro and its transcriptional and translational levels in vivo and by blocking its association with CDK4[19]. It also induced cell cycle arrest through the upregulation of CDKIs such as p21 and p27[20, 21]. Mitomycin C (MMC) is currently used as the third-line chemotherapeutic agent for breast cancers[22]. Previous studies have shown that there are cell cycle alterations in response to MMC treatment[23, 24]. However, it is not clear whether curcumin in combination with MMC impacts breast cancer cell proliferation and cell cycle progression. The present study sought to examine the combination treatment of curcumin and MMC-induced inhibition of cell growth and cell cycle arrest in vitro and in vivo. The p38 MAPK pathway was identified as a mechanism through which the combination treatment induced cell growth inhibition that was mediated via p21 and p27-induced cell cycle arrest.
Materials and methods
Materials MMC was purchased from ICN Company (USA), dissolved in physiological saline as a 1 mmol/L stock solution and stored at 4 °C away from light. Curcumin, with a purity of more than 98%, was obtained from the National Institute for the Control of Pharmaceutical and Biological Products in China. Curcumin was dissolved in dimethyl sulfoxide (DMSO) as 40 mmol/L solution for use in the treatment of cells, and it was dissolved in a solution of physiological saline with 1% DMSO and 10% Tween-80 for use in animal research. SB203580 was obtained from Biomol (Philadelphia, PA, USA). The antibodies against cyclin D1, cyclin E, cyclin A, CDK2, CDK4, p21 and p27 were obtained from Cell Signaling Inc (Boston, MA, USA). The antibody against phosphorylated p38 (p-p38) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Propidium iodide (PI) was purchased from Sigma (St Louis, MO, USA). Cell culture Human breast cancer MCF-7 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in RPMI-1640 medium from Gibco (San Francisco, CA, USA) supplemented with 10% heat-inactivated (56 °C, 30 min) fetal calf serum (PAA, Pasching, KA, Austria), 0.01 mg/mL insulin from Sigma (St Louis, MO, USA),
2 mmol/L glutamine from Gibco (San Francisco, CA, USA), penicillin (100 U/mL) and streptomycin (100 µg/mL). The cell culture was maintained at 37 °C with 5% CO2 in a humidified atmosphere. Cell viability assays MCF-7 cells were seeded in 96-well culture plates (5×104 cells/ mL). After overnight incubation, MCF-7 cells were treated with various concentrations of MMC, curcumin, or MMC plus curcumin (the ratio of two compounds was 1:1). DMSO was adjusted to the same final concentration of 0.01%. Following incubation, cell viability was measured at different time points using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) as described previously[25]. The formazan product was quantified by measuring the absorbance at 490 nm. The interaction between curcumin and MMC was analyzed by isobologram analysis described by Chou and Talalay[26]. The interaction of two compounds was quantified by determining the combination index (CI). From this analysis, the combined effects of the two drugs can be summarized as follows: the CI of less than, equal to or more than 1 indicate synergistic, additive or antagonistic effects, respectively. Cell cycle analysis MCF-7 cells were seeded into 10-cm dishes and treated with curcumin, MMC or a combination of the agents at the concentrations indicated. Cells were harvested 48 h after treatment. Detached cells were collected by centrifugation (100×g, 5 min). The attached cells were harvested by trypsinization. Cell pellets were washed with PBS and then fixed in ice-cold 70% ethanol. For cell cycle analysis, cells were recentrifuged and stained with propidium iodide (PI). Cell-cycle phase analysis was performed using a Becton Dickinson Facstar flow cytometer (San Jose, CA, USA) equipped with ModFit LT 3.0 software. Western blot analysis Whole cell lysate was loaded in each lane and separated by SDS-PAGE. Protein expression was detected using a primary antibody (1:1000, except p-p38 1:200) and secondary antibody (1:800) conjugated with horseradish peroxidase. Levels of cyclin D1, cyclin E, cyclin A, CDK2, CDK4, p21, p27, p38, p-p38, and GAPDH were analyzed in this manner. Chemiluminescence was observed by ECL (Pharmacia, Buckinghamshire, UK). Quantitative analysis of Western blotting was performed using Alpha Ease FC (FluorChem FC2) software. Using the analysis tools, we calculated the density ratio of each protein to GAPDH, the loading control. Human tumor xenografts Female nu/nu athymic mice (7 weeks old) were obtained from Chinese Academy of Sciences. The mice (five per cage) were housed in cages equipped with air filter lids and maintained under pathogen-limiting conditions. MCF-7 cells (1×107/mL) were inoculated into the mammary fat pads of the mice. Before inoculation, 17β-estradiol (1.7 mg) was intraperitoneActa Pharmacologica Sinica
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ally injected[27]. Once palpable tumors developed (approximately 2 weeks), mice were randomized to receive vehicle (physiological saline with 1% DMSO and 10% Tween-80 for curcumin, 100% physiological saline for MMC), curcumin (100 mg/kg), MMC (2 mg/kg), MMC (1.5 mg/kg), MMC (1 mg/kg) and a combination of both curcumin and MMC. All drugs were administered by intraperitoneal injection. There were eight tumor-bearing animals in each group and all tumors were harvested 28 d after treatment. The tumors were immediately removed, freed from connective and adipose tissue, and weighed. The animal protocols were preapproved by the ethical committee of our institution. Statistical analysis When appropriate, data were expressed as mean±SD. Data were analyzed by Student’s t-test where appropriate. Statistical significance was set at P
