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Pharmacological Reports Copyright © 2013 2013, 65, 453459 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Synergistic anti-cancer activity of the combination of dihydroartemisinin and doxorubicin in breast cancer cells Guo-Sheng Wu1, Jin-Jian Lu1,2, Jia-Jie Guo1, Ming-Qing Huang3, Li Gan2, Xiu-Ping Chen1, Yi-Tao Wang1 1 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China 2 College of Life Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, China 3 College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, 350108, China Correspondence: Jin-Jian Lu, e-mail: [email protected] and Yi-Tao Wang, e-mail: [email protected] Abstract: Background: Dihydroartemisinin (DHA) exhibits potent anti-malarial and anti-cancer activities. This study aimed to investigate the anti-proliferative effects of a combination of DHA and doxorubicin (DOX) on human breast cancer cells. Methods: MTT assay and the combination index (CI) were used to show the anti-proliferative effects and calculate the synergism potential, respectively. Flow cytometry assay was used to detect apoptosis and the intracellular accumulation of DOX. JC-1 staining was used to determine the mitochondrial membrane potential. Western blot analysis was used to detect the protein expression of some apoptosis-related molecules. Results: A synergistic anti-proliferative effect was found, and the enhanced anti-cancer activity was observed to be accompanied by the prompt onset of apoptosis in MCF-7 cells. The combinative treatment remarkably decreased the mitochondrial membrane potential and activated caspase cascades more than the mono-treatment. Pretreatment with DHA also did not influence the accumulation of DOX in MCF-7 cells. Conclusion: This study presented a new opportunity to enhance the effectiveness of future treatment regimens of breast cancer using DOX. Key words: dihydroartemisinin, doxorubicin, synergistic, anti-tumor, apoptosis; MCF-7 Abbreviations: ARTs – artemisinin and its derivatives, CI – combination index, DHA – dihydroartemisinin, DOX – doxorubicin, JC-1 – 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolylcarbocyanine iodide, MMP – mitochondrial membrane potential, MTT – 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide, PARP – poly (ADP-ribose) polymerase, PBS – phosphate buffered saline, P-gp – P-glycoprotein, TRAIL – tumor necrosis factor-related apoptosisinducing ligand Introduction Breast cancer is one of the most common cancers worldwide and the most common among women [8]. Doxorubicin (DOX), an anthracycline drug, is widely used as a chemotherapeutic agent for breast cancer. Despite the remarkable anti-cancer activity of DOX, Pharmacological Reports, 2013, 65, 453459 453 cells. DHA pretreatment was found to enhance DOXmediated apoptosis in MCF-7 cells significantly. This enhancement was accompanied by an increase in the cleavage of poly (ADP–ribose) polymerase (PARP) and the activation of caspase cascades. Our results revealed a new strategy for enhancing the effectiveness of future treatment regimens using DOX. Materials and Methods Reagents Fig. 1. Chemical structure of dihydroartemisinin (DHA) its practical therapeutic use is limited by toxicities such as cardiotoxicity [19]. Therefore, combined treatment with other drugs is desirable. Traditional Chinese medicinal herbs have a long history and are widely viewed as new, attractive sources of therapeutic regimens without severe toxicity. The herb Artemisia annua L., which has been used in China for centuries to treat fever and chills, contains artemisinin as its active constituent. Artemisinin and its derivatives (ARTs) are extensively used as anti-malarial drugs without severe side effects and have been found to inhibit the growth of tumor cells [4, 11, 17, 18]. One of the main active metabolites of ARTs is dihydroartemisinin (DHA) (Fig. 1), which is one of the most effective anti-cancer compounds both in vitro and in vivo. DHA mediates cell cycle arrest, induces apoptosis, blocks angiogenesis, and inhibits metastasis [1, 7, 10–12, 17]. DHA also selectively kills cancer cells with little effect on normal cells [6, 13]. DHA enhances as well the anti-cancer potential of gemcitabine in human hepatoma cells and cooperates with tumor necrosis factor-related apoptosisinducing ligand (TRAIL) to induce apoptosis in human prostate cancer cells [5, 6]. Herein, we hypothesized that the combination of DHA and DOX in human breast cancer cells exerts a synergistic anticancer effect. In this study, we found that the combination DHA and DOX exerted a synergistic effect on breast cancer 454 Pharmacological Reports, 2013, 65, 453459 DHA was provided by Prof. Ying Li of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). DOX was obtained from Sigma (St. Louis, MO, USA). 3-[4,5-Dimethyl-2thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were purchased from USB (OH, USA) and Molecular Probes (Eugene, OR, USA), respectively. Cell lines and culture MCF-7, MDA-MB-231, and T-47D human breast cancer cells were obtained from ATCC (Manassas, VA, USA). The cells were maintained at 37°C under an atmosphere of 5% CO2 and 95% air in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). MTT assay Exponentially growing MCF-7, MDA-MB-231, and T-47D cells were seeded into 96-well plates. The cells were treated with the indicated compounds, and cell viability was determined after 48 h of incubation by adding 20 µl of MTT (5 mg/ml). After slightly aspirating the MTT-containing medium after 4 h, 100 µl of dimethyl sulfoxide was added to solubilize the formazan followed by shaking 10 min in darkness. The absorbance at 570 nm was recorded using a Multilabel counter (PerkinElmer, Singapore). Synergistic activity of the combination of DHA and DOX Guo-Sheng Wu et al. Observation of morphologic changes MCF-7 cells were seeded into six-well plates and treated with 10 µM DOX for 24 h with or without pretreatment of 20 µM DHA for 6 h. Cellular morphology was observed with an AxioCam HRc CCD camera (Carl Zeiss, Germany). Flow cytometry assay MCF-7 cells seeded into six-well plates were treated with 10 µM DOX for 24 h with or without pretreatment of 20 µM DHA for 6 h. The cells were harvested, fixed in 70% ethanol, and stored at 4°C overnight. Then, the cells were stained in phosphatebuffered saline (PBS) containing 5 µg/ml RNase and 20 µg/ml propidium iodide (PI) in the dark at room temperature for 30 min. Flow cytometry (Becton Dickinson FACSCanto™, Franklin Lakes, NJ, USA) was used to analyze the cells. At least 10,000 events were counted for each sample, and the sub-G1 was analyzed. Mitochondrial membrane potential (MMP) assay The MMP of intact cells was measured by a fluorescent inverted microscope with the lipophilic probe JC-1 [15]. MCF-7 cells were seeded into six-well plates and treated with 10 µM DOX for 24 h with or without pretreatment of 20 µM DHA for 6 h. JC-1 fluorescence was observed with a fluorescent microscope, and pictures were taken with an Axiovert 200 fluorescent inverted microscope (Carl Zeiss) and an AxioCam HRc CCD camera (Carl Zeiss). Western blot analysis After treatment under desired conditions, the cells were harvested and total proteins were extracted with radioimmunoprecipitation assay lysis buffer containing 1% phenylmethanesulfonyl fluoride and 1% protease inhibitor cocktail for 30 min. Equal amounts of total protein were separated by appropriate SDSPAGE and transferred onto a poly (vinylidene fluoride) membrane. After blocking with 5% non-fat dried milk for 1 h, the membrane was incubated with the specific primary antibodies against PARP, caspase-7, caspase-9, and b-actin (Cell Signaling Technology, Beverly, MA, USA) followed by incubation with corresponding secondary antibodies at 37°C for 1 h. Specific protein bands were visualized with an ECL advanced western blot analysis detection kit. Intracellular accumulation of DOX MCF-7 cells were pre-cultured for 6 h with or without indicated concentrations of DHA and treated with different concentrations of DOX for 1 h. The cells were then harvested and resuspended in 1 ml of PBS. The cells were washed three times with PBS, and the mean fluorescence intensities of the cells were detected with a fluorescence-activated cell sorting (FACS)-Calibur cytometer (Becton Dickinson, San Jose, CA, USA). Statistical analyses The combination index (CI) is widely used to quantify drug synergism based on the multiple drug effect equation of Chou–Talalay [2, 3]. In our study, the CI values were determined for each concentration of DHA, DOX, and their combination in cell proliferation assays using CalcuSyn (Biosoft, Cambridge, UK). CI < 0.9 indicates synergism, CI = 0.9–1.10 indicates additive interaction, and CI > 1.10 indicates antagonism [20]. Results DHA synergistically increased the DOXinduced inhibition of cell proliferation We first examined the synergistic effects of DHA and DOX on the proliferation of MCF-7, MDA-MB-231, and T-47D human breast cancer cell lines. Figure 2A shows a schemate of the experimental protocol for the combined treatment. Cells in 96-well plates were pretreated with serial concentrations of DHA for 6 h and further treated with or without indicated DOX for 48 h. The cell proliferation inhibition bars to DHA, DOX, and DHA combined with DOX from three independent experiments are shown in Figures 2B–D. DHA exhibited moderate cytotoxicity against MCF-7 and T-47D cancer cells but had little effect on MDA-MB-231 cells, consistent with our previous reports [11, 12]. After the combined treatment, markedly stronger anti-proliferation abilities were achieved. Pharmacological Reports, 2013, 65, 453459 455 Fig. 2. Cytotoxicity of DOX + DHA in breast cancer cells. (A) Schematic of the experimental protocol + DHA treatment. (B, C, D) for DOX Dose re- sponse of MCF-7, MDA-MB-231, and T-47D cells to DHA, DOX, and DHA + DOX. (E) CI values in MCF-7, MDAMB-231and T-47D cells Subsequently, CI values were calculated using CalcuSyn at fixed-ratio concentrations of DHA and DOX. DHA plus DOX exhibited synergy in the tested breast cancer cell lines except at the highest dose in MDA-MB-231 cells, in which an additive effect was observed (Fig. 2E). DHA sensitized DOX-triggered apoptosis We further investigated the effects of exposure to DHA, DOX, or their combination on the apoptosisinducing abilities in MCF-7 cells. We first detected morphological changes after DHA, DOX, and DHA plus DOX treatment. A significantly higher number of 456 Pharmacological Reports, 2013, 65, 453459 round cells appeared in the combined-treatment group than in the other groups (Fig. 3A). We then used flow cytometry analysis with PI staining to detect the subG1 content, which is a characteristic of apoptosis. The cells treated with both DHA and DOX had the highest percentage in sub-G1. Given that mitochondria significantly influence DHA-induced apoptotic cell death [11] and mitochondrial changes including MMP collapse, the activation of caspases results in apoptosis. Therefore, we detected MMP changes after DHA, DOX, and DHA + DOX treatment. JC-1, which is a dual-emission fluorescent dye internalized and concentrated by respiring mitochondria, can reflect MMP changes in live cells [15]. In 24 h-treated MCF-7 cells, mitochondrial membrane depolarization as de- Synergistic activity of the combination of DHA and DOX Guo-Sheng Wu et al. Fig. 3. DOX + DHA triggers increased apoptosis in MCF-7 cells. MCF-7 cells seeded into six-well plates were treated with 20 µM DHA, 10 µM DOX, or their combination for 24 h; the morphological changes, sub-G1, MMP, and protein expression were detected. (A) Classical morphological changes. (B) Results of flow cytometry with PI staining. (C) JC-1 mitochondrial probe for MMP test. (D) Protein extracts were immunoblotted with the specified antibodies for PARP, caspase-7, and caspase-9 termined by JC-1 was moderately induced by DOX or DHA alone, whereas combined DOX and DHA resulted in a greater additive effect than the individual agents (Fig. 3C). We investigated further the effect of DHA, DOX, and their combination on PARP and caspases through western blot analysis. In MCF-7 cells, treatment with combined DOX (10 µM) and DHA (20 µM) for 24 h caused a much more significant cleavage of PARP and activation of caspase-7 than in mono-treated cells. This finding indicated the involvement of caspase-mediated apoptosis triggered by DHA + DOX, which favored the sensitizing effects of DHA on DOX-induced apoptosis (Fig. 3D). Besides, apoptosis induced by DHA + DOX was accompanied by the loss of MMP. This result prompted us to examine the involvement of caspase-9 in this process. In MCF-7 cells, DOX combined with DHA activated caspase-9 to a much higher extent than the monotreatments (Fig. 3D). Collectively, these data showed that DHA sensitized DOX in activating caspase cascades and triggering apoptosis. DHA pretreatment did not influence the intracellular concentration of DOX To determine whether DHA enhanced DOX activity by increasing the intracellular concentration of DOX, DOX was examined in the presence or absence of DHA. Given that DOX exhibited self-fluorescence, the intensity of intracellular fluorescence was adopted to reflect the intracellular concentration through FACS assay. MCF-7 cells were pretreated with DHA for 6 h and then with DOX for 1 h. We found that the fluoresPharmacological Reports, 2013, 65, 453459 457 Fig. 4. Intracellular accumulation of DOX in MCF-7 cells. (A) Cells were pretreated with 20 µM DHA for 6 h and incubated with 10 µM DOX for another 1 h. Intracellular fluorescence was then measured by flow cytometry analysis, and the fluorescence intensity indicated the intracellular concentration of DOX. Typical images of the flow cytometry assay are shown. (B) Semiquantitative analyses of the results in cence intensity in the DOX group was significantly higher than that in the blank and DHA-treated groups. However, the fluorescence intensity in the combinedtreatment group was identical to that in the DOX group, indicating that DHA did not influence the intracellular concentration of DOX (Figs. 4A and B). Discussion The CI is a widely accepted qualitative measure of the extent of drug interaction. In this study, the CI was used to evaluate the combination effects of DOX and DHA on the proliferation of breast cancer MCF-7, MDA-MB-231 and T-47D cells. DHA potentiated DOX-imposed cytotoxicity as revealed by the CI values (Fig. 2E) and the obvious enhancement of proliferation inhibition in the combined-treatment group (Figs. 2B–D). Compared with MCF-7 and T-47D cells, MDA-MB-231 cells were much more susceptible to DOX. However, after being combined with DHA, DOX exhibited similar potent antiproliferative capabilities especially at relatively high concentrations (Figs. 2B–D). More importantly, DHA did not 458 Pharmacological Reports, 2013, 65, 453459 A enhance intracellular DOX accumulation in MCF-7 cells (Figs. 4A and B). Thus, the same anti-cancer effect may be preferred at low doses of combined DOX and DHA or high doses of DOX alone. DHA is also widely used as an anti-malarial drug with few side effects, and the DHA-resistant cancer cell line does not present a multidrug-resistant phenotype [9]. Thus, DHA + DOX has tremendous latent capacity for clinical treatment in the future. Artemisinin, the mother compound of ARTs, reportedly induces resistance to DOX in human HT29 colon cancer cells and MCF-7 breast cancer cells partially by inducing Pglycoprotein (P-gp) expression [14]. Thus, artemisinin pretreatment reduces the intracellular DOX because of P-gp expression. The discrepancies may be due to the different compounds used. Although DHA is one of the main metabolites of ARTs, the pharmacological activities of artemisinin and DHA differ [6]. However, the detailed mechanisms require further investigation. The PI staining, JC-1 staining, and western blot analysis data indicated significant effects on apoptosis in the combined DOX and DHA groups compared with the mono-treated groups. This improvement partially explained the synergistic anti-proliferation ef- Synergistic activity of the combination of DHA and DOX Guo-Sheng Wu et al. fect of the DHA + DOX treatment. Actually, the enhancement of apoptosis is an effective way to improve the anti-proliferation potential in cancer cells [16]. However, how does DHA enhance DOXinduced apoptosis still warrants further study. In summary, DHA + DOX significantly improved the anti-cancer activity of each component, as revealed by the synergistic inhibitory effects on cancer cell proliferation. The synergism was partially due to the sensitized execution of apoptosis. Therefore, the superior pharmacological activities of DOX + DHA enabled their use as a promising anti-cancer strategy, although further research is needed. 8. 9. 10. 11. 12. Acknowledgments: This work was supported by Research Fund of Zhejiang Chinese Medicine University (No. 2009ZZ04), National Natural Science Foundation of China (No. 81001450), Research Fund of University of Macau (SRG026-ICMS13-LJJ, UL016/09-Y4/CMS/WYT01/ICMS) and Science and Technology Development Fund of Macau Special 13. Administrative Region (029/2007/A2). We greatly thank Prof. Ying Li form Shanghai Institute of Materia Medica for providing DHA and Dr. Hong Zhu from Zhejiang University for the statistical analyses. 14. References: 1. 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