Biomedicine & Pharmacotherapy 96 (2017) 371–377
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Original article
TanshinoneIIA enhances the chemosensitivity of breast cancer cells to doxorubicin through down-regulating the expression of MDR-related ABC transporters Kun Li, Hong Lai
T
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Department of Human Anatomy, China Medical University, Shenyang 110000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Tanshinone IIA Doxorubicin Chemosensitivity ABC transporters Breast cancer stem cells
As the first-line drug for breast cancer chemotherapy, doxorubicin (Dox) has strong cardiotoxicity. Meanwhile, prolonged Dox treatment of patients with breast cancer may result in resistance of breast cancer cells to Dox and an increased number of Dox-resistant breast cancer stem cells (BCSCs), thereby easily leading to breast cancer relapse. TanshinoneIIA (Tan IIA) has anti-tumor activity in addition to its cardiovascular protective effect. By preparing Dox resistant human breast cancer MCF-7 cells, here, we wanted to assess a new use of Tan IIA in enhancing the chemosensitivity of breast cancer cells to Dox and investigated its possible mechanisms. The results showed that Tan IIA could enhance the anti-tumor effect of Dox on MCF-7 and MCF-7/dox cells in a dosedependent manner, especially that of on MCF-7/dox cells. Even nontoxic dose of Tan IIA could also promote intracellular Dox accumulation of MCF-7 and MCF-7/dox cells through down-regulating the expression of efflux ABC transporters including P-gp, BCRP and MRP1, which can effectively eliminated cancerous cells including BCSCs, thereby enhancing the chemosensitivity of breast cancer. Therefore, Tan IIA can be used as a new potential chemotherapeutic sensitizer for the combination treatment of breast cancer.
1. Introduction Chemotherapy plays an important role in the systemic treatment of breast cancer. Anthracyclines such as doxorubicin (Dox) are often classified as the first-line drugs for breast cancer chemotherapy [1,2]. However, possible side effects of Dox such as the strong cardiotoxicity sometimes limit its use in clinic. Moreover, with the prolongation of chemotherapy cycle, breast cancer cells are not only prone to resist to Dox, but also develop to cross-resistance to a variety of chemotherapeutic drugs with different structure and function [3], thereby the breast cancer stem cell (BCSC) characteristics which may result in breast cancer relapse and metastasis are enhanced [4,5]. Therefore, a chemotherapeutic sensitizer found from cardiovascular protective agents may greatly improve the utilization efficiency of Dox. TanshinoneIIA (Tan IIA) is one of the effective active ingredient of Salvia miltiorrhiza and commonly used to treat ischemic heart disease in clinic. It has been shown to exert beneficial effects on cardiovascular system with minimal side effects, including improving the microcirculation of ischemic region [6], inhibiting platelet aggregation [7] and inflammatory responses [8], lowering blood lipids [9], retarding development of arteriosclerosis [10], ect. Meanwhile, it also has been
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showed to have a protective effect on myocardial damage induced by Dox [11]. Furthermore, several studies showed that Tan IIA had antitumor activity against multiple cancer cell types, including breast [12], gastric [13], colon [14], prostate [15], etc. Here, we wanted to assess a new use of Tan IIA in enhancing the chemosensitivity of breast cancer cells to Dox and investigated its possible mechanisms. 2. Materials and methods 2.1. Materials Tan IIA was purchased from Shanghai Jianglai Biological Technology Co Ltd (China). Doxorubicin (Dox) was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (China). Antibodies against P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-related protein 1 (MRP1) were purchased from antibody were purchased from Santa Cruz Inc. (USA). 2.2. Cell culture
Corresponding author. E-mail address:
[email protected] (H. Lai).
http://dx.doi.org/10.1016/j.biopha.2017.10.016 Received 9 August 2017; Received in revised form 19 September 2017; Accepted 2 October 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS.
RPMI-1640 supplemented with 10% FBS was used to culture MCF-7
Biomedicine & Pharmacotherapy 96 (2017) 371–377
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Fig. 1. Expression of efflux ABC transporters and CD44+/CD24−/low phenotype in MCF-7/dox cells. A. Expression of efflux ABC transporters including Pgp, BCRP and MRP1 in MCF-7/ dox cells was much higher than that of in MCF-7 cells. B. Expression of CD44+/CD24−/low phenotypel in MCF-7/dox cells was much higher than that in MCF-7 cells. *P < 0.05: significantly different from MCF-7 cells.
cells and MCF-7/dox cells. Medium for MCF7/dox cells was further supplemented with Dox (2 μg/mL). Before being used in experiments, MCF7/dox cells were cultured in Dox-free medium for 1 week.
ice-cold PBS to 400 μL and then analysed using flow cytometry.
2.3. Cell viability assay
MCF-7 and MCF-7/dox cells were respectively treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA for 24 h. The cells were then harvested and washed twice with PBS. As described previously [16], intracellular Dox accumulation was assessed using flow cytometry.
2.6. Intracellular Dox accumulation
MCF-7 and MCF-7/dox cells were respectively treated with Dox, Tan IIA, or Dox combined with Tan IIA for 24 h. MTS assay was performed according to the manufacturer's operating instructions. Firstly, cell viability of MCF-7 and MCF-7/dox cells treated with Tan IIA at different concentrations were assessed. Since less than 0.02 mg/L Tan IIA had little effect on cell viability (Fig. 2A), we selected this concentration of Tan IIA in the following experiments for better evaluation of its effect on anti-tumor activity of Dox. In our experiments, Cell proliferation ratio = (mean OD of experimental group − mean OD of blank)/(mean OD of control group − mean OD of blank) × 100%. Sensitizing fold = IC50 (Dox)/(combination of Dox and Tan IIA).
2.7. Western blot analysis MCF-7 and MCF-7/dox cells were respectively treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA for 24 h. The cells were washed twice and then collected. Total proteins were extracted. Equal amounts of protein were respectively separated with SDS-PAGE and then transferred onto PVDF membranes. The membranes were firstly incubated with primary antibodies GAPDH, P-gp, BCRP or MRP1 overnight at 4 °Cand then followed by second antibodies for 2 h at room temperature. Antibody detection was performed using chemiluminescence detection kit.
2.4. Cell apoptosis analyses MCF-7 and MCF-7/dox cells were respectively treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA for 24 h. The cells were collected and then washed twice with PBS and resuspended in the buffer solutions at a density of 1 × 106/mL. 100 μL of the cell suspensions were incubated with 5 μL of annexin V-FITC solution and 10 μL of dissolved PI for 15 min in the dark. 400 μL of PBS was added before the cell suspensions were analysed by flow cytometry.
2.8. Statistical analysis Each experiment was repeated at least three times. All data were presented as mean ± SD. Statistical analysis was performed using SPSS 13.0 software. P < 0.05 was considered as statistically significant.
2.5. CD44+/CD24−/low phenotype detection 3. Results MCF-7 and MCF-7/dox cells were respectively treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA for 24 h. The cells were washed twice with ice-cold PBS, harvested with trypsinization and then washed twice with ice-cold PBS again. 100 μL of cell suspensions with density of 1 × 106/mL were incubated with 1 μL of FITC-conjugated anti-CD44 and 5 μL of PE-conjugated anti-CD24 antibodies on ice for 20 min in the dark. The cells were resuspended with
3.1. Overexpression of efflux ABC transporters and CD44+/CD24−/low phenotype in MCF-7/dox cells Dox-resistant MCF-7/dox cells were constructed by long-term continuous exposure of its parental Dox-sensitive MCF-7 cells to Dox. As expected, the expression of MDR-related efflux ATP-binding cassette 372
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Fig. 2. Effects of Tan IIA on Dox cytotoxicity. A. Cytotoxicity of Tan IIA against MCF-7 and MCF-7/dox cells was assessed by MTS assay. Cells were treated with Tan IIA at different concentrations (ranging from 0 to 5 mg/L) for 24 h. B. Effects of different concentrations of Tan IIA on the killing effect of Dox on MCF-7 and MCF-7/dox cells. Cells were treated with 2 μg/mL Dox or 2 μg/mL Dox combined with different concentration of Tan IIA (ranging from 0 to 5 mg/L) for 24 h. C. Effects of nontoxic dose of Tan IIA on the anti-tumor activity of Dox on MCF-7 and MCF-7/dox cells. Cells were treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA for 24 h.
3.5. Tan IIA promoted intracellular accumulation of dox
(ABC) transporter proteins including P-gp, BCRP and MRP1 in MCF-7/ dox cells was much higher than that of in MCF-7 cells (Fig. 1A), meanwhile, the expression of BCSC surface marker CD44+/CD24−/ low was also the case (Fig. 1B).
In order to investigate how Tan IIA enhanced the anti-tumor activity of Dox, we analysed the intracellular Dox accumulation of MCF-7 and MCF-7/dox cells. The results showed that nontoxic dose of Tan IIA could promote intracellular Dox accumulation of MCF-7 and MCF-7/ dox cells, especially that of in MCF-7/dox cells (Fig. 5).
3.2. Tan IIA enhanced the cytotoxicity of dox Our results showed that Tan IIA could enhance the killing effect of Dox on MCF-7 and MCF-7/dox cells in a dose-dependent manner, especially that of on MCF-7/dox cells (Fig. 2B). Even nontoxic dose of Tan IIA could also enhance the killing effect of Dox on MCF-7 and MCF7/dox cells. Nontoxic dose of Tan IIA enhanced the cytotoxicity of Dox as 1.40-fold in MCF-7 cells and 1.78-fold in MCF-7/dox cells (Fig. 2C).
3.6. Combined treatment of Dox and Tan IIA down-regulated the expression of P-gp, BCRP and MRP1 In order to investigate whether ABC transporter proteins are involved in the effect of Tan IIA on intracellular Dox accumulation, we detected the expression of P-gp, BCRP and MRP1 in MCF-7 and MCF-7/ dox cells. Compared to treatment with Dox alone, combined treatment of Dox and nontoxic dose of Tan IIA could suppress the expression of Pgp, BCRP and MRP1 in MCF-7 and MCF-7/dox cells, especially that of in MCF-7/dox cells (Fig. 6).
3.3. Tan IIA increased cell apoptosis induced by dox In order to further study the effect of Tan IIA on the anti-tumor activity of Dox, we analysed the change in the apoptosis of MCF-7 and MCF-7/dox cells using flow cytometry. The results showed that nontoxic dose of Tan IIA could synergize with Dox in inducing cell apoptosis. Compared to treatment with Dox alone, combined treatment of Dox and Tan IIA could increase early apoptosis, late apoptosis and even necrosis of MCF- 7 and MCF-7/dox cells (Fig. 3).
4. Discussion Surgery is an important therapeutic method for breast cancer. Combined treatment of it with chemotherapy can lead to a long-term survival of patients. However, some patients still die of breast cancer relapse and metastasis. It is generally believed that conventional chemotherapy is incapable of killing all the tumor cells. A small group of highly tumorigenic ability cells may undergo adaptive changes after therapy, leading to drug resistance [18]. These cells with D44+/ CD24−/low makers, referred as BCSCs [19], are generally thought to be the source of breast cancer relapse and metastasis [20]. Hence, BCSCs are considered to be critical therapeutic targets, and elimination of BCSCs may improve the curative effect of chemotherapy [17]. Among many mechanisms mediating breast cancer resistance, one of the most studied is that of the overexpression of efflux ATP-binding cassette (ABC) transporter proteins mediated drug resistance [21]. Depending
3.4. Combined treatment of Dox and Tan IIA eliminates BCSCs more effectively BCSCs are considered to be critical therapeutic targets, and elimination of BCSCs may improve the outcomes of cancer chemotherapy [17]. In order to further study the effect of Tan IIA on the anti-tumor activity of Dox, we also analysed the change in the quantity of BCSCs through detection BCSC marker CD44+/CD24−/low using flow cytometry. Compared to treatment with Dox alone, combined treatment of Dox and nontoxic dose of Tan IIA could enhance the elimination effect of Dox on BCSCs in MCF- 7 and MCF-7/dox cells (Fig. 4). 373
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Fig. 3. Effects of Tan IIA on Dox inducing apoptosis. The apoptosis of MCF-7 and MCF-7/dox cells treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA were analysed by flow cytometry. A and B were respectively the typical flow cytometry test pictures of MCF- 7 and MCF-7/dox cells. C and D were respectively the statistical results of apoptosis in MCF- 7 and MCF-7/dox cells. *P < 0.05: significantly different from control group. #P < 0.05: significantly different from Dox alone.
Fig. 4. Effects of Tan IIA on the expression of BCSC surface marker. MCF-7 and MCF-7/dox cells were treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA for 24 h. Flow cytometry analysed BCSC surface marker CD44+/CD24−/low in MCF-7 cells (A) and MCF-7/dox cells (B). *P < 0.05: significantly different from Dox alone.
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Fig. 5. Effects of Tan IIA on intracellular Dox concentration. Dox concentration in MCF- 7 and MCF-7/dox cells treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA were analysed by flow cytometry. A and B were respectively the typical flow cytometry test pictures of MCF- 7 and MCF-7/dox cells. C and D were respectively the statistical results of Dox accumulation in MCF- 7 and MCF-7/dox cells. *P < 0.05: significantly different from Dox alone.
evaluate its effect on the anti-tumor activity of Dox. The results showed that Tan IIA could enhance the anti-tumor effect of Dox on MCF-7 and MCF-7/dox cells in a dose-dependent manner, especially that of on MCF-7/dox cells, even the nontoxic dose of Tan IIA had an obvious synergistic effect on the anti-tumor activity of Dox. Thus, we selected nontoxic dose of Tan IIA in the following experiments for better evaluating its effect on the chemosensitivity of MCF-7 and MCF-7/dox cells to Dox. As expected, Tan IIA could promote intracellular Dox accumulation of MCF-7 and MCF-7/dox cells through down-regulating the expression of efflux ABC transporters including P-gp, BCRP and MRP1, which can effectively eliminate cancerous cells including breast cancer stem cells, thereby increasing the chemosensitivity of breast cancer. Nontoxic dose of Tan IIA could synergize with Dox to eliminate breast cancer cells through suppressing the expression of efflux ABC transporters including P-gp, BCRP and MRP1, not to mention anti-tumor active doses.
on the energy released by the ATP decomposition, these efflux ABC transporters can transport drugs out of cells and limit drugs intake and absorption, thereby reducing the intracellular drug concentrations [22,23]. The tumor initiating and chemotherapy-resistant capabilities of BCSCs mentioned above are attributed to the overexpression of efflux ABC transporters. Therefore, suppressing the expression of efflux ABC transporters may certainly reverse breast cancer resistance, thereby effectively eliminating BCSCs. Among the 48 ABC transporters identified so far, Pgp, BCRP and MRP1 are the three main efflux transporters that have been shown to be associated with MDR of tumors [24]. However, despite their structural similarities, the substrate binding modes and binding sites of the three proteins are not well characterized. There is no common “pharmacophore” that can be identified as an inhibitor of these three ABC transporters [25]. Dox-resistant MCF-7/dox cells were constructed by long-term continuous exposure of its parental Dox-sensitive MCF-7 cells to Dox. As expected, the cells with high expression of BCSC surface marker CD44+/CD24−/low overexpressed MDR-related efflux ABC transporters including P-gp, BCRP and MRP1 [26,27]. As the first-line drug for breast cancer chemotherapy, Dox also has strong cardiotoxicity. Previous studies indicated that Tan IIA had anti-tumor activity [28] in addition to its cardiovascular protective effect [29,30]. Therefore, we chose Tan IIA as the study object to
5. Conclusions In conclusion, Tan IIA could increase the sensitivity of breast cancer cells to Dox through down-regulating the expression of efflux ABC transporters including P-gp, BCRP and MRP1. Since Tan IIA also has 375
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Fig. 6. Effects of Tan IIA on the expression of P-gp, BCRP and MRP1. MCF-7 and MCF-7/dox cells were treated with 2 μg/mL Dox or 2 μg/mL Dox combined with 0.02 mg/L Tan IIA for 24 h. Western blot analysed the expression of P-gp, BCRP and MRP1 in MCF-7 cells (A) and MCF-7/dox cells (B). *P < 0.05: significantly different from Dox alone.
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