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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 725-730

Investigation of simvastatin-induced apoptosis and cell cycle arrest in cancer stem cells of MCF-7


1 Department of Toxicology and Pharmacology, Faculty of Pharmacy and Virtual School, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Biology, College of Science, University of Tehran, Tehran, Iran
3 Department of Toxicology and Pharmacology, Faculty of Pharmacy and Poisoning Research Center, Tehran University of Medical Sciences, Tehran, Iran

Date of Web Publication25-Jul-2016

Correspondence Address:
Seyed Nasser Ostad
Department of Toxicology and Pharmacology, Faculty of Pharmacy and Poisoning Research Center, Tehran University of Medical Sciences, 14155/6451, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.146127

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 > Abstract 


Context: Recent studies have shown the association between statins use and cancer risk reduction. Furthermore the importance of cancer stem cells (CSCs) in tumor initiation, progression and migration has been firmly established in a variety of solid tumors. Hence, the effective targeting of breast CSCs has a potential to improve cancer treatment outcome significantly.
Aims: This study has been designed to investigation the anticancer effects of simvastatin on breast CSCs.
Settings and Design: In this study, MCF-7 CSCs were isolated from parent cells and cytotoxic effects of simvastatin were evaluated and compared in both cells.
Subjects and Methods: Stem cell isolation was done by flow cytometry technique and the effects of simvastatin on the stem cell viability, apoptosis and cell cycle were evaluated and compared with parent cells.
Statistical Analysis Used: The results were analyzed using one.way ANOVA, followed by Tukey.Kramer posttest. The P < 0.05 was considered as significant.
Results: Based on the result, simvastatin shows dose-dependent cytotoxic effects on both CSCs and parent MCF-7 cells, whereas the apoptosis induction and the elimination of nonapoptotic programmed death were increased in CSC compared with parent cells. In addition, simvastatin showed the reduction in DNA synthesis and induced cell cycle arrest in the G1 phase in MCF-7 CSCs.
Conclusions: This finding indicates that simvastatin with specific apoptotic effect on MCF-7 CSC may provide supporting reasons for future in vivo and in vitro statin trials.

Keywords: Apoptosis, breast cancer stem cell, flow cytometry, statin


How to cite this article:
Afzali M, Vatankhah M, Ostad SN. Investigation of simvastatin-induced apoptosis and cell cycle arrest in cancer stem cells of MCF-7. J Can Res Ther 2016;12:725-30

How to cite this URL:
Afzali M, Vatankhah M, Ostad SN. Investigation of simvastatin-induced apoptosis and cell cycle arrest in cancer stem cells of MCF-7. J Can Res Ther [serial online] 2016 [cited 2019 Sep 21];12:725-30. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/725/146127




 > Introduction Top


There is increasing evidence supporting the cancer stem cell (CSC) hypothesis. CSCs are a small population of stem-like cells that the abilities of tumor proliferation and propagation are attributed to them. Some features characterize this population, including ability of self-renewal, metastasis, proliferation, differentiation and drug resistance. These features might be the cause of tumor recurrence after the successful primary tumor-therapy.[1] Dr. Micheal Clarke succeeded in detecting CSCs in breast tumors in 2003.[2] Recent large studies have corroborated CSC hypothesis by isolating a subpopulation of CSCs in several solid tumors, including breast, brain, colon, pancreas, prostate, lung and head, and neck tumors.[1],[3],[4],[5],[6],[7],[8],[9]

In the case of breast cancer, as one of the most commonly diagnosed cancer among women, the evidences have proved the ability of CSCs to invade and proliferate at the metastatic site and resistance to chemotherapy and radiotherapy both in vitro and in vivo.[6],[10],[11],[12] Cell surface markers defined such as CD44+, CD24−/low, epithelial specific antigen + and lineage (lack of expression of CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b) have been used to identify these populations in breast cancer cell lines.[10]

Recent studies showed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of the mevalonate pathway, is a potential anticancer therapeutic target.[13] Based on these studies, statins (HMG-CoA reductase inhibitors) may induce programmed cell death or apoptosis in cancerous cells.[14],[15],[16],[17],[18] Statins have been approved and widely used for the treatment of lipid disorders and safety and availability of them have warranted. Furthermore several clinical trials have shown the effect of these agents in cancer prevention in patients who take statins as hypercholesterolemia therapy regularly.[14] Simvastatin is one of the most pharmacologically potent inhibitors of HMG-CoA reductase that showed apoptotic effect in MCF-7 human breast cancer cell line based on previous report.[19] However, the antitumor effect of simvastatin is incompletely characterized.

In this study, we first isolated CSCs population of MCF-7 cells and then tested the cytotoxic effect of the simvastatin on both parent and CSCs cells to characterize the anticancer mechanisms involved in apoptosis in CSCs, the subpopulation that are of particular importance for new anticancer treatments. Further comprehensive studies are needed to identify new effective drugs to prevent them.


 > Subjects and Methods Top


The human breast cancer MCF-7 cell line was obtained from Pasteur Institute Cell Bank of IRAN (Tehran, Iran). Cells were maintained in 89% RPMI-1640 (Biosera, UK) culture medium supplemented with 10% fetal bovine serum (Biosera, UK) and 1% Penicillin/streptomycin (Biosera, UK) and incubated at 37°C in humidified 5% CO2 atmosphere.

Pure simvastatin was kindly provided by Sobhan Daru Pharmaceutical Co., (Rasht, Iran), MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), dimethyl sulfoxide (DMSO) were purchased from Sigma (USA) and Merck (Germany) respectively. Annexin V-FITC and propidium iodide (PI) were purchased from eBioscience (USA). Antibodies (anti-CD44-conjugated microbeads, CD24-conjugated biotin and antibiotin-CD24), Miltenyi Running Buffer, LD column and MACS separator unit were obtained from Miltenyi Biotec (USA).

To fluorescence-activated cell sorting analysis (FACS) of MCF-7 cells, cells were cultured and pelleted by centrifugation, washed two times with PBS and stained with antibodies specific for human cell surface markers: CD44-PE-Cy7 (BD Pharmingen, USA), CD24-PHC (BD Pharmingen, USA) and EpCAM-FITC (BD Pharmingen, USA). 1 × 106 cells were incubated with antibodies for 30 min on ice. Unbounded antibody was washed and cells were analyzed on FACS Calibur Flow Cytomtry (BD, USA).

For CSC isolation 107 cells were suspended in 80 µl of PBS containing 2.5 mM ethylenediaminetetraacetic acid and 0.5% BSA (Miltenyi Running Buffer; MiltenyiBiotec, no. 130.091.221) and incubated with 20 µl MACS anti-CD44-conjugated microbeads (Miltenyi Biotec, USA) for 15 min at 4°C. The cells were resuspended in Miltenyi Buffer (500 µl) and applied to LD positive selection column (MiltenyiBiotec, no. 130.042.901) in the presence of a magnetic field (QuadroMACS Separator, MiltenyiBiotec, no. 130.091.051). The CD44 negative cells passed through the column and the CD44 positive cells remained in the column. The enriched CD44+ cells were collected and cultured to increasing the population of the cells. Then, 107 CD44+ cells were resuspended in 40 µl of Miltenyi Running Buffer and 10 µl of the secondary monoclonal antibody, CD24 conjugated biotin (Miltenyi Biotec, USA) for 15 min at 4°C. The cells washed and resuspended in 80 µl of Miltenyi Running Buffer and 20 µl of antibiotin-CD24 (Miltenyi Biotec, USA) for 15 min at 4°C. The cells were washed in PBS, resuspended in Miltenyi buffer (500 µl) and applied to LD depletion column in the presence of magnetic field of QuadroMACS separator unit. CD24 cells were passed through the column and collected. According to the company manual, there is no need to remove the microbeads from the cells. Hence, collected cells were CD44+ CD24 CSCs. In order to ensure the preservation of cellular properties of CSCs, the maximum splitting number was kept under 5 and the cells characteristics were reexamined at the end of the experiments.

For viability assay, the cells were cultured in a 96-well flat bottom plate approximately 7000 cells per well and were treated with different doses of simvastatin after 24 h incubation. Following 48 h of treatment, 25 µl of 5 mg/ml MTT solution in phosphate-buffered saline was added and incubated for 4 h at 37°C. The medium was removed and 100 μl DMSO was added to each well. The formazan salts were quantified by reading the absorbance at 570 nm with reference standard in 690 nm at micro plate reader.

Apoptosis detection and quantification was done by flow cytometry, double staining with Annexin V-FITC and PI. MCF-7 cells were seeded in 6-well plates (3 × 105 Cells/well) and treated with the concentration less than the IC50 values of simvastatin for 24 h. Cells were collected and resuspended in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) then were incubated with Annexin V-FITC and PI for 10 min at room temperature. 106 cells were measured for fluorescent intensity in FL1 (FITC) and FL2 (PI) for each assay.

Cell cycle was analyzed based on DNA labeling flow cytometry technique. The cells were centrifuged at 200 g for 5 min and the pellet was fixed in cold 70% ethanol on ice for 60 min. The cells were washed with PBS by following centrifugation at 300 g and then were resuspended in 1 ml of PI staining solution with RNase (0.1% (v/v) Triton X-100, 10 μg/mL PI, and 100 μg/mL DNase-free RNase in PBS) and were kept in the dark at 37°C for 10 min. The PI fluorescence of individual nucleic was measured using flow cytometer.


 > Results Top


Fluorescence-activated cell sorter was used to examine the expression profile of CSC markers in MCF-7 breast cancer cell line. MCF-7 derived CSCs were successfully isolated and characterized as epithelial specific antigen +, ABCG2+, CD44+ and CD24−/low using magnet-activated cell sorting method and LD positive selection column in presence of magnetic field. As shown in [Figure 1], the ratio of the cells with CD44+/CD24−/low phenotype increased from 0.96% [Figure 1]a to 28.6% [Figure 1]b.
Figure 1: Cell surface expression of CD44 and CD24 in MCF-7 cell line. Flow cytometry analysis was performed to detect CD44+/CD24 cell population. In part (a) 0.96% (R1) and in part (b) 28.6% of MCF-7 cells exhibit the characteristic CD44+/CD24

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According to the survival curve, simvastatin reduced cell viability of both parent and CSCs in a concentration and time dependent manner. As shown in [Figure 2], the doses of simvastatin higher than 50 µM significantly inhibited proliferation compared with control cells. IC50 values were determined 65.9 ± 12.3 and 69.4 ± 8.84 µM simvastatin, to reduce growth of parent cells and CSCs, respectively. The results show no significant difference between IC50 of parent cells and CSCs.
Figure 2: Effect of simvastatin on MCF-7 and cancer stem cells proliferation. Cells were incubated in medium containing simvastatin (0-100 μM), and cell viability was measured by MTT assay after 48 h. Values are expressed as mean ± standard deviation for three replicates

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To determine whether simvastatin could induce apoptosis in MCF-7 and stem cells, the cells were treated with 50 µM of simvastatin for 24 h, followed by staining with Annexin-V-FITC/PI and examination by flow cytometry. The apoptosis percentages are represented and compared to control group in [Figure 3]. According to the results, simvastatin increases nonspecific death in MCF-7 cells treated after 24 h [Figure 3]a, although a significant early apoptosis induction effect was observed in CSCs received the same treatment (P < 0.01) [Figure 3]b.
Figure 3: Effect of simvastatin on apoptosis. The cells were treated with simvastatin 50 μM for 24 h. Flow cytometry analysis was done using Annexin-V-FITC/PI and the results shown as percentage of cells (P < 0.05*, P < 0.01**). (a) MCF-7 parent cells. (b) MCF-7 cancer stem cells

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Cell cycle analysis by flow cytometry showed an increase in the distribution of MCF-7 cells in G0/G1 (P = 0.0504) beside the significant decrease in G2M phase after exposure to simvastatin 50 µM for 24 h compared to untreated cells. Furthermore a significant increase was observed in the percentage of stem cells in G0/G1 (P < 0.01) beside the decrease in the percentage of the cells in S phase (P = 0.059) compared to control group [Figure 4].
Figure 4: Flow cytometry analysis of simvastatin on cell cycle progression. The cells were treated with simvastatin 50 μM for 24 h and stained with PI and analyzed by flow cytometry. Data are subjected to ModFit analysis and are expressed as mean ± standard deviation for three replicates (P < 0.05*, P < 0.01**). (a) MCF-7 parent cells. (b) MCF-7 cancer stem cells

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 > Discussion Top


3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme acts as a catalyst for the formation of mevalonate from HMG-CoA that is required for the synthesis of sterols and nonsterol isoprenoids. The function of these compounds in cellular processes including membrane formation, hormone biosynthesis, and the activation of growth regulatory proteins and oncoproteins, has been proved.[20] Based on proved evidences, there is a significant increase in the activity of HMG-CoA reductase in malignant cells which due to increased expression and proportion of the active form and the catalytic efficiency of the enzyme.[21] The clinical use of statins, as HMG-CoA reductase inhibitors, results in depletion of mevalonate and prevents the biosynthesis of downstream products inhibiting sterol synthesis, protein isoprenylation, and disruption of N-glycosylation.[20] The apoptotic effect can be reversed by some intermediates of the mevalonate pathway.[17] On the other hand, an elevated mevalonate synthesis has been reported in malignant breast cancer cells because of increased levels and catalytic efficiency of 3-hydroxy-3-methylglutaryl-CoA reductase.[22],[23],[24] Moreover, a number of in vivo and in vitro studies have suggested that the targeting of HMG-CoA reductase leads to a pronounced tumor-specific apoptosis response that may introduce a novel approach in cancer treatment.[25],[26],[27] Beside, CSC hypothesis have been proved strongly and offers new therapeutic targets for cancer treatment.[1],[5],[6],[7],[10] In this study, we used MCF-7 cell line and isolated the CSCs to investigation the cytotoxic effects of simvastatin as a HMG-CoA reductase inhibitor. Some surface CD markers, including CD24, CD44, CD133 and CD166 have been used to CSC identification in vitro. The CSC population of MCF-7 cell line characterized by cell-surface CD44+/CD24−/low markers.[10] We isolated CD44+/CD24−/low cells from MCF-7 cell line with MACS and characterized them by flow cytometry technique that led to an increase in percentage of CSC population from 0.96% to 28.6% [Figure 1]. According to the MTT assay, we found that simvastatin decreases cell viability, in a dose-dependent manner in both cell types [Figure 2]. To determine if apoptosis is the major cell death pathway, the cell lines were examined by using Annexin-V affinity test. Based on our result, the apoptosis was not confirmed as the main mechanism of death in MCF-7 cells after 24 h, although the percentage of late apoptotic and necrotic cells had significantly increased [Figure 3]a. Previous studies reported different apoptotic effect of statins, which suggested that statin-induced apoptosis is dependent on the statin concentration and time to exposure as well as cell type.[26] In our study, Simvastatin was more successful in inducing apoptosis in CSCs. As shown in [Figure 4]b, there is a large increase in early apoptosis phase in simvastatin treated CSCs. These results indicate that breast CSCs are more sensitive to simvastatin compared to the parent cells.

Furthermore, a role of CD44 in breast cancer cell migration and invasion has been established [28],[29] and this is important to know whether CSCs are therapeutically sensitive to common antitumor agents.[30] The anticancer effects of statins as HMG-CoA reductase inhibitor have been established although the direct action of them on such CSCs has not been attempted as yet because of a lack of specific studies. However, statins increased the expression of p53, p21 and p27.[13],[14],[19],[26],[27] and decreased the expression of CD44 protein via a transcriptional mechanism.[31] Furthermore an inverse correlation between expression of p53 and CD44 has been demonstrated in vivo after simvastatin treatment.[31] An increase in the expression of tumor suppressor protein p53 indicates cell cycle arrest in G1.[32],[33],[34] According to our results, a significant increase of the percentage of G0/G1-phase cells and a decrease of the S-phase (P = 0.059), lead to cell cycle arrest in G1 in CSCs. In the case of MCF-7, the increase of cells in G0/G1 was not significant, although the percentage of G2M cells was significantly decreased [Figure 4]. Cell cycle analysis and apoptosis results confirm that the MCF-7 CSCs are more sensitive to simvastatin in comparison to parent cells.


 > Conclusion Top


Simvastatin showed cytotoxic effects in both MCF-7 parent and stem cells, but the patterns of cytotoxicity are a little different. Apoptosis was determined as major cell death pathway in MCF-7 CSCs and it was confirmed by cell cycle analysis and significant cell cycle arrest in G0/G1. The results of this study suggest that simvastatin not only may increase the success rate of anticancer treatment, but also may cause a decrease in cancer recurrence after an efficient CSCs treatment.


 > Acknowledgment Top


This project was supported by Tehran University of Medical Sciences (TUMS), grant No. 409/465/D/10 with thanks.

 
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