Evaluation of the Cytotoxic and Autophagic Effects of Atorvastatin on MCF-7 Breast Cancer Cells.
MATERIALS AND METHODS
Cell line and cell culture
For MCF-7 experiments, a human breast cancer cell line (DSMZ, Germany) was used. RPMI1640 (Sigma Chemical Co., St. Louis, MO, USA) was used for cell culture experiments. The culture media was maintained with 10% fetal calf serum, 1% penicillin-streptomycin, and 1% L-glutamine. Experiments were conducted at 37 [degrees]C, 100% humidity, and 5% C[O.sup.2] incubator conditions. MCF-7 cells were incubated with atorvastatin (Godecke/Parke-Davis: Freiburg, Germany) at 5, 10, 20, 40, and 80 M concentrations for 24 h and 48 h. The chemical structure of atorvastatin is represented in Figure 1 (Courtesy of Schachter M., published in Fundamental and Clinical Pharmacology, published by John Wiley and Sons).
Cell viability determination
Cell viability and cytotoxic effects caused by atorvastatin were determined by a WST-1 assay. This method depends on the cleavage of the stable tetrazolium salt to soluble formazan. The amount of formazan produced indicates the metabolically active cells. Therefore, quantifying the optical density of formazan can determine the cell viability with regard to both metabolic rate and cellular respiration. Each experiment was replicated three times. The percent viability represents the mean of two measurements of cells with respect to untreated control. MCF-7 cell lines (10x10 (3) cells/well) were seeded into plates, and after sub-confluency of cells, 100 uL of culture medium (control-without statin) and different concentrations of atorvastatin (5, 10, 20, 40, and 80 uM) were added and incubated for 24 h and 48 h. Subsequently, a WST-1 assay was performed as described in our previous work (15). Just as 10, 20, and 80 uM atorvastatin caused a statistically significant decrease in cell viability at 24 h, and 48 h, further evaluation of apoptotic and autophagic effects on MCF-7 cells were determined with these doses.
TUNEL analysis of apoptosis
Terminal deoxynucleotidyl transferase-kit conjugated with horseradish peroxidase (11684817001, ROCHE, Mannheim, Germany) was used for detection of apoptosis. MCF-7 cells seeded on chambered slides (Lab-Tec, Sigma, USA) were incubated with 10, 20, 80 uM concentration of atorvastatin for 24 h and 48 h. After the treatment, TUNEL staining was done as previously described (16). A light microscope (LeicaDM6000B, Germany) coupled with a camera (Leica DC490, Germany) was used for light microscopic evaluation of TUNEL-positive cells. TUNEL-positive cells were counted, and the apoptotic index [ratio of apoptotic (TUNEL-positive) cells to the total number of cells] was determined by the total number of 100 cells at 400X magnification.
Analysis of LC3B, and Beclin-1 immunoreactivity
The immunoreactivity of LC3B and Beclin-1 were detected by immunofluorescence labeling. MCF-7 cells were seeded on coverslips for immunocytochemistry evaluation. The doses of atorvastatin at 10, 20, and 80 uM concentrations were added and incubated for 24 h and 48 h. Then cells were fixed with acetone for 5 min at -20 [degrees]C and after washed with phosphate buffered saline (PBS) and kept under ventilator for 10 min for drying. After treating with 2% bovine serum albumin and 10% human serum for 1 h, the cells were labeled with anti-rabbit LC-3B (Cat no: ab63817, Abcam) and anti-rabbit Beclin-1 (Cat no: ab51031, Abcam) at a final dilution of 1:100 for 1 h in a humidified chamber. After removal of primary antibodies and washing with PBS, secondary antibody [Fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G (Chemicon 132F)] was applied for 30 min. After washing with PBS, they were mounted with fluorescence mounting medium. Photographs were taken using a DC490 camera (Leica, Germany) attached to a microscope (Leica DM6000B, Wetzlar, Germany).
Electron microscopic analysis
For ultrastructural analysis, MCF-7 breast cancer cells were incubated with 10, 20, and 80 uM concentrations of atorvastatin for 24 h and 48 h. Subsequently, both adherent and floating cells were fixed in phosphate buffered 2.5% glutaraldehyde solution, pH 7.4, for 4 h. Then, 1% osmium tetroxide in 0.1 M phosphate buffer solution was used for post-fixation. Cells were washed and placed in 2% agar solution, cooled at room temperature, and sliced into small sections. Then, specimens were dehydrated in an ethanol series and were rinsed in propylene oxide. Afterwards, cells were embedded in Araldite-Epon 812 (Cat no: 13940, EMS, PA, USA) and polymerized at 60 [degrees]C for 48 h. Semi-thin sections of 1 um thickness were obtained with an ultramicrotome (Leica ultracut R, Germany). Methylene blue-azure II was used to stain semi-thin sections. Ultrathin sections with 70 nm thickness were taken using an ultramicrotome and were contrasted with uranyl acetate-lead citrate. An electron microscope (JEOL-JEM 1400, Japan) was used to examine sections, and they were photographed with a chargecoupled device camera (Gatan Inc., CA, USA).
For multiple comparisons of cell viability and TUNEL analysis, we used an analysis of variance followed by Tukey's test and considered p<0.05 as statistically significant.
Atorvastatin induces cell death and decreases proliferation in dose- and time-dependent manner in breast cancer MCF-7 cells
Atorvastatin treatment at 5, 10, 20, 40, and 80 [j,M concentrations caused a decrease in cell viability of MCF-7 cells after 24 h and 48 h (Figure 2). The difference in the cell viability of untreated and treated MCF-7 cells with 40 and 80 [xM of atorvastatin was statistically significant at 24 h (p<0.001, p<0.001) and 5, 10, 20, 40, and 80 [xM of atorvastatin were also statistically significant at 48 h (p<0.05, p<0.05, p<0.001, p<0.001, p<0.001, respectively). Further analysis showed that the cell viability of MCF-7 incubated with 10 and 20 [M atorvastatin was statistically significantly lower at 48 h compared with 24 h (p<0.05, p<0.001, respectively; Figure 2).
Atorvastatin induces apoptosis in MCF-7 breast cancer cells
A TUNEL assay was used for the determination of apoptosis. The number of MCF-7 apoptotic cells increased after treatment with 10, 20, and 80 [xM atorvastatin at 24 h and 48 h (Figure 3). Atorvastatin caused a concentration-dependent increase in the apoptotic index at both 24 h and 48 h. There were statistically significant increases of 2-, 6-, and 7-fold in the apoptotic index with 10, 20, and 80 [xM concentration of atorvastatin at 24 h, respectively, compared with control (p<0.05, p<0.001, p<0.001, respectively). Atorvastatin caused a concentration-dependent increase in apoptotic index in 48 h only at concentrations of 10 and 20 [xM atorvastatin. The number of apoptotic cells decreased with 80 xM concentration of atorvastatin at 48 h as the number of cells in the plate decreased proportionally to increases in the concentration of atorvastatin treatment (Figure 4). The apoptotic index showed a statistically significant increase with 10, 20, and 80 xM atorvastatin at 48 h compared with control (p<0.05, p<0.001, p<0.05, respectively).
Atorvastatin induces autophagy in MCF-7 cells
Autophagy was determined by Beclin-1 and LC3B immunocytochemistry staining. Representative results from immunocytochemistry staining are shown in Figures 5, 6. MCF-7 cells treated with 10, 20, and 80 xM atorvastatin showed immunoreactivity for Beclin-1 and LC3B (Figure 5, 6, respectively). Staining for both LC3B and Beclin-1 was observed as fine granular, punctate forms in intense immunoreactivity. Autophagy was more evident in treatment groups of 10 and 20 xM atorvastatin when compared with that in the control group.
Electron microscopy results
We observed untreated MCF-7 cells as round cells with a prominent nucleus and nucleolus at different phases of mitosis in semi-thin sections (Figure 7a-7e). A few untreated MCF-7 cells showed vacuoles in their cytoplasm (Figure 7e). MCF-7 cells treated with atorvastatin, especially with 10 and 20 xM atorvastatin for 24 h, had an increased number of cytoplasmic vacuoles in their cytoplasm (Figure 7b, 7c). Some MCF-7 cells treated with 10 and 20 xM atorvastatin for 48 h appeared as giant cells indicating mitotic catastrophe (Figure 7f, 7g). Moreover, MCF-7 cells treated with 10, 20, and 80 xM atorvastatin revealed condensed nuclei of apoptotic cells. Apoptosis was more evident in MCF-7 cells treated with 20 xM atorvastatin for 48 h when compared with cells receiving lower concentrations at shorter time intervals (Figure 7g). An increase in both concentrations and time intervals of atorvastatin treatments resulted in a gradual decrease in the number of cells. After treatment with 80 xM atorvastatin for 24 h and 48 h, most cells lost their normal morphology and showed necrosis (Figure 7d, 7h). Electron microscopy enabled us to detect euchromatic nuclei, prominent nucleoli, vesicles, secondary lysosomes, and numerous rough-endoplasmic reticulum in the cytoplasm of untreated MCF-7 cells (Figure 8). Moreover, indentation and enlargement of the nucleus were prominent resulting in an increased nucleus to cytoplasm ratio (Figure 8a). On the other hand, MCF-7 cells exposed to 10, 20, and 80 xM atorvastatin for 24 h and 48 h showed apoptotic and autophagic degenerative changes (Figure 8b-8d, 8f-8h). After 24 h, MCF-7 cells exposed to 10 and 20 xM atorvastatin showed small vesicles and some vacuolization in their cytoplasm (Figure 8b, 8c). Lipid droplets and vesicles were also increased in MCF-7 cells exposed to 10 and 20 xM atorvastatin for 48 h (Figure 8f, 8g). We observed enlarged pleomorphic vacuoles containing moderate electron-dense material in the cytoplasm of these autophagic cells. However, the presence of both an intact nucleus and nucleolus implied undisturbed cellular integrity (Figure 8g). Some MCF-7 cells exposed to 80 xM atorvastatin for 24 h showed apoptotic changes with apoptotic bodies and nuclear material condensation (Figure 8d-left inset). Prominent cell death was detected in MCF-7 cells treated with 80 xM atorvastatin for 48 h (Figure 8h). Most MCF-7 cells exposed to 80 xM atorvastatin for 48 h completely lost the integrity of their cellular membrane and showed extremely degenerated cytoplasmic content (Figure 8h). Additionally, phagocytic vacuoles with membranous residues and widened perinuclear cisterna were observed in a few apoptotic cells (Figure 8h-inset).
In our study, we examined the mechanism behind the anticancer effects of statins on breast cancer. Recently, beyond lipid-lowering effects, statins were suggested as potential cancer therapeutics (6). In vitro studies showed that statins have potent anti-tumor effects in several human cancers including breast cancer (15,17-19). Although preclinical evidence demonstrated tumor-suppressive effects, the clinical reports investigating the association between statin usage and breast cancer have yielded mixed results. Therefore, at present, there is a debate about the preventive effects of statins on breast cancer (20,21). Studies conducted with atorvastatin also show contradictory results. For example, in the study by Ji et al. (22) biomarker assessments were not changed by atorvastatin application. However, more randomized clinical trials are needed to investigate these associations. In this study, we showed that atorvastatin displayed anti-tumor activities on breast cancer MCF-7 cells by inhibiting cell proliferation. According to our results, cell viability decreased 60% in MCF-7 cells in a dose- and time-dependent manner in accordance with previous studies, which reported anti-tumor effects for lipophilic statins (7,23). The lipophilicity of statins is an important factor that determines their cellular effects because only lipophilic statins can penetrate the plasma membrane and affect cellular proliferation. Thus, hydrophilic statins do not induce significant anti-proliferative and anti-tumor effects in breast cancer cells (23). For this reason, in our study, we chose atorvastatin, a commonly prescribed lipophilic statin, to evaluate the cytotoxic effects of statins. Furthermore, our results indicated that atorvastatin promoted apoptosis in MCF-7 cells. In this study, various methods like the TUNEL assay and electron microscopic examination for ultrastructural analysis were used for determining apoptosis. It is well known that statins induce apoptosis in various cells lines such as colon, lung, pancreatic, melanoma, prostate, leukemia, neuroblastoma, and breast cancer (5,13,18,19,24). Anti-proliferative and apoptotic effects of statins in breast tumor cells are triggered by inhibiting the enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This enzyme is the rate-limiting step in mevalonate synthesis. In addition to the effects on cholesterol biosynthesis, statins regulate the synthesis of various other major products such as dolichol, geranyl pyrophosphate, and farnesyl pyrophosphate. These agents play important roles in cellular functions, including both DNA synthesis and cell cycle progression, and inhibition of their synthesis by statins may induce anti-tumor responses (25,26). However, the anticancer effect of statins acting through a mevalonate-independent pathway is also under investigation. Following atorvastatin treatment, mevalonate and pro-apoptotic pathways are up-regulated in gene expression analyses of breast cancer cell lines (27). To address the biological mechanisms underlying the anticancer effect of statins further, the present study examined subsequent processes of autophagy, apoptosis, and necrosis. In these cells, the activation of autophagy may contribute to apoptosis and/or necrosis in a dose- and time-dependent manner similar to the study in which rottlerin was explored in bladder cancer (11). Autophagy is characterized by lysosomal degradation and recycling of cytoplasmic contents. The cellular homeostasis can be maintained by autophagy with degradation of misfolded proteins and organelles (9). Although autophagy is known as a defensive mechanism in response to cellular stress, the proceeding stimulation of autophagy may also cause cell death, by inducing apoptosis or autophagy (12). Our study demonstrated that atorvastatin-induced autophagy in breast cancer MCF-7 cells. Breast cancer cells showed Beclin-1 and LC3B immunoreactivity and widened perinuclear cisterna, induced by stress depending atorvastatin treatment. Increased vacuolization and engulfment of membrane residues and/or cytoplasm by autophagic vesicles may be attributed to autophagy, and these changes were more prominently observed in MCF-7 cells treated with 10 and 20 uM atorvastatin for 48 h. Our results confirm previous studies that showed autophagic effects of atorvastatin on different kinds of cancer cell lines (10-14). Atorvastatin induced autophagic alterations in MCF-7 cells even at the lowest doses used. In addition, a shift from autophagic changes to both apoptosis and necrosis is detected with gradually increasing atorvastatin concentrations. The results revealed that treatment of atorvastatin induced autophagy and subsequent apoptosis. Importantly, findings of our study highlight an additional mechanism for the anti-proliferative effect of statins on breast cancer cells. Many studies showing autophagy in both tumor and normal cells suggest that statins play a role in the regulation of cancer treatments (13,14,25). Statins are reasonably safe and relatively inexpensive drugs. Once the preventive or treatment effects of statins on breast cancer cells are verified, statins would be available for breast cancer patients as a cost-effective, low-toxicity option for adjuvant therapy in the short term. This study shows atorvastatin as a promising treatment option for breast cancer by activating both autophagy and apoptosis and resulting in the death of tumor cells. In conclusion, several preclinical studies described the anti-proliferative and pro-apoptotic effects of statins on breast cancer cells. Statins are widely considered to have therapeutic potential in breast cancer, although further questions should be answered about their clinical significance. Identification of the precise mechanisms by which statins elicit anti-proliferative effects will directly affect the role of statins in the treatment of breast cancer. The present study revealed that atorvastatin treatment has anti-proliferative, proapoptotic, and autophagic effects on breast cancer cells. Statins can lead to autophagy and cell death in breast cancer cells. Further studies should be conducted to understand the relationship between autophagy and apoptosis; however, both the clinical significance and relevance of these findings need to be verified. The inclusion of statins in combination therapies for breast cancer can be a promising new treatment strategy for the future.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: This research was supported partly by a Grant of Hacettepe University Research Foundation (Grant No: 010 D03 101 002).
(1.) World Health Organization. Breast Cancer. http://www.who.int/cancer/prevention/diagnosis-screening/breast-cancer/en/.
(2.) Austreid E, Lonning PE, Eikesdal HP. The emergence of targeted drugs in breast cancer to prevent resistance to endocrine treatment and chemotherapy. Expert Opin Pharmacother 2014;15:681-700.
(3.) Zeybek ND, Gulcelik NE, Kaymaz FF, Sarisozen C, Vural I, Bodur E, et al. Rosuvastatin induces apoptosis in cultured human papillary thyroid cancer cells. J Endocrinol 2011;210:105-15.
(4.) Alegret M, Silvestre JS. Pleiotropic effects of statins and related pharmacological experimental approaches. Methods Find Exp Clin Pharmacol 2006;28:627-56.
(5.) Pisanti S, Picardi P, Ciaglia E, D'Alessandro A, Bifulco M. Novel prospects of statins as therapeutic agents in cancer. Pharmacol Res 2014;88:84-98.
(6.) Chan KK, Oza AM, Siu LL. The statins as anticancer agents. Clin Cancer Res 2003;9:10-9.
(7.) Sanchez CA, Rodriguez E, Varela E, Zapata E, Paez A, Masso FA, et al. Statininduced inhibition of MCF-7 breast cancer cell proliferation is related to cell cycle arrest and apoptotic and necrotic cell death mediated by an enhanced oxidative stress. Cancer Invest 2008;26:698-707.
(8.) Gao Y, Lu XC, Yang HY, Liu XF, Cao J, Fan L. The molecular mechanism of the anticancer effect of atorvastatin: DNA microarray and bioinformatic analyses. Int J Mol Med 2012;30:765-74.
(9.) Gozuacik D, Kimchi A. Autophagy and cell death. Curr Top Dev Biol 2007;78:217-45.
(10.) He Z, Yuan J, Qi P, Zhang L, Wang Z. Atorvastatin induces autophagic cell death in prostate cancer cells in vitro. Mol Med Rep 2015;11:4403-8.
(11.) Qi P, He Z, Zhang L, Fan Y, Wang Z Rottlerin-induced autophagy leads to apoptosis in bladder cancer cells. Oncol Lett 2016;12:4577-83.
(12.) Hippert MM, O'Toole PS, Thorburn A. Autophagy in cancer: good, bad, or both? Cancer Res 2006;66: 9349-51.
(13.) Parikh A, Childress C, Deitrick K, Lin Q, Rukstalis D, Yang W. Statin-induced autophagy by inhibition of geranylgeranyl biosynthesis in prostate cancer PC3 cells. Prostate 2010;70:971-81.
(14.) Yang PM, Liu YL, Lin YC, Shun CT, Wu MS, Chen CC. Inhibition of autophagy enhances anticancer effects of atorvastatin in digestive malignancies. Cancer Res 2010;70:7699-709.
(15.) Kucukkaya S, Gorduysus MO, Zeybek ND, Muftuoglu SF. In Vitro Cytotoxicity of Calcium Silicate-Based Endodontic Cement as Root-End Filling Materials. Scientifica (Cairo) 2016;2016:9203932.
(16.) Zeybek ND, Inan S, Ekerbicer N, Vatansever HS, Karakaya J, Muftuoglu SF. The effects of Gemcitabine and Vinorelbine on inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) distribution of MCF-7 breast cancer cells. Acta Histochem 2011;113:62-7.
(17.) Park YH, Jung HH, Ahn JS, Im YH. Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway. Biochem Biophys Res Commun 2013;439:275-9.
(18.) Kusama T, Mukai M, Iwasaki T, Tatsuta M, Matsumoto Y, Akedo H, et al. 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors reduce human pancreatic cancer cell invasion and metastasis. Gastroenterology 2002;122:308-17.
(19.) Hawk MA, Cesen KT, Siglin JC, Stoner GD, Ruch RJ. Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin. Cancer Lett 1996;109:217-22.
(20.) Boudreau DM, Yu O, Miglioretti DL, Buist DS, Heckbert SR, Daling JR. Statin use and breast cancer risk in a large population-based setting. Cancer Epidemiol Biomarkers Prev 2007;16:416-21.
(21.) Cardwell CR, Hicks BM, Hughes C, Murray LJ. Statin use after diagnosis of breast cancer and survival: a population-based cohort study. Epidemiol 2015;26:68-78.
(22.) Ji Y, Rounds T, Crocker A, Sussman B, Hovey RC, Kingsley F, et al. The Effect of Atorvastatin on Breast Cancer Biomarkers in High-Risk Women. Cancer Prev Res (Phila) 2016;9:379-84.
(23.) Kang S, Kim ES, Moon A. Simvastatin and lovastatin inhibit breast cell invasion induced by H-Ras. Oncol Rep 2009;21:1317-22.
(24.) Campbell MJ, Esserman LJ, Zhou Y, Shoemaker M, Lobo M, Borman E, et al. Breast cancer growth prevention by statins. Cancer Res 2006;66:8707-14.
(25.) Sane KM, Mynderse M, Lalonde DT, Dean IS, Wojtkowiak JW, Fouad F, et al. A novel geranylgeranyl transferase inhibitor in combination with lovastatin inhibits proliferation and induces autophagy in STS-26T MPNST cells. J Pharmacol Exp Ther 2010;333:23-33.
(26.) Denoyelle C, Vasse M, Korner M, Mishal Z, Ganne F, Vannier JP, et al. Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits the signaling pathways involved in the invasiveness and metastatic properties of highly invasive breast cancer cell lines: an in vitro study. Carcinogenesis 2001;22:1139-48.
(27.) Bjarnadottir O, Kimbung S, Johansson I, Veerla S, Jonsson M, Bendahl PO, et al. Global Transcriptional Changes Following Statin Treatment in Breast Cancer. Clin Cancer Res 2015;21:3402-11.
Tugba Alarcon Martinez (1,2), Naciye Dilara Zeybek (1), Sevda Muftuoglu (1)
(1) Department of Histology and Embryology, Hacettepe University School of Medicine, Ankara, Turkey
(2) Department of Pediatrics, Hacettepe University School of Medicine, Ankara, Turkey
Address for Correspondence: Dr. Sevda Muftuoglu, Department of Histology and Embryology, Hacettepe University School of Medicine, Ankara, Turkey
Phone: +90 312 305 14 05
ORCID ID: orcid.org/0000-0003-0664-0204
Received: 6 July 2017 Accepted: 23 February 2018
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|Title Annotation:||Original Article|
|Author:||Martinez, Tugba Alarcon; Zeybek, Naciye Dilara; Muftuoglu, Sevda|
|Publication:||Balkan Medical Journal|
|Date:||Jun 1, 2018|
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