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Fluvastatin is effective against thymic carcinoma
Life Sciences 240 (2020) 117110
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Fluvastatin is effective against thymic carcinoma a,⁎
b
a
T a
a
Keitaro Hayashi , Yoshimasa Nakazato , Noriaki Morito , Mizuki Sagi , Tomoe Fujita , Naohiko Anzaia,c, Masayuki Chidad a
Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Shimotsuga, Japan Department of Diagnostic Pathology, Dokkyo Medical University School of Medicine, Shimotsuga, Japan Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan d Department of General Thoracic Surgery, Dokkyo Medical University School of Medicine, Shimotsuga, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Thymic carcinoma HMG-CoA reductase Fluvastatin Isoprenylation ERK
Aims: Thymic carcinoma is a rare epithelial tumor, for which, optimal pharmacotherapeutic methods have not yet been established. To develop new drug treatments for thymic carcinoma, we investigated the effects of fluvastatin-mediated pharmacological inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) on thymic carcinoma. Main methods: Thymic carcinoma tissue was surgically excised and HMGCR expression was assessed by immunohistochemistry. Ty82 human thymic carcinoma cells were treated with fluvastatin (1–10 μM) and their growth was monitored. Key findings: HMGCR was expressed on carcinoma cells but not on normal epithelial cells in thymic tissue. Inhibition of HMGCR by fluvastatin suppressed cell proliferation and induced the death of Ty-82 human thymic carcinoma cells. Fluvastatin mediated its antitumor effects by blocking the production of geranylgeranyl-pyrophosphate (GGPP), an isoprenoid that is produced from mevalonate and binds to small GTPases, which promotes cell proliferation. Significance: Fluvastatin showed marked antitumor effects on thymic carcinoma. The results suggest that the statin has clinical benefits in thymic carcinoma management.
1. Introduction
administration of statins has been shown to improve survival in patients with various cancers, including breast, head and neck, colorectal, esophageal and pancreatic cancers [8–15]. Statins have been clinically used for a considerable period time; noticeable side effects such as liver function disorder and muscle weakness have been reported in only a small proportion of patients [16]. Therefore, statins are considered to be the potentially attractive options for cancer management. However, the effects of statins on thymic carcinoma have not been investigated. In this study, we evaluated the inhibitory effects of fluvastatin on thymic carcinoma.
Thymic carcinoma is a rare but an aggressive malignancy [1,2]. Owing to its low incidence rate, drug development efforts for thymic carcinoma treatment have not received much attention, although these patients frequently develop drug resistance. Therefore, improved strategies of drug therapy for thymic carcinoma are required. Statins are widely used for the hypercholesterolemia treatment [3,4]. They reduce blood cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR) [5,6]. HMGCR catalyzes the conversion of HMG-CoA to mevalonate, which is a rate-limiting step for cholesterol biosynthesis. Although statins are used for the treatment of hyperlipidemia because of their cholesterollowering effects, attention is now being paid to them because of their antitumor effects. Previous studies have shown that statins have antitumor effects in various cancer cells [7]. Furthermore, the
2. Material and methods 2.1. Reagents Fluvastatin was purchased from wako (Osaka, Japan). Mevalonate
Abbreviations: DAB, 3–3′ diaminobenzidine; GGPP, geranylgeranyl-pyrophosphate; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; PI, propidium iodide ⁎ Corresponding author at: Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Shimotsuga, Tochigi 321-0293, Japan. E-mail address: [email protected] (K. Hayashi). https://doi.org/10.1016/j.lfs.2019.117110 Received 25 September 2019; Received in revised form 21 November 2019; Accepted 26 November 2019 Available online 28 November 2019 0024-3205/ © 2019 Elsevier Inc. All rights reserved.
Life Sciences 240 (2020) 117110
K. Hayashi, et al.
Fig. 1. Expression of HMGCR in thymic carcinoma. Thymic carcinoma specimens were immunostained with anti-HMGCR antibody and visualized with DAB (yellow signals). Arrows, arrows with dashed line and arrowheads indicate carcinoma cells, normal epithelial cells and lymphoid cells, respectively. Lower panel is an enlarged view of carcinoma specimens. Dashed lines delimit carcinoma intercellular boundaries. Data are representative of five patients. Bars, 100 μm; original magnification, ×200. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4. Apoptosis and cell cycle analysis
was purchased from Sigma (St. Louis, MO, USA). Geranylgeranyl-pyrophosphate was purchased from Cayman Chemical (Ann Arbor, MI, USA). Squalene was purchased from Tokyo Chemical Industry (Tokyo, Japan). U0126 was purchased from Promega (Madison, WI, USA).
To perform apoptosis analysis, the cells were stained with annexin V and propidium iodide (PI) using Mebcyto apoptosis kit (Medical and Biological Laboratory, Nagoya, Japan), according to the manufacturer's instructions. To perform cell cycle analysis, the cells were incubated in 70% ethanol for 30 min at 4 °C, and then stained with PI using Tali cell cycle kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Stained cells were analyzed with FACS (Becton Dickinson, Franklin Lakes, NJ, USA).
2.2. Immunohistochemistry The study using human tissue was approved by Dokkyo Medical University Bioethics Committee. Informed written consent was obtained from each subject. Surgically excised thymus from thymic carcinoma patients was fixed with formalin, embedded with paraffin and sectioned at 4 μm. The sections were deparaffinized and dehydrated. For antigen retrieval, the slides were heated in citrate buffer (pH 6.0) using a microwave oven for 20 min. The endogenous peroxidase activity was blocked with 0.3% H2O2. Anti-HMGCR antibody (NBP191996, Novus Biologicals, Centennial, CO, USA) was applied to the slides at room temperature for 60 min. The slides were then incubated with biotinylated rabbit anti-IgG followed by biotinylated peroxidaseavidin complex using Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA). The samples were visualized with 3–3′ diaminobenzidine (DAB) (Vector Laboratories). The slides were counterstained with hematoxylin.
2.5. Western blot The cells were lysed with Laemmli Sample Buffer (Bio-Rad) containing 5% 2-mercaptoethanol and heated at 95 °C for 5 min. After evaluating protein concentration with XL-Bradford (Apro Science, Tokushima, Japan), the lysate containing 10 μg protein was used for western blotting. Electrophoresis with SDS-PAGE and immunoblotting were performed according to a standard protocol. Anti-HMGCR (ab174830) and Anti-ERK1/2 antibody (EPR17526) were purchased from Abcam (Cambridge, UK). Anti-phospho-ERK1/2 antibody (Thr202/Tyr204, D13.14.4E) was purchased from Cell Signaling Technology (Danvers, MA, USA).
2.3. Cell culture Ty82 human thymic carcinoma cells were purchased from Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan). The cells were cultured in RPMI1640 containing 10% FCS. The cell viability was measured with alamarblue (Bio-Rad, Hercules, CA, USA). The correlation of the cell number with alamarblue fluorescence intensity was confirmed. Trypan blue was used for detection of dead cells.
2.6. Statistics Results are expressed as arithmetic mean ± SEM. Data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. p < 0.05 was considered indicative of statistical significance. 2
Life Sciences 240 (2020) 117110
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3. Results
through by the lowering of cholesterol.
3.1. Predominant expression of HMGCR on carcinoma cells in the thymus
3.4. Fluvastatin prevents ERK phosphorylation
Antitumor effects of statins have been investigated in various types of cancers; however, only few studies have reported the differences in HMGCR expression between cancer and normal tissues. To investigate the role of HMGCR in human thymic carcinoma, we initially performed immunostaining analysis on surgically excised lesions from thymic carcinoma patients. The expression of HMGCR was clearly observed in carcinoma cells in the thymus (Fig. 1). However, its expression in normal epithelial cells was almost undetectable (Fig. 1). These results suggest that HMGCR is predominantly expressed in carcinoma cells in the human thymus.
Ras is a small GTPase and its activation is enhanced by isoprenylation [19,20]. Its signaling induces phosphorylation and activation of ERK [21], which is crucial for the proliferation of many cancer cells [22]. To investigate the influence of fluvastatin on ERK activity in thymic carcinoma cells, we analyzed ERK phosphorylation in Ty82 cells cultured with fluvastatin. ERK phosphorylation was reduced by fluvastatin (Fig. 4A). Furthermore, this reduction was rescued by GGPP but not by squalene (Fig. 4A). These results suggest that inhibition of GGPP production by fluvastatin results in impaired ERK activation. ERK plays an important role in cell cycle progression in many cancers. Reduction of ERK phosphorylation by fluvastatin prompted us to hypothesize that ERK could promote the growth of Ty82 cells. To test this hypothesis, we cultured Ty82 cells with U0126, an inhibitor of MEK that directly phosphorylates ERK [23]. Ty82 cell growth was significantly inhibited by U0126 in a dose-dependent manner (Fig. 4B). The combination of fluvastatin and U0126 showed a stimulatory effect on Ty82 cell growth (Fig. 4C). These results suggest that ERK activation is critical for the growth of Ty82 cells and the inactivation of ERK is, at least partly, the cause of suppression of Ty82 cells growth by fluvastatin.
3.2. Suppressive effects of fluvastatin on human thymic carcinoma cell The expression pattern of HMGCR observed in thymic carcinoma tissues prompted us to investigate the functional significance of HMGCR in thymic cancer cell proliferation. To examine this, we evaluated the effects of fluvastatin on the growth of Ty82 human thymic carcinoma cells, which highly expressed HMGCR (Fig. 2A). The cells were cultured in the presence or absence of fluvastatin and viability was measured. Treatment with 1 μM fluvastatin for 6 days decreased cell viability to 30% as compared to the control group, and 10 μM fluvastatin treatment for 6 days resulted in almost 0% viability and little change in HMGCR levels (Fig. 2B). This result suggests that HMGCR is critical for the continued viability of thymic carcinoma cells. To determine whether fluvastatin affects the cell cycle progression of Ty82 cells, we performed cell cycle analysis. With increase in fluvastatin concentration, the population of S-phase cells was also increased (Fig. 2C). Although G1 and G2/M phase were decreased by 10 μM fluvastatin, no changes of G1 or G2/M phase were observed with 1 μM and 3 μM fluvastatin-treated cells, suggesting that a high fluvastatin concentration is required to produce discernible induction of Sphase arrest in Ty82 cells. We therefore examined the effects of fluvastatin on the death of Ty82 cells. The cells were cultured in the presence of various concentrations of fluvastatin, stained with trypan blue, and the number of positively stained cells was determined. Fluvastatin treatment caused increased cell death in a concentration-dependent manner (Fig. 2D). The number of annexin V+ propidium iodide (PI)− cells was also increased by fluvastatin at any tested concentration (Fig. 2E), indicating that fluvastatin induced cell death via apoptosis. Taken together, these results suggest that fluvastatin prevents the growth of thymic carcinoma cells by either arresting the cell cycle progression or exerting cytotoxic effects, depending on its concentration.
4. Discussion The suppressive effects of statins on cell viability have been recently reported in various types of cancers, but there have been no reports on their effect on thymic carcinoma. Only few studies have investigated the differences of HMGCR expression between cancer and normal tissues. In this study, we demonstrated that HMGCR was predominantly expressed in thymus carcinoma cells; fluvastatin induced cell cycle arrest and cell death in these cells. These findings suggest that HMGCR is an attractive target molecule for the treatment of thymic carcinoma. Remarkably, fluvastatin at 1 μM (433 ng/ml) concentration, which is within its Cmax (490–100 ng/ml) range in human serum when it is administered for the treatment of hyperlipidemia, markedly suppressed thymic carcinoma. This indicates that clinically used concentration of fluvastatin could be effective for the prevention of thymic carcinoma. Some previous studies have also reported antitumor effects of statins in vivo [24–26]. These data suggest that antitumor effect of statins is not limited to in vitro experiments, but has potential clinical benefit for cancer treatment. Although our study suggests statins' potential antitumor activity against thymic carcinoma in vitro, in vivo efficacy validation is required to provide sufficient evidence of statins' clinical value for thymic carcinoma treatment. Future investigation along this direction is warranted. It is noteworthy that HMGCR was predominantly detected in cancer cells but not in the normal cells in the thymus. This could be beneficial, since statins would cause minimal side effects and produce a favorable outcome when used for the treatment of thymic carcinoma. These results warrant further work on evaluating the potential clinical value of statins for the treatment of thymic carcinoma. Although cholesterol is an indispensable component for cell survival, results of the present study showed that GGPP but not squalene restored the viability of statin-treated Ty-82 cells, indicating that impaired isoprenylation is a major cause of anticancer effects. GGPP is produced from mevalonate and used as a substrate for protein isoprenylation. Small GTPases are well-documented targets for isoprenylation with GGPP [19,20]. The covalent binding of GGPP to small GTPases promotes membrane localization to activate their downstream signals such as MAP kinase pathway. In this study, phosphorylation of ERK, a main downstream of Ras, was inhibited by fluvastatin but was reversed by GGPP, indicating that the critical pathway affected by statins in Ty-82 cells was the small GTPase-MAP kinase axis. Although
3.3. Suppression of thymic carcinoma cell by fluvastatin is mediated by isoprenylation inhibition The inhibition of mevalonate synthesis by statins results in lowering of blood cholesterol and a reduction in isoprenoid lipids including geranylgeranyl-pyrophosphate (GGPP) (Fig. 3A) [17,18]. GGPP binds to signal transduction factors such as small GTPase and generally positively modifies their activity [19,20]. Statins could thereby inhibit the growth of thymic carcinoma cells by impairing small GTPase signaling by blocking the production of GGPP and preventing isoprenylation. To test this, we examined whether externally added isoprenoids could restore Ty82 cell growth impaired by the statins. Briefly, cells were cultured with fluvastatin in the presence of GGPP or squalene, a precursor of cholesterol (Fig. 3A). Incubation with GGPP rescued the impaired cell viability induced by fluvastatin, whereas incubation with squalene had no effect (Fig. 3B). These results suggest that the antitumor effect of fluvastatin is principally mediated through by the prevention of GGPP synthesis, which is predominantly enforced by HMGCR, and not 3
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Fig. 2. Suppressive effects of fluvastatin on thymic carcinoma cells. (A) Western blot analysis for HMGCR expression in Ty82 cells. Arrow indicates signal of HMGCR. (B) Ty82 cells were cultured with fluvastatin for the indicated days. Cell viability was determined by alamarBlue assay. Western blotting was performed to determine the HMGCR expression levels in Ty82 cells that had been treated with fluvastatin for 6 days. (C) Representative FACS profile and graph of cell cycle analysis. Ty82 cells were cultured with fluvastatin for 3 days and the cell cycle was analyzed using propidium iodide (PI). The numbers on the top of the histogram indicate the percentages of cells in G1, S and G2/M phase from the left. (D) Ty82 cells were cultured with fluvastatin for 3 days and cell death was measured by trypan blue staining. (E) Representative FACS profile and graph of apoptosis analysis. Ty82 cells were cultured with fluvastatin for 1 day and stained with annexin V and propidium iodide (PI). The numbers in the dot blot indicate the percentages of each subpopulation. FACS data was acquired from the analysis of at least 10,000 cells. Data are mean ± SEM (n = 3) for representative of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control.
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Fig. 3. GGPP rescues impaired cell viability caused by fluvastatin. (A) A schematic of the mevalonate pathway. (B) Ty82 cells were cultured with 3 μM fluvastatin, 0.5 M mevalonate, 1 μM GGPP or 3 μM squalene for 72 h, and cell viability was determined. Data are mean ± SEM (n = 3) for representative of at least 3 independent experiments. ***p < 0.001 vs control.
Fig. 4. Fluvastatin down-regulates ERK activity. (A) Ty82 cells were cultured in the presence of the indicated reagents for 24 h. Western blots were performed to evaluate ERK phosphorylation. (B) Ty82 cells were cultured with U0126 for 6 days and cell viability was determined. (C) Synergic effects of fluvastatin and U0126 on Ty82 cells. The cells were cultured with fluvastatin and/or U0126 for 3 days and cell viability was determined. Data are mean ± SEM (n = 3) for representative of at least 3 independent experiments. **p < 0.01, ***p < 0.001 vs control.
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there have been limited studies on the role of small GTPases in thymic carcinoma, genetic alterations in Kras were detected in some thymic carcinoma patients [27]. These findings suggest that the activation of Ras family protein by isoprenylation and subsequent ERK activation are involved in thymic carcinoma progression. We also demonstrated that the combination of U0126 and fluvastatin further reduced Ty82 cell viability. Therefore, a concomitant use of small GTPase inhibitors and statins could have synergistic effects in thymic carcinoma treatment. However, further in vivo studies will be needed to provide insights into this hypothesis. In conclusion, this study demonstrated the potential therapeutic effects of statins against thymic carcinoma and could provide the basis for the development of a novel potential strategy for thymic carcinoma management.
[9] S.F. Nielsen, B.G. Nordestgaard, S.E. Bojesen, Statin use and reduced cancerrelated mortality, N. Engl. J. Med. 367 (2012) 1792–1802, https://doi.org/10.1056/ NEJMoa1201735. [10] S. Borgquist, A. Giobbie-Hurder, T.P. Ahern, et al., Cholesterol, cholesterol-lowering medication use, and breast cancer outcome in the BIG 1-98 study, J. Clin. Oncol. 35 (2017) 1179–1188, https://doi.org/10.1200/JCO.2016.70.3116. [11] T.P. Ahern, T.L. Lash, P. Damkier, P.M. Christiansen, D.P. Cronin-Fenton, Statins and breast cancer prognosis: evidence and opportunities, Lancet Oncol. 15 (2014) e461–e468, https://doi.org/10.1016/S1470-2045(14)70119-6. [12] A. Gupta, W. Stokes, M. Eguchi, et al., Statin use associated with improved overall and cancer specific survival in patients with head and neck cancer, Oral Oncol. 90 (2019) 54–66, https://doi.org/10.1016/j.oraloncology.2019.01.019. [13] Y. Li, X. He, Y. Ding, H. Chen, L. Sun, Statin uses and mortality in colorectal cancer patients: an updated systematic review and meta-analysis, Cancer Med. 8 (2019) 3305–3313, https://doi.org/10.1002/cam4.2151. [14] O. Lacroix, A. Couttenier, E. Vaes, C.R. Cardwell, H. De Schutter, A. Robert, Statin use after diagnosis is associated with an increased survival in esophageal cancer patients: a Belgian population-based study, Cancer Causes Control 30 (2019) 385–393, https://doi.org/10.1007/s10552-019-01149-3. [15] Y. Zhang, M. Liang, C. Sun, et al., Statin use and risk of pancreatic cancer: an updated meta-analysis of 26 studies, Pancreas 48 (2019) 142–150, https://doi.org/10. 1097/MPA.0000000000001226. [16] D.H. Katz, S.S. Intwala, N.J. Stone, Addressing statin adverse effects in the clinic: the 5 Ms, J. Cardiovasc. Pharmacol. Ther. 19 (2014) 533–542, https://doi.org/10. 1177/1074248414529622. [17] M.S. Brown, J.L. Goldstein, Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth, J. Lipid Res. 21 (1980) 505–517. [18] N. Berndt, A.D. Hamilton, S.M. Sebti, Targeting protein prenylation for cancer therapy, Nat. Rev. Cancer 11 (2011) 775–791, https://doi.org/10.1038/nrc3151. [19] J.F. Hancock, A.I. Magee, J.E. Childs, C.J. Marshall, All ras proteins are polyisoprenylated but only some are palmitoylated, Cell 57 (1989) 1167–1177. [20] P.J. Casey, P.A. Solski, C.J. Der, J.E. Buss, p21ras is modified by a farnesyl isoprenoid, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 8323–8327. [21] R. Roskoski Jr., ERK1/2 MAP kinases: structure, function, and regulation, Pharmacol. Res. 66 (2012) 105–143, https://doi.org/10.1016/j.phrs.2012.04.005. [22] L. Santarpia, S.M. Lippman, A.K. El-Naggar, Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy, Expert Opin. Ther. Targets 16 (2012) 103–119, https://doi.org/10.1517/14728222.2011.645805. [23] R. Roskoski Jr., MEK1/2 dual-specificity protein kinases: structure and regulation, Biochem. Biophys. Res. Commun. 417 (2012) 5–10, https://doi.org/10.1016/j. bbrc.2011.11.145. [24] M.J. Campbell, L.J. Esserman, Y. Zhou, et al., Breast cancer growth prevention by statins, Cancer Res. 66 (2006) 8707–8714. [25] Y. Kobayashi, H. Kashima, R.C. Wu, et al., Mevalonate pathway antagonist suppresses formation of serous tubal intraepithelial carcinoma and ovarian carcinoma in mouse models, Clin. Cancer Res. 21 (2015) 4652–4662, https://doi.org/10. 1158/1078-0432.CCR-14-3368. [26] A. Mohammed, L. Qian, N.B. Janakiram, S. Lightfoot, V.E. Steele, C.V. Rao, Atorvastatin delays progression of pancreatic lesions to carcinoma by regulating PI3/AKT signaling in p48Cre/+ LSL-KrasG12D/+ mice, Int. J. Cancer 131 (2012) 1951–1962, https://doi.org/10.1002/ijc.27456. [27] H. Sasaki, M. Yano, Y. Fujii, Evaluation of Kras gene mutation and copy number in thymic carcinomas and thymomas, J. Thorac. Oncol. 5 (2010) 1715–1716, https:// doi.org/10.1097/JTO.0b013e3181f1cab3.
Acknowledgments The authors thank Noriko Ohshima and Mio Maekawa for their technical assistance. This research was supported by the Japan Society for the Promotion of Science KAKENHI (Grant No. 17K10810). Declaration of competing interest The authors have no conflict of interest. References [1] T.W. Shields, Thymic tumors, in: T.W. Shields, J. LoCicero, IIIC.E. Reed, R.H. Feins (Eds.), General Thoracic Surgery, 7th ed., Lippincott Williams & Wilkins, Philadelphia, PA, 2009, pp. 2323–2362. [2] U. Ahmad, X. Yao, F. Detterbeck, et al., Thymic carcinoma outcomes and prognosis: results of an international analysis, J. Thorac. Cardiovasc. Surg. 149 (2015) 95–100, https://doi.org/10.1016/j.jtcvs.2014.09.124. [3] P.P. Toth, M. Banach, Statins: then and now, Methodist Debakey Cardiovasc. J. 15 (2019) 23–31, https://doi.org/10.14797/mdcj-15-1-23. [4] J.L. Goldstein, M.S. Brown, Regulation of the mevalonate pathway, Nature 343 (1990) 425–430. [5] O. Larsson, HMG-CoA reductase inhibitors: role in normal and malignant cells, Crit. Rev. Oncol. Hematol. 22 (1996) 197–212. [6] E.S. Istvan, J. Deisenhofer, Structural mechanism for statin inhibition of HMG-CoA reductase, Science 292 (2001) 1160–1164. [7] J. Sopková, E. Vidomanová, J. Strnádel, H. Škovierová, E. Halašová, The role of statins as therapeutic agents in cancer, Gen. Physiol. Biophys. 36 (2017) 501–511, https://doi.org/10.4149/gpb_2017045. [8] Z. Mei, M. Liang, L. Li, Y. Zhang, Q. Wang, W. Yang, Effects of statins on cancer mortality and progression: a systematic review and meta-analysis of 95 cohorts including 1,111,407 individuals, Int. J. Cancer 140 (2017) 1068–1081, https://doi. org/10.1002/ijc.30526.
6
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Fluvastatin is effective against thymic carcinoma a,⁎
b
a
T a
a
Keitaro Hayashi , Yoshimasa Nakazato , Noriaki Morito , Mizuki Sagi , Tomoe Fujita , Naohiko Anzaia,c, Masayuki Chidad a
Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Shimotsuga, Japan Department of Diagnostic Pathology, Dokkyo Medical University School of Medicine, Shimotsuga, Japan Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan d Department of General Thoracic Surgery, Dokkyo Medical University School of Medicine, Shimotsuga, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Thymic carcinoma HMG-CoA reductase Fluvastatin Isoprenylation ERK
Aims: Thymic carcinoma is a rare epithelial tumor, for which, optimal pharmacotherapeutic methods have not yet been established. To develop new drug treatments for thymic carcinoma, we investigated the effects of fluvastatin-mediated pharmacological inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) on thymic carcinoma. Main methods: Thymic carcinoma tissue was surgically excised and HMGCR expression was assessed by immunohistochemistry. Ty82 human thymic carcinoma cells were treated with fluvastatin (1–10 μM) and their growth was monitored. Key findings: HMGCR was expressed on carcinoma cells but not on normal epithelial cells in thymic tissue. Inhibition of HMGCR by fluvastatin suppressed cell proliferation and induced the death of Ty-82 human thymic carcinoma cells. Fluvastatin mediated its antitumor effects by blocking the production of geranylgeranyl-pyrophosphate (GGPP), an isoprenoid that is produced from mevalonate and binds to small GTPases, which promotes cell proliferation. Significance: Fluvastatin showed marked antitumor effects on thymic carcinoma. The results suggest that the statin has clinical benefits in thymic carcinoma management.
1. Introduction
administration of statins has been shown to improve survival in patients with various cancers, including breast, head and neck, colorectal, esophageal and pancreatic cancers [8–15]. Statins have been clinically used for a considerable period time; noticeable side effects such as liver function disorder and muscle weakness have been reported in only a small proportion of patients [16]. Therefore, statins are considered to be the potentially attractive options for cancer management. However, the effects of statins on thymic carcinoma have not been investigated. In this study, we evaluated the inhibitory effects of fluvastatin on thymic carcinoma.
Thymic carcinoma is a rare but an aggressive malignancy [1,2]. Owing to its low incidence rate, drug development efforts for thymic carcinoma treatment have not received much attention, although these patients frequently develop drug resistance. Therefore, improved strategies of drug therapy for thymic carcinoma are required. Statins are widely used for the hypercholesterolemia treatment [3,4]. They reduce blood cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR) [5,6]. HMGCR catalyzes the conversion of HMG-CoA to mevalonate, which is a rate-limiting step for cholesterol biosynthesis. Although statins are used for the treatment of hyperlipidemia because of their cholesterollowering effects, attention is now being paid to them because of their antitumor effects. Previous studies have shown that statins have antitumor effects in various cancer cells [7]. Furthermore, the
2. Material and methods 2.1. Reagents Fluvastatin was purchased from wako (Osaka, Japan). Mevalonate
Abbreviations: DAB, 3–3′ diaminobenzidine; GGPP, geranylgeranyl-pyrophosphate; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; PI, propidium iodide ⁎ Corresponding author at: Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Shimotsuga, Tochigi 321-0293, Japan. E-mail address: [email protected] (K. Hayashi). https://doi.org/10.1016/j.lfs.2019.117110 Received 25 September 2019; Received in revised form 21 November 2019; Accepted 26 November 2019 Available online 28 November 2019 0024-3205/ © 2019 Elsevier Inc. All rights reserved.
Life Sciences 240 (2020) 117110
K. Hayashi, et al.
Fig. 1. Expression of HMGCR in thymic carcinoma. Thymic carcinoma specimens were immunostained with anti-HMGCR antibody and visualized with DAB (yellow signals). Arrows, arrows with dashed line and arrowheads indicate carcinoma cells, normal epithelial cells and lymphoid cells, respectively. Lower panel is an enlarged view of carcinoma specimens. Dashed lines delimit carcinoma intercellular boundaries. Data are representative of five patients. Bars, 100 μm; original magnification, ×200. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4. Apoptosis and cell cycle analysis
was purchased from Sigma (St. Louis, MO, USA). Geranylgeranyl-pyrophosphate was purchased from Cayman Chemical (Ann Arbor, MI, USA). Squalene was purchased from Tokyo Chemical Industry (Tokyo, Japan). U0126 was purchased from Promega (Madison, WI, USA).
To perform apoptosis analysis, the cells were stained with annexin V and propidium iodide (PI) using Mebcyto apoptosis kit (Medical and Biological Laboratory, Nagoya, Japan), according to the manufacturer's instructions. To perform cell cycle analysis, the cells were incubated in 70% ethanol for 30 min at 4 °C, and then stained with PI using Tali cell cycle kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Stained cells were analyzed with FACS (Becton Dickinson, Franklin Lakes, NJ, USA).
2.2. Immunohistochemistry The study using human tissue was approved by Dokkyo Medical University Bioethics Committee. Informed written consent was obtained from each subject. Surgically excised thymus from thymic carcinoma patients was fixed with formalin, embedded with paraffin and sectioned at 4 μm. The sections were deparaffinized and dehydrated. For antigen retrieval, the slides were heated in citrate buffer (pH 6.0) using a microwave oven for 20 min. The endogenous peroxidase activity was blocked with 0.3% H2O2. Anti-HMGCR antibody (NBP191996, Novus Biologicals, Centennial, CO, USA) was applied to the slides at room temperature for 60 min. The slides were then incubated with biotinylated rabbit anti-IgG followed by biotinylated peroxidaseavidin complex using Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA). The samples were visualized with 3–3′ diaminobenzidine (DAB) (Vector Laboratories). The slides were counterstained with hematoxylin.
2.5. Western blot The cells were lysed with Laemmli Sample Buffer (Bio-Rad) containing 5% 2-mercaptoethanol and heated at 95 °C for 5 min. After evaluating protein concentration with XL-Bradford (Apro Science, Tokushima, Japan), the lysate containing 10 μg protein was used for western blotting. Electrophoresis with SDS-PAGE and immunoblotting were performed according to a standard protocol. Anti-HMGCR (ab174830) and Anti-ERK1/2 antibody (EPR17526) were purchased from Abcam (Cambridge, UK). Anti-phospho-ERK1/2 antibody (Thr202/Tyr204, D13.14.4E) was purchased from Cell Signaling Technology (Danvers, MA, USA).
2.3. Cell culture Ty82 human thymic carcinoma cells were purchased from Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan). The cells were cultured in RPMI1640 containing 10% FCS. The cell viability was measured with alamarblue (Bio-Rad, Hercules, CA, USA). The correlation of the cell number with alamarblue fluorescence intensity was confirmed. Trypan blue was used for detection of dead cells.
2.6. Statistics Results are expressed as arithmetic mean ± SEM. Data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. p < 0.05 was considered indicative of statistical significance. 2
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3. Results
through by the lowering of cholesterol.
3.1. Predominant expression of HMGCR on carcinoma cells in the thymus
3.4. Fluvastatin prevents ERK phosphorylation
Antitumor effects of statins have been investigated in various types of cancers; however, only few studies have reported the differences in HMGCR expression between cancer and normal tissues. To investigate the role of HMGCR in human thymic carcinoma, we initially performed immunostaining analysis on surgically excised lesions from thymic carcinoma patients. The expression of HMGCR was clearly observed in carcinoma cells in the thymus (Fig. 1). However, its expression in normal epithelial cells was almost undetectable (Fig. 1). These results suggest that HMGCR is predominantly expressed in carcinoma cells in the human thymus.
Ras is a small GTPase and its activation is enhanced by isoprenylation [19,20]. Its signaling induces phosphorylation and activation of ERK [21], which is crucial for the proliferation of many cancer cells [22]. To investigate the influence of fluvastatin on ERK activity in thymic carcinoma cells, we analyzed ERK phosphorylation in Ty82 cells cultured with fluvastatin. ERK phosphorylation was reduced by fluvastatin (Fig. 4A). Furthermore, this reduction was rescued by GGPP but not by squalene (Fig. 4A). These results suggest that inhibition of GGPP production by fluvastatin results in impaired ERK activation. ERK plays an important role in cell cycle progression in many cancers. Reduction of ERK phosphorylation by fluvastatin prompted us to hypothesize that ERK could promote the growth of Ty82 cells. To test this hypothesis, we cultured Ty82 cells with U0126, an inhibitor of MEK that directly phosphorylates ERK [23]. Ty82 cell growth was significantly inhibited by U0126 in a dose-dependent manner (Fig. 4B). The combination of fluvastatin and U0126 showed a stimulatory effect on Ty82 cell growth (Fig. 4C). These results suggest that ERK activation is critical for the growth of Ty82 cells and the inactivation of ERK is, at least partly, the cause of suppression of Ty82 cells growth by fluvastatin.
3.2. Suppressive effects of fluvastatin on human thymic carcinoma cell The expression pattern of HMGCR observed in thymic carcinoma tissues prompted us to investigate the functional significance of HMGCR in thymic cancer cell proliferation. To examine this, we evaluated the effects of fluvastatin on the growth of Ty82 human thymic carcinoma cells, which highly expressed HMGCR (Fig. 2A). The cells were cultured in the presence or absence of fluvastatin and viability was measured. Treatment with 1 μM fluvastatin for 6 days decreased cell viability to 30% as compared to the control group, and 10 μM fluvastatin treatment for 6 days resulted in almost 0% viability and little change in HMGCR levels (Fig. 2B). This result suggests that HMGCR is critical for the continued viability of thymic carcinoma cells. To determine whether fluvastatin affects the cell cycle progression of Ty82 cells, we performed cell cycle analysis. With increase in fluvastatin concentration, the population of S-phase cells was also increased (Fig. 2C). Although G1 and G2/M phase were decreased by 10 μM fluvastatin, no changes of G1 or G2/M phase were observed with 1 μM and 3 μM fluvastatin-treated cells, suggesting that a high fluvastatin concentration is required to produce discernible induction of Sphase arrest in Ty82 cells. We therefore examined the effects of fluvastatin on the death of Ty82 cells. The cells were cultured in the presence of various concentrations of fluvastatin, stained with trypan blue, and the number of positively stained cells was determined. Fluvastatin treatment caused increased cell death in a concentration-dependent manner (Fig. 2D). The number of annexin V+ propidium iodide (PI)− cells was also increased by fluvastatin at any tested concentration (Fig. 2E), indicating that fluvastatin induced cell death via apoptosis. Taken together, these results suggest that fluvastatin prevents the growth of thymic carcinoma cells by either arresting the cell cycle progression or exerting cytotoxic effects, depending on its concentration.
4. Discussion The suppressive effects of statins on cell viability have been recently reported in various types of cancers, but there have been no reports on their effect on thymic carcinoma. Only few studies have investigated the differences of HMGCR expression between cancer and normal tissues. In this study, we demonstrated that HMGCR was predominantly expressed in thymus carcinoma cells; fluvastatin induced cell cycle arrest and cell death in these cells. These findings suggest that HMGCR is an attractive target molecule for the treatment of thymic carcinoma. Remarkably, fluvastatin at 1 μM (433 ng/ml) concentration, which is within its Cmax (490–100 ng/ml) range in human serum when it is administered for the treatment of hyperlipidemia, markedly suppressed thymic carcinoma. This indicates that clinically used concentration of fluvastatin could be effective for the prevention of thymic carcinoma. Some previous studies have also reported antitumor effects of statins in vivo [24–26]. These data suggest that antitumor effect of statins is not limited to in vitro experiments, but has potential clinical benefit for cancer treatment. Although our study suggests statins' potential antitumor activity against thymic carcinoma in vitro, in vivo efficacy validation is required to provide sufficient evidence of statins' clinical value for thymic carcinoma treatment. Future investigation along this direction is warranted. It is noteworthy that HMGCR was predominantly detected in cancer cells but not in the normal cells in the thymus. This could be beneficial, since statins would cause minimal side effects and produce a favorable outcome when used for the treatment of thymic carcinoma. These results warrant further work on evaluating the potential clinical value of statins for the treatment of thymic carcinoma. Although cholesterol is an indispensable component for cell survival, results of the present study showed that GGPP but not squalene restored the viability of statin-treated Ty-82 cells, indicating that impaired isoprenylation is a major cause of anticancer effects. GGPP is produced from mevalonate and used as a substrate for protein isoprenylation. Small GTPases are well-documented targets for isoprenylation with GGPP [19,20]. The covalent binding of GGPP to small GTPases promotes membrane localization to activate their downstream signals such as MAP kinase pathway. In this study, phosphorylation of ERK, a main downstream of Ras, was inhibited by fluvastatin but was reversed by GGPP, indicating that the critical pathway affected by statins in Ty-82 cells was the small GTPase-MAP kinase axis. Although
3.3. Suppression of thymic carcinoma cell by fluvastatin is mediated by isoprenylation inhibition The inhibition of mevalonate synthesis by statins results in lowering of blood cholesterol and a reduction in isoprenoid lipids including geranylgeranyl-pyrophosphate (GGPP) (Fig. 3A) [17,18]. GGPP binds to signal transduction factors such as small GTPase and generally positively modifies their activity [19,20]. Statins could thereby inhibit the growth of thymic carcinoma cells by impairing small GTPase signaling by blocking the production of GGPP and preventing isoprenylation. To test this, we examined whether externally added isoprenoids could restore Ty82 cell growth impaired by the statins. Briefly, cells were cultured with fluvastatin in the presence of GGPP or squalene, a precursor of cholesterol (Fig. 3A). Incubation with GGPP rescued the impaired cell viability induced by fluvastatin, whereas incubation with squalene had no effect (Fig. 3B). These results suggest that the antitumor effect of fluvastatin is principally mediated through by the prevention of GGPP synthesis, which is predominantly enforced by HMGCR, and not 3
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Fig. 2. Suppressive effects of fluvastatin on thymic carcinoma cells. (A) Western blot analysis for HMGCR expression in Ty82 cells. Arrow indicates signal of HMGCR. (B) Ty82 cells were cultured with fluvastatin for the indicated days. Cell viability was determined by alamarBlue assay. Western blotting was performed to determine the HMGCR expression levels in Ty82 cells that had been treated with fluvastatin for 6 days. (C) Representative FACS profile and graph of cell cycle analysis. Ty82 cells were cultured with fluvastatin for 3 days and the cell cycle was analyzed using propidium iodide (PI). The numbers on the top of the histogram indicate the percentages of cells in G1, S and G2/M phase from the left. (D) Ty82 cells were cultured with fluvastatin for 3 days and cell death was measured by trypan blue staining. (E) Representative FACS profile and graph of apoptosis analysis. Ty82 cells were cultured with fluvastatin for 1 day and stained with annexin V and propidium iodide (PI). The numbers in the dot blot indicate the percentages of each subpopulation. FACS data was acquired from the analysis of at least 10,000 cells. Data are mean ± SEM (n = 3) for representative of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control.
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Fig. 3. GGPP rescues impaired cell viability caused by fluvastatin. (A) A schematic of the mevalonate pathway. (B) Ty82 cells were cultured with 3 μM fluvastatin, 0.5 M mevalonate, 1 μM GGPP or 3 μM squalene for 72 h, and cell viability was determined. Data are mean ± SEM (n = 3) for representative of at least 3 independent experiments. ***p < 0.001 vs control.
Fig. 4. Fluvastatin down-regulates ERK activity. (A) Ty82 cells were cultured in the presence of the indicated reagents for 24 h. Western blots were performed to evaluate ERK phosphorylation. (B) Ty82 cells were cultured with U0126 for 6 days and cell viability was determined. (C) Synergic effects of fluvastatin and U0126 on Ty82 cells. The cells were cultured with fluvastatin and/or U0126 for 3 days and cell viability was determined. Data are mean ± SEM (n = 3) for representative of at least 3 independent experiments. **p < 0.01, ***p < 0.001 vs control.
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there have been limited studies on the role of small GTPases in thymic carcinoma, genetic alterations in Kras were detected in some thymic carcinoma patients [27]. These findings suggest that the activation of Ras family protein by isoprenylation and subsequent ERK activation are involved in thymic carcinoma progression. We also demonstrated that the combination of U0126 and fluvastatin further reduced Ty82 cell viability. Therefore, a concomitant use of small GTPase inhibitors and statins could have synergistic effects in thymic carcinoma treatment. However, further in vivo studies will be needed to provide insights into this hypothesis. In conclusion, this study demonstrated the potential therapeutic effects of statins against thymic carcinoma and could provide the basis for the development of a novel potential strategy for thymic carcinoma management.
[9] S.F. Nielsen, B.G. Nordestgaard, S.E. Bojesen, Statin use and reduced cancerrelated mortality, N. Engl. J. Med. 367 (2012) 1792–1802, https://doi.org/10.1056/ NEJMoa1201735. [10] S. Borgquist, A. Giobbie-Hurder, T.P. Ahern, et al., Cholesterol, cholesterol-lowering medication use, and breast cancer outcome in the BIG 1-98 study, J. Clin. Oncol. 35 (2017) 1179–1188, https://doi.org/10.1200/JCO.2016.70.3116. [11] T.P. Ahern, T.L. Lash, P. Damkier, P.M. Christiansen, D.P. Cronin-Fenton, Statins and breast cancer prognosis: evidence and opportunities, Lancet Oncol. 15 (2014) e461–e468, https://doi.org/10.1016/S1470-2045(14)70119-6. [12] A. Gupta, W. Stokes, M. Eguchi, et al., Statin use associated with improved overall and cancer specific survival in patients with head and neck cancer, Oral Oncol. 90 (2019) 54–66, https://doi.org/10.1016/j.oraloncology.2019.01.019. [13] Y. Li, X. He, Y. Ding, H. Chen, L. Sun, Statin uses and mortality in colorectal cancer patients: an updated systematic review and meta-analysis, Cancer Med. 8 (2019) 3305–3313, https://doi.org/10.1002/cam4.2151. [14] O. Lacroix, A. Couttenier, E. Vaes, C.R. Cardwell, H. De Schutter, A. Robert, Statin use after diagnosis is associated with an increased survival in esophageal cancer patients: a Belgian population-based study, Cancer Causes Control 30 (2019) 385–393, https://doi.org/10.1007/s10552-019-01149-3. [15] Y. Zhang, M. Liang, C. Sun, et al., Statin use and risk of pancreatic cancer: an updated meta-analysis of 26 studies, Pancreas 48 (2019) 142–150, https://doi.org/10. 1097/MPA.0000000000001226. [16] D.H. Katz, S.S. Intwala, N.J. Stone, Addressing statin adverse effects in the clinic: the 5 Ms, J. Cardiovasc. Pharmacol. Ther. 19 (2014) 533–542, https://doi.org/10. 1177/1074248414529622. [17] M.S. Brown, J.L. Goldstein, Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth, J. Lipid Res. 21 (1980) 505–517. [18] N. Berndt, A.D. Hamilton, S.M. Sebti, Targeting protein prenylation for cancer therapy, Nat. Rev. Cancer 11 (2011) 775–791, https://doi.org/10.1038/nrc3151. [19] J.F. Hancock, A.I. Magee, J.E. Childs, C.J. Marshall, All ras proteins are polyisoprenylated but only some are palmitoylated, Cell 57 (1989) 1167–1177. [20] P.J. Casey, P.A. Solski, C.J. Der, J.E. Buss, p21ras is modified by a farnesyl isoprenoid, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 8323–8327. [21] R. Roskoski Jr., ERK1/2 MAP kinases: structure, function, and regulation, Pharmacol. Res. 66 (2012) 105–143, https://doi.org/10.1016/j.phrs.2012.04.005. [22] L. Santarpia, S.M. Lippman, A.K. El-Naggar, Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy, Expert Opin. Ther. Targets 16 (2012) 103–119, https://doi.org/10.1517/14728222.2011.645805. [23] R. Roskoski Jr., MEK1/2 dual-specificity protein kinases: structure and regulation, Biochem. Biophys. Res. Commun. 417 (2012) 5–10, https://doi.org/10.1016/j. bbrc.2011.11.145. [24] M.J. Campbell, L.J. Esserman, Y. Zhou, et al., Breast cancer growth prevention by statins, Cancer Res. 66 (2006) 8707–8714. [25] Y. Kobayashi, H. Kashima, R.C. Wu, et al., Mevalonate pathway antagonist suppresses formation of serous tubal intraepithelial carcinoma and ovarian carcinoma in mouse models, Clin. Cancer Res. 21 (2015) 4652–4662, https://doi.org/10. 1158/1078-0432.CCR-14-3368. [26] A. Mohammed, L. Qian, N.B. Janakiram, S. Lightfoot, V.E. Steele, C.V. Rao, Atorvastatin delays progression of pancreatic lesions to carcinoma by regulating PI3/AKT signaling in p48Cre/+ LSL-KrasG12D/+ mice, Int. J. Cancer 131 (2012) 1951–1962, https://doi.org/10.1002/ijc.27456. [27] H. Sasaki, M. Yano, Y. Fujii, Evaluation of Kras gene mutation and copy number in thymic carcinomas and thymomas, J. Thorac. Oncol. 5 (2010) 1715–1716, https:// doi.org/10.1097/JTO.0b013e3181f1cab3.
Acknowledgments The authors thank Noriko Ohshima and Mio Maekawa for their technical assistance. This research was supported by the Japan Society for the Promotion of Science KAKENHI (Grant No. 17K10810). Declaration of competing interest The authors have no conflict of interest. References [1] T.W. Shields, Thymic tumors, in: T.W. Shields, J. LoCicero, IIIC.E. Reed, R.H. Feins (Eds.), General Thoracic Surgery, 7th ed., Lippincott Williams & Wilkins, Philadelphia, PA, 2009, pp. 2323–2362. [2] U. Ahmad, X. Yao, F. Detterbeck, et al., Thymic carcinoma outcomes and prognosis: results of an international analysis, J. Thorac. Cardiovasc. Surg. 149 (2015) 95–100, https://doi.org/10.1016/j.jtcvs.2014.09.124. [3] P.P. Toth, M. Banach, Statins: then and now, Methodist Debakey Cardiovasc. J. 15 (2019) 23–31, https://doi.org/10.14797/mdcj-15-1-23. [4] J.L. Goldstein, M.S. Brown, Regulation of the mevalonate pathway, Nature 343 (1990) 425–430. [5] O. Larsson, HMG-CoA reductase inhibitors: role in normal and malignant cells, Crit. Rev. Oncol. Hematol. 22 (1996) 197–212. [6] E.S. Istvan, J. Deisenhofer, Structural mechanism for statin inhibition of HMG-CoA reductase, Science 292 (2001) 1160–1164. [7] J. Sopková, E. Vidomanová, J. Strnádel, H. Škovierová, E. Halašová, The role of statins as therapeutic agents in cancer, Gen. Physiol. Biophys. 36 (2017) 501–511, https://doi.org/10.4149/gpb_2017045. [8] Z. Mei, M. Liang, L. Li, Y. Zhang, Q. Wang, W. Yang, Effects of statins on cancer mortality and progression: a systematic review and meta-analysis of 95 cohorts including 1,111,407 individuals, Int. J. Cancer 140 (2017) 1068–1081, https://doi. org/10.1002/ijc.30526.
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