Cancer Letters

Cancer Letters

Volume 288, Issue 1, 1 February 2010, Pages 57-67
Cancer Letters

Statins potentiate cytostatic/cytotoxic activity of sorafenib but not sunitinib against tumor cell lines in vitro

https://doi.org/10.1016/j.canlet.2009.06.022Get rights and content

Abstract

The aim of this study was to investigate the potential cytostatic/cytotoxic effects of HMG-CoA reductase inhibitors and two orphan drugs registered for the treatment of advanced renal cell carcinoma, i.e. sorafenib and sunitinib against several different tumor cell lines. Cytostatic/cytotoxic effects were measured using crystal violet or MTT reduction assays. Cell cycle regulation was investigated using flow cytometry and Western blotting. The combination of lovastatin and sorafenib (but not sunitinib) produced synergistic cytostatic/cytotoxic effects against all tested tumor cell lines. In this study, an impairment of the protein prenylation, especially geranylgeranylation, resulted predominantly in the potentiation of the cytostatic/cytotoxic activity of sorafenib, in cell cycle arrest in G1 phase, and, in poor induction of apoptosis. Moreover, due to the fact that it has been well documented that sorafenib compromises the heart function, we studied the interaction of lovastatin and sorafenib using rat cardiomyoblast line H9c2. The combination showed strong synergistic cardiotoxic effects. Statins and tyrosine kinase inhibitors were used at doses that are achievable clinically, which makes the combination promising for future studies, especially in urooncology, bearing in mind possible cardiotoxic effects.

Introduction

3-Hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, i.e. statins, demonstrate potent cholesterol-lowering properties. Their approval for use in humans has been a crucial step in the prevention of atherosclerosis-related diseases, such as heart infarction or stroke. Moreover, these drugs not only inhibit cholesterol synthesis, but also affect production of many other compounds within mevalonic acid pathway, such as ubiquinone, dolichol, farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophospahte (GGPP). The last two compounds participate in protein prenylation, which is a process of posttranslational modification of many proteins, e.g. Ras, Rho or nuclear lamins, which enables their correct localization and participation in signal transduction processes [1]. Therefore, it is not surprising that statins, beyond decreasing the lipid levels, may exhibit important anti-inflammatory and antitumor activities [2]. Dozen of studies have demonstrated that in vitro statins induce antiproliferative and proapoptotic effects [3], [4], [5] and they have the ability to sensitize tumor cell lines of diverse origin to many chemotherapeutics [6], [7], [8], [9], [10], [11], [12]. These observations were also confirmed in the murine model [13]. However, there are also suggestions that statins diminish the effectiveness of anticancer drugs [14]. Interestingly, the debate on whether regular use of statins increases or reduces cancer incidence has not been settled yet [15], [16], [17], [18], [19].

Sorafenib and sunitinib are multitargeted tyrosine kinase inhibitors that exert potent anti-angiogenic and antitumor activities. Sorafenib (BAY 43-9006) is approved for the treatment of advanced renal cell carcinoma (RCC) and unresectable hepatocellular carcinoma (HCC). Initially, it was identified as an effective inhibitor of Raf serine/threonine kinases, especially Raf-1 and B-Raf, but later it was found that it inhibits many receptor tyrosine kinases, such as Flt-3, c-Kit and RET. In addition, sorafenib potently inhibits many proangiogenic growth factor receptor tyrosine kinases: VEGFR-1, VEGFR-2, VEGFR-3 and PDGFR-β [20], [21]. It was also observed that sorafenib induces apoptosis via down-regulation of Mcl-1, which is the anti-apoptotic member of the Bcl-2 family [22]. Because sorafenib exerts potent cytostatic/cytotoxic effects against many different types of human cancer cells, it has been evaluated in a number of phase II trials in thyroid [23], prostate [24], breast [25] or head and neck cancer [26]. Moreover, due to the good tolerability of sorafenib in single-agent trials it has been applied in the combination with different anticancer drugs, such as paclitaxel and carboplatin [27], interferon α [28], monoclonal antibody – bevacizumab [29] or selective EGFR inhibitor – erlotinib [30].

Sunitinib (SU11248) is another multitargeted tyrosine kinase inhibitor, registered for the treatment of advanced renal cell carcinoma, and it was also proved to be effective in imatinib-resistant gastrointestinal stromal tumors (GIST). Similarly to sorfenib, it inhibits the activity of VEGFR1, VEGFR2, VEGFR3, PDGFRβ, c-Kit, Flt-3 and RET. Additionally, sunitinib also impairs signal transduction via PDGFα and CSF1 receptors [31]. It demonstrates potent anti-angiogenic activity, e.g. it may inhibit VEGF-dependent mitogenic response of human umbilical vein endothelial cells and has potent cytostatic/cytotoxic and anti-metastatic effects [32], [33]. Therefore, sunitinib has been tested in many types of cancers both as a single agent [34], [35], [36] and in combinations with other drugs [37], [38], [39]. Recently, it has been shown that inhibition of the MEK/ERK signaling pathway, especially using inhibitor of MEK1/2 kinases, increases both cytostatic and proapoptotic activity of sunitinib [40].

Progression of many tumors is dependent on multiple mutations in genes regulating multiple signaling pathways. Therefore, simultaneous targeted inhibition of many signaling pathways could be more effective than inhibiting a single pathway. Considering the observations that blocking of the Raf/MEK/ERK pathway sensitizes cells to cytotoxic effect of statins [41], we decided therefore, to investigate whether the combination of receptor tyrosine kinases (sorafenib or sunitinib) and statins could produce potentiated effects in tumor cell lines.

Section snippets

Cell lines and cell culture

Human bladder carcinoma (T24), human ovarian carcinoma (MDAH-2774), human lymphoma (Raji), murine renal carcinoma (Renca), murine melanoma (B78), rat bladder carcinoma (AY-27) and rat cardiomyoblast (H9c2) cell lines were purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEM (T24, MDAH-2774, Renca, B78) or in RPMI 1640 (Raji, H9c2) (Invitrogen, Co., Paisley, UK), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 μg/ml streptomycin and 250 ng/ml amphoterycin B

Statins potentiate activity of sorafenib but not sunitinib in vitro via synergistic cytostatic/cytotoxic effects

We have evaluated cytostatic/cytotoxic effects exerted by two tyrosine kinase inhibitors (sorafenib or sunitinib) and statins as well as by their combination against bladder carcinoma (T24, AY-27), ovarian carcinoma (MDAH-2744), renal cell carcinoma (Renca), Burkitt’s lymphoma (Raji) or melanoma (B78) using crystal violet staining, except for Raji cells, in which MTT reduction assay was applied. The effects of the combination against normal rat cardiomyoblasts (H9c2) were evaluated using both

Discussion

Statins are among the most frequently prescribed drugs all over the world. It is estimated that only in the US there are nearly 30 million people taking statins regularly [43], and it should be emphasized that the indications for this group of drugs are still widening. The majority of statin users comprises elderly people who take those drugs because of cardiovascular diseases. Because of their age these patients are also at high risk for developing tumors. Apart from the influence on the

Conflict of interest

None declared.

Acknowledgements

This work was supported in part by the Grant: N405 3028 36 (M.J.) from the Ministry of Science and Higher Education in Poland. J.G. is a recipient of the Mistrz Award from the Foundation for Polish Science. J.B. is the recipient of the Mistrz Stipend from the Foundation for Polish Science.

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