Elsevier

European Journal of Pharmacology

Volume 813, 15 October 2017, Pages 161-171
European Journal of Pharmacology

Full length article
Vorinostat and Simvastatin have synergistic effects on triple-negative breast cancer cells via abrogating Rab7 prenylation

https://doi.org/10.1016/j.ejphar.2017.08.022Get rights and content

Abstract

Since the lack of targeted treatment, triple-negative breast cancer (TNBC) has poor outcomes. Histone deacetylase inhibitors (HDACi) blocking the activity of specific HDACs have emerged as cancer therapeutic agents. However, the therapeutic efficiency is still not satisfactory for patients with solid tumor. We thus performed screening for the synergistic agents of Vorinostat (SAHA). The resulting candidate Simvastatin was obtained. The efficacy and mechanism of combination have been studied in TNBC cells. The synergism of SAHA and Simvastatin was evaluated by IC50 of proliferation and combination index (CI). The antitumor activities of combination were further evaluated in TNBC cells. The pro-apoptotic effects were determined by flow cytometry and Western blot. Autophagosome-lysosome fusion was monitored using confocal microscope. The underlying mechanism was further studied by over-expressing of wild-type or inactive (C205S/C207S) Rab7 in compounds treated cells. The in vivo efficacy was also evaluated in mice. The combination of SAHA and Simvastatin had potent synergism in apoptosis of TNBC cells. It exerted pro-apoptosis effect by compromising the fusion between autophagosome and lysosome. Over-expressing of wild-type, but not inactive Rab7 rescued cells from apoptosis induced by the combinatory treatments. Mevalonate supplementation also decreased the combinatory treatment-induced apoptosis. These results indicate that the combinatory treatment enhances the apoptosis of TNBC cells by interrupting Rab7 prenylation and obstructing autophagosome-lysosome fusion. Combination between SAHA and Simvastatin could also significantly decrease the tumor growth in xenografted mice by inducing apoptosis and inhibiting Rab7 prenylation. Rab7 is a potential target for the combined effects of Simvastatin and SAHA.

Introduction

Breast cancer with high mortality is considered as the second leading cause of cancer-related deaths among women in the United States, and 255,180 new diagnoses are expected in 2017 (Giuliano et al., 2017). Several targeted therapies are available for breast cancers over-expressing either hormone receptors or growth-promoting protein HER2 (Giuliano et al., 2017). However, there is about ~ 20% of breast cancers are triple negative (TNBC) (Bianchini et al., 2016, Lehmann et al., 2011). TNBC lacking targeted therapies, have the poorest overall prognosis compared with other breast cancer types (Blows et al., 2010). Thus new approaches are remained to be developed for TNBC treatment. Histone deacetylase inhibitors (HDACi) have attracted more and more attentions in the recent clinical trials for therapy of TNBC (Chiu et al., 2016, Schech et al., 2015, Tate et al., 2012).

Suberoylanilide hydroxamic acid (Vorinostat, SAHA) depresses the de-acetylating activity of all 11 known human class I and class II HDACs (Grant et al., 2007), and shows moderate inhibition on TNBC cells (Palmieri et al., 2009). It not only induces apoptosis of TNBC cells via caspase activation and poly ADP-ribose polymerase (PARP) cleavage, but also exhibits potent cell growth inhibition and cell-cycle arrest in cancer cells (Deming et al., 2014; Schelman el al, 2013; Zibelman et al., 2015).

Autophagy allows the degradation and recycling of cellular materials. Cancer cells up-regulate autophagy under cellular stress to facilitate chemotherapeutic resistance (Mowers et al., 2016, Wei et al., 2014, Dupere-Richer et al., 2013). Autophagosome-lysosome fusion is a critical stage of autophagy mediated by the late endosome-/lysosome-associated small GTPase Rab7 (Wang et al., 2011, Wen et al., 2017). Failure of autophagosome-lysosome fusion causes the accumulation of malfunctioning proteins in cells, impairs the recycling of materials and energy, and thus exerts cytotoxic effects (Gandesiri et al., 2012). Autophagy is becoming an attractive target to enhance the proficiency of existing cancer treating strategy (Dupere-Richer et al., 2013). Unfortunately, SAHA is an inducer of autophagy. It may weaken its antitumor activity (Dupere-Richer et al., 2013), and agents blocking autophagic flux may have synergism with SAHA in cancer treatment (Patel et al., 2016, Torgersen et al., 2013).

The present study identified that Simvastatin, a well-known 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor of mevalonate pathway (Åberg et al., 2008, Gopalan et al., 2013), could enhance the pro-apoptotic effect of SAHA on TNBC via interrupting Rab7 prenylation and autophagosome-lysosome fusion.

Section snippets

Cells and culture conditions

TNBC cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-453 were cultured in RPMI-1640 medium (Wisent, Canada) containing 10% fetal bovine serum (FBS, Gibco, Australia). TNBC cell line Hs578T and non-malignant fibrocystic disease cell line MCF-10A were maintained in DMEM (Wisent, Canada) supplemented with 10% FBS. All cell lines were obtained from the American Type Culture Collection (ATCC). Cells were grown at 37 °C in a humidified incubator with 95% air and 5% CO2.

Chemicals and reagents

Dimethyl sulfoxide (DMSO), SAHA,

The inhibition of TNBC cells by SAHA and Simvastatin

SAHA had moderate inhibitory effect on several TNBC cells. To find out whether other agents could enhance its efficacy, we employed chemicals from LOPAC library as combination partners to study the anti-proliferation activity. Four active compounds were identified to have synergistic effects with SAHA on MDA-MB-231 (Table 1). Among them, it is interesting that Mevastatin is synergistic with SAHA in antitumor effect. Thus we also tested the IC50 values of other two statins, Simvastatin and

Discussion

TNBC is a subset of breast cancer defined by the absence of ER, PR, and HER2, and represents a panel of heterogeneous tumors based on gene-expressing profiling (Lehmann et al., 2011). Despite great efforts were made in adjuvant chemotherapy regimens, the overall survival and prognosis for patients with TNBC is significantly lower than those who with either ER+ or HER2+ breast cancers (Bianchini et al., 2016). New treatment strategies are needed for patients with TNBC emergently. SAHA inhibits

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81272391 and 81572721 to Y.Y.). The authors declare no conflicts of interest.

References (30)

  • D.A. Deming et al.

    A Phase I study of intermittently dosed vorinostat in combination with bortezomib in patients with advanced solid tumors

    Investig. New Drugs

    (2014)
  • D. Dupere-Richer et al.

    Vorinostat-induced autophagy switches from a death-promoting to a cytoprotective signal to drive acquired resistance

    Cell Death Dis.

    (2013)
  • M. Gandesiri et al.

    DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions

    Apoptosis

    (2012)
  • A.E. Giuliano et al.

    Breast Cancer-major changes in the American joint committee on cancer eighth edition cancer staging manual

    CA Cancer J. Clin.

    (2017)
  • S. Grant et al.

    Vorinostat

    Nat. Rev. Drug Discov.

    (2007)
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