Intensified antineoplastic effect by combining an HDAC-inhibitor, an mTOR-inhibitor and low dosed interferon alpha in prostate cancer cells

Abstract

A significant proportion of men diagnosed with prostate cancer (PCa) eventually develop metastatic disease, which progresses to castration resistance, despite initial response to androgen deprivation. As anticancer therapy has become increasingly effective, acquired drug resistance has emerged, limiting efficacy. Combination treatment, utilizing different drug classes, exemplifies a possible strategy to foil resistance development. The effects of the triple application of the histone deacetylase (HDAC) inhibitor valproic acid (VPA), the mammalian target of rapamycin inhibitor everolimus and low dosed interferon alpha (IFNα) on PCa cell growth and dissemination capacity were investigated. For that purpose, the human PCa cell lines, PC-3, DU-145 and LNCaP were treated with the combined regimen or separate single agents. Cell growth was investigated by the MTT dye reduction assay. Flow cytometry served to analyse cell cycle progression. Adhesion to vascular endothelium or immobilized collagen, fibronectin and laminin was quantified. Migration and invasion characteristics were determined by the modified Boyden chamber assay. Integrin α and β subtypes were investigated by flow cytometry, western blotting and RT-PCR. Integrin related signalling, Epidermal Growth Factor Receptor (EGFr), Akt, p70S6kinase and extracellular signal-regulated kinases (ERK)1/2 activation were also assessed. The triple application of VPA, everolimus and low dosed IFNα blocked tumour cell growth and dissemination significantly better than any agent alone. Antitumour effects were associated with pronounced alteration in the cell cycle machinery, intracellular signalling and integrin expression profile. Combining VPA, everolimus and low dosed IFNα might be a promising option to counteract resistance development and improve outcome in PCa patients.

Keywords: combination therapy; everolimus; interferon alpha; prostate cancer; valproic acid.

Figure 1

(A) Cell growth analysis of PC-3, DU-145 and LNCaP cells. Tumour cells were treated with either 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. Cells were counted after 24, 48 and 72 hrs. One representative experiment of six is shown. * indicates significant difference to controls, # indicates significant difference to single drug treatment. (B) Adhesion of prostate cancer cells to HUVEC. PC-3, DU-145 and LNCaP cells were treated with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. Tumour cells were added at a density of 0.5 × 10⁶ cells/well to HUVEC monolayers for 1, 2 or 4 hrs. Non-adherent tumour cells were washed off and the remaining cells fixed and counted in five different fields (5 × 0.25 mm²). Mean values were calculated from five counts. One representative of six experiments is shown. * indicates significant difference to controls, ^# indicates significant difference to single drug treatment.

Figure 2

(A) Cell cycle analysis of PC-3 cells. Tumour cells were treated either with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. Cell cycle analysis was carried out after 24 hrs. The cell population at each checkpoint is expressed as percentage of total analysed cells. One representative experiment of three is shown. (B) Western blot of cell cycle proteins. PC-3 cells were treated either with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. β-actin served as the internal control. The figure shows one representative from three separate experiments.

Figure 3

(A) Adhesion of prostate cancer cells to extracellular matrix proteins. PC-3 cells were treated with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Cells were added to immobilized collagen, laminin or fibronectin at a density of 0.5 × 10⁶ cells/well for 60 min. Plastic dishes were used to evaluate unspecific binding (background control). One representative of six experiments is shown. * indicates significant difference to controls, ^# indicates significant difference to single drug treatment. (B) PC-3 cell migration (left) and invasion (right). Cells treated with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls were set to 100%. * = significant difference to controls. # = significant difference to single drug treatment.

Figure 4

(A) Integrin expression on cell surface. PC-3 cells were treated with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. Mean fluorescence units are given in percentage difference to the controls. One of three independent experiments is shown. * indicates significant difference to controls, # indicates significant difference to single drug treatment. (B) Western blot analysis of integrin protein expression. PC-3 cells were treated either with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. β-actin served as the internal control. The figure shows one representative from three separate experiments. (C) Integrin gene expression. PC-3 cells were treated with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. One representative from three separate experiments is shown. * indicates significant difference to controls, ^# indicates significant difference to single drug treatment (i.e. to VPA, IFNα or everolimus).

Figure 5

Western blot analysis of cell signalling proteins. PC-3, DU-145 or LNCaP cells were treated with 100 U/ml IFNα, 1 nM everolimus or 1 mM VPA, or with all compounds simultaneously (TD). Controls remained untreated. Cells were kept for 2 hrs in serum-free cell culture medium and subsequently stimulated for 30 min. with EGF (100 ng/ml). β-actin served as the internal control. The figure shows one representative from three separate experiments.