Abstract
Histone deacetylase inhibitors (HDACi) are agents capable of inducing growth arrest and apoptosis in different tumour cell types. Previously, we reported a series of novel HDACi obtained by hybridizing SAHA or oxamflatin with 1,4-benzodiazepines. Some of these hybrids proved effective against haematological and solid cancer cells and, above all, compound (S)-8 has emerged for its activities in various biological systems. Here, we describe the effectiveness of (S)-8 against highly metastatic human A375 melanoma cells by using normal PIG1 melanocytes as control. (S)-8 prompted: acetylation of histones H3/H4 and α-tubulin; G0/G1 and G2/M cell cycle arrest by rising p21 and hypophos-phorylated RB levels; apoptosis involving the cleavage of PARP and caspase 9, BAD protein augmentation and cytochrome c release; decrease in cell motility, invasiveness and pro-angiogenic potential as shown by results of wound-healing assay, down-regulation of MMP-2 and VEGF-A/VEGF-R2, besides TIMP-1/TIMP-2 up-regulation; and also intracellular accumulation of melanin and neutral lipids. The pan-caspase inhibitor Z-VAD-fmk, but not the antioxidant N-acetyl-cysteine, contrasted these events. Mechanistically, (S)-8 allows the disruption of cytoplasmic HDAC6-protein phosphatase 1 (PP1) complex in A375 cells thus releasing the active PP1 that dephosphorylates AKT and blocks its downstream pro-survival signalling. This view is consistent with results obtained by: inhibiting PP1 with Calyculin A; using PPP1R2-transfected cells with impaired PP1 activity; monitoring drug-induced HDAC6-PP1 complex re-shuffling; and, abrogating HDAC6 expression with specific siRNA. Altogether, (S)-8 proved very effective against melanoma A375 cells, but not normal melanocytes, and safe to normal mice thus offering attractive clinical prospects for treating this aggressive malignancy.
Highlights
Samples of soft tissues such as liver, kidney and spleen were fixed in 10% (v/v) phosphate-buffered formalin (PBF, pH 7.4) and embedded in paraffin; bones were fixed in PBF and decalcified with acidified ethylenedia-minetetra-acetic acid (EDTA) according to standard procedures before paraffin embedding
The Calyculin A (CA)-mediated effects in A375 cells treated without/with either (S)-8 or trichostatin A (TSA) have been comparatively examined on the same blot and showed that the chemically-induced inhibition of phosphatase 1 (PP1) activity was capable of abrogating pro-apoptotic potential of both hydroxamic Histone deacetylase inhibitors (HDACi) (Fig. 7B)
(S)-8 is safe to normal mice in vivo up to very high dosages as we reported for hydroxamicbased analogue (S)-2 that, instead of undergoing degradation upon ip injection, was capable of reaching the tumor masses on the flanks of immuno-suppressed mice xenografted with prostate cancer cells and contrasting tumor growth [15]
Summary
Histone deacetylases (HDACs) and histone acetyl-transferases (HATs) play an opposite and balanced role in chromatin remodel-. We reported a series of new HDACi characterized by a 1,4-benzodiazepine ring (BDZ) hybridized with either SAHA or oxamflatin [13] to yield compounds capable of inducing H3/H4 histone acetylation in cell-based-assays; and especially one, termed (S)-2, displayed interesting anticancer properties towards various subtypes of cultured and primary acute myeloid leukaemia cells [14] and prostate adenocarcinoma cells [15]. Results reported aim at describing the anti-tumour properties of (S)-8 on A375 metastatic melanoma cells as the primary model (and on other melanoma cell lines and normal immortalized melanocytes) and understanding its fine mechanism of action to provide additional pharmacological support for therapy of this heterogeneous and lifethreatening human cancer.
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