The histone deacetylase inhibitors (HDACis) are a class of chemically heterogeneous anticancer agents of which suberoylanilide hydroxamic acid (SAHA) is a prototypical member. SAHA derivatives may be obtained by the three-dimensional manipulation of the SAHA aryl cap, such as the incorporation of a ferrocene unit like that present in Jay Amin hydroxamic acid (JAHA) and homo-JAHA (Spencer et al., 2011). These metal-based SAHA analogues have been tested for their cytotoxic activity toward triple-negative MDA-MB231 breast cancer cells. The results obtained indicate that of the two compounds tested, only JAHA was prominently active on breast cancer cells with an IC50 of 8.45 μM at 72 h of treatment. For this reason JAHA only was used for the subsequent experiment at 8.45 μM concentration. Biological assays showed that exposure of MDA-MB231 cells to the HDACi resulted in cell cycle perturbation with an alteration of S phase entry and a delay at G2/M transition and in an early production of reactive oxygen species followed by mitochondrial membrane potential (MMP) dissipation and autophagy inhibition. No annexin binding was observed after short-(5 h) and longer (24 and 48 h) term incubation with JAHA, thereby excluding the promotion of apoptosis by the HDACi (Librizzi et al., 2012). An in vitro “scratch assay” has also been performed to measure migration of cells treated with JAHA for 24 h, but preliminary indications suggested that JAHA had no effect on the motile behaviour of MDA-MB231 cells. Subsequently, in order to identify protein signatures associated to its cytotoxic activity, we utilized a proteomic approach to reveal protein expression changes after 18, 24 and 48 h of exposure. Protein identification was performed by mass spectrometry, and a total of eleven differentially-expressed proteins were visualized. In parallel, Differential Display (DD) gene expression analysis was used to identify gene signatures in the MDA-MB231 human breast cancer cell line after exposure to JAHA. The result obtained by DD-PCR were confirmed by Real Time PCR analysis. Further study were required to compare the reported signature pattern with that obtained after exposure of MDA-MB231 cells with the parental molecule SAHA, and to understand the biological implications of the expression changes found. A further set of assays was designed to check the effect of JAHA on the intracellular signaling pathways of MDA-MB231 breast cancer cells. Concerning the MEK pathway JAHA repressed MAP kinase (ERK) activation after 18 h and up to 30 h of treatment, and also down-regulated DNA (cytosine-5-)-methyltransferase 1 (DNMT1), a downstream ERK target, already at 18 h with an increase up to 48 h of exposure. To check the occurrence of changes in the extent of global DNA methylation, genomic DNA was submitted to MeSAP (Methylation Sensitive Restriction Arbitrarily-Primed) PCR (Naselli et al., 2014) using Afa and then HpaII enzymes followed by PCR amplification with an arbitrary primer binding preferentially to guanine and cytosine (GC)-rich regions of DNA, including CpG islands. Preliminary indications suggest the ability of JAHA to induce hypomethylation patterns in tumoral breast cancer cells after 30 h of the treatment. Collectively, these data demonstrate that the HDACi JAHA, by inhibiting ERK activity, regulates DNMT1 expression and ultimately DNA methylation. Although caution must be exercised in extrapolation of the vitro results to the in vivo situation for which research on animals and human trials are needed, nevertheless JAHA treatment possesses the potential for its development as an agent for prevention and/or therapy of “aggressive” breast carcinoma, thus prompting us to get more insight into the molecular basis of its anti-breast cancer activity.
Librizzi, M.BIOLOGICAL EFFECTS OF JAHA, A NEW HISTONE DEACETYLASE INHIBITOR, ON CANCER CELLS FROM HUMAN BREAST EPITHELIUM.
|Titolo:||BIOLOGICAL EFFECTS OF JAHA, A NEW HISTONE DEACETYLASE INHIBITOR, ON CANCER CELLS FROM HUMAN BREAST EPITHELIUM|
|Citazione:||Librizzi, M.BIOLOGICAL EFFECTS OF JAHA, A NEW HISTONE DEACETYLASE INHIBITOR, ON CANCER CELLS FROM HUMAN BREAST EPITHELIUM.|
|Appare nelle tipologie:||4.2 Tesi di dottorato|