Effect of transfection with PLP2 antisense oligonucleotides on gene expression of cadmium-treated MDA-MB231 breast cancer cells

Emerging evidence indicates that cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in human normal and pathological cells. We have already shown that exposure of MDA-MB231 breast cancer cells to 5 uM CdCl2 for 96 h, apart from affecting significantly mitochondrial metabolism, induces modifications of the expression level of genes coding for members of stress response-, mitochondrial respiration-, MAP kinase-, NF-kB and apoptosis-related pathways. In the present study, we have expanded the knowledge on the biological effects of Cd-breast cancer cell interactions, indicating PLP2 (proteolipid protein-2) as a novel member of the list of Cd-upregulated genes by MDA-MB231 cancer cells and, through the application of transfection techniques with specific antisense oligonucleotides, we have demonstrated that such over-expression may be an upstream event to some of the changes of gene expression levels already observed in Cd-treated cells, thus unveiling new possible molecular relationship between PLP2 and genes linked to the stress- and apoptotic responses

BIOLOGICAL EFFECTS OF JAHA, A NEW HISTONE DEACETYLASE INHIBITOR, ON CANCER CELLS FROM HUMAN BREAST EPITHELIUM

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

Cytotoxicity of the urokinase-plasminogen activator inhibitor carbamimidothioic acid (4-boronophenyl) methyl ester hydrobromide (BC-11) on triple-negative MDA-MB231 breast cancer cells

Abstract: BC-11 is an easily synthesized simple thiouronium-substituted phenylboronic acid, which has been shown to be cytotoxic on triple negative MDA-MB231 breast cancer cells by inducing a perturbation of cell cycle when administered at a concentration equal to its ED50 at 72 h (117 μM). Exposure of cells to BC-11, either pre-absorbed with a soluble preparation of the N-terminal fragment of urokinase-plasminogen activator (uPa), or in co-treatment with two different EGFR inhibitors, indicated that: (i) BC-11 acts via binding to the N-terminus of the enzyme where uPa- and EGF receptor-recognizing sites are present, thereby abrogating the growth-sustaining effect resulting from receptor binding; and (ii) the co-presence of the EGFR inhibitor PD153035 potentiates BC-11’s cytotoxicity. Exposure of cells to a higher concentration of BC-11 corresponding to its ED75 at 72 h (250 μM) caused additional impairment of mitochondrial activity, the production of reactive oxygen species and promotion of apoptosis. Therefore, BC-11 treatment appears to show potential for the development of this class of compounds in the prevention and/or therapy of “aggressive” breast carcinoma

Biological effect of a hybrid anticancer agent based on kinase and histone deacetylase inhibitors on triple-negative (MDA-MB231) breast cancer cells

We examined the effects of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) combined with the vascular endothelial growth factor receptor-1/2 inhibitor (3Z)-5-hydroxy-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol-2-one on MDA-MB-231 breast cancer cells (triple-negative) in the form of both a cocktail of the separate compounds and a chemically synthesized hybrid (N-hydroxy-N'-[(3Z)-2-oxo-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol- 5-yl]octanediamide). Comparative flow cytometric and Western blot analyses were performed on cocktail- and hybrid-treated cells to evaluate cell cycle distribution, autophagy/apoptosis modulation, and mitochondrial metabolic state in order to understand the cellular basis of the cytotoxic effect. Cell cycle analysis showed a perturbation of the rate of progression through the cycle, with aspects of redistribution of cells over different cycle phases for the two treatments. In addition, the results suggest that the two distinct classes of compounds under investigation could induce cell death by different preferential pathways, i.e., autophagy inhibition (the cocktail) or apoptosis promotion (the hybrid), thus confirming the enhanced potential of the hybrid approach vs. the combination approach in finely tuning the biological activities of target cells and also showing the hybrid compound as an additional promising drug-like molecule for the prevention or therapy of “aggressive” breast carcinoma

The histone deacetylase inhibitor JAHA down-regulates pERK and global DNA methylation in MDA-MB231 breast cancer cells

The histone deacetylase inhibitor N1-(ferrocenyl)-N8-hydroxyoctanediamide (JAHA) down-regulates extracellular-signal-regulated kinase (ERK) and its activated form in triple-negative MDA-MB231 breast cancer cells after 18 h and up to 30 h of treatment, and to a lesser extent AKT and phospho-AKT after 30 h and up to 48 h of treatment. Also, DNA methyltransferase 1 (DNMT1), 3b and, to a lesser extent, 3a, downstream ERK targets, were down-regulated already at 18 h with an increase up to 48 h of exposure. Methylation-sensitive restriction arbitrarily-primed (MeSAP) polymerase chain reaction (PCR) analysis confirmed the ability of JAHA to induce genome-wide DNA hypomethylation at 48 h of exposure. Collective data suggest that JAHA, by down-regulating phospho-ERK, impairs DNMT1 and 3b expression and ultimately DNA methylation extent, which may be related to its cytotoxic effect on this cancer cytotype

Natural Anticancer Peptides from Marine Animal Species: Evidence from In Vitro Cell Model Systems

Anticancer peptides are short and structurally heterogeneous aminoacidic chains, which display selective cytotoxicity mostly against tumor cells, but not healthy cells, based on their different cell surface properties. Their anti-tumoral activity is carried out through interference with intracellular homeostasis, such as plasmalemma integrity, cell cycle control, enzymatic activities and mitochondrial functions, ultimately acting as angiogenesis-, drug resistance- and metastasis-inhibiting agents, immune stimulators, differentiation inducers and necrosis or extrinsic/intrinsic apoptosis promoters. The marine environment features an ever-growing level of biodiversity, and seas and oceans are poorly exploited mines in terms of natural products of biomedical interest. Adaptation processes to extreme and competitive environmental conditions led marine species to produce unique metabolites as a chemical strategy to allow inter-individual signalization and ensure survival against predators, infectious agents or UV radiation. These natural metabolites have found broad use in various applications in healthcare management, due to their anticancer, anti-angiogenic, anti-inflammatory and regeneration abilities. The aim of this review is to pick selected studies that report on the isolation of marine animal-derived peptides and the identification of their anticancer activity in in vitro cultures of cancer cells, and list them with respect to the taxonomical hierarchy of the source organism

Synthesis of hybrid anticancer agents based on kinase and histone deacetylase inhibitors

Fragments based on the VEGFR2i Semaxanib (SU5416, (vascular endothelial growth factor receptor-2 inhibitor) and the HDACi (histone deacetylase inhibitor) SAHA (suberanilohydroxamic acid) have been merged to form a range of low molecular weight dual action hybrids. Vindication of this approach is provided by SAR, docking studies, in vitro cancer cell line and biochemical enzyme inhibition data as well as in vivo Xenopus data for the lead molecule (Z)-N1-(3-((1H-pyrrol-2-yl)methylene)-2-oxoindolin-5-yl)- N8-hydroxyoctanediamide 6