Epigenetic processes, which affect DNA accessibility, impact on multifactorial function in normal and transformed cells. [1, 2]. In particular, DNA methylation and histone modifications, through the enzymatic action of histone acetyltransferase (HAT) and histone deacetylase (HDAC) modulate a large number of cellular responses and play a relevant role in tumorigenicity [3].

Several studies have shown that in many tumor types, high HDAC enzyme expressions or complete loss of histone acetylation well correlate with a poorer prognosis [4, 5]. Based on structural homology, HDACs have been grouped into 4 major classes. Class I and II, which include HDACs 1, 2, 3 and 8, are recognized as critical for tumorigenesis. Numerous studies have suggested a correlation between the expression levels of class I HDACs and breast cancer subtypes, aggressiveness and the presence of ER, PR and HER-2. HDACs class II enzymes are involved in breast cancer progression and response to therapeutic treatments [6,7,8,9].

Numerous HDAC inhibitors have been studied and several of them have shown to influence growth, apoptosis and invasiveness in cancer cells both in vitro and in vivo. In breast cancer, the genes that regulate these events are often hypermethylated, such as Cyclin D2, thus causing a perturbation of the cell cycle and tumor progression [10,11,12,13,14,15,16].

Indeed, some HDAC inhibitors, alone or in combination with other chemotherapeutic agents, are used in many clinical trials for both hematologic and solid tumors [17]. Valproic acid (VA), a short-chain fatty acid, which has been used for the past two decades in the treatment of epilepsy and other neuropsychiatric diseases, is considered a class I HDAC inhibitor with strong antitumoral activity [18].

Other studies have shown that VA impairs tumor progression through inhibition of cell proliferation, cell cycle regulation, DNA repair, and apoptosis [19, 20], as well as it can alter differentiation process and angiogenesis in prostatic tumoral cells [21, 22].

VA has also been studied in breast cancer, which represents the second leading cause of death among women after lung cancer [23]. Despite the increase and improvement of preventive screening, breast cancer still remains a threat to both pre- and postmenopausal women, due to the varied histopathological features and hormonal status of tumor that determine a different effectiveness of response to treatments. The HDAC inhibitor VA showed anti tumoral effects according to the dose and cell type used. Regarding breast cancer, VA has been reported to affect hormone receptor and to up-regulate the expression of cyclin dependent kinase inhibitors, such as p21 and p57, with a consequent arrest of cell progression [24].

In accordance with the above findings, depending on the receptor status, breast cancers are treated with surgery and adjuvant chemotherapy, which includes endocrine-based agents, such as SERMS (tamoxifen), SERDs (fulvestran) and aromatase inhibitors (letrazole and anastrazole). However, it is known that over time, breast cancer often develops resistance to drug therapy, that can be traced to several mechanisms, such as mutations in the estrogen receptor, altered expression of the same receptor (epigenetic modifications), changes in the levels of metabolic enzymes involved in hormone synthesis and aberrant activation of signal transduction pathways (up- regulation of PI3K, MAPK, CDKs) [25, 26].

To overcome the mechanisms of resistance, novel agents, that negatively act on target molecules important in cell growth, such as EGF receptor (lapatinib) and CDK4/6 receptor (ribociclib and palbociclib) inhibitors, have been developed and used in the clinic in conjunction with endocrine therapy and in particular, with the aromatase inhibitor letrazol.

VA has emerged as an active compound in the treatment of resistant or metastatic breast cancer associated with chemotherapy or endocrine therapy [27]. Indeed, the association of VA and cisplatin induces apoptosis in breast cancer cells and this improves the efficacy of the response compared to monotherapy alone [28]. Interestingly, synergy between VA and SERMs leads to a better breast cancer prognosis. However, the anticancer activity and the specificity of VA need to be further investigated, given the results not always unique in this context.

Regarding breast cancer cells, it has been reported that VA shows more satisfactory responses in hormonal receptor positive cells than those receptor-negative. This highlights that the action of the molecule is aimed at different target signaling pathways that account for the cell-type specific effect. Considering the use of VA, as promising anticancer agent, we further focused our attention on signaling pathways mainly involved in breast cancer cells viability, using ER-α positive MCF-7 and triple negative (ER-α, PR, HER2) MDA-MB-231 cells.

Methods reagents

Valproic Acid sodium salt was purchased from Sigma-Aldrich (Merck)(Cod. p4543-10G).

Cell culture

Human breast cancer epithelial cell line MCF-7 (estrogen receptor (ERα-positive) and triple-negative human breast cancer cell line MDA-MB-231 (ER-, PR-, HER-2-negative) were cultured in DMEM/F12 containing 10% fetal bovine serum (FBS) (Life Technologies) at 37 °C with 5% CO2 air. Human normal breast epithelial cell line MCF-10A was grown in DMEM-F12 medium containing 5% horse serum (Life Technologies).

Cell viability assay

MCF-7, MDA-MB-231 and MCF-10A cells (5 × 103 cells/mL) were grown in 96well plates and incubated to allow attachment. Then cells were incubated in phenol red free medium (PRF-SFM DMEM/F12) for 24 h and treated with VA at different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5 mM) for 48 and 72 h. At the end of incubation 100 µl of 2 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium) (Sigma-Aldrich, Merck) was added to each well and incubated at 37 °C for 4 h. Subsequently, the medium was removed and 100μl/well DMSO was added to solubilize the formazan. Finally, the optical density of the soluble formazan was read at 570 nm with a plate reader (Multiskan EX, Thermofisher System).

Cell cycle analysis

To determine cell cycle distribution analysis, MCF-7 and MDA-MB-231 cells were cultured in regular medium in 6 well plates and shifted in medium without serum for 24 h. Next, both cells were exposed to treatments with 2 mM of VA for 24 and 48 h. At the end of incubation, the cells were pelleted, once washed with PBS and fixed in 50% methanol overnight at  − 20 °C and stained with a solution containing 50 μg/ml propidium iodide (PI), 20 U/ml RNAse-A and 0.1% Triton (Merck Life Science, Milan, Italy). Cell phases were estimated as a percentage of a total of 10 000 events. The DNA content was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and the data acquired using CellQuest software. Cell cycle profiles were determined using ModFit LT [29].

Annexin V/PI assay

The Annexin V-FITC Kit-Apotosis Detection (Beckman Coulter) was used to perform the annexin V/PI assay. The breast cancer cells (2 × 105/well in 2 ml of medium) were seeded in 6-well plates. Next, the cells were treated with VA for 12 and 24 h. At the end of the treatment the cells were collected with trypsin, centrifuged at 1000/1200 rpm for 5 min, resuspended in PBS and counted. Cells (1 × 106 cells/ml of buffer) were resuspended in 1 × binding buffer provided by the kit. 100 µl (containing 105 cells) were transferred into a tube and incubated with 2 µl of Annexin V and 5 µl of PI for 15 min in the dark at room temperature. At the end of the incubation, 400 µl of Binding Buffer 1 × were added to each tube and the samples were analyzed by FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and the data were acquired using CellQuest software.

Immunoblotting analysis

MCF-7 and MDA-MB-231 cells were grown to 70–80% confluence and treated in PRF-SFM DMEM/F12, with VA for 24 and 48 h. At the end of each treatment, cells were lysed with 500 μl of RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS) with protease inhibitors (1.7 mg/ml aprotinin, 1 mg/ml leupeptin, 200 mmol/l phenylmethylsulfonyl fluoride, 200 mmol/l sodium orthovanadate and 100 mmol/lsodium fluoride; Sigma-Aldrich, Merck).

Equal amounts of proteins were resolved on 8% and 12% SDS/polyacrylamide gel, transferred to a nitrocellulose membrane and probed with primary antibodies against: Cyclin D1, Cyclin B1, p21, p-38, p-ERK (Invitrogen, Thermo Fisher Scientific); Bcl2, Bad, p-Bad, cythocrome C, Survivin, COX2, Catalase, SOD1, ERK2, Actin (Santa Cruz Biotechnology, DBA, Milan, Italy); p-STAT3, STAT3, JNK and p-p38 (Cell Signaling Technology, Euroclone, Milan, Italy); p-JNK, Bax (Bios, Massachusetts, USA); GAPDH (ProteinTech).

The antigen-antibody complex was detected by incubation of the membranes with peroxidase- coupled goat anti-mouse or goat anti-rabbit antibodies and then revealed using the chemiluminescent substrate for Western Blotting, ECL System (Amersham Pharmacia, Buckinghamshire UK) [29].

Evaluation of mitochondrial mass and mitochondrial membrane potential

Mitochondrial mass and membrane potential were measured by FACS analysis of cells stained with MitoTracker® Deep Red (mitochondrial mass evaluation) or MitoTracker® Orange CM-H2TMRos (mitochondrial membrane potential evaluation) (Life Technologies), as previously reported [30].

Briefly, MCF-7 and MDA-MB-231 cells were plated and treated or untreated in PRF-SFM-DMEM/F12 for 48 h with VA. The cells were collected and incubated with MitoTracker staining solution (10 nM final concentration in PBS) for 30–60 min at 37 °C. Cells were then harvested, re-suspended in PBS and analyzed by flow cytometry (CytoFLEX Beckman, Beckman Coulter, Milan, Italy). Data analysis was performed using CytExpert Beckman Coulter software (Beckman Coulter, Milan, Italy).

Reactive oxygen species (ROS) assessment

ROS were quantified using the chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Thermo Fisher Scientific, Waltham, MA, USA), as the manifacturer’s recommendations. Briefly, MCF-7 and MDA-MB-231 cells were plated and treated or untreated in PRF- SFM-DMEM/F12 for 24 and 48 h with 2 mM of VA. Then, the treated cells were rinsed with PBS, harvested, resuspended in 5 μM CM-H2DCFDA, a fluorescent probe used as an indicator for ROS, in PBS and incubated at 37 °C, for 30–40 min. Subsequently, the stained cells were harvested by centrifugation and maintained in a fresh medium and incubated at 37 °C for 20 min. The cells were analyzed by flow cytometry (CytoFLEX Beckman, Beckman Coulter, Milan, Italy). Data analysis was performed using CytExpert Beckman Coulter software (Beckman Coulter, Milan, Italy).

Statistical analysis

Data obtained from multiple independent experiments are expressed as the mean ± standard deviation (SD). Data were analyzed for statistical significance using the Bonferroni post-test. Student’s t test for unpaired data (2-tailed) was used to test the probability of significant differences between two groups of samples. Differences were considered significant when p ≤ 0.05 and p ≤ 0.005. Statistical tests were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).