2-hexyl-4-pentynoic acid, a potential therapeutic for breast carcinoma by influencing RPA2 hyperphosphorylation-mediated DNA repair

Highlights

• At low dose (15 μM HPTA) it has the same anti-cancer effects as 500 μM Valproic acid.

• HPTA can sensitize breast tumor cells to HU by augmenting HU-induced DNA DSBs.

• HPTA can inhibit RPA2-p mediated Rad51 dependent HR pathway after HU-induced DNA replication arrest.

Abstract

Breast carcinoma is one of the most common malignancies in women. Previous studies have reported that 500 μM valproic acid can sensitize breast tumor cells to the anti-neoplastic agent hydroxyurea. However, the dose requirements for valproic acid is highly variable due to the wide inter-individuals clinical characteristics. High therapeutic dose of valproic acid required to induce anti-tumor activity in solid tumor was associated with increased adverse effects. There are attempts to locate suitably high-efficient low-toxicity valproic acid derivatives. We demonstrated that lower dose of 2-hexyl-4-pentynoic acid (HPTA; 15 μM) has similar effects as 500 μM VPA in inhibiting breast cancer cell growth and sensitizing the tumor cells to hydroxyurea on MCF7 cells, EUFA423 cells, MCF7 cells with defective RPA2-p gene and primary culture cells derived from tissue-transformed breast tumor cells. We discovered HPTA resulted in more DNA double-strand breaks, the homologous recombination was inhibited through the interference of the hyperphosphorylation of replication protein A2 and recombinase Rad51. Our data postulate that HPTA may be a potential novel sensitizer to hydroxyurea in the treatment of breast carcinoma.

Introduction

Many tumors display overexpression or mutated histone deacetylases [1], hence there has been much interest in the roles histone deacetylase inhibitors (HDACi) may play in targeted tumor therapies. As an epigenetic modulator, HDACi can inhibit DNA repair, alter gene expression and making post-translational modifications to proteins, stop proliferation of transformed tumor cells, stimulate apoptotic cell death and arrest the cell cycle [2]. The US Food and Drug Administration has already approved four HDACi: vorinostat (Zolinza by Merck), romidepsin (Istodax by Gloucester), belinostat (Beleodaq by Spectrum) and panobinostat (Farydak by Novartis) for the treatment of T-cell lymphoma and myeloma. Several other HDACi of natural and synthetic origin are under clinical trials for the evaluation of their safety and efficiency. Despite HDACi having held great promise, HDACi have shown limited success in treating solid tumors, such as breast carcinoma [3]. The use of HDACi as an adjuvant or neoadjuvant to radiotherapy [1,[4], [5], [6]] and chemotherapy [7] is also being explored [3,8].

The low-cost anti-convulsant, valproic acid (VPA; 2-propylvaleric acid, 2-propylpentanoic acid or n-dipropylacetic acid) is a selective class I and II HDACi. Since VPA was used clinically over two decades ago, the pharmacology and adverse effects of this therapeutic agent have been studied in detail. As expected for HDACi, VPA was shown to alter the proliferation, survival, cell migration, and hormone receptor expression of breast tumor cells in both pre-clinical and clinical settings [8]. A clinical window-of-opportunity study on 30 women with breast carcinoma reported a strong correlation between the doses of VPA and its effectiveness; however, such high therapeutic dose required to induce anti-cancer activity in solid cancer was also associated with increased reports of adverse effects [9], thereby limits VPA clinical usefulness. There is a wide inter-individual variability with VPA dosing due to clinical characteristics such as sodium channel polymorphism, body weight, age and drug-to-drug interactions [10]. The recommended therapeutic range of 50 – 100 μg/mL (347 – 693 μM) VPA was based on findings from 54 patients [11].

There are recent attempts to locate a suitable high-efficiency, low-toxicity VPA derivatives. It was reported that some VPA derivatives exceed the HDAC-inhibition potential 40 folds as compared to VPA [12]. Depending on the atom or groups of atoms that are substituted in their core chemical structure, VPA derivatives are classified as unsaturated monocarboxylic acids, organic sulfurs, VPA sulfonamides and valproyl glycine hydrazide schiff bases [12]. 2-hexyl-4-pentynoic acid (HPTA), an unsaturated monocarboxylic VPA derivative, has emerged as a promising novel candidate as it was shown in vitro to be more potent than VPA in inducing histone hyperacetylation [13,14], with lower half-maximal inhibitory concentration (IC50; 11-15μM as compared to 334-448μM for VPA) [15,16].

Hydroxyurea (HU) is a common antimetabolite used in chemotherapy to treat hematologic malignancies, melanoma, refractory ovarian tumor and breast carcinoma; and as a radiation-enhancing agent with concomitant radiotherapy in squamous cell carcinoma of the head and neck [7,[17], [18], [19], [20], [21]]. Although the prevalence study of HU use reported about 6.5% of drug failure and 10.5% of adverse effects, including symptomatic macrocytic anemia, myelodysplasia and painful leg ulcers [22]; long term use of HU, such as in sickle cells anemia, has shown the drug to be safe [23]. The use of HU in the treatment of breast carcinoma had somewhat fallen out of favor in resource-rich countries [24] but it remained a mainstay of chemotherapy for breast carcinoma in resource-constraint countries. It was previously reported that VPA sensitized breast tumor cells to HU by inhibiting RPA2 hyperphosphorylation-mediated DNA repair pathway [25]. We aim to investigate whether HPTA may have a similar HU-sensitizing effect as VPA in breast tumor cells, thereby affording a safe and cheap alternative chemotherapy regimen for breast carcinoma.