VPA inhibitor – Selective PI3K inhibitor 1

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

Wenwen Dinga, David Limb,c, Zhendong Wanga, Zuchao Caia, Guochao Liua, Fengmei Zhanga,Zhihui Fenga,*

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.

Keywords:
HPTA, 2-hexyl-4-pentynoic acid
VPA, valproic acid
HU, hydroxyurea
HDACi, histone deacetylase inhibitor
Breast cancer

1. 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 [1,4–6] and chemotherapy [7] is also
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, lowtoxicity 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 [7,17–21]. 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.

2. Materials and methods

2.1. Cell culture and cell lines

The MCF7 and EUFA423 breast tumor cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle Medium supplemented with 10% fetal bovine serum (Gibco). MCF7 cell lines expressing the HA-RPA2 wild type (wtRPA2) and HA-RPA2-phospho mutant (S4A/S8A/S11A/ S12A/S13A/T21A/S33A) with endogenous RPA2 silenced by shRNA (muRPA2) as described elsewhere [26,27] was a gift from Dr. Junran Zhang, Ohio State University (Columbus, OH, USA).

2.2. Treatment of in vitro breast tumor cells

The MCF7 and EURA423 cell lines were pre-treated with 15 μM HPTA (Trichemicals) or 500 μM VPA (Sigma) for 24 hours, and subsequently with 2 mM HU (Sigma) for another 18 hours, before further experimental analysis.

2.3. Primary culture of chemically induced breast tumor in Sprague-Dawley (SD) rats

This was outlined in previous studies [4]. The studies of animal tissue were conducted in accordance with the terms outlined in the Shandong University Human and Animal Ethics Research Committee approval 20140315, based on international best practices [28]. In brief, breast tumors were induced in 50 days old SD rats with 7,12-dimethylbenz[α]anthracene (DMBA) (Sigma). The morphological structure of the tissue was observed by hematoxylin and eosin staining (see Fig. 6A). The cells were used subsequently used in the immunofluorescence analysis of γH2AX, RPA2 and Rad51.

2.4. Immunoblotting and immunofluorescence analysis

As was outlined in previous studies [5,25,29], the primary antibodies used were: anti-phospho-RPA2 Ser4/Ser8 (Bethyl), anti-RPA2 (NA18,Calbiochem), anti-γH2AX (Millipore), anti-GAPDH (ZSGB-Bio), anti-Rad51 (F-11, sc-398587, Santa Cruz Biotechnology), anti-53BP1 antibody (NB100-304, Novus). The secondary antibodies included goat anti-rabbit IgG horseradish peroxidase conjugated and goat anti-mouse IgG-horseradish peroxidase conjugated IgG (Pierce), goat anti-mouse IgG (AlexaFluor 594) and chicken anti-rabbit IgG (AlexaFluor 488; Molecular Probe).

2.5. Co-immunoprecipitation

The method was described in previous study [29]. In brief, the treated MCF7 cells were harvested and lysed in buffer. The protein lysates were then incubated with anti-phospho-RPA2 Ser4/Ser8 antibody (Bethyl). Immunoprecipitated proteins were eluted and supernatants subsequently analyzed by immunoblotting.

2.6. Comet assay

The neutral and alkaline comet assays were performed using the respective comet assay kits from Trevigen following manufacturer’s instructions. Comets were analyzed using CometScore software (TriTek, Sumerduck, VA, USA) licensed to the university.

2.7. Clonogenic survival assay

As detailed elsewhere [5,29], the cell lines were cultured for 14 days after the experimental treatments until cell colonies appeared. Colonies were stained with crystal violet and the number of cell colonies was counted with a target number of surviving colonies at 50–100 per dish. Survival fractions were calculated as the ratio of the plating efficiency of treated cells to untreated cells.

2.8. MTT Assay for cell viability

The MTT assay (Sigma) was employed. In brief, after the individual cell lines were treated with either VPA or HTPA, replaced with fresh medium and incubated for 48 hours, the MTT solution (5 mg/mL) was added to the treated cells and incubated for a further 4 hours, washed and absorbance of the solution measured using an enzyme immunoassay analyzer at 490 nm.

2.9. Homologues recombination assays

The HR was measured in MCF7-pDR-green fluorescent protein cells (GFP) as outlined elsewhere [5,29]. In brief, the plasmid containing pDR–GFP (gifted by Prof Maria Jasin, Memorial Sloan Kattering Cancer Centre, NY, USA) was transferred into the MCF7 cells by the standard lipofectamine transfection process using Lipofectamine 2000 and OptiMEM (Gibco). The MCF-7/pDR–GFP cells were cultured in complete Dulbecco’s modified Eagle’s medium containing puromycin. The cells which emit GFP were detected by flow cytometry. At least 10 × 105 cells in each group were processed.

3. Results

3.1. HPTA can sensitize breast tumor cells to HU

Our previous studies demonstrated that the therapeutic-equivalent dose of 500 μM VPA can sensitize breast tumor cells to 2 mM HU [5,25]. To investigate whether HPTA has a similar effect as VPA in inhibiting tumor cell growth in response to HU-induced DNA replication arrest, we compared 15 μM HPTA – its half maximal inhibitory concentration [30] – to 500 μM VPA in HU-induced replication arrest in both the breast tumor cell lines of MCF7 [31] and EUFA423 cells with a biallelic mutation in the BRCA2 gene [32].

3.1.1. Clonogenic survival assay of breast tumor cells pre-treated with either VPA or HTPA, and in combination with HU

The clonogenic assay in Fig. 1A showed that the survival fractions of MCF7 cells treated with 500 μM VPA or 15 μM HPTA alone were 78.28% (SD = 17.62) and 67.72% (SD = 12.59) respectively, compared to the untreated control group. There was no statistically significant differences between the VPA-only and HPTA-only groups (P = 0.84). The survival fraction of the 2 mM HU-only positive control was 37.49% (SD=7.36). The survival fraction in the two combined treatment groups showed similar reduction; specifically, the survival fraction of HU+VPA was 11.72% (SD =3.76) and HU+HPTA was 19.37%. (SD =6.30).
Similar findings were also observed in the EUFA423 cell line (see Fig. 1B). The survival fractions for the negative controls 500 μM VPA and 15 μM HTPA were 99.29% (SD = 6.37) and 96.78% (SD = 7.41) respectively, and the survival fractions for the 2 mM HU positive control was 65.18% (SD = 8.00). The survival fraction of EUFA423 cells treated with HU + VPA was 51.89% (SD = 2.79) and 53.52% (SD = 7.33) for HU + HPTA; these were lower than the HU-only positive control and the respective VPA- or HTPA-only negative control (P < 0.01 and P < 0.01). Our findings demonstrated that in vitro 15 μM HTPA has a similar HU-sensitizing effect as 500 μM VPA in the two popular breast tumor cell lines employed for this study. 3.1.2. MTT viability assay of tumor cells pre-treated with either VPA or HTPA, and in combination with HU To triangulate the data, the MTT assay was used on treated MCF7 and EUFA423 cells. The results as presented in Fig. 1C showed that the relative cell survival of the VPA-only and HPTA-only negative controls were 93.60% (SD = 8.10) and 78.50% (SD = 5.44) respectively as compared to the untreated control MCF7 cells. The relatively cell survival of the HU-only positive control was 71.70% (SD = 6.38). The combination of HU + VPA and HU + HPTA resulted in statistically less cell viability: 65.60% (SD = 4.08; P < 0.05) and 50.10% (SD =5.41; P < 0.05) respectively. The results in Fig. 1D showed that the relative cell survival of the VPA-only, HPTA-only and HU-only was 96.97% (SD = 4.87), 88.48% (SD = 5.47) and 51.71% (SD = 4.38) in EUFA423 cells, respectively. The cell viability in the combination of HU + VPA and HU + HPTA significantly further decreased to 42.98% (SD = 3.48, P < 0.05) and 35.16% (SD = 3.51, P < 0.05), respectively. We observed that HPTA resulted in statistically less cell survival as compared to VPA; both by itself only and in combination with 2 mM HU, suggesting that HTPA may have better sensitizing effects than VPA to HU in breast tumor. 3.2. HPTA augment HU-induced DNA DSBs We and others have previously demonstrated that VPA may have inhibited the growth of breast tumor cells and enhance the effects of HU through DNA DSBs. We next investigate the likely HPTA’s mechanisms of actions. Both HU-only and HPTA-only were included as positive control. 3.2.1. Effects of HPTA on HU-induced DNA DSBs Previous studies have reported that HU led to replication forks collapse which in turn generate DSBs [27,33]. Therefore we explored whether a potential action by which HPTA sensitized the above tumor cell lines to HU may be through inducing DNA DSBs. The neutral comet assay employed in MCF7 (see Fig. 2A Upper) showed that compared with the controls, the comet tail length of the combined HU + HPTA treatment group was significantly longer: 37.16% (SD = 2.41) vs. 11.21% HPTA-only control (SD = 1.48; P < 0.01); vs. 17.61% HU-only control (SD =1.48, P < 0.01). Similarly, the alkaline comet assay showed that the comet tail length of the combined HPTA-HU treatment group was longer than the HPTA-only and HU-only controls (Fig. 2A Lower). In EUFA423 cells, the neutral comet assay results showed that compared with the untreated control group, the comet tail length of the HPTA-treated group and HU-treated group increased by 7.74% (SD = 2.69, P > 0.05) and 22.67% (SD =3.60, P < 0.05), respectively (Fig. 2B Upper). The combination group of HPTA and HU effected cell tail length further increased significantly by 33.20% (SD = 5.30, P < 0.01) (Fig. 2B Upper). The alkaline comet assay data showed that HU also increased the comet tail length as compared to the control group, and further enhanced the tail length in combination with HPTA (Fig. 2B Lower, P < 0.01). The results from both neutral and alkaline comet assay were consistent. To validate these findings, immunofluorescence assays of two DNA DSB markers, γH2AX (Figs. 2C and 2E) and 53BP1 (Figs. 2D and 2F), were utilized. The percentages of MCF7 cells with γH2AX foci in HPTA-only control and HU-only control were 13.07% (SD = 1.06) and 35.10% (SD = 1.63), respectively (Fig. 2C Upper). The combination of HU + HPTA resulted in more γH2AX foci: 52.27% (SD = 1.83; P < 0.05). In the EUFA423cells, compared with the untreated control group (7.63%, SD = 0.69), the positive rate of cells containing γH2AX foci formation in the HPTA-only control increased to 16.23% (SD = 1.52, P < 0.01), while significantly increased to 55.77% (SD =1.35, P < 0.01) in the HU-only control (Fig. 2E Upper). Importantly, for cells that were pretreated with HPTA for 24 hours and then combined with HU for another 18 hours, the positive rate of the cells with γH2AX foci formation further increased to 75.72% (SD = 1.68, P < 0.01) as compared to either the HU-alone or HTPA-alone controls (Fig. 2E Upper). The western blot confirmed the increased expression of γH2AX in HU + HPTA as compared to the negative and positive controls: MCF7 (Fig. 2C Lower): untreated control 0.18 (SD = 0.11), HPTAonly control 0.27 (SD = 0.15), HU-only control 0.40 (SD = 0.15), HU + HPTA 0.91 (SD = 0.20, P < 0.01); and, EUFA423 (Fig. 2E Lower): untreated control 0 (SD = 0), HPTA-only control 1.82 (SD = 0.42), HU-only control 2.84 (SD = 0.70), HU + HPTA 4.67 (SD = 0.88, P < 0.01). Similar observations of increased expression of 53BP1 in HU + HTPA were noted: The percentages of MCF7 cells with 53BP1 foci in untreated control and HPTA-only control were 10.40% (SD = 1.2) and 15.82% (SD = 1.5), respectively (Fig. 2D Upper). Moreover, there was a statistically increase in the percentage of cells containing 53BP1 foci with HUtreatment 30.34% (SD = 1.5; P < 0.01), and the combination of HU +HPTA resulted in more 53BP1 foci: 48.57% (SD =3.4; P < 0.01; Fig. 2D Upper). The positive rate of EUFA423 cells containing 53BP1 foci formation in the untreated control group was 3.21% (SD = 0.53) and HPTA-only control increased to 8.85% (SD = 1.10, P > 0.05), while significantly increased to 32.74% (SD =5.19, P < 0.01) in the HU-only control (Fig. 2F Upper). When then combined with HPTA pretreatment for 24 hours, the percentage of the cells containing 53BP1 foci further increased to 45.16% (SD = 4.88, P < 0.01) (Fig. 2F Upper) as compared to either the HU-alone or HTPA-alone controls (Fig. 2F Upper). The amount of protein 53BP1 in MCF7cells and EUFA423 cells with both HPTA and HU treatment was higher than individual treatments: MCF7 (Fig. 2D Lower): untreated control 0.79 (SD = 0.11), HPTAonly control 1.05 (SD = 0.12), HU-only control 2.16 (SD = 0.21), HU + HPTA 2.61 (SD = 0.08, P < 0.01); and, EUFA423 (Fig. 2F Lower): untreated control 0.99 (SD = 0.01), HPTA-only control 1.35 (SD = 0.08), HU-only control 1.46 (SD = 0.03), HU + HPTA 1.82 (SD = 0.07, P < 0.01). The above findings confirmed that the combination of HU + HTPA resulted in more DNA DSBs as compared to HU-only or HTPA-only. 3.2.2. HPTA inhibit HR activity after HU-induced DNA replication arrest To test the hypothesis that HPTA augments HU-induced DNA DSBs through the similar pathway as VPA in the disruption of DNA repair function [25], the MCF7/ pDR-GFP cell system was utilized to test HR repair efficiency [5,25,29]. At least 5 × 105 cells were used to measure the HR frequency by flow cytometry (see Fig. 3). The HR repair frequency in the HPTA-only control was not statistically different from the untreated control group: 65 × 10-6 vs. 60 × 10-6 (Fig. 3, P > 0.05). The HR frequency for HU-only control was 100 × 10-6. The addition of HPTA in the treatment group (HU + HPTA: 66 × 10-6, P < 0.01) resulted in a reduction in HR frequency as compared to the HU-only control, suggesting that HPTA may suppress the HR activity induced by HU. 3.2.3. HPTA inhibit RPA2 phosphorylation and Rad51 in HU-induced DNA replication arrest Previous study has demonstrated that RPA2 hyperphosphorylation and its subsequent recruitment of recombinase protein Rad51 plays an important role in genomic stability especially after HU treatment [27]. Our own previous studies also demonstrated that VPA in combination with HU inhibited the growth of breast tumor cells by impairing the RPA2-Rad51 mediated HR repair pathway [25,27]. Therefore, the following series of six experiments examine whether the HR activity observed above is associated with changes in the RPA2 phosphorylation. Firstly, the expression of RPA2-p in MCF7 (see Fig. 4A) and EUFA423 cells (see Fig. 4E Upper) was detected using western blot. Our results in Figs. 4A and 4E showed that HU-induced more RPA2 phosphorylation as compared to HPTA-only (MCF7: 0.24; EUFA423: 0.18, P < 0.01). The addition of HPTA to HU-treated cells (HU+HPTA) significantly reduced the density of RPA2 phosphorylation. In the MCF7 cell lines, the reduction in RPA2 hyperphosphorylation was substantial and comparable with HPTA-only control cells (0.24, P < 0.01, Fig. 4A Upper). Whilst in the EUFA423 cell lines, the reduction in RPA2 hyperphosphorylation was significant and comparable with the HPTAonly control (0.18, P < 0.05, Fig. 4E Upper). We repeated the western blot experiments with the regular RPA2 antibody, the results were consistent (see Fig. 4A and Fig. 4E Lower, P < 0.01). The findings demonstrated that HPTA can reduce the hyperphosphorylation of RPA2 in response to HU-treatment. Secondly, to confirm the above findings, we detected the RPA2-p foci formation using immunofluorescence assay. The results showed that the addition of HPTA to HU-treatment (HU + HPTA) statistically reduced RPA2-p foci formulation (MCF7: HU + HPTA 35.87% [SD = 1.13] vs. HU-only 51.99% [SD = 2.33], P < 0.01, Fig. 4B; EUFA423: HU + HPTA 17.10% [SD = 1.81] vs. HU-only 42.28% [SD = 1.92], P < 0.01, Fig. 4E). The findings from the western blot and immunofluorescence were consistent, suggesting that HPTA augments HU-induced DNA DSBs through RPA2-p mediated HR pathway. Thirdly, we previously demonstrated that hyperphosphorylated RPA2 was associated with the recombinase protein Rad51 in response to the HU-induced replication arrest [27], therefore to affirm the findings, western blot was performed on both MCF7 and EUFA423 cells to test the effects of HPTA in HU treatment. In the MCF7 cell lines, it was found that the amount of Rad51 protein levels in the HU-treated group alone was only slightly increased (0.69, P < 0.01), and decreased in combination with HPTA (0.50, P < 0.05; Fig. 4C). In the EUFA423 cells, the decline in Rad51 protein level was also statistically significant (HU + HPTA 0.70; HU-only 0.89; P < 0.05, Fig. 4F). Fourthly: some studies indicated Rad51 foci formation is more important than its protein levels to represent HR activity in response to DNA damage [34,35], therefore we next investigate whether HU- (caption on next page) induced Rad51 foci formation was affected by HPTA. The immunofluorescence presented in Fig. 4D showed lesser Rad51 foci with HU + HPTA as compared to HU-only (HU + HPTA 19.25% [SD = 1.45], HU-only 33.45% [SD = 2.50], P < 0.01]. Taken together, our results demonstrated that HPTA may have an inhibitory effect on the activity of RPA2-p and Rad51. Fifthly: our previous research found that both RPA2-p and Rad51 foci formation were co-localized in the cell nuclei in response to replication arrest [25], therefore a co-immunoprecipitation experiment was conducted using MCF7 cells. The results in Fig. 4G demonstrated that both RPA2-p and Rad51 proteins were present in the same protein complex precipitate, suggesting that HPTA’s augmentation of HU-induced DNA DSBs involved both RPA2-p and Rad51 (Fig. 4G Upper). In addition, we used MCF7 cells for immunofluorescence assay, RPA2-p and Rad51 foci formation are co-localized in response to replication arrest (see Fig. 4G Lower), thus support the proposition of a RPA2-p mediated Rad51 dependent HR pathway. Sixthly, to verify the role of RPA2-p in this process, MCF7 cells were used to establish an isogenic pair cell line with the expression of wtRPA2 and mutRPA2 [27]. To test whether our mutRPA2-cells were successfully established, the results in Fig. 5A showed that in response to HU-induced replication arrest, the expression of RPA2 protein in mutRPA2-cells was reduced as compared to the wtRPA2-cells (mutRPA2-cells: 0.46 vs. wtRPA2-cells: 0.98; P < 0.01, Fig. 5A Upper). Furthermore, the RPA2 expression in the mutRPA2-cells was demonstrated by the regular anti-RPA2 antibody in Fig. 5A Lower, indicating that the defective RPA2 cell line was successfully established. This was also affirmed in the clonogenic assay (Fig. 5B) where it was observed that the mutRPA2 cells were more sensitive to HU treatment as compared with wt-RPA2 cells (mutRPA2 cells: 22.89% vs. wt-RPA2 cells: 38.75%, P < 0.01). We found that the clonogenic assay’s survival fractions of HPTApretreated wtRPA2-cells were significantly reduced after HU treatment (18.20%, P < 0.01), these were similarly observed in the mutRPA2cells (13.60%, P > 0.05 Fig. 5B). However, there were no significant differences between the survival fractions of HU-HPTA treated wtRPA2cells and mutRPA2-cells (wtRPA2-cells: 18.20% vs. mutRPA2-cells: 13.60%; P > 0.05).
The above results further confirmed that the HPTA’s mechanism of action in HU-induced cell death involved RPA2.

3.3. HPTA can sensitize primary breast tumor cells to HU

We have so far experimented on two popular breast tumor cell lines to ascertain HPTA effects on HU-induced cell death, to progress the findings we next confirm the sensitization effect of HPTA on HU using a previously published primary breast tumor culture [4,36] (see Fig. 6A).
The immunofluorescence staining of our primary breast tumor tissue culture in Fig. 6B using γH2AX demonstrated an elevated amount of DNA DSBs with HU-only and HU + HPTA treatment. There were more γH2AX foci in the HU + HPTA treatment group as compared to the HU-only positive control (P < 0.01), consistent with the findings above from the two popular breast tumor cell lines. Likewise, we found an expected increase in RPA2-p (Fig. 6C) and Rad51 (Fig. 6D) with HU-only treatment (positive control). There was a significant reduction in the RPA2-p foci and Rad51 foci with the addition of HPTA pre-treatment prior to HU: RPA2-p: HU + HPTA: 40.50% vs. HU: 34.87%; different 5.63%; P < 0.01. Rad51: HU + HPTA: 32.30% vs. HU: 27.00%; different 5.30%; P < 0.01. Taken together, the findings from the primary-cultured tumor cells were consistent with those obtained from the tumor cell lines and support the hypothesis that the combination of HPTA and HU can lead to an increase in DNA DSBs and interference in DNA repair activity via the RPA2 mediated Rad51 pathway. 4. Discussion According to the World Health Organization, breast carcinoma is the most commonly diagnosed form of malignancies in women and some 627,000 women died from breast carcinoma per year [37]. Breast carcinoma is a group of very heterogeneous diseases, different subtypes of breast carcinoma has various prognoses and responses to therapy. In general, systemic therapy is active at the beginning of therapy in 90% of primary breast tumor and 50% of metastases, but resistance to therapy is common and to be expected [38]. More recently, multimodal treatment has been recognized as an important treatment strategy for breast carcinoma to improve the effectiveness of current therapeutic agents, decrease the emergence of resistance, and increase disease-free survival. Numerous agents have been investigated for use in combination with existing therapies, these include VPA which has been shown to inhibit the growth of tumor cells and various tissue-transformed cells [25]. However, the therapeutic dose of VPA required can produce adverse events including liver and kidney toxicity and teratogenicity. In our series of experiment above, we demonstrated that at a much lower dose of 15μM, the VPA derivative, HPTA significantly inhibites breast tumor cell growth. Although we are not able to verify the toxicity of HPTA at 15μM, there has been reports that 10-25μM HPTA have no significant adverse effects and in contrary exhibit significant neuroprotective effects [39] and increase the expression level of heat shock proteins [39]; postulating that HPTA may be the best substitute of VPA derivatives. HU’s mechanism of action includes breaking the DNA replication fork to cause serious damage to the DNA. The HR is one of the important mechanisms for repairing DNA DSBs. The process usually uses the same sister chromosome that is not damaged as a template to accurately repair the damaged DNA through base pairing [40]. If this HR mechanism is inhibited at the same time, it will cause more accumulation of DNA DSBs damage, leading to more cell death. In response to replication arrest, there are reasons to suggest that RPA2 hyperphosphorylation may play a key role in the regulation of HR function in repairing DNA damage: (1) under conditions of replication stress, the ssDNA bound by RPA actives ATM- and Rad3- related (ATR) and subsequently the RPA2 hyperphosphorylation induced by ATR promotes HR [41]; (2) phosphorylations of RPA not only frees RPA during DNA damage response (DDR) but also allows RPA to more efficiently recruit Rad51 to the DSB sites during an early step of HR, Rad51 did directly interact with RPA with relatively higher affinities to hyperphosphorylated RPA than to unphosphorylated RPA [42]; (3) Rad51 interaction with ssDNA-bound RPA plays an important role in promoting Rad51 presynaptic filament assembling at DSBs [43]; (4)the hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by HU treatment[41]. Based on the above information, RPA2 hyperphosphorylation is likely to be a main factor for regulating Rad51-dependent HR during DNA replication arrest process, consequently it is necessary to investigate how HPTA inhibits RPA2 hyperphosphorylation. Our previous study found that HU-induced one-end DSBs are specifically repaired by the RPA2mediated HR mechanism [33].VPA augment HU-induced DNA DSBs and this mechanism by which VPA sensitizes breast tumor cells to HU was RPA2 dependent [33]. In this study, we observed that the pretreatment with HPTA resulted in reduced HR efficiency, resulted in more DNA DSBs in the breast tumor cells when subsequently treated with HU. The HPTA inhibited breast tumor cells growth by inhibiting RPA2 phosphorylation and Rad51 in response to HU-induced DNA damage. 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