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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 2  |  Page : 86-92

Valproic acid, a histone deacetylase inhibitor, enhances radiosensitivity in breast cancer cell line


1 Biochemistry and Metabolic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran
2 Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran
3 Babol University of Medical Sciences, Babol, Iran
4 Department of Biochemistry and Biophysics, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran

Date of Web Publication22-May-2018

Correspondence Address:
Dr. Alireza Khoshbin Khoshnazar
Department of Biochemistry and Biophysics, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_37_17

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  Abstract 

Purpose: Valproic acid (VPA) is used mainly for the treatment of epilepsy and other seizure disorders, however, it is known to be one of histone deacetylase (HDAC) inhibitors. HDACIs have represented roles in radiation-sensitizing of cancer cells. This study is aimed to study to evaluate the radiosensitizing capability of VPA in MCF-7 breast cancer cell line. Materials and Methods: Cell viability and apoptosis were assayed using MTT and TUNEL assays, respectively and caspase-8 and caspase-9 activities were measured by commercially available kits. Results: Our finding showed that pre treatment of cells with VPA, notably enhanced apoptotic cell death in MCF-7 cell line. Our results showed that VPA sensitizes cancer cells against radiation. Conclusion: Valproic acid could be a beneficial radio-sensitizer in breast cancer radiotherapy.

Keywords: MCF-7 cell, radiosensitizer, valproic acid


How to cite this article:
Yarmohamadi A, Asadi J, Gharaei R, Mir M, Khoshnazar AK. Valproic acid, a histone deacetylase inhibitor, enhances radiosensitivity in breast cancer cell line. J Radiat Cancer Res 2018;9:86-92

How to cite this URL:
Yarmohamadi A, Asadi J, Gharaei R, Mir M, Khoshnazar AK. Valproic acid, a histone deacetylase inhibitor, enhances radiosensitivity in breast cancer cell line. J Radiat Cancer Res [serial online] 2018 [cited 2018 Aug 21];9:86-92. Available from: http://www.journalrcr.org/text.asp?2018/9/2/86/232983


  Introduction Top


Breast cancer is the second prevalent cancer worldwide after lung cancer, while it is the most prevalent and the first cause of cancer-related mortality among females.[1],[2]

Valproic acid (VPA, 2- pentatonic propyl acid) is a branched eight-carbon fatty acid, which is often used for the treatment of epilepsy, bipolar, and migraine's diseases.[3] Recently, it has been shown that VPA is a histone deacetylase (HDAC) inhibitor.[4],[5] HDAC inhibitors are considered as a new class of anticancer agents. In fact, several HDACI including hydroxamate, suberoylanilide hydroxamic acid (SAHA), depsipeptide, and the cyclic tetrapeptide have shown acceptable anticancer functions in both hematologic and solid cancers in clinical trials.[6],[7] HDAC inhibitors inhibit proliferation and induce cell growth arrest, cell-cycle arrest, and apoptosis in cancer cell lines in both cell culture and in vivo.[8],[9] Selective toxicity on numerous cancer cell lines is one of the potential benefits of HDAC inhibitors so that they are nontoxic to normal cells.[10] There are two suggested pathways which define HDACI function: modulation of tertiary chromatin structure and acetylation and alteration of nonhistone proteins such as transcription factor p53. It is a protein which affects the expression of more than 150 genes that contribute in the arrest of cell-cycle checkpoints or apoptosis induction.[11] These findings accelerated the evaluation of VPA as an anticancer treatment factor.

Radiotherapy is commonly used for cancer treatment. Loss of clonogenic potential is the main mechanism which radiation kills most of the tumor cells.[12] Ionizing radiation (IR) imposes one of the worst DNA impairments, DNA double-stranded breaks.[12],[13] Ataxia telangiectasia mutated (ATM) kinase exhibits the first response to DNA damage. ATM kinase triggers a cascade of response through signal transduction to downstream elements for cell-cycle arrest and induction of repair and finally cell death if the damage is irreparable.[14] However, IR induces apoptosis or cell-cycle arrest, but its effects are heterogeneous in various cell lines.[15]

Radiation therapy is an accepted approach in cancer therapy, and worldwide data indicate that approximately 50% of cancer patients are treated with radiation either for therapeutic or palliative purposes.[16] However, relapse of tumors is still an issue of concern and so on suggests the necessity of using other agents to sensitize cancer cells and improve the therapeutic idea of radiotherapy.[16],[17],[18] Therefore, the development of such compounds has been actively pursued to reach better clinical results.

It was demonstrated that HDACI can serve as a radiation sensitizer in different types of tumor cells both in vivo and in vitro.[19],[20],[21] It seems that the mechanism which HDACI can act as a radiation sensitizer is to block HDAC activity and therefore acetylation of core histone resulting in chromatin structure decondense. DNA in decondensed chromatin is more sensitive to DNA damage caused by radiotherapy.[22] Trichostatin A (TSA) is a radiation sensitizer in K562 cells. Treatment of cells by TSA before irradiation decreases cell viability.[23] Furthermore, SAHA decreases cell viability and increases induced apoptosis caused by irradiation in prostate and glioma cancer cell lines.[24] In this study, we proposed to assess potent radiation enhancer properties of VPA on MCF-7 breast cancer cell line. In this context, we evaluated the effect of VPA and IR alone and combined on cytotoxicity and apoptosis including the makers of extrinsic and intrinsic apoptotic pathways (caspases 8 and 9).


  Materials and Methods Top


Cell culture and treatments: MCF-7 human breast cancer cells (obtained from Pasteur Institute, Tehran, Iran) were grown in a humidified atmosphere of 95% air and 5% CO2 (v/v) at 37°C Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 100 U/ml streptomycin, 100 U/ml penicillin, and 10% heat-inactivated fetal bovine serum. All reagents were purchased from Gibco, Germany. Cells were maintained in an exponential growth phase, and experiments were performed on cells that were collected at a density of 4–7 × 105 cell/ml. The cell lines in a maximum range of up to 20 passages were used for this study. In typical experiments, MCF-7 cells (5 × 105 ml) in complete DMEM medium were treated with 0.5, 2, 4, 8, 16, 32, and 64 mM VPA (Sigma Aldrich, Germany), for 24, 48, and 72 h at 37°C. The VPA was dissolved in phosphate buffer saline (PBS) to a stock concentration of 100 mM and stored at −20°C. The exponentially growing, untreated MCF-7 cells and cells that had been pretreated with VPA, irradiated using g-rays from Cobalt 60 source (Theratron teletherapy unit) at a dose rate of 0.63 Gy/min. Cells were exposed to doses of 0, 2, 4, and 6 Gy of g-rays.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

To determine cell viability, specified numbers (8 × 103) of cells were seeded in individual wells of 96well plates and incubated for 24 h at 37°C before treatment with various concentrations of VPA for 24, 48, and 72 h. Subsequently, cells were washed with PBS, and 20 μl of sterile 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml, Sigma Aldrich) was added to each well which were incubated at 37°C for 4 h. Supernatant media containing MTT were discarded, and 200 μl dimethyl sulfoxide was added in each well. Plate was gently mixed at darkness for 15 min, and absorbance was recorded at 492 nm to evaluate cell proliferation. The percentage cell prolifeartion was determined by comparing the optical density (OD) of the drug-treated cells with that of untreated controls. All experiments were repeated at least thrice.

Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay

For apoptosis detection, adherent and floating cells were harvested and analyzed for DNA fragmentation by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay and propidium iodide (PI) staining with the APO-BrdU TUNEL assay kit (Invitrogen) according to the manufacturer's recommendation. Briefly, MCF-7 cells were fixed in paraformaldehyde and were stored in 70% (v/v) ethanol at −20°C for 24 h. Then, ethanol was removed by centrifugation, and cells were resuspended in washing buffer, mixed with 50 μl of DNA labeling solution and incubated for 60 min at 37°C. After addition of rinse buffer, the cell suspension was centrifuged to remove the buffer; 100 L of antibody solution was added to each cell pellet and incubated for 30 min in darkness at room temperature. Then, 60 μl PI/RNase A solution was added and incubated for 30 min in darkness at room temperature. Then, 20 μl of the homogeneous mixture of cell suspension was dropped onto a slide before being covered with a cover slip. The apoptotic cells were determined by counting cells showing green fluorescence staining over an orange–red PI counter-staining. Viable and apoptotic cells were observed and quantified. All assays were performed in triplicate.

Caspase-8 and caspase-9 activity assays

Induction of apoptosis in MCF-7 cells was determined by measuring the activities of caspase-8 and caspase-9 with the colorimetric assay kits (R and D systems) according to the protocols suggested by the manufacturer. Briefly, after treatment, cells were washed with cold PBS and harvested by scraping, then centrifuged (250 × g for 10 min) and incubated for 10 min in cell lysis buffer. Cell lysates were centrifuged (10,000 × g for 1 min), and 10 μl of that was loaded in triplicate on 96-well plate, then 50 μl of caspase assay buffer was added to each one. Then, 5 μl of the colorimetric caspase-8 and caspase-9 substrates (IETD-pNA and LEHD-pNA, respectively) was added separately to each well. The plates were incubated for 2 h at room temperature in darkness. Finally, colorimetric signal was determined by measuring the absorbance at 405 nm. The protocol was repeated thrice for each assay.

Statistical analysis

The data were analyzed using SPSS (version 16) software (SPSS Inc, Chicago, USA). Kolmogorov–Smirnov test was used to measure the normality of data distribution. In the cases of normality, one-way analysis of variance was used followed by post hoc Tukey's test. The statistical significance was considered at P < 0.05 level.


  Results Top


Evaluation of growth inhibition by valproic acid

The effect of VPA on the viability of the MCF-7 cell line was assessed by the MTT assay. VPA inhibited the proliferation of MCF-7 cells in a time and dosedependent manner, as shown in [Figure 1].
Figure 1: Effect of valproic acid on cell viability. MCF-7 cell was treated for 24, 48, and 72 h with valproic acid (0–64 mM), and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after the indicated times. (a) MCF-7 cells after 24 h treatment, (b) MCF-7 cells after 48 h treatment, and (c) MCF-7 cells after 72 h treatment. The results are the means of three independent experiments with six repetitive treatments (*P < 0.05 and ***P < 0.0001)

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A Concentration of 0.5 mM VPA resulted in a cell survival equal to 90%; however, at higher concentrations, the reduction in viability was obtained at the highest level of statistical significance. In summary, the results indicated that the cells treated with VPA showed marked decrease in proliferation, which was found to be statistically significant (P < 0.0001) as compared to control [Figure 1]. Using LD50 obtained in our previous work,[25] the doses 4 mM and 8 mM at 48 h period were selected for the current experiment.

Deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling

Increased apoptosis of MCF-7 cells using combined IR and VPA. To determine the effect of IR and/or VPA on MCF-7 cells, apoptosis was evaluated by TUNEL assay. MCF-7 cells were treated with VPA (4 and 8 mM) for 48 h than irradiated with, IR (2 and 4 Gy), apoptotic cells showed green fluorescence, while the red fluorescence was an indication of viable cells [26] [Figure 2].
Figure 2: The effect of valproic acid and ionizing radiation treatment on MCF-7 cell line, assessed by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining assay technique (Apo-BrdU), double stained with fluorescein Br-deoxyuridine triphosphate and PI solution: (a) untreated cells (b) treatment of cells with 4 mM (c) 8 mM (d) 2 Gy (e) 4 Gy (f) 4 mm + 2 Gy (g) 4 mm + 4 Gy (h) 8 mm + 2 Gy (i) 8 mm + 4 Gy. Viable cells show red nuclei (yellow arrow) whereas apoptotic cells show yellow to greenish nuclei (white arrow) (Mag × 20)

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Based on these figures, there were no observable apoptotic cells seen in the untreated control cells (99% viability) [Figure 2]a. An induction of TUNEL positive cells was observed in the presence of each single factor, while the combined treatment produced a remarkable increase of apoptosis compared to the untreated control cells (P > 0.0001). Reduced size and impaired morphology of cells nuclei were obviously seen in treated cells in comparison to control cells. All of the mentioned alterations are indicating chromatin condensation and DNA fragmentation which are the underlying mechanisms of cell apoptosis.[27]

Quantification of apoptotic cells was reported as the percentage of apoptotic cells among total cells. Five to six high-power fields were selected randomly, and the total number of cells was counted manually [Figure 3].
Figure 3: The percentage of apoptotic cells in the treated group in comparison to control cells assayed by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling method. V4: 4 mM valproic acid, V8: 8 mM valproic acid, R2: 2 Gy, R4: 4 Gy, V4 + R2: 4 mM valproic acid + 2 Gy, V4 + R4: 4 mM valproic acid +4 Gy V8 + R2: 8 Mm valproic acid + 2 Gy, V8 + R4: 8 mM + 4 Gy (***P < 0.0001)

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Caspase activity

Mechanism of apoptosis induction to further elucidate the mechanism of VPA and IR on the increase of apoptosis or its enhancement of IR exposure, caspase-8 and caspase-9 activities were measured as the markers of extrinsic and internal apoptosis pathways, respectively. Caspase-8 activity increased about 4.4- and 6.4-fold for 4 and 8 mM concentrations of VPA, also 3.4- and 5-fold for 2 and 4 Gy, respectively, as compared to untreated cells [Figure 4]. Caspase-8 activity increased about 7.5-fold for 4 mM concentration combined with 4 Gy radiation, also 7.2-fold for 8 mM concentration of VPA together with 4 Gy of radiation, compared with control group.
Figure 4: Effect of valproic acid on caspase-8 and caspase-9 induction in MCF-7 cells. Caspase 8 (a) and 9 (b) activities in cells treated with valproic acid and irradiation; Activities (absorbance/mg protein) were measured spectrophotometrically at a wavelength of 405 nm. V4:4 mM valproic acid, V8: 8 mm valproic acid, R2: 2 Gy, R4: 4 Gy, V4 + R2: 4 mm valproic acid + 2 Gy, V4 + R4: 4 mm valproic acid + 4 Gy V8 + R2: 8 mm valproic acid + 2 Gy, V8 + R4: 8 mm + 4 Gy. Caspase activities are presented as mean ± standard deviation (n = 3). ***P < 0.0001

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On the other hand, caspase-9 activity increased about 4.5- and 7.4-fold for 4 and 8 mM concentrations of VPA also 3.7- and 4.9-fold for 2 and 4 Gy, respectively, as compared to untreated cells. Caspase-9 activity increased about 5.6-fold for 4 mM concentration combined with 2 Gy radiation, also 7.6-fold for 4 mM concentration of VPA together with 4 Gy of radiation and finally 8.5-fold for synchronous usage of 8 mM concentration of VPA together with 4 Gy of radiation compared with control group [Figure 4]. This concentration-dependent increase was statistically significant (P < 0.0001).


  Discussion Top


In this study, we aimed to survey the effects of VPA and radiation on viability and apoptosis of MCF-7 cell line. Our results showed that VPA is an antiproliferative agent in MCF-7 cell line which notably decreases cell viability in a dose- and time-dependent manner. These results are concordant with the previous surveys which claimed the reduction of cell viability in case of VPA treatment.[25],[28] HDACIs are suggested to exhibit antiproliferative actions through several mechanisms, including: cell-cycle arrest, differentiation, and apoptosis induction.[29],[30] TSA, oxamflatin, MS-275, butyrate, and SAHA are considered as the members of HDACIs family, which induce the expression of CDKN1A gene, resulting in p21 encoding and finally cell-cycle arrest G1.[31],[32],[33],[34]

Apoptosis is the other selective mechanism by anticancer agents. This process is triggered by extrinsic (receptor-dependent) or intrinsic (mitochondrial) pathways modulated by caspase-8 and caspase-9 enzymatic activity, respectively.[35] The results of the TUNEL assay showed that VPA strongly induced apoptosis in MCF-7 cell line. These findings were confirmed when performing caspase-8 and caspase-9 activity evaluation for mentioned cells. It seems that HDACI itself and cell type are the two major factors which determine the specific pathway to be used in apoptosis induction. For instance, SAHA includes intrinsic pathway through cleavage and activation of Bcl-2 which results in mitochondrial membrane damage and ROS production.[36] On the other hand, apicidin exploits fas/fas ligand expression in leukemia cells and desipeptide involve TNF to activate caspase-8 and trigger apoptosis extrinsic pathway.[37],[38] Vandermeers et al. reported that VPA activated both intrinsic and extrinsic pathways by hyperactivation of histone H3, p21 overexpression, bid cleavage and cytochrome C release from mitochondria in malignant mesothelioma [39] whereas Catalano et al. could not find the effect of VPA on caspase-8 pathway in thyroid tumor cells and their results limited just in caspase-9 pathway in this issue.[28]

We also assessed the radiation sensitizing potency of VPA in MCF-7 cell line by a 48 h period of VPA pretreatment, before radiation. We understood that VPA is an efficient radiation sensitizer in MCF-7 cells and therefore, are in concurrence with those from the previous report.[40],[41],[42] Our data from TUNEL assay demonstrated that the treatment of cells with VPA before irradiation enhances apoptotic cell from 32% to 45% at 4 mM and from 43% to 58% at 8 mM in 2 Gy; these changes were 32%–52% at 4 mM and 34%–66% at 8 mM for 4 Gy radiation [Figure 3]. Similarly, caspase-8 and caspase-9 assays indicate that pretreatment with VPA potentates radiation-induced apoptosis in MCF-7 cells in a pattern just like what we concluded in the TUNEL assay [Figure 4]. Irradiation can cause an increase in levels of p53 by recruiting ATM proteins, and ultimately results in an increase in the level of the cyclin-dependent kinase inhibitory protein p21.[43]

Some of the reported mechanisms which claim the synergistic effect of HDACIs on radiation enhancement include inhibition of DNA damage repair pathways, resulting in radiation sensitizing actions by involving the protein kinases, ATM, ataxia telangiectasia-related proteins and 53PB1 which all have important roles in DNA damage cascades.[23],[42],[44],[45] HDACIs also can induce gene expression alteration or changing the acetylation status of p53 protein, which is a known substrate for HATs and HDACs.[46] Acetylated p53 can stimulate its sequence-specific DNA binding activity to modulate proapoptotic responses in cancer cells.[47]

One of the common problems of clinical oncology is the occurrence of tumors relapse in radiotherapy.[17] Hence, sensitizing the cancer cell to radiation-induced DNA damage and apoptosis by radiation-sensitizing agents such as VPA, seem to be an efficient approach to conflict against this problem. Our findings showed that VPA is a potent radiation-sensitizer in MCF-7 breast cancer cell line. We could show that VPA utilization before radiation can enhance cell death and apoptosis in vitro that could be an application strategy to enhance radiosensitivity of breast cancer cells in the clinic.

Acknowledgments

The authors would like to thank biochemistry laboratory of faculty of medicine of Golestan University of medical sciences. We would like thank specially Mr. Mohamad Mostakhdem Hashemi for his useful comments.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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