|Year : 2018 | Volume
| Issue : 1 | Page : 11-16
Valproic acid reduces tumor cell survival and proliferation with inhibitors of downstream molecules of epidermal growth factor receptor pathway
Bhuvanesh Sukhlal Kalal1, Vinitha Ramanath Pai2, Dinesh Upadhya3
1 Yenepoya Research Centre; Department of Biochemistry, Yenepoya Medical College, Yenepoya University, Mangalore, Karnataka, India
2 Department of Biochemistry, Yenepoya Medical College, Yenepoya University, Mangalore, Karnataka, India
3 Yenepoya Research Centre, Yenepoya University, Mangalore; Department of Anatomy, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India
|Date of Submission||04-Oct-2017|
|Date of Decision||04-Jan-2018|
|Date of Acceptance||05-Jun-2018|
|Date of Web Publication||11-Jul-2018|
Department of Anatomy, Kasturba Medical College, Manipal Academy of Higher Education, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: To evaluate the beneficial effect of treating tumor cells with valproic acid (VPA) in combination with inhibitors of various downstream molecules of epidermal growth factor receptor pathway for limiting tumor cell growth. Materials and Methods: Cytotoxic effect of VPA was tested with various combinations of inhibitors of PI3K-AKT, ERK1/2, Jun N-terminal kinases (JNK) as well as p38 kinases in A375 cells using methyl-thiazol-tetrazolium assay, clonogenic assay, and caspase assay. Antiproliferative effect of the combination was tested with ethynyl-2′-deoxyuridine incorporation assay. Results: Among the combinations tested, a combination of VPA with PI3K-AKT inhibitor showed enhanced tumor cell death and reduced tumor cell proliferation compared to the combination of VPA with ERK1/2, JNK, and p38 inhibitors at lower doses. Conclusions: Combination of VPA with PI3K-AKT inhibitor at lower concentration reduced tumor cell growth.
Keywords: Cancer, histone deacetylase inhibitors, Ly294002, valproic acid
|How to cite this article:|
Kalal BS, Pai VR, Upadhya D. Valproic acid reduces tumor cell survival and proliferation with inhibitors of downstream molecules of epidermal growth factor receptor pathway. J Pharmacol Pharmacother 2018;9:11-6
|How to cite this URL:|
Kalal BS, Pai VR, Upadhya D. Valproic acid reduces tumor cell survival and proliferation with inhibitors of downstream molecules of epidermal growth factor receptor pathway. J Pharmacol Pharmacother [serial online] 2018 [cited 2020 Sep 25];9:11-6. Available from: http://www.jpharmacol.com/text.asp?2018/9/1/11/236311
| Introduction|| |
Valproic acid (VPA) is a histone deacetylase inhibitor (HDACi) with a branched short chain fatty acid, widely used as a generic low-cost antiepileptic and mood stabilizer. It has been in medical use since 1962 and has a good long-term safety profile in the therapeutic doses comparable to other HDACi, with limited toxicities.,, Because of this, it has been considered as a good candidate for anticancer therapy. Recently, its anticancer activity is being evaluated in different cancer models by several groups either as a monotherapy or in combination with chemotherapeutic agents both in vitro and in vivo. Although as a monotherapy agent VPA failed to show good response, in combination with other chemotherapeutic agents, it demonstrated good therapeutic efficacy both in cell culture and animal studies. Because of this, multiple translational clinical trials were undertaken to find the effect of adding VPA in treating multiple types of solid tumors in combination with other conventional chemotherapeutic agents.,,,,,,,,,, Most of the studies showed moderate therapeutic responses while none of the studies showed exciting outcomes.
Targeted therapeutics have gained much importance for treating multiple cancer types in recent years. Among the targetable molecules, epidermal growth factor receptor (EGFR) and its downstream molecules occupies a central role since this pathway plays a critical role in driving tumor cell proliferation, migration, and their survival. ERK1/2, PI3K-AKT, Jun N-terminal kinases (JNK), and p38 are well-known downstream molecules of EGFR pathway. In the present study, we analyzed the antiproliferative and cell killing effect of VPA in combination with inhibitors of ERK1/2, PI3K-AKT, JNK, and p38 kinase using A375 cells.
| Materials and Methods|| |
Cell culture and inhibitors
Human melanoma cell line A375 was purchased from the National Centre for Cell Science (Pune, Maharashtra, India), cultured in Dulbecco's modified Eagle's medium (DMEM) media (Himedia, India) supplemented with 2 mM L-glutamine 10% fetal bovine serum (Himedia, India) and 1% antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin), (Himedia, India), and maintained at 37°C in a humidified incubator of 5% CO2. U0126 (ERK1/2 inhibitor), LY294002 (PI3K-AKT kinase inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 mitogen-activated protein kinase inhibitor) were procured from Calbiochem, USA. All these inhibitors were dissolved in dimethyl sulfoxide (DMSO) (Himedia, India), aliquoted for single time use and stored at − 20°C. VPA was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMEM medium immediately before use.
Measurement of cell viability
Cell viability was measured by methyl-thiazol-tetrazolium (MTT) assay. In triplicates, cells were seeded into 96-well plates (Nunclon Cat no: 167008, Thermo Scientific); at 3 × 103 cells per well (100 μl) for 24 h. The next day, 100 μl of fresh medium was added containing different concentrations of drugs or vehicle (DMSO). VPA was used at a concentration of 0.5–6 mM; U0126 and LY294002 at 0.5–5 μM; SP600125 and SB203580 at 2.5–30 μM. For combination therapy, VPA was used with 0.5–2 mM with different concentrations of U0126, LY294002, SP600125, and SB203580 with multiple combinations. After incubation for 72 h, 100 μl MTT (3- [4, 5-dimethylthiazol-2-yl]-3, 5-diphenyltetrazolium bromide) solution (Himedia, India) was added to each well, at a final concentration of 1 mg/ml, and incubated at 37°C for 4 h. After incubation, the media/MTT solution was aspirated and Formazan crystals were dissolved in 100 μl DMSO (Himedia, India). The absorbance at 570nm was measured using a multi-mode microplate reader (FLUOstar Omega; BMG Labtech).
A 375 cells were seeded at a density of 105 cells in 30 mm tissue culture dish and allowed to adhere overnight. Cells were re-fed with fresh media containing either 2 mM of VPA or 2 μM of LY294002 or combination of 2 mM VPA and 2 μM LY294002. After 24 h of incubation, cells were trypsinized and plated onto new culture dishes at the density of 500 cells/dish and kept without the drug for additional 10 days. At completion of 10 days, culture dishes were washed with 1X phosphate buffered saline (PBS) (Himedia, India), fixed with methanol: Acetone (1:1 v/v) and stained with Giemsa (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. The remaining staining solution was removed, and culture dishes were washed with distilled water and dried at room temperature. Colonies (>50 cells) were counted under stereomicroscope (Stemi DV4, Carl Zeiss, Germany).
To detect the incidence of cell death, caspase assay was performed using the caspase assay kit (EMD Millipore, Cat. No 4500–0540) and quantified using the flow cytometry system (Guava EasyCyte, EMD Millipore, Hayward, CA) according to the manufacturers' instructions. Cells were seeded in 6-well plates (2 × 104 cells per well). Following 24 h, cells were treated with either 2 mM VPA or 2 μM LY294002 or a combination of both. Twenty-four hours after treatment cells were detached and harvested, washed in 1X PBS and suspended in 50 μL media. 10 μl of 10X FLICA reagent was added, mixed, well and incubated in dark for 1 h at 37°C and 5% CO2. Cells were washed twice and 150 μl master mix propidium iodide was added and incubated at room temp for 5 min. Cell samples were analyzed in the Guava EasyCyte (EMD Millipore).
Ethynyl-2′-deoxyuridine incorporation assay for cell proliferation
For cell proliferation, Click-iT ® ethynyl-2′-deoxyuridine (EdU) Microplate Assay (Invitrogen kit; Catalog no. C10214) was used. This assay utilizes the nucleoside analog EdU (5-ethynyl-2′-deoxyuridine) for DNA incorporation during active DNA synthesis. Cells were seeded into 96-well plates in triplicates at 3 × 103 cells per well (100 μl) for 24 h in black 96-well plates (Nunclon Delta Black Microwell; Cat no: 137,101, Thermo Scientific) and after overnight attachment, treated with inhibitor/vehicle for 24 h. Next day, 10 μM EdU was added in each well containing inhibitor/vehicle-medium and incubated for another 24 h. Following incubation the assay performed according to manufacturer's instruction. The Fluorescence (520 ex/590 em) was measured using multi-mode microplate reader (FLUOstar Omega; BMG Labtech).
Data were presented as the mean ± standard error from three independent experiments. Statistical analyses were performed by Student's t-test as well as one way ANOVA. P<0.05 was considered to indicate a statistically significant difference.
| Results|| |
Reduction in cell viability with valproic acid, U0126, LY294002, SP600125, and SB203580
In order to determine the influence of the VPA, U0126, LY294002, SP600125, and SB203580 on cell viability, A375 cells were incubated in the presence of different concentrations (0.5–6 mM) of VPA. The effect of VPA on 72 h of incubation is shown in [Figure 1]. VPA showed the concentration-dependent reduction in cell viability. However, more than 50% reduction in cell viability was observed only at 2 mM or higher doses. U0126 was effective even at lower doses while LY294002, SP600125, and SB203580 are effective only at higher doses [Figure 1].
|Figure 1: Cytotoxic effect of valproic acid and different inhibitors of downstream molecules of epidermal growth factor receptor pathway. Valproic acid as well as inhibitors of ERK1/2(U0126), PI3K-AKT (LY294002), Jun N-terminal kinases (SP600125) as well as p38 kinase (SB203580) were tested for their cytotoxicity on A375 cells using methyl-thiazol-tetrazolium assay|
Click here to view
Combination of valproic acid with LY294002 significantly reduces cell viability
To find a combination that could reduce tumor cell viability, 1 and 2 mM concentrations of VPA was treated with 0.5, 1 and 2 μM concentrations of U0126 and LY294002. Further, 0.5, 1, and 2 mM concentrations of VPA were treated with 5 and 10 μM concentrations of SP600125 and SB203580. Although VPA with U0126 showed a significant reduction in cell viability, it is not different from cell viability observed with U0126 alone [Figure 1] and [Figure 2]. However, we observed a significant reduction with cell viability in VPA with LY294002 compared to the effect of these two individual drugs. Even the combination of VPA with SP600125 produced a significant reduction in cell viability although at higher concentrations [Figure 2]. No significant reduction was observed with the combination of VPA with SB203580 [Figure 2].
|Figure 2: Cytotoxic effect of valproic acid in combination with different inhibitors of downstream molecules of epidermal growth factor receptor pathway. Cytotoxic effect of valproic acid was tested in combination with inhibitors of ERK1/2, PI3K-AKT, Jun N-terminal kinases as well as p-38 kinase at multiple concentrations on A375 cells using methyl-thiazol-tetrazolium assay|
Click here to view
Reduced cell survival was evident from clonogenic assay
Clonogenic assay was performed to study the effect of VPA and LY294002 combination on survival of A375 cells. Scoring of colonies containing more than 50 cells under a stereo microscope revealed that VPA (2 mM) and LY 294002 (2 μM) individual drugs showed significant reduction (P < 0.01 and P< 0.05, respectively) in survival of A375 cells compared to untreated A375 cells. Further, the combination of these at this specified concentration further reduced (P < 0.0001) the survival of the A375 cells [Figure 3].
|Figure 3: Clonogenic cell survival assay. A representative image of the clonogenic assay (left panels). Valproic acid (2mM) as well as LY294002 (2μM) individual drugs showed a significant reduction (P < 0.01 and P < 0.05, respectively) in survival of A375 cells compared to untreated A375 cells. Combination of valproic acid and LY294002 at the same concentrations further reduced (P < 0.0001) the survival of the A375 cells|
Click here to view
Enhanced cytotoxic effect of valproic acid with LY294002 was confirmed by caspase assay
To validate the cytotoxic effect of the combination of VPA and LY294002, we have performed caspase assay for the evaluation of caspase activation in dying A375 cells. For this, control A375 cells, cells treated with 2 mM VPA, 2 μM LY294002 and their combination was tested [Figure 3]. Lower levels of caspase activity were observed in control A375 cells. Treating cells with either VPA or LY294002 resulted in increased cell death while combined treatment further enhanced the caspase activity [Figure 4] suggesting the beneficial effects of this combined treatment.
|Figure 4: Cytotoxic effect of valproic acid in combination with PI3K-AKT inhibitor LY294002. Cytotoxic effect of valproic acid was tested in combination with PI3K-AKT inhibitor LY294002 on A375 cells using caspase assay. Box label: Upper left: Necrotic cells; Lower left: viable cells; Lower right: Early/mid apoptotic cells; Upper right: Late apoptotic cells|
Click here to view
Antiproliferative effect of combination of valproic acid with LY294002
While results from MTT assay is a measure of cell proliferation as well as cell death, to check whether the combination of VPA (2 mM) with LY294002 (2 μM) possess any antiproliferative effect, A375 cell proliferation was assessed by EdU incorporation assay. EdU detection is based on a click reaction-a copper-catalyzed covalent reaction between an azide and an alkyne. In this assay, the EdU contains the alkyne and Oregon Green ® 488 dye contains the azide. VPA (2 mM) significantly reduced A375 cell proliferation compared to control A375 cells (P < 0.05) whereas LY294002 (2 μM) failed to show any antiproliferative effect at the tested concentration. However, when a combination of VPA (2 mM) was used with LY294002 (2 μM), cell proliferation was significantly reduced (P < 0.01) [Figure 5].
|Figure 5: Evaluation of cell proliferation using ethynyl-2′-deoxyuridine incorporation assay. Anti-proliferative effect of valproic acid was tested in combination with PI3K-AKT inhibitor LY294002 on A375 cells using ethynyl-2′-deoxyuridine incorporation assay. Valproic acid (2 mM) significantly (P < 0.05) reduced proliferation of A375 cells while cell proliferation is unaffected with LY294002 (2μM). However, a combination of valproic acid (2 mM) and LY294002 (2μM) showed enhanced (P < 0.01) antiproliferative effects on A375 cells|
Click here to view
| Discussion|| |
The strong rationale for using epigenetic agents such as VPA for treating cancer is based on the fact that epigenetic changes such as aberrant DNA methylation and histone acetylation are common in cancer. Since EGFR and its downstream molecules play central roles in tumor cell survival, proliferation as well as migration, targeting them with VPA is expected to provide a clear advantage for controlling the robust growth of tumor cells. Among these downstream molecules, overexpression of ERK1/2 and PI3K-AKT were observed in multiple type of tumors whereas JNK and p-38 kinases are upregulated on induction of stress. In the present study, as monotherapy agent, VPA provided better cytotoxicity only at higher doses, similar to previous observation. However, combined treatment of VPA with multiple inhibitors of EGFR downstream molecules provided varying amounts of cytotoxicity. Among the combinations, VPA with PI3K-AKT inhibitor LY294002 offered enhanced cytotoxicity compared to other combinations.
We have three observations from the present study. First of all, cell death induced by combination of VPA with ERK1/2 inhibitor is not significantly higher compared to ERK1/2 inhibition alone. Second, cell death induced by combination of VPA with PI3K-AKT inhibitor is significantly higher compared to inhibition of PI3K-AKT alone. Third, combination of VPA with JNK or p38 kinase inhibitor showed enhanced cell death only at higher concentrations. Taken together, these results indicate that combination of VPA with PI3K-AKT inhibitor is a better option if a combined treatment is required. These can be explained on the basis of differential potential of VPA in inhibiting ERK1/2 and AKT apart from its HDACi activity. From our results, it appears that VPA failed to enhance ERK1/2 inhibitor-induced cell death while it enhanced PI3K-AKT inhibitor-induced cell death. This indicates that VPA mostly inhibits ERK1/2 activity apart from its HDACi activity. This is explained based on the well-known fact that tumor cells can grow when one of the PI3K-AKT or ERK1/2 pathway is active, and also ERK1/2 contribution for cell proliferation is higher while PI3K–AKT contribution for cell survival is high. When both pathways are inhibited more cytotoxicity is expected, which is seen in the combination of VPA with LY294002, confirmed through MTT assay, clonogenic assay as well as caspase assay. ERK1/2 inhibition by VPA is further confirmed from our antiproliferative assay with EdU, where significant reduction in cell proliferation was observed with VPA and LY294002 combination whereas LY294002 at the tested concentration failed to show significant antiproliferative activity. In support of this hypothesis, an earlier study showed the up-regulation of AKT on inhibition of tumor cell growth with VPA  while another study  showed the beneficial effect of treating target of rapamycin inhibitor RAD001with VPA.
Financial support and sponsorship
The work was supported by funding from BRNS 2013/34/8, sanctioned to DU.
BSK received financial assistance as Senior Research Fellowship (Ref No. 08/652(001)/2017- EMR-l) from Council for Scientific and Industrial Research (CSIR), India.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mazurkiewicz-Bełdzińska M, Szmuda M, Matheisel A. Long-term efficacy of valproate versus lamotrigine in treatment of idiopathic generalized epilepsies in children and adolescents. Seizure 2010;19:195-7.
Chavez-Blanco A, Segura-Pacheco B, Perez-Cardenas E, Taja-Chayeb L, Cetina L, Candelaria M, et al.
Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A phase I study. Mol Cancer 2005;4:1-9.
Atmaca A, Al-Batran SE, Maurer A, Neumann A, Heinzel T, Hentsch B, et al.
Valproic acid (VPA) in patients with refractory advanced cancer: A dose escalating phase I clinical trial. Br J Cancer 2007;97:177-82.
Arce C, Pérez-Plasencia C, González-Fierro A, de la Cruz-Hernández E, Revilla-Vázquez A, Chávez-Blanco A, et al.
A proof-of-principle study of epigenetic therapy added to neoadjuvant doxorubicin cyclophosphamide for locally advanced breast cancer. PLoS One 2006;1:e98.
Münster P, Marchion D, Bicaku E, Schmitt M, Lee JH, DeConti R, et al.
Phase I trial of histone deacetylase inhibition by valproic acid followed by the topoisomerase II inhibitor epirubicin in advanced solid tumors: A clinical and translational study. J Clin Oncol 2007;25:1979-85.
Munster P, Marchion D, Bicaku E, Lacevic M, Kim J, Centeno B, et al.
Clinical and biological effects of valproic acid as a histone deacetylase inhibitor on tumor and surrogate tissues: Phase I/II trial of valproic acid and epirubicin/FEC. Clin Cancer Res 2009;15:2488-96.
Chateauvieux S, Morceau F, Dicato M, Diederich M. Molecular and therapeutic potential and toxicity of valproic acid. J Biomed Biotechnol 2010;2010. pii: 479364.
Avallone A, Piccirillo MC, Delrio P, Pecori B, Di Gennaro E, Aloj L, et al.
Phase 1/2 study of valproic acid and short-course radiotherapy plus capecitabine as preoperative treatment in low-moderate risk rectal cancer-V-shoRT-R3 (Valproic acid – Short radiotherapy – Rectum 3rd
trial). BMC Cancer 2014;14:875.
Wheler JJ, Janku F, Falchook GS, Jackson TL, Fu S, Naing A, et al.
Phase I study of anti-VEGF monoclonal antibody bevacizumab and histone deacetylase inhibitor valproic acid in patients with advanced cancers. Cancer Chemother Pharmacol 2014;73:495-501.
Coronel J, Cetina L, Pacheco I, Trejo-Becerril C, González-Fierro A, de la Cruz-Hernandez E, et al.
A double-blind, placebo-controlled, randomized phase III trial of chemotherapy plus epigenetic therapy with hydralazine valproate for advanced cervical cancer. Preliminary results. Med Oncol 2011;28 Suppl 1:S540-6.
Bilen MA, Fu S, Falchook GS, Ng CS, Wheler JJ, Abdelrahim M, et al.
Phase I trial of valproic acid and lenalidomide in patients with advanced cancer. Cancer Chemother Pharmacol 2015;75:869-74.
Issa JP, Garcia-Manero G, Huang X, Cortes J, Ravandi F, Jabbour E, et al.
Results of phase 2 randomized study of low-dose decitabine with or without valproic acid in patients with myelodysplastic syndrome and acute myelogenous leukemia. Cancer 2015;121:556-61.
Scherpereel A, Berghmans T, Lafitte JJ, Colinet B, Richez M, Bonduelle Y, et al.
Valproate-doxorubicin: Promising therapy for progressing mesothelioma. A phase II study. Eur Respir J 2011;37:129-35.
Daud AI, Dawson J, DeConti RC, Bicaku E, Marchion D, Bastien S, et al.
Potentiation of a topoisomerase I inhibitor, karenitecin, by the histone deacetylase inhibitor valproic acid in melanoma: Translational and phase I/II clinical trial. Clin Cancer Res 2009;15:2479-87.
Citri A, Yarden Y. EGF-ERBB signalling: Towards the systems level. Nat Rev Mol Cell Biol 2006;7:505-16.
Brodie SA, Brandes JC. Could valproic acid be an effective anticancer agent? The evidence so far. Expert Rev Anticancer Ther 2014;14:1097-100.
Chodurek E, Orchel A, Gawlik N, Kulczycka A, Gruchlik A, Dzierzewicz Z, et al.
Proliferation and cellular death of A375 cell line in the presence of HDACs inhibitors. Acta Pol Pharm 2010;67:686-9.
Zhao Y, You W, Zheng J, Chi Y, Tang W, Du R, et al.
Valproic acid inhibits the angiogenic potential of cervical cancer cells via HIF-1α/VEGF signals. Clin Transl Oncol 2016;18:1123-30.
Juengel E, Makarević J, Tsaur I, Bartsch G, Nelson K, Haferkamp A, et al.
Resistance after chronic application of the HDAC-inhibitor valproic acid is associated with elevated akt activation in renal cell carcinoma in vivo
. PLoS One 2013;8:e53100.
Wedel S, Hudak L, Seibel JM, Juengel E, Tsaur I, Wiesner C, et al.
Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001. Life Sci 2011;88:418-24.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]