Evaluation of the Molecular Effects of the Anticancer Adjuvant Valproic Acid on HEK293T Cells

Abstract

Objective: Valproic acid (VPA) can induce apoptosis while inhibiting proliferation in various cancer cells. This study adopted a novel strategy for investigating the molecular effects of VPA on non-cancerous cells. Building upon our previous work, which examined the effects of VPA in PANC-1 cells, we now turn our attention to HEK293T cells to determine the effect of VPA in non-cancerous cells. Materials and Methods: HEK293T cells were treated with 2.5 and 5-mM VPA. Flow cytometry measurements were performed on the 24th, 48th, 72nd, and 96th h. Assays for apoptosis and proliferation were conducted using annexin V/ propidium iodide (PI) staining and carboxyfluorescein-succinimidyl ester (CFSE), respectively. Results: No statistical significance was detected between the two different doses of VPA-treated cells and the controls at any time point. While early apoptosis values remained stable until 72 h in all groups, an increase was monitored in cells subjected to VPA for 96 h. Conclusion: Preliminary results indicated no proliferative effect of VPA treatment. However, it may induce apoptosis in long-term incubations. Nevertheless, additional doses of VPA at increased concentrations should be administered to explore cytotoxic levels and their impact on proliferation and apoptosis.

Keywords

• HEK293T
• VPA
• apoptosis
• proliferation

References

1. 1. Alenzi FQ. Links between apoptosis, proliferation and the cell cycle. Br J Biomed Sci 2004; 61(2): 99-102. google scholar
2. 2. Williams GH, Stoeber K. The cell cycle and cancer. J Pathol 2012; 226(2): 352-64. google scholar
3. 3. Perri F, Longo F, Giuliano M, Sabbatino F, Favia G, Ionna F, et al. Epigenetic control of gene expression: Potential implications for cancer treatment. Crit Rev Oncol Hematol 2017; 111: 166-72. google scholar
4. 4. Chen QW, Zhu XY, Li YY, Meng ZQ. Epigenetic regulation and cancer (review). Oncol Rep 2014; 31(2): 523-32. google scholar
5. 5. Cooper GM. The Cell: A molecular approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. google scholar
6. 6. Yagi Y, Fushida S, Harada S, Kinoshita J, Makino I, Oyama K, et al. Effects of valproic acid on the cell cycle and apoptosis through acetylation of histone and tubulin in a scirrhous gastric cancer cell line. J Exp Clin Cancer Res 2010; 29(1): 149. google scholar
7. 7. Bruserud O, Tsykunova G, Hernandez-Valladares M, Reikvam H, Tvedt THA. Therapeutic use of valproic acid and all-trans retinoic acid in Acute Myeloid Leukemia-literature review and discussion of possible use in relapse after allogeneic stem cell transplantation. Pharmaceuticals (Basel) 2021; 14(5): 423. google scholar
8. 8. Jankovic SM, Jankovic SV. Lessons learned from the discovery of sodium valproate and what has this meant to future drug discovery efforts? Expert Opin Drug Discov 2020; 15(11):1355-64. google scholar

9. 9. Xie Z, Ago Y, Okada N, Tachibana M. Valproic acid attenuates immunosuppressive function of myeloid-derived suppressor cells. J Pharmacol Sci 2018; 137(4): 359-65. google scholar
10. 10. Russo R, Kemp M, Bhatti UF, Pai M, Wakam G, Biesterveld B, et al. Life on the battlefield: Valproic acid for combat applications. J Trauma Acute Care Surg 2020; 89(2S Suppl 2): S69-S76. google scholar
11. 11. Gotfryd K, Skladchikova G, Lepekhin EA, Berezin V, Bock E, Walmod PS. Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation. BMC Cancer 2010; 10: 383. google scholar
12. 12. Ekici Y, Yilmaz A, Kucuksezer UC, Gazioglu SB, Yamalioglu ZD, Gurol AO, et al. Combined evaluation of proliferation and apoptosis to calculate IC(50) of VPA-induced PANC-1 cells and assessing its effect on the Wnt signaling pathway. Med Oncol 2021; 38(9): 109. google scholar
13. 13. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184(1): 39-51. google scholar
14. 14. Raz S, Sheban D, Gonen N, Stark M, Berman B, Assaraf YG. Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest. Cell Death Dis 2014; 5(2): e1067. google scholar
15. 15. Ahlen MT, Husebekk A, Killie MK, Skogen B, Stuge TB. T-cell responses associated with neonatal alloimmune thrombocytopenia: isolation of HPA-1a-specific, HLA-DRB3*0101-restricted CD4+ T cells. Blood 2009; 113(16): 3838-44. google scholar
16. 16. Zini R, Norfo R, Ferrari F, Bianchi E, Salati S, Pennucci V, et al. Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression. Exp Hematol 2012; 40(12): 1043-54.e6. google scholar
17. 17. Stapnes C, Ryningen A, Hatfield K, 0yan AM, Eide GE, Corbascio M, et al. Functional characteristics and gene expression profiles of primary acute myeloid leukaemia cells identify patient subgroups that differ in susceptibility to histone deacetylase inhibitors. Int J Oncol 2007; 31(6): 1529-38. google scholar
18. 18. Li XN, Shu Q, Su JM, Perlaky L, Blaney SM, Lau CC. Valproic acid induces growth arrest, apoptosis, and senescence in medulloblastomas by increasing histone hyperacetylation and regulating expression of p21Cip1, CDK4, and CMYC. Mol Cancer Ther 2005; 4(12): 1912-22. google scholar