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Combined evaluation of proliferation and apoptosis to calculate IC50 of VPA-induced PANC-1 cells and assessing its effect on the Wnt signaling pathway

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly cancers. Since most patients develop resistance to conventional treatments, new approaches are in urgency. Valproic acid (VPA) was shown to induce apoptosis and reduce proliferation in PANC-1 cells. Wnt signaling pathway is known to be involved in apoptosis and PDAC onset. However, VPA-induced apoptosis and its impact on Wnt signaling in PDACs are not linked, yet. We aimed to calculate IC50 of VPA-induced PANC-1 cells by combined analyses of proliferation and apoptosis, while assessing its effect on Wnt signaling pathway. PANC-1 was induced with increased VPA doses and time points. Three independent proliferation and apoptosis assays were performed utilizing carboxyfluorescein succinimidyl ester and Annexin V/PI staining, respectively. Flow cytometry measurements were analyzed by CellQuest and NovoExpress. Taqman hydrolysis probes and SYBR Green PCR Mastermix were assessed in expression analyses of Wnt components utilizing 2−ΔΔCt method. Cell proliferation was inhibited by 50% at 2.5 mM VPA that evoked a significant apoptotic response. Among the screened Wnt components and target genes, only LEF1 exhibited significant four-fold upregulation at this concentration. In conclusion, cancer studies mostly utilize MTT or BrdU assays in estimating cell proliferation and calculating IC50 of drugs, which provided conflicting VPA dosages utilizing PANC-1 cells. Our novel combined approach enabled specific, accurate and reproducible IC50 calculation at single cell basis with no apparent effect on Wnt signaling components. Future studies are needed to clarify the role of LEF1 in this model.

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References

  1. McGuire S, World Cancer Report. Geneva, Switzerland: World Health Organization, International Agency for Research on Cancer, WHO Press, 2015. Adv Nutr. 2014. https://doi.org/10.3945/an.116.012211.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018. https://doi.org/10.3322/caac.21492.

    Article  PubMed  Google Scholar 

  3. Gilardini Montani MS, Granato M, Santoni C, Del Porto P, Merendino N, D’Orazi G, et al. Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells. Cell Oncol (Dordr). 2017. https://doi.org/10.1007/s13402-017-0314-z.

    Article  Google Scholar 

  4. Kleger A, Perkhofer L, Seufferlein T. Smarter drugs emerging in pancreatic cancer therapy. Ann Oncol. 2014. https://doi.org/10.1093/annonc/mdu013.

    Article  PubMed  Google Scholar 

  5. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002. https://doi.org/10.1038/nrg816.

    Article  PubMed  Google Scholar 

  6. Spange S, Wagner T, Heinzel T, Krämer OH. Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol. 2009. https://doi.org/10.1016/j.biocel.2008.08.027.

    Article  PubMed  Google Scholar 

  7. Blaheta RA, Cinatl J Jr. Anti-tumor mechanisms of valproate: a novel role for an old drug. Med Res Rev. 2002. https://doi.org/10.1002/med.10017.

    Article  PubMed  Google Scholar 

  8. Michaelis M, Michaelis UR, Fleming I, Suhan T, Cinatl J, Blaheta RA, Hoffmann K, Kotchetkov R, Busse R, Nau H, Cinatl J Jr. Valproic acid inhibits angiogenesis in vitro and in vivo. Mol Pharmacol. 2004. https://doi.org/10.1124/mol.65.3.520.

    Article  PubMed  Google Scholar 

  9. Blaheta RA, Michaelis M, Driever PH, Cinatl J Jr. Evolving anticancer drug valproic acid: insights into the mechanism and clinical studies. Med Res Rev. 2005. https://doi.org/10.1002/med.20027.

    Article  PubMed  Google Scholar 

  10. Duenas-Gonzalez A, Candelaria M, Perez-Plascencia C, Perez-Cardenas E, de la Cruz-Hernandez E, Herrera LA. Valproic acid as epigenetic cancer drug: preclinical, clinical and transcriptional effects on solid tumors. Cancer Treat Rev. 2008. https://doi.org/10.1016/j.ctrv.2007.11.003.

    Article  PubMed  Google Scholar 

  11. Li Y, Liu T, Ivan C, Huang J, Shen DY, Kavanagh JJ, et al. Enhanced cytotoxic effects of combined valproic acid and the aurora kinase inhibitor VE465 on gynecologic cancer cells. Front Oncol. 2013. https://doi.org/10.3389/fonc.2013.00058.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kostrouchová M, Kostrouch Z, Kostrouchová M. Valproic acid, a molecular lead to multiple regulatory pathways. Folia Biol (Praha). 2007;53:37–49.

    Google Scholar 

  13. Jamieson C, Sharma M, Henderson BR. Targeting the β-catenin nuclear transport pathway in cancer. Semin Cancer Biol. 2014. https://doi.org/10.1016/j.semcancer.2014.04.012.

    Article  PubMed  Google Scholar 

  14. Sarkar S, Mandal C, Sangwan R, Mandal C. Coupling G2/M arrest to the Wnt/β-catenin pathway restrains pancreatic adenocarcinoma. Endocr Relat Cancer. 2014. https://doi.org/10.1530/ERC-13-0315.

    Article  PubMed  Google Scholar 

  15. Parish CR. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol. 1999. https://doi.org/10.1046/j.1440-1711.1999.00877.x.

    Article  PubMed  Google Scholar 

  16. 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. https://doi.org/10.1038/cddis.2014.39.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 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. https://doi.org/10.1182/blood-2008-09-178475.

    Article  PubMed  Google Scholar 

  18. 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. https://doi.org/10.1016/0022-1759(95)00072-i.

    Article  PubMed  Google Scholar 

  19. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010;60:277–300.

    Article  Google Scholar 

  20. Romoli M, Mazzocchetti P, D’Alonzo R, Siliquini S, Rinaldi VE, Verrotti A, et al. Valproic acid and epilepsy: from molecular mechanisms to clinical evidences. Curr Neuropharmacol. 2019. https://doi.org/10.2174/1570159X17666181227165722.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Lichun S, David H. Anti-convulsant drug valproic acid in cancers and in combination anti-cancer therapeutics. Mod Chem Appl. 2014. https://doi.org/10.4172/2329-6798.1000118.

    Article  Google Scholar 

  22. 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. https://doi.org/10.1200/JCO.2006.08.6165.

    Article  PubMed  Google Scholar 

  23. Lehner B, Sandner B, Marschallinger J, Lehner C, Furtner T, Couillard-Despres S, et al. The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival. Cell Tissue Res. 2011. https://doi.org/10.1007/s00441-011-1213-7.

    Article  PubMed  Google Scholar 

  24. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983. https://doi.org/10.1016/0022-1759(83)90303-4.

    Article  PubMed  Google Scholar 

  25. Han M, Li JF, Tan Q, Sun YY. Limitations of the use of MTT assay for screening in drug discovery. J Chin Pharmaceutical Sci. 2010. https://doi.org/10.5246/jcps.2010.03.027.

    Article  Google Scholar 

  26. Lü L, Zhang L, Wai MS, Yew DT, Xu J. Exocytosis of MTT formazan could exacerbate cell injury. Toxicol In Vitro. 2012. https://doi.org/10.1016/j.tiv.2012.02.006.

    Article  PubMed  Google Scholar 

  27. Miyashita T, Miki K, Kamigaki T, Makino I, Tajima H, Nakanuma S, et al. Low-dose valproic acid with low-dose gemcitabine augments MHC class I-related chain A/B expression without inducing the release of soluble MHC class I-related chain A/B. Oncol Lett. 2017. https://doi.org/10.3892/ol.2017.6943.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Li H, Zhang Z, Gao C, Wu S, Duan Q, Wu H, et al. Combination chemotherapy of valproic acid (VPA) and gemcitabine regulates STAT3/Bmi1 pathway to differentially potentiate the motility of pancreatic cancer cells. Cell Biosci. 2019. https://doi.org/10.1186/s13578-019-0312-0.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Venkataramani V, Rossner C, Iffland L, Schweyer S, Tamboli IY, Walter J, et al. Histone deacetylase inhibitor valproic acid inhibits cancer cell proliferation via down-regulation of the alzheimer amyloid precursor protein. J Biol Chem. 2010. https://doi.org/10.1074/jbc.M109.057836.

    Article  PubMed  PubMed Central  Google Scholar 

  30. 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;4:1912–22.

    Article  CAS  Google Scholar 

  31. Garnett MJ, Edelman EJ, Heidorn SJ, Greenman CD, Dastur A, Lau KW, et al. Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature. 2012. https://doi.org/10.1038/nature11005.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012. https://doi.org/10.1038/nature11003.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Heiser PW, Cano DA, Landsman L, Kim GE, Kench JG, Klimstra DS, et al. Stabilization of beta-catenin induces pancreas tumor formation. Gastroenterology. 2008. https://doi.org/10.1053/j.gastro.2008.06.089.

    Article  PubMed  Google Scholar 

  34. Wang L, Heidt DG, Lee CJ, Yang H, Logsdon CD, Zhang L, et al. Oncogenic function of ATDC in pancreatic cancer through Wnt pathway activation and beta-catenin stabilization. Cancer Cell. 2009. https://doi.org/10.1016/j.ccr.2009.01.018.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Jesse S, Koenig A, Ellenrieder V, Menke A. Lef-1 isoforms regulate different target genes and reduce cellular adhesion. Int J Cancer. 2010. https://doi.org/10.1002/ijc.24802.

    Article  PubMed  Google Scholar 

  36. Santiago L, Daniels G, Wang D, Deng FM, Lee P. Wnt signaling pathway protein LEF1 in cancer, as a biomarker for prognosis and a target for treatment. Am J Cancer Res. 2017;7:1389–406.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Pasca di Magliano M, Biankin AV, Heiser PW, Cano DA, Gutierrez PJ, Deramaudt T, et al. Common activation of canonical Wnt signaling in pancreatic adenocarcinoma. PLoS ONE. 2007. https://doi.org/10.1371/journal.pone.0001155.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

PANC-1 cell line was kindly provided by Thomas Linn, M.D. and Ali Osman Gurol, M.D Ph.D. in support of their joint project TÜBİTAK-INTENC Project No. 113S029.

Funding

This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University. Project No.: 32763.

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Authors and Affiliations

  1. Department of Genetics, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Vakif Gureba Caddesi, Capa, 34093, Istanbul, Turkey

    Yeliz Ekici & Feyza Nur Tuncer

  2. Graduate School of Health Sciences, Istanbul University, Istanbul, Turkey

    Yeliz Ekici

  3. Department of Immunology, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey

    Abdullah Yilmaz, Umut Can Kucuksezer, Sema Bilgic Gazioglu & Ali Osman Gurol

  4. Cell Processing Unit, Bone Marrow Transplantation Center, Yeni Yuzyil University Gaziosmanpasa Hospital, Istanbul, Turkey

    Zeynep Dogusan Yamalioglu

  5. Department of Medical Microbiology, Medical Faculty, Yeni Yuzyil University, Istanbul, Turkey

    Zeynep Dogusan Yamalioglu

  6. Diabetes Application and Research Center, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey

    Ali Osman Gurol & Feyza Nur Tuncer

  7. Clinical Research Unit, Center of Internal Medicine, Justus-Liebig-University, Giessen, Germany

    Thomas Linn

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  1. Yeliz Ekici
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Ekici, Y., Yilmaz, A., Kucuksezer, U.C. et al. Combined evaluation of proliferation and apoptosis to calculate IC50 of VPA-induced PANC-1 cells and assessing its effect on the Wnt signaling pathway. Med Oncol 38, 109 (2021). https://doi.org/10.1007/s12032-021-01560-4

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