{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:53:15Z","timestamp":1785426795961,"version":"3.56.0"},"reference-count":54,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T00:00:00Z","timestamp":1780272000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001325","name":"Cancer Association of South Africa","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001325","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>Colorectal cancer is the predominant and recurring cancer leading to the second largest cause of mortality, globally. Abnormal regulation of oncogenes and deactivation of tumor suppressor genes over an extended period were key mechanisms underlying colon tumorigenesis. As a result, an effective drug targeting these genes should be explored to combat this disease. We aim to explore the effects of Valproic acid (VPA), a histone deacetylase inhibitor (HDACi), and Zebularine (ZEB), DNA methyltransferase inhibitor, on the expression of the Kr\u00fcppel-like factor 4 (KLF4) and CTNNB1 (\u03b2-catenin) in an early-stage colon cells (SW480 cells) and a late-stage colon cell (DLD-1 cells). Using several in-vitro assays including cell viability, proliferation assay, single cell imaging and analysis and molecular assays including qPCR, protein expression analysis, we have assessed the anticancer properties of both drugs, VPA and ZEB. The synergistic effect of VPA and ZEB could effectively inhibit the proliferation of colon cells than their independent doses. The gene expression profile revealed 2-fold increase in the expression of KLF4 in SW480 cells, with increase to about 22-fold in DLD-1 cells. Notably, CTNNB1 was downregulated than KLF4 in a dose dependent manner, potentially leading to antitumorigenic and anti-proliferative properties of the combinatorial drug. Cellular localization of KLF4 and CTNNB1 protein expression in cytoplasm and nucleus with morphological changes of the cancer cells revealed the onset of programmed cell death. qPCR analysis also showed the downregulation of KLF4 and CTNNB1 upon synergistic activity of ZEB and VPA treated colon cancer cells. Microscopic analysis confirmed the upregulation of KLF4 in ZEB treatment cells leading to DNA methylation. Finally, single cell image analysis has shown the reduced expression of both KLF4 and CTNNB1. Overall, the analysis confirmed that synergistic effect of ZEB and VPA served as a potential anti-colon cancer agent.<\/jats:p>","DOI":"10.1371\/journal.pone.0348215","type":"journal-article","created":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T17:44:58Z","timestamp":1780335898000},"page":"e0348215","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Combinatorial drug repurposing of Valproic acid and Zebularine regulates Kr\u00fcppel-like factor 4 and \u03b2-catenin expression in colon cancer cells"],"prefix":"10.1371","volume":"21","author":[{"given":"Jeyalakshmi","family":"Kandhavelu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kumar","family":"Subramanian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natanya","family":"Moodley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kasim S.","family":"Abass","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chandrabose","family":"Sureka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4986-055X","authenticated-orcid":true,"given":"Meenakshisundaram","family":"Kandhavelu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1055-1730","authenticated-orcid":true,"given":"Paul","family":"Ruff","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clement","family":"Penny","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,6,1]]},"reference":[{"key":"pone.0348215.ref001","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1093\/clinchem\/hvad202","article-title":"Epidemiology of Cancer","volume":"70","author":"SM Schwartz","year":"2024","journal-title":"Clinical Chemistry"},{"key":"pone.0348215.ref002","first-page":"209","article-title":"Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries","volume":"71","author":"H Sung","year":"2021","journal-title":"CA: A Cancer Journal for Clinicians"},{"issue":"1","key":"pone.0348215.ref003","doi-asserted-by":"crossref","first-page":"204","DOI":"10.1186\/s12876-022-02275-0","article-title":"Burden and trend of colorectal cancer in 54 countries of Africa 2010-2019: a systematic examination for Global Burden of Disease","volume":"22","author":"AF Awedew","year":"2022","journal-title":"BMC Gastroenterol"},{"issue":"8","key":"pone.0348215.ref004","doi-asserted-by":"crossref","first-page":"2374","DOI":"10.3748\/wjg.v21.i8.2374","article-title":"Retrospective study of colorectal cancer in Zimbabwe: colonoscopic and clinical correlates","volume":"21","author":"L Katsidzira","year":"2015","journal-title":"World J Gastroenterol"},{"key":"pone.0348215.ref005"},{"key":"pone.0348215.ref006","first-page":"2449","article-title":"Molecular origins of cancer","volume":"361","author":"SD Markowitz","year":"2009","journal-title":"N Engl J Med"},{"key":"pone.0348215.ref007","article-title":"Cancer statistics, 2009","author":"A Jemal","year":"2009","journal-title":"CA: A Cancer Journal for Clinicians"},{"issue":"2","key":"pone.0348215.ref008","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/S0092-8674(03)01075-4","article-title":"Principles of tumor suppression","volume":"116","author":"CJ Sherr","year":"2004","journal-title":"Cell"},{"issue":"32","key":"pone.0348215.ref009","doi-asserted-by":"crossref","first-page":"30423","DOI":"10.1074\/jbc.M101194200","article-title":"Kr\u00fcppel-like factor 4 (gut-enriched Kr\u00fcppel-like factor) inhibits cell proliferation by blocking G1\/S progression of the cell cycle","volume":"276","author":"X Chen","year":"2001","journal-title":"J Biol Chem"},{"issue":"6","key":"pone.0348215.ref010","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1002\/bies.20581","article-title":"The diverse functions of Kr\u00fcppel-like factors 4 and 5 in epithelial biology and pathobiology","volume":"29","author":"BB McConnell","year":"2007","journal-title":"Bioessays"},{"key":"pone.0348215.ref011","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.gene.2017.02.025","article-title":"Kr\u00fcppel-like factor 4 (KLF4): What we currently know","volume":"611","author":"AM Ghaleb","year":"2017","journal-title":"Gene"},{"issue":"2","key":"pone.0348215.ref012","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1128\/MCB.00063-09","article-title":"KLF4 interacts with beta-catenin\/TCF4 and blocks p300\/CBP recruitment by beta-catenin","volume":"30","author":"PM Evans","year":"2010","journal-title":"Mol Cell Biol"},{"issue":"6","key":"pone.0348215.ref013","doi-asserted-by":"crossref","first-page":"2055","DOI":"10.1128\/MCB.26.6.2055-2064.2006","article-title":"Novel cross talk of Kruppel-like factor 4 and beta-catenin regulates normal intestinal homeostasis and tumor repression","volume":"26","author":"W Zhang","year":"2006","journal-title":"Mol Cell Biol"},{"issue":"10","key":"pone.0348215.ref014","doi-asserted-by":"crossref","first-page":"1489","DOI":"10.1634\/stemcells.2005-0034","article-title":"Defining the role of Wnt\/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells","volume":"23","author":"G Dravid","year":"2005","journal-title":"Stem Cells"},{"issue":"3","key":"pone.0348215.ref015","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/j.cell.2006.10.018","article-title":"Wnt\/beta-catenin signaling in development and disease","volume":"127","author":"H Clevers","year":"2006","journal-title":"Cell"},{"key":"pone.0348215.ref016","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1042\/bj20021321","article-title":"Histone deacetylases (HDACs): characterization of the classical HDAC family","volume":"370","author":"AJM de Ruijter","year":"2003","journal-title":"Biochem J"},{"issue":"24","key":"pone.0348215.ref017","doi-asserted-by":"crossref","first-page":"6969","DOI":"10.1093\/emboj\/20.24.6969","article-title":"Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells","volume":"20","author":"M G\u00f6ttlicher","year":"2001","journal-title":"EMBO J"},{"issue":"7","key":"pone.0348215.ref018","doi-asserted-by":"crossref","first-page":"2537","DOI":"10.1158\/0008-5472.CAN-04-3011","article-title":"Valproic acid stimulates proliferation and self-renewal of hematopoietic stem cells","volume":"65","author":"G Bug","year":"2005","journal-title":"Cancer Res"},{"issue":"5","key":"pone.0348215.ref019","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1093\/jnci\/95.5.399","article-title":"Inhibition of DNA methylation and reactivation of silenced genes by zebularine","volume":"95","author":"JC Cheng","year":"2003","journal-title":"J Natl Cancer Inst"},{"issue":"10","key":"pone.0348215.ref020","doi-asserted-by":"crossref","first-page":"3862","DOI":"10.1158\/1078-0432.CCR-04-2406","article-title":"Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine","volume":"11","author":"JL Holleran","year":"2005","journal-title":"Clin Cancer Res"},{"key":"pone.0348215.ref021","doi-asserted-by":"crossref","first-page":"3219","DOI":"10.1038\/srep03219","article-title":"Zebularine inhibits tumorigenesis and stemness of colorectal cancer via p53-dependent endoplasmic reticulum stress","volume":"3","author":"P-M Yang","year":"2013","journal-title":"Sci Rep"},{"issue":"6","key":"pone.0348215.ref022","first-page":"689","article-title":"HDAC inhibitor, valproic acid, induces p53-dependent radiosensitization of colon cancer cells","volume":"24","author":"X Chen","year":"2009","journal-title":"Cancer Biother Radiopharm"},{"issue":"10","key":"pone.0348215.ref023","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1016\/j.bcp.2005.02.012","article-title":"Histone deacetylases inhibition and tumor cells cytotoxicity by CNS-active VPA constitutional isomers and derivatives","volume":"69","author":"S Eyal","year":"2005","journal-title":"Biochem Pharmacol"},{"key":"pone.0348215.ref024","first-page":"479364","article-title":"Molecular and therapeutic potential and toxicity of valproic acid","volume":"2010","author":"S Chateauvieux","year":"2010","journal-title":"BioMed Research International"},{"issue":"2","key":"pone.0348215.ref025","doi-asserted-by":"crossref","first-page":"513","DOI":"10.3892\/ijmm.2014.1795","article-title":"Valproic acid, an anti-epileptic drug and a histone deacetylase inhibitor, in combination with proteasome inhibitors exerts antiproliferative, pro-apoptotic and chemosensitizing effects in human colorectal cancer cells: underlying molecular mechanisms","volume":"34","author":"M-SI Abaza","year":"2014","journal-title":"Int J Mol Med"},{"issue":"2","key":"pone.0348215.ref026","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.ccr.2004.06.023","article-title":"Preferential response of cancer cells to zebularine","volume":"6","author":"JC Cheng","year":"2004","journal-title":"Cancer Cell"},{"issue":"3","key":"pone.0348215.ref027","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1007\/s10549-009-0420-3","article-title":"Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells","volume":"120","author":"M Billam","year":"2010","journal-title":"Breast Cancer Res Treat"},{"issue":"18","key":"pone.0348215.ref028","doi-asserted-by":"crossref","first-page":"2161","DOI":"10.1038\/onc.2010.591","article-title":"Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion","volume":"30","author":"F Yu","year":"2011","journal-title":"Oncogene"},{"key":"pone.0348215.ref029","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/j.fob.2015.02.003","article-title":"Kr\u00fcppel-like factor 4 induces apoptosis and inhibits tumorigenic progression in SK-BR-3 breast cancer cells","volume":"5","author":"B Wang","year":"2015","journal-title":"FEBS Open Bio"},{"key":"pone.0348215.ref030","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1007\/s10495-022-01735-y","article-title":"Programmed cell death detection methods: a systematic review and a categorical comparison","volume":"27","author":"S Kari","year":"2022","journal-title":"Apoptosis"},{"issue":"10","key":"pone.0348215.ref031","doi-asserted-by":"crossref","DOI":"10.1186\/gb-2006-7-10-r100","article-title":"CellProfiler: image analysis software for identifying and quantifying cell phenotypes","volume":"7","author":"AE Carpenter","year":"2006","journal-title":"Genome Biol"},{"issue":"1","key":"pone.0348215.ref032","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1109\/TSMC.1979.4310076","article-title":"A Threshold Selection Method from Gray-Level Histograms","volume":"9","author":"N Otsu","year":"1979","journal-title":"IEEE Trans Syst, Man, Cybern"},{"issue":"3","key":"pone.0348215.ref033","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1083\/jcb.200109094","article-title":"DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death","volume":"157","author":"B Inbal","year":"2002","journal-title":"J Cell Biol"},{"issue":"2","key":"pone.0348215.ref034","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S0959-437X(99)80026-4","article-title":"Histone acetylation and cancer","volume":"9","author":"SY Archer","year":"1999","journal-title":"Curr Opin Genet Dev"},{"key":"pone.0348215.ref035","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1111\/j.1749-6632.2003.tb05977.x","article-title":"Histone Acetylation and Gastrointestinal Carcinogenesis","volume":"983","author":"W Yasui","year":"2003","journal-title":"Ann N Y Acad Sci"},{"key":"pone.0348215.ref036","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1038\/icb.2011.100","article-title":"HDAC inhibitors in cancer biology: emerging mechanisms and clinical applications","volume":"90","author":"O Khan","year":"2012","journal-title":"Immunology & Cell Biology"},{"issue":"5","key":"pone.0348215.ref037","doi-asserted-by":"crossref","first-page":"1492","DOI":"10.3892\/ol.2013.1565","article-title":"Inhibitory effect of valproic acid on bladder cancer in combination with chemotherapeutic agents in vitro and in vivo","volume":"6","author":"D Wang","year":"2013","journal-title":"Oncol Lett"},{"issue":"11","key":"pone.0348215.ref038","doi-asserted-by":"crossref","first-page":"1148","DOI":"10.1056\/NEJMra072067","article-title":"Epigenetics in cancer","volume":"358","author":"M Esteller","year":"2008","journal-title":"N Engl J Med"},{"issue":"54","key":"pone.0348215.ref039","doi-asserted-by":"crossref","DOI":"10.1126\/scitranslmed.3001338","article-title":"An integrated genomic and epigenomic approach predicts therapeutic response to zebularine in human liver cancer","volume":"2","author":"JB Andersen","year":"2010","journal-title":"Sci Transl Med"},{"issue":"3","key":"pone.0348215.ref040","doi-asserted-by":"crossref","first-page":"1270","DOI":"10.1128\/MCB.24.3.1270-1278.2004","article-title":"Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells","volume":"24","author":"JC Cheng","year":"2004","journal-title":"Mol Cell Biol"},{"issue":"3","key":"pone.0348215.ref041","doi-asserted-by":"crossref","first-page":"1079","DOI":"10.1158\/0008-5472.CAN-03-0799","article-title":"Histone deacetylase is a target of valproic acid-mediated cellular differentiation","volume":"64","author":"N Gurvich","year":"2004","journal-title":"Cancer Res"},{"issue":"1","key":"pone.0348215.ref042","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/1476-4598-4-22","article-title":"Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A phase I study","volume":"4","author":"A Chavez-Blanco","year":"2005","journal-title":"Mol Cancer"},{"issue":"4","key":"pone.0348215.ref043","first-page":"1327","article-title":"DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors","volume":"61","author":"WG Zhu","year":"2001","journal-title":"Cancer Res"},{"issue":"8","key":"pone.0348215.ref044","doi-asserted-by":"crossref","first-page":"2709","DOI":"10.1158\/1078-0432.CCR-03-0484","article-title":"Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer","volume":"10","author":"AY Pandya","year":"2004","journal-title":"Clin Cancer Res"},{"issue":"8","key":"pone.0348215.ref045","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1007\/s00432-008-0353-y","article-title":"Dynamic down-regulation of Kr\u00fcppel-like factor 4 in colorectal adenoma-carcinoma sequence","volume":"134","author":"J Xu","year":"2008","journal-title":"J Cancer Res Clin Oncol"},{"issue":"22","key":"pone.0348215.ref046","first-page":"6488","article-title":"Increase of GKLF messenger RNA and protein expression during progression of breast cancer","volume":"60","author":"KW Foster","year":"2000","journal-title":"Cancer Res"},{"issue":"11","key":"pone.0348215.ref047","doi-asserted-by":"crossref","first-page":"1701","DOI":"10.1136\/gut.2004.052704","article-title":"Apoptosis and colorectal cancer","volume":"53","author":"AJM Watson","year":"2004","journal-title":"Gut"},{"key":"pone.0348215.ref048","doi-asserted-by":"crossref","first-page":"750230","DOI":"10.1155\/2011\/750230","article-title":"Apoptotic Cell Death and Inhibition of Wnt\/\u03b2-Catenin Signaling Pathway in Human Colon Cancer Cells by an Active Fraction (HS7) from Taiwanofungus camphoratus","volume":"2011","author":"C-T Yeh","year":"2011","journal-title":"Evid Based Complement Alternat Med"},{"issue":"1","key":"pone.0348215.ref049","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/S1476-5586(04)80048-4","article-title":"A monoclonal antibody against Wnt-1 induces apoptosis in human cancer cells","volume":"6","author":"B He","year":"2004","journal-title":"Neoplasia"},{"key":"pone.0348215.ref050","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1101\/gad.324319.119","article-title":"KLF4 protein stability regulated by interaction with pluripotency transcription factors overrides transcriptional control","volume":"33","author":"NK Dhaliwal","year":"2019","journal-title":"Genes Dev"},{"issue":"22","key":"pone.0348215.ref051","doi-asserted-by":"crossref","first-page":"10394","DOI":"10.1158\/0008-5472.CAN-05-2059","article-title":"Destabilization of Kr\u00fcppel-like factor 4 protein in response to serum stimulation involves the ubiquitin-proteasome pathway","volume":"65","author":"ZY Chen","year":"2005","journal-title":"Cancer Res"},{"issue":"9","key":"pone.0348215.ref052","doi-asserted-by":"crossref","first-page":"6890","DOI":"10.1074\/jbc.M110.179952","article-title":"Regulation of Kr\u00fcppel-like factor 4 by the anaphase promoting complex pathway is involved in TGF-beta signaling","volume":"286","author":"D Hu","year":"2011","journal-title":"J Biol Chem"},{"issue":"10","key":"pone.0348215.ref053","doi-asserted-by":"crossref","first-page":"5077","DOI":"10.1158\/0008-5472.CAN-05-2629","article-title":"Silencing of bidirectional promoters by DNA methylation in tumorigenesis","volume":"66","author":"J Shu","year":"2006","journal-title":"Cancer Res"},{"issue":"2","key":"pone.0348215.ref054","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1038\/nrc1799","article-title":"Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction?","volume":"6","author":"SB Baylin","year":"2006","journal-title":"Nat Rev Cancer"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0348215","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T17:45:09Z","timestamp":1780335909000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0348215"}},"subtitle":[],"editor":[{"given":"Suresh","family":"Yenugu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,6,1]]},"references-count":54,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6,1]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0348215","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,1]]}}}