title: In vitro effects of phenytoin and DAPT on MDA-MB-231 breast cancer cells.
abstract: Voltage-gated sodium channel (VGSC) activity enhances cell behaviors related to metastasis, such as motility, invasion, and oncogene expression. Neonatal alternative splice form of Nav1.5 isoform is expressed in metastatic breast cancers. Furthermore, aberrant Notch signaling pathway can induce oncogenesis and may promote the progression of breast cancers. In this study, we aimed to analyze the effect of the nNav1.5 inhibitor phenytoin and Notch signal inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester (DAPT) on triple negative breast cancer cell line (MDA-MB-231) via inhibition of nNav1.5 VGSC activity and Notch signaling, respectively. In order to determine the individual and combined effects of these inhibitors, the 4-[3-(4-iyodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) test, wound healing assay, and zymography were performed to detect the proliferation, lateral motility, and matrix metalloproteinase-9 (MMP9) activity, respectively. The expressions of nNav1.5, Notch4, MMP9, and tissue inhibitor of metalloproteinases-1 (TIMP1) were also detected by quantitative real-time reverse transcriptase-polymerase chain reaction. DAPT caused an antiproliferative effect when the doses were higher than 10 µM, whereas phenytoin showed no inhibitory action either alone or in combination with DAPT on the MDA-MB-231 cells. Furthermore, it was found that the lateral motility was inhibited by both inhibitors; however, this inhibitory effect was partially rescued when they were used in combination. Meanwhile, the results showed that the MMP9 activity and the ratio of MMP9 mRNA to TIMP1 mRNA were only decreased by DAPT. Thus, we conclude that the combined effect of DAPT and phenytoin is not as beneficial as using DAPT alone on MDA-MB-231 breast cancer cells.
authors.affiliation: Department of Medical Biochemistry, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey.
authors.forename: Canan Cakir
authors.initials: CC
authors.lastname: Aktas
authors.affiliation: Department of Histology and Embryology, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey.
authors.forename: N Dilara
authors.initials: ND
authors.lastname: Zeybek
authors.affiliation: Department of Medical Biochemistry, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey akpiskin@gmail.com.
authors.forename: A Kevser
authors.initials: AK
authors.lastname: Piskin
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004151:Dipeptides / Q000494:pharmacology*; D004347:Drug Interactions; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000235:genetics; D010672:Phenytoin / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000235:genetics; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics
chemical_list: D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D062554:NAV1.5 Voltage-Gated Sodium Channel; C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D000074562:Receptor, Notch4; D051880:Receptors, Notch; C568320:SCN5A protein, human; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D010672:Phenytoin; D020780:Matrix Metalloproteinase 9
keywords: Notch4 receptor; breast cancer; matrix metalloproteinase 9; tissue inhibitor of metalloproteinase 1; voltage-gated Na+ channel