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• W2920415219 abstract "Notch signaling is reported to be deregulated in several malignancies, including breast, and the enzyme γ-secretase plays an important role in the activation and nuclear translocation of Notch intracellular domain (NICD). Hence, pharmacological inhibition of γ-secretase might lead to the subsequent inhibition of Notch signaling in cancer cells. In search of novel γ-secretase inhibitors (GSIs), we screened a series of triazole-based compounds for their potential to bind γ-secretase and observed that 3-(3′4′,5′-trimethoxyphenyl)-5-(N-methyl-3′-indolyl)-1,2,4-triazole compound (also known as NMK-T-057) can bind to γ-secretase complex. Very interestingly, NMK-T-057 was found to inhibit proliferation, colony-forming ability, and motility in various breast cancer (BC) cells such as MDA-MB-231, MDA-MB-468, 4T1 (triple-negative cells), and MCF-7 (estrogen receptor (ER)/progesterone receptor (PR)–positive cell line) with negligible cytotoxicity against noncancerous cells (MCF-10A and peripheral blood mononuclear cells). Furthermore, significant induction of apoptosis and inhibition of epithelial-to-mesenchymal transition (EMT) and stemness were also observed in NMK-T-057–treated BC cells. The in silico study revealing the affinity of NMK-T-057 toward γ-secretase was further validated by a fluorescence-based γ-secretase activity assay, which confirmed inhibition of γ-secretase activity in NMK-T-057–treated BC cells. Interestingly, it was observed that NMK-T-057 induced significant autophagic responses in BC cells, which led to apoptosis. Moreover, NMK-T-057 was found to inhibit tumor progression in a 4T1-BALB/c mouse model. Hence, it may be concluded that NMK-T-057 could be a potential drug candidate against BC that can trigger autophagy-mediated cell death by inhibiting γ-secretase–mediated activation of Notch signaling. Notch signaling is reported to be deregulated in several malignancies, including breast, and the enzyme γ-secretase plays an important role in the activation and nuclear translocation of Notch intracellular domain (NICD). Hence, pharmacological inhibition of γ-secretase might lead to the subsequent inhibition of Notch signaling in cancer cells. In search of novel γ-secretase inhibitors (GSIs), we screened a series of triazole-based compounds for their potential to bind γ-secretase and observed that 3-(3′4′,5′-trimethoxyphenyl)-5-(N-methyl-3′-indolyl)-1,2,4-triazole compound (also known as NMK-T-057) can bind to γ-secretase complex. Very interestingly, NMK-T-057 was found to inhibit proliferation, colony-forming ability, and motility in various breast cancer (BC) cells such as MDA-MB-231, MDA-MB-468, 4T1 (triple-negative cells), and MCF-7 (estrogen receptor (ER)/progesterone receptor (PR)–positive cell line) with negligible cytotoxicity against noncancerous cells (MCF-10A and peripheral blood mononuclear cells). Furthermore, significant induction of apoptosis and inhibition of epithelial-to-mesenchymal transition (EMT) and stemness were also observed in NMK-T-057–treated BC cells. The in silico study revealing the affinity of NMK-T-057 toward γ-secretase was further validated by a fluorescence-based γ-secretase activity assay, which confirmed inhibition of γ-secretase activity in NMK-T-057–treated BC cells. Interestingly, it was observed that NMK-T-057 induced significant autophagic responses in BC cells, which led to apoptosis. Moreover, NMK-T-057 was found to inhibit tumor progression in a 4T1-BALB/c mouse model. Hence, it may be concluded that NMK-T-057 could be a potential drug candidate against BC that can trigger autophagy-mediated cell death by inhibiting γ-secretase–mediated activation of Notch signaling. Breast cancer (BC) 6The abbreviations used are: BCbreast cancerNICDNotch intracellular domainGSIγ-secretase inhibitorNMK-T-0573-(3′4′,5′-trimethoxyphenyl)-5-(N-methyl-3′-indolyl)-1,2,4-triazole compoundERestrogen receptorPRprogesterone receptorPBMCperipheral blood mononuclear cellTNBCtriple-negative breast cancerPSEN-1Presenilin-1APH-1anterior pharynx-defective 1PTENphosphatase and tensin homologNMKNMK-T-057PIpropidium iodideBWbody weightSGOTserum glutamate oxaloacetate transaminaseSGPTserum glutamate pyruvate transaminaseALPalkaline phosphataseEMTepithelial-to-mesenchymal transitionALDHaldehyde dehydrogenaseCSCcancer stem cellTICtumor-initiating cellpAktphosphorylated AktDAPTN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl esterdnp2,4-dinitrophenylNMAN-methyl aspartic acidLC3light chain 3MDCmonodansylcadaverine3-MA3-methyladenineZbenzyloxycarbonylfmkfluoromethyl ketonePCNAproliferating cell nuclear antigenTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labelingDMEMDulbecco’s modified Eagle’s mediumFBSfetal bovine serumMTT3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromideHRPhorseradish peroxidasePEphycoerythrinPEN-2Presenilin enhancer 2. is the most common cancer in women and accounts for almost 15% of all cancer-related deaths in women worldwide (1Ferlay J. Soerjomataram I. Dikshit R. Eser S. Mathers C. Rebelo M. Parkin D.M. Forman D. Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.Int. J Cancer. 2015; 136 (25220842): E359-E38610.1002/ijc.29210Crossref PubMed Scopus (21366) Google Scholar, 2Torre L.A. Siegel R.L. Ward E.M. Jemal A. Global cancer incidence and mortality rates and trends—an update.Cancer Epidemiol. Biomarkers Prev. 2016; 25 (26667886): 16-2710.1158/1055-9965.EPI-15-0578Crossref PubMed Scopus (2365) Google Scholar). In recent years, the rate and incidence of BC have increased in India at an alarming rate. Presently, BC is the most common cancer in the female population, preceding cervical cancer, in India with a very high mortality rate (3Ghoncheh M. Momenimovahed Z. Salehiniya H. Epidemiology, incidence and mortality of breast cancer in Asia.Asian Pac. J. Cancer Prev. 2016; 17 (27165207): 47-5210.7314/APJCP.2016.17.S3.47Crossref PubMed Scopus (65) Google Scholar, 4Gupta S. Breast cancer: Indian experience, data, and evidence.South Asian J. Cancer. 2016; 5 (27606287): 85-8610.4103/2278-330X.187552Crossref PubMed Google Scholar). BC may be classified into distinct molecular subtypes, including normal-like, luminal A and B, HER2+, and basal-like (5Sørlie T. Perou C.M. Tibshirani R. Aas T. Geisler S. Johnsen H. Hastie T. Eisen M.B. van de Rijn M. Jeffrey S.S. Thorsen T. Quist H. Matese J.C. Brown P.O. Botstein D. et al.Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc. Natl. Acad. Sci. U.S.A. 2001; 98 (11553815): 10869-1087410.1073/pnas.191367098Crossref PubMed Scopus (8545) Google Scholar). The basal-like subtype, also known as triple-negative breast cancer (TNBC) due to the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2, is characterized by resistance to chemotherapy, acquisition of stemness, and poor prognosis (6Rakha E.A. Tan D.S. Foulkes W.D. Ellis I.O. Tutt A. Nielsen T.O. Reis-Filho J.S. Are triple-negative tumours and basal-like breast cancer synonymous?.Breast Cancer Res. 2007; 9 (18279542): 40410.1186/bcr1827Crossref PubMed Scopus (104) Google Scholar, 7Rodríguez-Pinilla S.M. Sarrió D. Honrado E. Hardisson D. Calero F. Benitez J. Palacios J. Prognostic significance of basal-like phenotype and fascin expression in node-negative invasive breast carcinomas.Clin. Cancer Res. 2006; 12 (16533778): 1533-153910.1158/1078-0432.CCR-05-2281Crossref PubMed Scopus (297) Google Scholar). Hence, development of novel drug candidates against BCs as well as identification of novel cellular targets has become a major thrust area of research. breast cancer Notch intracellular domain γ-secretase inhibitor 3-(3′4′,5′-trimethoxyphenyl)-5-(N-methyl-3′-indolyl)-1,2,4-triazole compound estrogen receptor progesterone receptor peripheral blood mononuclear cell triple-negative breast cancer Presenilin-1 anterior pharynx-defective 1 phosphatase and tensin homolog NMK-T-057 propidium iodide body weight serum glutamate oxaloacetate transaminase serum glutamate pyruvate transaminase alkaline phosphatase epithelial-to-mesenchymal transition aldehyde dehydrogenase cancer stem cell tumor-initiating cell phosphorylated Akt N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester 2,4-dinitrophenyl N-methyl aspartic acid light chain 3 monodansylcadaverine 3-methyladenine benzyloxycarbonyl fluoromethyl ketone proliferating cell nuclear antigen terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling Dulbecco’s modified Eagle’s medium fetal bovine serum 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide horseradish peroxidase phycoerythrin Presenilin enhancer 2. The Notch signaling pathway has been reported to be highly deregulated in several human malignancies, including lung, cervical, colon, pancreatic, and renal carcinoma to name a few (8Haque I. De A. Majumder M. Mehta S. McGregor D. Banerjee S.K. Van Veldhuizen P. Banerjee S. The matricellular protein CCN1/Cyr61 is a critical regulator of Sonic Hedgehog in pancreatic carcinogenesis.J. Biol. Chem. 2012; 287 (23027863): 38569-3857910.1074/jbc.M112.389064Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar9Koch U. Radtke F. Notch and cancer: a double-edged sword.Cell. Mol. Life Sci. 2007; 64 (17687513): 2746-276210.1007/s00018-007-7164-1Crossref PubMed Scopus (283) Google Scholar, 10Sriuranpong V. Borges M.W. Ravi R.K. Arnold D.R. Nelkin B.D. Baylin S.B. Ball D.W. Notch signaling induces cell cycle arrest in small cell lung cancer cells.Cancer Res. 2001; 61 (11306509): 3200-3205PubMed Google Scholar11Wang Z. Li Y. Banerjee S. Sarkar F.H. Exploitation of the Notch signaling pathway as a novel target for cancer therapy.Anticancer Res. 2008; 28 (19189643): 3621-3630PubMed Google Scholar). It has also been reported that elevated expression levels of Notch-1 and its ligand Jagged-1 are associated with the poor prognosis, lower survival rate, and development of chemoresistance in breast cancer (12Cho S. Lu M. He X. Ee P.L. Bhat U. Schneider E. Miele L. Beck W.T. Notch1 regulates the expression of the multidrug resistance gene ABCC1/MRP1 in cultured cancer cells.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (22143792): 20778-2078310.1073/pnas.1019452108Crossref PubMed Scopus (76) Google Scholar13Reedijk M. Odorcic S. Chang L. Zhang H. Miller N. McCready D.R. Lockwood G. Egan S.E. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival.Cancer Res. 2005; 65 (16166334): 8530-853710.1158/0008-5472.CAN-05-1069Crossref PubMed Scopus (619) Google Scholar, 14Stylianou S. Clarke R.B. Brennan K. Aberrant activation of notch signaling in human breast cancer.Cancer Res. 2006; 66 (16452208): 1517-152510.1158/0008-5472.CAN-05-3054Crossref PubMed Scopus (459) Google Scholar, 15Wang Z. Li Y. Ahmad A. Azmi A.S. Banerjee S. Kong D. Sarkar F.H. Targeting Notch signaling pathway to overcome drug resistance for cancer therapy.Biochim. Biophys. Acta. 2010; 1806 (20600632): 258-26710.1016/j.bbcan.2010.06.001PubMed Google Scholar, 16Bolós V. Mira E. Martínez-Poveda B. Luxán G. Cañamero M. Martínez-A C. Mañes S. de la Pompa J.L. Notch activation stimulates migration of breast cancer cells and promotes tumor growth.Breast Cancer Res. 2013; 15 (23826634): R5410.1186/bcr3447Crossref PubMed Scopus (100) Google Scholar17Guo S. Liu M. Gonzalez-Perez R.R. Role of Notch and its oncogenic signaling crosstalk in breast cancer.Biochim. Biophys. Acta. 2011; 1815 (21193018): 197-21310.1016/j.bbcan.2010.12.002PubMed Google Scholar). Moreover, Notch signaling is also known to be the key regulator of stemness in breast cancer (18Boyle S.T. Gieniec K.A. Gregor C.E. Faulkner J.W. McColl S.R. Kochetkova M. Interplay between CCR7 and Notch1 axes promotes stemness in MMTV-PyMT mammary cancer cells.Mol. Cancer. 2017; 16 (28137279): 1910.1186/s12943-017-0592-0Crossref PubMed Scopus (22) Google Scholar, 19Pal D. Kolluru V. Chandrasekaran B. Baby B.V. Aman M. Suman S. Sirimulla S. Sanders M.A. Alatassi H. Ankem M.K. Damodaran C. Targeting aberrant expression of Notch-1 in ALDH+ cancer stem cells in breast cancer.Mol. Carcinog. 2017; 56 (27753148): 1127-113610.1002/mc.22579Crossref PubMed Scopus (31) Google Scholar20Zhong Y. Shen S. Zhou Y. Mao F. Lin Y. Guan J. Xu Y. Zhang S. Liu X. Sun Q. NOTCH1 is a poor prognostic factor for breast cancer and is associated with breast cancer stem cells.Onco Targets Ther. 2016; 9 (27853380): 6865-687110.2147/OTT.S109606Crossref PubMed Scopus (47) Google Scholar). Hence, targeting Notch signaling has gained immense importance in the development of novel anticancer regimes against breast cancer (19Pal D. Kolluru V. Chandrasekaran B. Baby B.V. Aman M. Suman S. Sirimulla S. Sanders M.A. Alatassi H. Ankem M.K. Damodaran C. Targeting aberrant expression of Notch-1 in ALDH+ cancer stem cells in breast cancer.Mol. Carcinog. 2017; 56 (27753148): 1127-113610.1002/mc.22579Crossref PubMed Scopus (31) Google Scholar, 21Al-Hussaini H. Subramanyam D. Reedijk M. Sridhar S.S. Notch signaling pathway as a therapeutic target in breast cancer.Mol. Cancer Ther. 2011; 10 (20971825): 9-1510.1158/1535-7163.MCT-10-0677Crossref PubMed Scopus (117) Google Scholar, 22Grudzien P. Lo S. Albain K.S. Robinson P. Rajan P. Strack P.R. Golde T.E. Miele L. Foreman K.E. Inhibition of Notch signaling reduces the stem-like population of breast cancer cells and prevents mammosphere formation.Anticancer Res. 2010; 30 (21036696): 3853-3867PubMed Google Scholar). Activation of Notch signaling requires the interaction between Notch receptor and its ligand Jagged-1 or Jagged-2 followed by a series of proteolytic cleavages. Notch receptors are transmembrane heterodimeric proteins consisting of an extracellular domain, a transmembrane domain, and a cytoplasmic domain (17Guo S. Liu M. Gonzalez-Perez R.R. Role of Notch and its oncogenic signaling crosstalk in breast cancer.Biochim. Biophys. Acta. 2011; 1815 (21193018): 197-21310.1016/j.bbcan.2010.12.002PubMed Google Scholar). The initial cleavage, or S1, of the Notch precursor is initiated in the Golgi network by a furin-like convertase to generate a heterodimeric receptor at the cell surface (23Borggrefe T. Oswald F. The Notch signaling pathway: transcriptional regulation at Notch target genes.Cell. Mol. Life Sci. 2009; 66 (19165418): 1631-164610.1007/s00018-009-8668-7Crossref PubMed Scopus (453) Google Scholar). Binding of Notch receptor to its ligands triggers the second, or S2, cleavage. This is catalyzed by a family of metalloendopeptidases known as ADAM (a disintegrin and metalloproteinase) or TACE (tumor necrosis factor-α–converting enzyme), which cleaves the Notch receptor at an extracellular site (S2) between Ala (1710) and Val (1711) residues, thus releasing a fragment, known as Notch extracellular domain (24Zhang Y.W. Luo W.J. Wang H. Lin P. Vetrivel K.S. Liao F. Li F. Wong P.C. Farquhar M.G. Thinakaran G. Xu H. Nicastrin is critical for stability and trafficking but not association of other presenilin/γ-secretase components.J. Biol. Chem. 2005; 280 (15711015): 17020-1702610.1074/jbc.M409467200Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The remaining receptor fragment then undergoes the third proteolytic cleavage (S3) by a membrane-bound protease known as γ-secretase complex to release the activated Notch intracellular domain (NICD) into the cytosol (24Zhang Y.W. Luo W.J. Wang H. Lin P. Vetrivel K.S. Liao F. Li F. Wong P.C. Farquhar M.G. Thinakaran G. Xu H. Nicastrin is critical for stability and trafficking but not association of other presenilin/γ-secretase components.J. Biol. Chem. 2005; 280 (15711015): 17020-1702610.1074/jbc.M409467200Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). γ-Secretase is a multisubunit enzyme complex consisting of four subunits, namely Presenilin-1 (PSEN-1), Nicastrin, anterior pharynx-defective 1 (APH-1), and Presenilin enhancer 2 (PEN-2). Presenilin is an aspartyl protease that forms the catalytic core, whereas Nicastrin is required for substrate recognition (24Zhang Y.W. Luo W.J. Wang H. Lin P. Vetrivel K.S. Liao F. Li F. Wong P.C. Farquhar M.G. Thinakaran G. Xu H. Nicastrin is critical for stability and trafficking but not association of other presenilin/γ-secretase components.J. Biol. Chem. 2005; 280 (15711015): 17020-1702610.1074/jbc.M409467200Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The activated NICD then translocates to the nucleus and modulates the transcription of its downstream targets genes such as Hes-1, Hey-1, Akt, and PTEN (21Al-Hussaini H. Subramanyam D. Reedijk M. Sridhar S.S. Notch signaling pathway as a therapeutic target in breast cancer.Mol. Cancer Ther. 2011; 10 (20971825): 9-1510.1158/1535-7163.MCT-10-0677Crossref PubMed Scopus (117) Google Scholar, 25Palomero T. Dominguez M. Ferrando A.A. The role of the PTEN/AKT pathway in NOTCH1-induced leukemia.Cell Cycle. 2008; 7 (18414037): 965-97010.4161/cc.7.8.5753Crossref PubMed Scopus (192) Google Scholar). Due to its important role in the activation of Notch signaling, γ-secretase complex has become a potential target for specific inhibitors, known as γ-secretase inhibitor (GSIs), which are reported to show significant anticancer properties against several carcinomas, including breast, and a few of them have also entered phase I and phase II clinical trials (26Schott A.F. Landis M.D. Dontu G. Griffith K.A. Layman R.M. Krop I. Paskett L.A. Wong H. Dobrolecki L.E. Lewis M.T. Froehlich A.M. Paranilam J. Hayes D.F. Wicha M.S. Chang J.C. Preclinical and clinical studies of gamma secretase inhibitors with docetaxel on human breast tumors.Clin. Cancer Res. 2013; 19 (23340294): 1512-152410.1158/1078-0432.CCR-11-3326Crossref PubMed Scopus (193) Google Scholar, 27Yuan X. Wu H. Xu H. Xiong H. Chu Q. Yu S. Wu G.S. Wu K. Notch signaling: an emerging therapeutic target for cancer treatment.Cancer Lett. 2015; 369 (26341688): 20-2710.1016/j.canlet.2015.07.048Crossref PubMed Scopus (281) Google Scholar). In search of novel GSIs, we investigated the Notch inhibitory properties of a triazole-based compound, 3-(3′4′,5′-trimethoxyphenyl)-5-(N-methyl-3′-indolyl)-1,2,4-triazole (also known as NMK-T-057), consisting of 1,2,4-triazole ring linked with an indole moiety (Fig. 1A). The presence of an indole ring system is known to attribute diverse medicinal and pharmacological properties to small molecules of both natural and synthetic origins (28de Sá Alves F.R. Barreiro E.J. Fraga C.A. From nature to drug discovery: the indole scaffold as a ’privileged structure’.Mini Rev. Med. Chem. 2009; 9 (19519503): 782-79310.2174/138955709788452649Crossref PubMed Scopus (475) Google Scholar). When the indole ring is linked with five-member heterocyclic triazoles, the resultant compounds have been reported to gain potent anticancer properties (29Kumar D. Narayanam M.K. Chang K.H. Shah K. Synthesis of novel indolyl-1,2,4-triazoles as potent and selective anticancer agents.Chem. Biol. Drug Des. 2011; 77 (21251232): 182-18810.1111/j.1747-0285.2010.01051.xCrossref PubMed Scopus (58) Google Scholar). Moreover, available literature indicates that 1,2,4-triazole derivatives possess potent anticancer properties (29Kumar D. Narayanam M.K. Chang K.H. Shah K. Synthesis of novel indolyl-1,2,4-triazoles as potent and selective anticancer agents.Chem. Biol. Drug Des. 2011; 77 (21251232): 182-18810.1111/j.1747-0285.2010.01051.xCrossref PubMed Scopus (58) Google Scholar, 30Sztanke K. Pasternak K. Rzymowska J. Sztanke M. Kandefer-Szerszeń M. Synthesis, structure elucidation and identification of antitumoural properties of novel fused 1,2,4-triazine aryl derivatives.Eur. J. Med. Chem. 2008; 43 (17868955): 1085-109410.1016/j.ejmech.2007.07.009Crossref PubMed Scopus (67) Google Scholar31Sztanke K. Tuzimski T. Rzymowska J. Pasternak K. Kandefer-Szerszeń M. Synthesis, determination of the lipophilicity, anticancer and antimicrobial properties of some fused 1,2,4-triazole derivatives.Eur. J. Med. Chem. 2008; 43 (17531354): 404-41910.1016/j.ejmech.2007.03.033Crossref PubMed Scopus (252) Google Scholar) and are also effective against breast cancer cells (32Genc M. Karagoz Genc Z. Tekin S. Sandal S. Sirajuddin M. Hadda Taibi B. Sekerci M. Design, synthesis, in vitro antiproliferative activity, binding modeling of 1,2,4,-triazoles as new anti-breast cancer agents.Acta Chim. Slov. 2016; 63 (28004087): 726-737Crossref PubMed Scopus (16) Google Scholar, 33Mioc M Soica C Bercean V Avram S Balan-Porcarasu M Coricovac D Ghiulai R. Muntean D. Andrica F. Dehelean C. Spandidos D.A. Tsatsakis A.M. Kurunczi L. Design, synthesis and pharmaco-toxicological assessment of 5-mercapto-1,2,4-triazole derivatives with antibacterial and antiproliferative activity.Int. J. Oncol. 2017; 50 (28350123): 1175-118310.3892/ijo.2017.3912Crossref Scopus (27) Google Scholar). Hence, in the present study, we have investigated the anticancer mechanism of the novel indolyl triazole derivative NMK-T-057 against several breast cancer cell lines by monitoring different oncogenic parameters, including migratory properties, epithelial-to-mesenchymal transition, and stemness. Because Notch signaling is known to be a regulator of epithelial-to-mesenchymal transition (EMT) and stemness in BC (34Espinoza I. Miele L. Deadly crosstalk: Notch signaling at the intersection of EMT and cancer stem cells.Cancer Lett. 2013; 341 (23973264): 41-4510.1016/j.canlet.2013.08.027Crossref PubMed Scopus (160) Google Scholar, 35Suman S. Das T.P. Damodaran C. Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells.Br. J. Cancer. 2013; 109 (24129237): 2587-259610.1038/bjc.2013.642Crossref PubMed Scopus (109) Google Scholar), we also investigated whether NMK-T-057 can target Notch signaling in BC cells. To delineate the specific mechanism of Notch inhibition by NMK-T-057, we monitored γ-secretase activity in NMK-treated cells. Furthermore, it has been reported that inhibition of Notch signaling leads to autophagic cell death in BC cells (36Ray A Vasudevan S Sengupta S. 6-Shogaol inhibits breast cancer cells and stem cell-like spheroids by modulation of Notch signaling pathway and induction of autophagic cell death.PLoS One. 2015; 10 (26355461)e013761410.1371/journal.pone.0137614Crossref PubMed Scopus (69) Google Scholar, 37Wang Y Wang H Ge H Yang Z. AG-1031 induced autophagic cell death and apoptosis in C6 glioma cells associated with Notch-1 signaling pathway.J. Cell. Biochem. 2018; 119 (29575026): 5893-590310.1002/jcb.26781Crossref PubMed Scopus (15) Google Scholar). Hence, we have also investigated whether inhibition of γ-secretase–mediated Notch activation by NMK-T-057 leads to autophagic cell death in BCs. Thus, the present study highlighted a very specific mechanism by which the triazole-based drugs can inhibit BC cells. Treatment of TNBC cells such as MDA-MB-231, MDA-MB-468, and 4T1 and non-TNBC cell type MCF-7 with NMK-T-057 for 24 h resulted in the loss of viability in a dose-dependent manner (Fig. 1, B and C). However, comparatively lesser cytotoxicity was observed when noncancerous cell types such as MCF-10A and peripheral blood mononuclear cells (PBMCs) were treated with NMK-T-057, with respective IC50 values >50 μm. Effects of NMK-T-057 on TNBCs such as MDA-MB-231, MDA-MB-468, and 4T1 cells were more or less similar, with the observed IC50 values ranging between 8 and 12 μm, whereas MCF-7 was found to be most susceptible to NMK-T-057, with the IC50 observed at 5 μm (Fig. 1C). The colony-forming ability of BC cells such as MDA-MB-231 and MCF-7 cells in the presence or absence of NMK-T-057 was determined by in vitro clonogenic assay, following the protocol described under “Experimental procedures.” Viable cells were seeded at a density of 5000 cells/ml for colony formation and simultaneously treated with different concentrations of NMK-T-057 (0–10 μm) from the 2nd to the 6th day. Crystal violet staining of the viable colonies revealed that NMK-T-057 significantly inhibited the colony-forming properties of MDA-MB-231 and MCF-7 cells in a dose-dependent fashion (Fig. 1, D and E). To determine the mode of cell death in NMK-treated BC cells we checked the induction of apoptosis by annexin V/propidium iodide (PI) double staining method using FACS. We observed that treatment of MDA-MB-231, MDA-MB-468, and MCF-7 cells with different concentrations of NMK-T-057 resulted in a dose-dependent induction of apoptosis (Fig. 1F and Fig. S1). In the presence of 5 μm compound, the apoptotic population was found to increase 25% from 2% in untreated MDA-MB-231 cells, whereas in MDA-MB-468 cells, the apoptotic population increased from 1.5 to 35%. Similarly, when treated with 10 μm compound, the apoptotic population increased to 37% in MDA-MB-231 cells and 42% in MDA-MB-468 cells, respectively. Consistent with the cell viability results, MCF-7 cells showed higher responsiveness to NMK-T-057–induced apoptosis. In the presence of 3 μm compound, the apoptotic population increased to 30% as compared with 1.2% in control cells, whereas in the presence of 5 μm compound, the apoptotic population increased to 45%. Migratory ability of various BC cells in the presence and absence of NMK-T-057 was assessed by Boyden chamber assay. Migratory activities of BC cells were found to be significantly decreased by NMK-T-057 in a dose-dependent fashion (Fig. 1G). In the presence of 5 μm NMK-T-057, the extent of migration was decreased by 1.9- and 1.5-fold for MDA-MB-231 and MDA-MB-468 cells, respectively. Similarly, treatment with 10 μm NMK-T-057 resulted in a decrease of migration by 2.7- and 2.4-fold, respectively. Consistent with our previous results, treatment of MCF-7 cells with NMK-T-057 resulted in drastic inhibition of cell migration. In the presence of 3 and 5 μm NMK-T-057, migration of MCF-7 cells was decreased by 2.7- and 4.8-fold, respectively (Fig. 1G). After demonstrating the noncytotoxic effect of NMK-T-057 in noncancer cells, we further assessed its toxicity in female Swiss albino mice. NMK-T-057 was injected intraperitoneally in female mice at different doses of (10–100 mg/kg-BW). On the 7th day of i.p. treatment, different hematological and clinical parameters were determined to assess the toxicity of NMK-T-057. We observed that NMK treatments did not significantly affect the red blood cell count or hemoglobin levels in mice compared with the control group. Only minor alterations in the total white blood cell counts were observed, particularly the differential counts for neutrophils and lymphocytes in NMK-treated groups compared with the control group. Additionally, we also observed that NMK-T-057 had no added toxic effects on the liver, as revealed by measuring serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), and alkaline phosphatase (ALP) levels in serum from each experimental set. No significant alterations in serum SGOT, SGPT, and ALP levels were observed in NMK-treated mice compared with control mice (Table S1), and all the values fell within the normal range. Thus, consistent with in vitro results, NMK-T-057 showed limited toxicity in in vivo conditions as well. Epithelial-to-mesenchymal transition is an important physiological process responsible for the acquisition of migratory and invasive phenotype by BC cells that enhances their ability to invade the surrounding tissues (38Christofori G. New signals from the invasive front.Nature. 2006; 441 (16724056): 444-45010.1038/nature04872Crossref PubMed Scopus (814) Google Scholar). It has been reported that remodeling of the actin cytoskeleton plays an important role in the EMT process (39Haynes J. Srivastava J. Madson N. Wittmann T. Barber D.L. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression.Mol. Biol. Cell. 2011; 22 (22031288): 4750-476410.1091/mbc.e11-02-0119Crossref PubMed Scopus (168) Google Scholar). Actin stress fibers are found in abundance in mesenchymal cells, whereas few stress fibers are observed in epithelial cells (39Haynes J. Srivastava J. Madson N. Wittmann T. Barber D.L. Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression.Mol. Biol. Cell. 2011; 22 (22031288): 4750-476410.1091/mbc.e11-02-0119Crossref PubMed Scopus (168) Google Scholar). MDA-MB-231 cells, which are known to be highly aggressive and invasive, possess a spindle-shaped morphology similar to the mesenchymal type. Staining the actin cytoskeleton with phalloidin-FITC revealed an organized network of F-actin filaments in the untreated cells. However, on treatment with sublethal concentrations of NMK-T-057 (3–5 μm), we observed that the mesenchymal morphology of MDA-MB-231 cells was altered to epithelial type accompanied by disruption of the actin stress fibers (Fig. 2A). We further investigated the status of several EMT markers in NMK-T-057–treated MDA-MB-231 cells. Interestingly, we observed that proteins like vimentin, N-cadherin, and TWIST, which are essential for maintaining the mesenchymal phenotype, were significantly down-regulated by NMK-T-057 in a dose-dependent fashion. Conversely, epithelial markers such as E-cadherin and cytokeratin-19 were also found to be significantly up-regulated in NMK-T-057–treated MDA-MB-231 cells (Fig. 2, B and C). We also observed that the CD44high/CD24low population, responsible for the metastatic potential and stemness properties of BC cells (40Jin J. Krishnamachary B. Mironchik Y. Kobayashi H. Bhujwalla Z.M. Phototheranostics of CD44-positive cell populations in triple negative breast cancer.Sci. Rep. 2016; 6 (27302409)2787110.1038/srep27871Crossref PubMed Scopus (59) Google Scholar, 41Sheridan C. Kishimoto H. Fuchs R.K. Mehrotra S. Bhat-Nakshatri P. Turner C.H. Goulet Jr., R. Badve S. N" @default.
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• W2920415219 date "2019-04-01" @default.
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• W2920415219 title "A novel triazole, NMK-T-057, induces autophagic cell death in breast cancer cells by inhibiting γ-secretase–mediated activation of Notch signaling" @default.
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• W2920415219 doi "https://doi.org/10.1074/jbc.ra119.007671" @default.
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