Activation of Intrinsic Apoptosis and G1 Cell Cycle Arrest by a Triazole Precursor, N-(4-chlorophenyl)-2-(4-(3,4,5-trimethoxybenzyloxy)benzoyl)-hydrazinecarbothioamide in Breast Cancer Cell Line

Author(s): Stephanie B. Arulnathan, Kok H. Leong*, Azhar Ariffin*, Huda S. Kareem, Kevin K.H. Cheah

Journal Name: Anti-Cancer Agents in Medicinal Chemistry
(Formerly Current Medicinal Chemistry - Anti-Cancer Agents)

Volume 20 , Issue 9 , 2020

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Abstract:

Background: Oxadiazoles, triazoles, and their respective precursors have been shown to exhibit various pharmacological properties, namely antitumour activities. Cytotoxic activity was reported for these compounds in various cancer cell lines.

Aim and Objectives: In this study, we aim at investigating the mechanism of apoptosis by N-(4-chlorophenyl)-2-(4- (3,4,5-trimethoxybenzyloxy)benzoyl)-hydrazinecarbothioamide, a triazole precursor, henceforth termed compound P7a, in breast cancer cell line, MCF-7. We first screen a series of analogues containing (3,4,5-trimethoxybenzyloxy) phenyl moiety in breast cancer cell lines (MCF-7 and MDA-MB-231) to select the most cytotoxic compound and demonstrate a dose- and time-dependent cytotoxicity. Then, we unravel the mechanism of apoptosis of P7a in MCF-7 as well as its ability to cause cell cycle arrest.

Methods: Synthesis was performed as previously described by Kareem and co-workers. Cytotoxicity of analogues containing (3,4,5-trimethoxybenzyloxy)phenyl moiety against MCF-7 and MDA-MB-231 cell lines was evaluated using the MTS assay. Flow cytometric analyses was done using Annexin V/PI staining, JC-1 staining and ROS assay. The activity of caspases using a chemoluminescence assay and western blot analysis was conducted to study the apoptotic pathway induced by the compound in MCF-7 cells. Lastly, cell cycle analysis was conducted using flow cytometry.

Results: Upon 48 hours of treatment, compound P7a inhibited the proliferation of human breast cancer cells with IC50 values of 178.92 ± 12.51μM and 33.75 ± 1.20μM for MDA-MB-231 and MCF-7, respectively. Additionally, compound P7a showed selectivity towards the cancer cell line, MCF-7 compared to the normal breast cell line, hTERT-HME1, an advantage against current anticancer drugs (tamoxifen and vinblastine). Flow cytometric analyses using different assays indicated that compound P7a significantly increased the proportion of apoptotic cells, increased mitochondria membrane permeabilisation and caused generation of ROS in MCF-7. In addition, cell cycle analysis showed that cell proliferation was arrested at the G1 phase in the MCF-7 cell line. Furthermore, upon treatment, the MCF-7 cell line showed increased activity of caspase-3/7, and caspase-9. Lastly, the western blot analysis showed the up-regulation of pro-apoptotic proteins along with up-regulation of caspase-7 and caspase-9, indicating that an intrinsic pathway of apoptosis was induced.

Conclusion: The results suggest that compound P7a could be a potential chemotherapeutic agent for breast cancer.

Keywords: Apoptosis, breast cancer, flow cytometry, cell cycle, ROS, mechanism studies, triazole.

[1]
Cancer http://www.who.int/news-room/fact-sheets/detail/cancer [Accessed November 20, 2019].
[2]
Siegel, R.; Ma, J.; Zou, Z.; Jemal, A. Cancer statistics, 2014. CA Cancer J. Clin., 2014, 64(1), 9-29.
[http://dx.doi.org/10.3322/caac.21208] [PMID: 24399786]
[3]
Rahman, N.A.; Yazan, L.S.; Wibowo, A.; Ahmat, N.; Foo, J.B.; Tor, Y.S.; Yeap, S.K.; Razali, Z.A.; Ong, Y.S.; Fakurazi, S. Induction of apoptosis and G2/M arrest by ampelopsin E from Dryobalanops towards triple negative breast cancer cells, MDA-MB-231. BMC Complement. Altern. Med., 2016, 16, 354.
[http://dx.doi.org/10.1186/s12906-016-1328-1] [PMID: 27609190]
[4]
Loganathan, R.; Radhakrishnan, A.K.; Selvaduray, K.R.; Nesaretnam, K. Selective anti-cancer effects of palm phytonutrients on human breast cancer cells. RSC Advances, 2015, 5, 1745-1753.
[http://dx.doi.org/10.1039/C4RA12343C]
[5]
Hilmi: 30,000 new cancer cases reported yearly.,. http://www.cancer.org.my/in-the-news/ [Accessed November 20, 2017];
[6]
Nussbaumer, S.; Bonnabry, P.; Veuthey, J.L.; Fleury-Souverain, S. Analysis of anticancer drugs: a review. Talanta, 2011, 85(5), 2265-2289.
[http://dx.doi.org/10.1016/j.talanta.2011.08.034] [PMID: 21962644]
[7]
Zhang, H.Z.; Kasibhatla, S.; Kuemmerle, J.; Kemnitzer, W.; Ollis-Mason, K.; Qiu, L.; Crogan-Grundy, C.; Tseng, B.; Drewe, J.; Cai, S.X. Discovery and structure-activity relationship of 3-aryl-5-aryl-1,2,4-oxadiazoles as a new series of apoptosis inducers and potential anticancer agents. J. Med. Chem., 2005, 48(16), 5215-5223.
[http://dx.doi.org/10.1021/jm050292k] [PMID: 16078840]
[8]
Armania, N.; Yazan, L.S.; Ismail, I.S.; Foo, J.B.; Tor, Y.S.; Ishak, N.; Ismail, N.; Ismail, M. Dillenia Suffruticosa extract inhibits proliferation of human breast cancer cell lines (MCF-7 and MDA-MB-231) via induction of G2/M arrest and apoptosis. Molecules, 2013, 18(11), 13320-13339.
[http://dx.doi.org/10.3390/molecules181113320] [PMID: 24172241]
[9]
Medarde, M.; Maya, A.B.; Pérez-Melero, C. Naphthalene combretastatin analogues: synthesis, cytotoxicity and antitubulin activity. J. Enzyme Inhib. Med. Chem., 2004, 19(6), 521-540.
[http://dx.doi.org/10.1080/14756360412331280473] [PMID: 15662956]
[10]
Kareem, H.S.; Ariffin, A.; Nordin, N.; Heidelberg, T.; Abdul-Aziz, A.; Kong, K.W.; Yehye, W.A. Correlation of antioxidant activities with theoretical studies for new hydrazone compounds bearing a 3,4,5-trimethoxy benzyl moiety. Eur. J. Med. Chem., 2015, 103, 497-505.
[http://dx.doi.org/10.1016/j.ejmech.2015.09.016] [PMID: 26402727]
[11]
Terzioglu, N.; Gürsoy, A. Synthesis and anticancer evaluation of some new hydrazone derivatives of 2,6-dimethylimidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazide. Eur. J. Med. Chem., 2003, 38(7-8), 781-786.
[http://dx.doi.org/10.1016/S0223-5234(03)00138-7] [PMID: 12932910]
[12]
Cheng, L.X.; Tang, J.J.; Luo, H.; Jin, X.L.; Dai, F.; Yang, J.; Qian, Y.P.; Li, X.Z.; Zhou, B. Antioxidant and antiproliferative activities of hydroxyl-substituted Schiff bases. Bioorg. Med. Chem. Lett., 2010, 20(8), 2417-2420.
[http://dx.doi.org/10.1016/j.bmcl.2010.03.039] [PMID: 20346660]
[13]
Jessen, K.A.; English, N.M.; Yu Wang , J. Maliartchouk, S.; Archer, S.P.; Qiu, L.; Brand, R.; Kuemmerle, J.; Zhang, H.Z.; Gehlsen, K.; Drewe, J.; Tseng, B.; Cai, S.X.; Kasibhatla, S.. The discovery and mechanism of action of novel tumor-selective and apoptosis-inducing 3,5-diaryl-1,2,4-oxadiazole series using a chemical genetics approach. Mol. Cancer Ther., 2005, 4(5), 761-771.
[http://dx.doi.org/10.1158/1535-7163.MCT-04-0333] [PMID: 15897240]
[14]
Kumar, D.; Maruthi Kumar, N.; Chang, K.H.; Shah, K. Synthesis and anticancer activity of 5-(3-indolyl)-1,3,4-thiadiazoles. Eur. J. Med. Chem., 2010, 45(10), 4664-4668.
[http://dx.doi.org/10.1016/j.ejmech.2010.07.023] [PMID: 20692741]
[15]
Kareem, H.S.; Nordin, N.; Heidelberg, T.; Abdul-Aziz, A.; Ariffin, A. Conjugated oligo-aromatic compounds bearing a 3,4,5-trimethoxy moiety: investigation of their antioxidant activity correlated with a DFT study. Molecules, 2016, 21(2), 224.
[http://dx.doi.org/10.3390/molecules21020224] [PMID: 26901175]
[16]
Malki, A.; Elbayaa, R.Y.; Ashour, H.M.; Loffredo, C.A.; Youssef, A.M. Novel thiosemicarbazides induced apoptosis in human MCF-7 breast cancer cells via JNK signaling. J. Enzyme Inhib. Med. Chem., 2015, 30(5), 786-795.
[http://dx.doi.org/10.3109/14756366.2014.971781] [PMID: 25363687]
[17]
Ruiz, J.; Vicente, C.; de Haro, C.; Bautista, D. Novel bis-C,N-cyclometalated iridium(III) thiosemicarbazide antitumor complexes: interactions with human serum albumin and DNA, and inhibition of cathepsin B. Inorg. Chem., 2013, 52(2), 974-982.
[http://dx.doi.org/10.1021/ic302219v] [PMID: 23301676]
[18]
Gupta, R.K.; Banerjee, A.; Pathak, S.; Sharma, C.; Singh, N. Induction of mitochondrial-mediated apoptosis by Morinda citrifolia (Noni) in human cervical cancer cells. Asian Pac. J. Cancer Prev., 2013, 14(1), 237-242.
[http://dx.doi.org/10.7314/APJCP.2013.14.1.237] [PMID: 23534730]
[19]
Looi, C.Y.; Arya, A.; Cheah, F.K.; Muharram, B.; Leong, K.H.; Mohamad, K.; Wong, W.F.; Rai, N.; Mustafa, M.R. Induction of apoptosis in human breast cancer cells via caspase pathway by vernodalin isolated from Centratherum anthelminticum (L.) seeds. PLoS One, 2013, 8(2)e56643
[http://dx.doi.org/10.1371/journal.pone.0056643] [PMID: 23437193]
[20]
Rosilio, C.; Lounnas, N.; Nebout, M.; Imbert, V.; Hagenbeek, T.; Spits, H.; Asnafi, V.; Pontier-Bres, R.; Reverso, J.; Michiels, J.F.; Sahra, I.B.; Bost, F.; Peyron, J.F. The metabolic perturbators metformin, phenformin and AICAR interfere with the growth and survival of murine PTEN-deficient T cell lymphomas and human T-ALL/T-LL cancer cells. Cancer Lett., 2013, 336(1), 114-126.
[http://dx.doi.org/10.1016/j.canlet.2013.04.015] [PMID: 23612073]
[21]
Bohnert, M.; Dahse, H.; Gibson, D.M.; Krasnoff, S.B.; Hoffmeister, D. The fusarin analog NG-391 impairs nucleic acid formation in K-562 leukemia cells. Phytochem. Lett., 2013, 6(2), 189-192.
[http://dx.doi.org/10.1016/j.phytol.2013.01.001]
[22]
Holliday, D.L.; Speirs, V. Choosing the right cell line for breast cancer research. Breast Cancer Res., 2011, 13(4), 215.
[http://dx.doi.org/10.1186/bcr2889] [PMID: 21884641]
[23]
Lewis-Wambi, J.S.; Jordan, V.C. Estrogen regulation of apoptosis: how can one hormone stimulate and inhibit? Breast Cancer Res., 2009, 11(3), 206.
[http://dx.doi.org/10.1186/bcr2255] [PMID: 19519952]
[24]
Motaghed, M. Cytotoxic, cytostatic and anti-estrogenic effect of Thymoquinone on estrogen receptor-positive breast cancer MCF7 cell line. Am. J. Life Sci, 2015, 3(2), 7-14.
[http://dx.doi.org/10.11648/j.ajls.s.2015030202.12]
[25]
Suffness, M.; Pezzuto, J.M. Assays related to cancer drug discovery.Methods in Plant Biochemistry: Assays for Bioactivity; Hostettmann, K., Ed.; Academic Press: London, 1990, Vol. 6, pp. 71-133.
[26]
Oliveira, P.F.; Alves, J.M.; Damasceno, J.L.; Oliveira, R.A.; Dias, H.J.; Crotti, A.M.; Tavares, D.C. Cytotoxicity screening of essential oils in cancer cell lines. Rev. Bras. Farmacogn., 2015, 25(2), 183-188.
[http://dx.doi.org/10.1016/j.bjp.2015.02.009]
[27]
Demchenko, A.P. Beyond annexin V: fluorescence response of cellular membranes to apoptosis. Cytotechnology, 2013, 65(2), 157-172.
[http://dx.doi.org/10.1007/s10616-012-9481-y] [PMID: 22797774]
[28]
Sinha, A.; Banerjee, K.; Banerjee, A.; Sarkar, A.; Ahir, M.; Adhikary, A.; Chatterjee, M.; Choudhuri, S.K. Induction of apoptosis in human colorectal cancer cell line, HCT-116 by a vanadium- Schiff base complex. Biomed. Pharmacother., 2017, 92, 509-518.
[http://dx.doi.org/10.1016/j.biopha.2017.05.108] [PMID: 28575808]
[29]
Cummings, B.S.; Schnellmann, R.G.; Wills, L.P. Measurement of cell death in mammalian cells.; Curr. Protocols Pharmacol., 2004, Chapter 12 , p. 8.
[http://dx.doi.org/10.1002/0471141755.ph1208s25] [PMID: 22294120]
[30]
Chang, H.Y.; Huang, H.C.; Huang, T.C.; Yang, P.C.; Wang, Y.C.; Juan, H.F. Ectopic ATP synthase blockade suppresses lung adenocarcinoma growth by activating the unfolded protein response. Cancer Res., 2012, 72(18), 4696-4706.
[http://dx.doi.org/10.1158/0008-5472.CAN-12-0567] [PMID: 22822083]
[31]
Pelicano, H.; Carney, D.; Huang, P. ROS stress in cancer cells and therapeutic implications. Drug Resist. Updat., 2004, 7(2), 97-110.
[http://dx.doi.org/10.1016/j.drup.2004.01.004] [PMID: 15158766]
[32]
Wang, G.W.; Lv, C.; Shi, Z.R.; Zeng, R.T.; Dong, X.Y.; Zhang, W.D.; Liu, R.H.; Shan, L.; Shen, Y.H. Abieslactone induces cell cycle arrest and apoptosis in human hepatocellular carcinomas through the mitochondrial pathway and the generation of reactive oxygen species. PLoS One, 2014, 9(12)e115151
[http://dx.doi.org/10.1371/journal.pone.0115151] [PMID: 25502685]
[33]
Kwan, Y.P.; Saito, T.; Ibrahim, D.; Al-Hassan, F.M.; Ein Oon, C.; Chen, Y.; Jothy, S.L.; Kanwar, J.R.; Sasidharan, S. Evaluation of the cytotoxicity, cell-cycle arrest, and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells. Pharm. Biol., 2016, 54(7), 1223-1236.
[PMID: 26154521]
[34]
Earnshaw, W.C.; Martins, L.M.; Kaufmann, S.H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem., 1999, 68, 383-424.
[http://dx.doi.org/10.1146/annurev.biochem.68.1.383] [PMID: 10872455]
[35]
Hu, H.; Ahn, N.S.; Yang, X.; Lee, Y.S.; Kang, K.S. Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell. Int. J. Cancer, 2002, 102(3), 250-253.
[http://dx.doi.org/10.1002/ijc.10707] [PMID: 12397644]
[36]
Ricci, M.S.; Zong, W.X. Chemotherapeutic approaches for targeting cell death pathways. Oncologist, 2006, 11(4), 342-357.
[http://dx.doi.org/10.1634/theoncologist.11-4-342] [PMID: 16614230]
[37]
Jakimov, D.S.; Kojić, V.V.; Aleksić, L.D.; Bogdanović, G.M.; Ajduković, J.J.; Djurendić, E.A.; Penov Gaši, K.M.; Sakač, M.N.; Jovanović-Šanta, S.S. Androstane derivatives induce apoptotic death in MDA-MB-231 breast cancer cells. Bioorg. Med. Chem., 2015, 23(22), 7189-7198.
[http://dx.doi.org/10.1016/j.bmc.2015.10.015] [PMID: 26494582]
[38]
Kim, E.M.; Jung, C.H.; Kim, J.; Hwang, S.G.; Park, J.K.; Um, H.D. The p53/p21 complex regulates cancer cell invasion and apoptosis by targeting Bcl-2 family proteins. Cancer Res., 2017, 77(11), 3092-3100.
[http://dx.doi.org/10.1158/0008-5472.CAN-16-2098] [PMID: 28377455]
[39]
Ramakrishna, S.C. Role of leptomycin in p53 induced apoptosis. J. Cancer Sci. Ther., 2013, 5, 158-181.
[http://dx.doi.org/10.4172/1948-5956.1000203]


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Article Details

VOLUME: 20
ISSUE: 9
Year: 2020
Published on: 20 August, 2020
Page: [1072 - 1086]
Pages: 15
DOI: 10.2174/1871520620666200318100051
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