Effect of Salinomycin on Expression Pattern of Genes Associated with Apoptosis in Endometrial Cancer Cell Line

Author(s): Kamil Kiełbasiński*, Wojciech Peszek, Beniamin O. Grabarek, Dariusz Boroń, Magdalena Wierzbik-Strońska, Marcin Oplawski

Journal Name: Current Pharmaceutical Biotechnology

Volume 21 , Issue 12 , 2020


Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Abstract:

Background: Salinomycin is part of a group of ionophore antibiotics characterized by an activity towards tumor cells. To this day, the mechanism through which salinomycin induces their apoptosis is not fully known yet. The goal of this study was to assess the expression pattern of genes and the proteins coded by them connected with the process of programmed cell death in an endometrial cancer cell Ishikawa culture exposed to salinomycin and compared to the control.

Materials and Methods: Analysis of the effect of salinomycin on Ishikawa endometrial cancer cells (ECACC 99040201) included a cytotoxicity MTT test (with a concentration range of 0.1-100 μM), assessment of the induction of apoptosis and necrosis by salinomycin at a concentration of 1 μM as well the assessment of the expression of the genes chosen in the microarray experiment (microarray HG-U 133A_2) and the proteins coded by them connected with apoptosis (RTqPCR, ELISA assay). The statistical significance level for all analyses carried out as part of this study was p<0.05.

Results: It was observed that salinomycin causes the death of about 50% of cells treated by it (50.74±0.80% of all cells) at a concentration of 1μM. The decrease in the number of living cells was determined directly after treatment of the cells with the drug (time 0). The average percent of late apoptotic cells was 1.65±0.24% and 0.57±0.01% for necrotic cells throughout the entire observation period.

Discussion: Microarray analysis indicated the following number of mRNA differentiating the culture depending on the time of incubation with the drug: H_12 vs C = 114 mRNA, H_8 vs C = 84 mRNA, H_48 vs. C = 27 mRNA, whereas 5 mRNAs were expressed differently at all times. During the whole incubation period of the cells with the drug, the following dependence of the expression profile of the analyzed transcripts was observed: Bax>p53>FASL>BIRC5>BCL2L.

Conclusion: The analysis carried out indicated that salinomycin, at a concentration of 1 μM, stopped the proliferation of 50% of endometrial cancer cells, mainly by inducing the apoptotic process of the cells. The molecular exponent of the induction of programmed cell death was an observed increase in the transcriptional activity of pro-apoptotic genes: Bax;p53;FASL and a decrease in the expression of anti-apoptotic genes: BCL2L2; BIRC5.

Keywords: Salinomycin, apoptosis, microarray technique, RTqPCR, ELISA, assayed pro- and anti-apoptotic genes, MTT assay.

[1]
Wu, A.Y.; Kong, N.C.; de Leon, F.A.; Pan, C.Y.; Tai, T.Y.; Yeung, V.T.; Yoo, S.J.; Rouillon, A.; Weir, M.R. An alarmingly high prevalence of diabetic nephropathy in Asian type 2 diabetic patients: the MicroAlbuminuria Prevalence (MAP) Study. Diabetologia, 2005, 48(1), 17-26.
[http://dx.doi.org/10.1007/s00125-004-1599-9 ] [PMID: 15616801]
[2]
Shaw, J.E.; Sicree, R.A.; Zimmet, P.Z. lobal estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res. Clin. Pract., 2010, 87(1), 4-14.
[http://dx.doi.org/10.1016/j.diabres.2009.10.007] [PMID: 19896746]
[3]
Loeffler, I.; Wolf, G. Epithelial-to-mesenchymal transition in diabetic nephropathy: Fact or fiction? Cells, 2015, 4(4), 631-652.
[http://dx.doi.org/10.3390/cells4040631 ] [PMID: 26473930]
[4]
Valcourt, U.; Kowanetz, M.; Niimi, H.; Heldin, C.H.; Moustakas, A. TGF-β and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Mol. Biol. Cell, 2005, 16(4), 1987-2002.
[http://dx.doi.org/10.1091/mbc.e04-08-0658 ] [PMID: 15689496]
[5]
Du, F.; Li, S.; Wang, T.; Zhang, H.Y.; Li, D.T.; Du, Z.X.; Wang, H.Q.; Li, D.; Du, Z.; Wang, H. Implication of Bcl-2-associated athanogene 3 in fibroblast growth factor-2-mediated epithelial-mesenchymal transition in renal epithelial cells. Exp. Biol. Med. (Maywood), 2015, 240(5), 566-575.
[http://dx.doi.org/10.1177/1535370214558023 ] [PMID: 25361773]
[6]
He, L.; Lou, W.; Ji, L.; Liang, W.; Zhou, M.; Xu, G.; Zhao, L.; Huang, C.; Li, R.; Wang, H.; Chen, X.; Sun, S. Serum response factor accelerates the high glucose-induced Epithelial-to-Mesenchymal Transition (EMT) via snail signaling in human peritoneal mesothelial cells. PLoS One, 2014, 9(10)e108593
[http://dx.doi.org/10.1371/journal.pone.0108593 ] [PMID: 25303231]
[7]
Sharma, S.; Sirin, Y.; Susztak, K. The story of Notch and chronic kidney disease. Curr. Opin. Nephrol. Hypertens., 2011, 20(1), 56-61.
[http://dx.doi.org/10.1097/MNH.0b013e3283414c88] [PMID: 21088575]
[8]
Wang, Y.; Zhong, Y.; Hou, T.; Liao, J.; Zhang, C.; Sun, C.; Wang, G. PM2.5 induces EMT and promotes CSC properties by activating Notch pathway in vivo and in vitro. Ecotoxicol. Environ. Saf., 2019, 178, 159-167.
[http://dx.doi.org/10.1016/j.ecoenv.2019.03.086 ] [PMID: 31002970]
[9]
Huang, C.C.; Cheng, S.H.; Wu, C.H.; Li, W.Y.; Wang, J.S.; Kung, M.L.; Chu, T.H.; Huang, S.T.; Feng, C.T.; Huang, S.C.; Tai, M.H. Delta-like 1 homologue promotes tumorigenesis and epithelial-mesenchymal transition of ovarian high-grade serous carcinoma through activation of Notch signaling. Oncogene, 2019, 38(17), 3201-3215.
[http://dx.doi.org/10.1038/s41388-018-0658-5 ] [PMID: 30626939]
[10]
Tian, D.; Zeng, X.; Wang, W.; Wang, Z.; Zhang, Y.; Wang, Y. Protective effect of rapamycin on endothelial-to-mesenchymal transition in HUVECs through the Notch signaling pathway. Vascul. Pharmacol., 2019, 113, 20-26.
[http://dx.doi.org/10.1016/j.vph.2018.10.004 ] [PMID: 30336218]
[11]
Bonegio, R.; Susztak, K. Notch signaling in diabetic nephropathy. Exp. Cell Res., 2012, 318(9), 986-992.
[http://dx.doi.org/10.1016/j.yexcr.2012.02.036 ] [PMID: 22414874]
[12]
Bielesz, B.; Sirin, Y.; Si, H.; Niranjan, T.; Gruenwald, A.; Ahn, S.; Kato, H.; Pullman, J.; Gessler, M.; Haase, V.H.; Susztak, K. Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans. J. Clin. Invest., 2010, 120(11), 4040-4054.
[http://dx.doi.org/10.1172/JCI43025 ] [PMID: 20978353]
[13]
Sun, G.D.; Li, C.Y.; Cui, W.P.; Guo, Q.Y.; Dong, C.Q.; Zou, H.B.; Liu, S.J.; Dong, W.P.; Miao, L.N. Review of herbal traditional Chinese medicine for the treatment of diabetic nephropathy. J. Diabetes Res., 2016.20165749857
[http://dx.doi.org/10.1155/2016/5749857 ] [PMID: 26649322]
[14]
Xu, Z.J.; Shu, S.; Li, Z.J.; Liu, Y.M.; Zhang, R.Y.; Zhang, Y. Liuwei Dihuang pill treats diabetic nephropathy in rats by inhibiting of TGF-β/SMADS, MAPK, and NF-kB and upregulating expression of cytoglobin in renal tissues. Medicine (Baltimore), 2017, 96(3), e5879.
[http://dx.doi.org/10.1097/MD.0000000000005879] [PMID: 28099346]
[15]
Tang, F.; Hao, Y.; Zhang, X.; Qin, J. Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy. Drug Des. Devel. Ther., 2017, 11, 2813-2826.
[http://dx.doi.org/10.2147/DDDT.S143805 ] [PMID: 29033543]
[16]
Chen, L.; Qi, J.; Chang, Y.X.; Zhu, D.; Yu, B. Identification and determination of the major constituents in Traditional Chinese Medicinal formula Danggui-Shaoyao-San by HPLC-DAD-ESI-MS/MS. J. Pharm. Biomed. Anal., 2009, 50(2), 127-137.
[http://dx.doi.org/10.1016/j.jpba.2009.03.039 ] [PMID: 19411155]
[17]
Wang, Y.; Li, G.; Zhou, Y.; Yin, D.; Tao, C.; Han, L.; Yue, X.; Pan, Y.; Yao, Y.; Peng, D.; Xu, F. The difference between blood-associated and water-associated herbs of Danggui-Shaoyao San in theory of TCM, based on serum pharmacochemistry. Biomed. Chromatogr., 2016, 30(4), 579-587.
[http://dx.doi.org/10.1002/bmc.3586 ] [PMID: 26270156]
[18]
Zhang, L.Y.; Zhao, Y.F.; Wang, Y.H.; Yang, Q.P. Study on the Mechanism of Protection About JDSS on the Early Diabetic Nephropathy. Praction. Chinese Internal Med., 2011, 25, 26-28.
[19]
Chen, H.P.; Song, E.F.; Mei, S.S. Clinical observation of non-decocted Chinese Herb of Dangguishaoyao powder in treatment of diabetic nephropathy in early stage. Hubei Univ Chin Med., 2015, 17, 68-70.
[20]
Li, X.; Miao, X.; Wang, L.; Zhang, W.; Fu, C.; Zhao, M.; Chen, W. Cai, Rui.; Chen, Y.; Hou, L. Effect of Danggui-Shaoyao-San on renal macrophages in STZ-induced DN rats. J. King Saud Univ.-. Sci., 2020, 32, 1778-1784.
[http://dx.doi.org/10.1016/j.jksus.2020.01.016]
[21]
Li, X.; Hou, L.; Sheng, G.; You, H.; Zhang, X.; Chen, Y.; Cai, R.; Chen, W.; Miao, X. Danggui shaoyao san exerts anti-inflammatory and anti-oxident effects to alleviates diabetic nephropathy. Acta Med. Mediter., 2020, 36, 539-543.
[22]
Chen, S.; Wang, Z.; Wan, S.; Huang, H.; Liang, H. Effect of modified Xiaochaihu decoction-containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway. Mol. Med. Rep., 2017, 16, 74166-77422.
[http://dx.doi.org/10.3892/mmr.2017.7561]
[23]
Yang, S.; Li, L.; Zhu, L.; Zhang, C.; Li, Z.; Guo, Y.; Nie, Y.; Luo, Z. Bu-Shen-Huo-Xue-Fang modulates nucleus pulposus cell proliferation and extracellular matrix remodeling in intervertebral disk degeneration through miR-483 regulation of Wnt pathway. J. Cell. Biochem., 2019, 120(12), 19318-19329.
[http://dx.doi.org/10.1002/jcb.26760 ] [PMID: 29393545]
[24]
Wei, P.Z.; Fung, W.W.; Ng, J.K.; Lai, K.B.; Luk, C.C.; Chow, K.M.; Li, P.K.; Szeto, C.C. author correction: metabolomic changes of human proximal tubular cell line in high glucose environment. Sci. Rep., 2020, 10(1), 1733.
[http://dx.doi.org/10.1038/s41598-020-57576-9 ] [PMID: 31992732]
[25]
Bernardo-Bermejo, S.; Sánchez-López, E.; Castro-Puyana, M.; Benito-Martínez, S.; Lucio-Cazaña, F.J.; Marina, M.L. A non-targeted capillary electrophoresis-mass spectrometry strategy to study metabolic differences in an in vitro model of high-glucose induced changes in human proximal tubular HK-2 Cells. Molecules, 2020, 25(3), 1-16.
[http://dx.doi.org/10.3390/molecules25030512 ] [PMID: 31991659]
[26]
Huang, H.; Zheng, F.; Dong, X.; Wu, F.; Wu, T.; Li, H. Allicin inhibits tubular epithelial-myofibroblast transdifferentiation under high glucose conditions in vitro. Exp. Ther. Med., 2017, 13(1), 254-262.
[http://dx.doi.org/10.3892/etm.2016.3913 ] [PMID: 28123498]
[27]
Gu, L.; Gao, Q.; Ni, L.; Wang, M.; Shen, F. Fasudil inhibits epithelial-myofibroblast transdifferentiation of human renal tubular epithelial HK-2 cells induced by high glucose. Chem. Pharm. Bull. (Tokyo), 2013, 61(7), 688-694.
[http://dx.doi.org/10.1248/cpb.c13-00066 ] [PMID: 23812394]
[28]
Zhuang, W.; Li, Z.; Dong, X.; Zhao, N.; Liu, Y.; Wang, C.; Chen, J. Schisandrin B inhibits TGF-β1-induced epithelial-mesenchymal transition in human A549 cells through epigenetic silencing of ZEB1. Exp. Lung Res., 2019, 45(5-6), 157-166.
[http://dx.doi.org/10.1080/01902148.2019.1631906] [PMID: 31268360]
[29]
El-Dawla, N.M.Q.; Sallam, A.M.; El-Hefnawy, M.H.; El-Mesallamy, H.O. E-cadherin and periostin in early detection and progression of diabetic nephropathy: epithelial-to-mesenchymal transition. Clin. Exp. Nephrol., 2019, 23(8), 1050-1057.
[http://dx.doi.org/10.1007/s10157-019-01744-3 ] [PMID: 31104272]
[30]
Wang, S.; Yan, Y.; Cheng, Z.; Hu, Y.; Liu, T. Sotetsuflavone suppresses invasion and metastasis in non-small-cell lung cancer A549 cells by reversing EMT via the TNF-α/NF-κB and PI3K/AKT signaling pathway. Cell Death Discov., 2018, 4, 26-36.
[http://dx.doi.org/10.1038/s41420-018-0026-9 ] [PMID: 29531823]
[31]
Yi, Y.E.; Li, S.Y.; Nie, Y.N.; Jia, D.X.; Zhang, Z.H.; Wang, Y.F.; Wang, Q. Effect of astragalus injection on renal tubular epithelial transdifferentiation in type 2 diabetic mice. BMC Complement. Altern. Med., 2016, 16, 222.
[http://dx.doi.org/10.1186/s12906-016-1208-8 ] [PMID: 27422712]
[32]
Siar, C.H.; Ng, K.H. Epithelial-to-mesenchymal transition in ameloblastoma: focus on morphologically evident mesenchymal phenotypic transition. Pathology, 2019, 51(5), 494-501.
[http://dx.doi.org/10.1016/j.pathol.2019.04.004 ] [PMID: 31262562]
[33]
Ji, Y.; Dou, Y.N.; Zhao, Q.W.; Zhang, J.Z.; Yang, Y.; Wang, T.; Xia, Y.F.; Dai, Y.; Wei, Z.F. Paeoniflorin suppresses TGF-β mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway. Acta Pharmacol. Sin., 2016, 37(6), 794-804.
[http://dx.doi.org/10.1038/aps.2016.36 ] [PMID: 27133302]
[34]
Wei, M.G.; Sun, W.; He, W.M.; Ni, L.; Yang, Y.Y. Ferulic acid attenuates TGF-β1-induced renal cellular fibrosis in NRK-52E cells by inhibiting Smad/ILK/snail pathway. Evid. Based Complement. Alternat. Med., 2015.2015619720
[http://dx.doi.org/10.1155/2015/619720 ] [PMID: 25949265]
[35]
Bocci, F.; Jolly, M.K.; Tripathi, S.C.; Aguilar, M.; Hanash, S.M.; Levine, H.; Onuchic, J.N. Numb prevents a complete epithelial-mesenchymal transition by modulating Notch signalling. J. R. Soc. Interface, 2017, 14(136), 1-11.
[http://dx.doi.org/10.1098/rsif.2017.0512 ] [PMID: 29187638]
[36]
Liu, W.; Wu, Y.; Yu, F.; Hu, W.; Fang, X.; Hao, W. The implication of Numb-induced Notch signaling in endothelial-mesenchymal transition of diabetic nephropathy. J. Diabetes Complications, 2018, 32(10), 889-899.
[http://dx.doi.org/10.1016/j.jdiacomp.2018.06.011 ] [PMID: 30097225]
[37]
Zang, M.D.; Hu, L.; Fan, Z.Y.; Wang, H.X.; Zhu, Z.L.; Cao, S.; Wu, X.Y.; Li, J.F.; Su, L.P.; Li, C.; Zhu, Z.G.; Yan, M.; Liu, B.Y. Luteolin suppresses gastric cancer progression by reversing epithelial-mesenchymal transition via suppression of the Notch signaling pathway. J. Transl. Med., 2017, 15(1), 52.
[http://dx.doi.org/10.1186/s12967-017-1151-6 ] [PMID: 28241766]
[38]
Karlsson, C.; Jonsson, M.; Asp, J.; Brantsing, C.; Kageyama, R.; Lindahl, A. Notch and HES5 are regulated during human cartilage differentiation. Cell Tissue Res., 2007, 327(3), 539-551.
[http://dx.doi.org/10.1007/s00441-006-0307-0 ] [PMID: 17093926]
[39]
van Tetering, G.; Vooijs, M. Proteolytic cleavage of Notch: “HIT and RUN. Curr. Mol. Med., 2011, 11(4), 255-269.
[http://dx.doi.org/10.2174/156652411795677972 ] [PMID: 21506924]
[40]
Nishad, R.; Mukhi, D.; Tahaseen, S.V.; Mungamuri, S.K.; Pasupulati, A.K. Growth hormone induces Notch1 signaling in podocytes and contributes to proteinuria in diabetic nephropathy. J. Biol. Chem., 2019, 294(44), 16109-16122.
[http://dx.doi.org/10.1074/jbc.RA119.008966 ] [PMID: 31511328]
[41]
Ito, T.; Kudoh, S.; Ichimura, T.; Fujino, K.; Hassan, W.A.; Udaka, N. Small cell lung cancer, an epithelial to mesenchymal transition (EMT)-like cancer: Significance of inactive Notch signaling and expression of achaete-scute complex homologue 1. Hum. Cell, 2017, 30(1), 1-10.
[http://dx.doi.org/10.1007/s13577-016-0149-3 ] [PMID: 27785690]
[42]
Zhang, J.; Yuan, G.; Dong, M.; Zhang, T.; Hua, G.; Zhou, Q.; Shi, W. Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition. Histochem. Cell Biol., 2017, 147(3), 367-375.
[http://dx.doi.org/10.1007/s00418-016-1484-x ] [PMID: 27600720]


open access plus

Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 21
ISSUE: 12
Year: 2020
Published on: 21 October, 2020
Page: [1269 - 1277]
Pages: 9
DOI: 10.2174/1389201021666200513074022

Article Metrics

PDF: 24
HTML: 1