Impacts of theaflavin on biological behavior of oral squamous cell carcinoma cells by regulating Snail/Slug signaling pathway

doi:10.11958/20230202

Abstract

Abstract:

Objective To study the impact of theaflavin on the proliferation, apoptosis and epithelial mesenchymal transformation of oral squamous cell carcinoma (OSCC) cells by regulating Snail/Slug signaling pathway. Methods MTT method was used to detect the survival rate of human OSCC cells SCC-25 treated with 25, 50, 100, 150 and 175 mg/L theaflavin, and the appropriate concentration of theaflavin was selected. SCC-25 cells were cultured in vitro and OSCC transplanted tumor model was constructed. Cells were randomly divide into the control group, the low dose theaflavin group, the high dose theaflavin group, the high dose theaxanthin+empty group and the high dose theaflavin+Snail overexpression group. The expression levels of Snail and Slug in cells of each group were detected by real-time fluorescent quantitative PCR and Western blot assay. MTT assay and flow cytometry were applied to detect cell proliferation and apoptosis. The migration and invasion of SCC-25 cells in each group were detected by cell scratch and Transwell invasion tests. The apoptosis of SCC-25 cells and the expression of epithelial mesenchymal transformation related proteins were detected by Western blot assay. The tumor volume and tumor weight of nude mice in each group were detected. Results Compared with the control group, the cell survival rate, migration rate, invasion number, Snail mRNA and protein expression, Slug mRNA and protein expression, Bcl-2, N-cadherin protein expression, tumor weight and volume of nude mice were decreased in the low dose theaflavin group and the high dose theaxanthin group (P<0.05), and the apoptosis rate, Bax, ZO-1 and E-cadherin protein expression increased (P<0.05). There was no significant difference in each index between the high-dose theaxanthin group and the high-dose theaflavin+no-load group (P>0.05). Overexpression of Snail can reverse the effect of theaflavin on cells and nude mice. Conclusion Theaflavin can down regulate the expression of Snail/Slug pathway, thus inhibit the epithelial mesenchymal transformation, proliferation, migration and invasion of OSCC cells, promote their apoptosis, and delay the growth of transplanted tumors in nude mice.

Key words: theaflavins, mouth neoplasms, carcinoma, squamous cell, Snail family transcription factors, cell proliferation, apoptosis, epithelial-mesenchymal transition

CLC Number:

R739.8

Figures/Tables 12

References 20

[1]	NIU Q, SUN Q, BAI R, et al. Progress of nanomaterials-based photothermal therapy for oral squamous cell carcinoma[J]. Int J Mol Sci, 2022, 23(18):10428. doi:10.3390/ijms231810428.
[2]	FERREIRA E COSTA R, LEÃO M, SANT'ANA M, et al. Oral squamous cell carcinoma frequency in young patients from referral centers around the world[J]. Head Neck Pathol, 2022, 16(3):755-762. doi:10.1007/s12105-022-01441-w.
[3]	O'NEILL E J, TERMINI D, ALBANO A, et al. Anti-cancer properties of theaflavins[J]. Molecules, 2021, 26(4):987-1011. doi:10.3390/molecules26040987.
[4]	XU J, WANG S J, BU S S, et al. Theaflavin promoted apoptosis in nasopharyngeal carcinoma unexpectedly via inducing autophagy in vitro[J]. Iran J Basic Med Sci, 2022, 25(1):68-74. doi:10.22038/IJBMS.2021.59190.13143.
[5]	GHOSH A, LAHIRI A, MUKHERJEE S, et al. Prevention of inorganic arsenic induced squamous cell carcinoma of the skin in Swiss albino mice by black tea through epigenetic modulation[J]. Heliyon, 2022, 8(8):e10341. doi:10.1016/j.heliyon.2022.e10341.
[6]	WANG H, LI Q F, CHOW H Y, et al. Arginine deprivation inhibits pancreatic cancer cell migration,invasion and EMT via the down regulation of Snail,Slug,Twist,and MMP1/9[J]. J Physiol Biochem, 2020, 76(1):73-83. doi:10.1007/s13105-019-00716-1.
[7]	CHEN D D, CHENG J T, CHANDOO A, et al. microRNA-33a prevents epithelial-mesenchymal transition,invasion,and metastasis of gastric cancer cells through the Snail/Slug pathway[J]. Am J Physiol Gastrointest Liver Physiol, 2019, 317(2):G147-G160. doi:10.1152/ajpgi.00284.2018.
[8]	CHO Y A, KIM E K, CHO B C, et al. Twist and Snail/Slug expression in oropharyngeal squamous cell carcinoma in correlation with lymph node metastasis[J]. Anticancer Res, 2019, 39(11):6307-6316. doi:10.21873/anticanres.13841.
[9]	DANTAS R, GUIMARÃES V, DE SOUZA R O, et al. Immunodetection of epithelial-mesenchymal transition and tumor proliferation markers in GLi-1-positive oral squamous cell carcinoma[J]. Appl Immunohistochem Mol Morphol, 2021, 29(5):335-344. doi:10.1097/PAI.0000000000000866.
[10]	BHATTACHARYA R, CHATTERJEE R, MANDAL A, et al. Theaflavin-containing black tea extract:A potential DNA methyltransferase inhibitor in human colon cancer cells and ehrlich ascites carcinoma-induced solid tumors in mice[J]. Nutr Cancer, 2021, 73(11/12):2447-2459. doi:10.1080/01635581.2020.1828943.
[11]	邓力, 蔡婷, 王静雷, 等. 黄芪多糖通过JAK/STAT3通路抑制口腔鳞癌SCC-25裸鼠移植瘤[J]. 中国临床解剖学杂志, 2019, 37(2):169-173.
	DENG L, CAI T, WANG J L, et al. Astragalus polysaccharide inhibits oral squamous cell carcinoma cell line SCC-25 xenograft tumor by suppressing JAK/STAT3 signaling pathway[J]. Chinese Journal of Clinical Anatomy, 2019, 37(2):169-173. doi:10.13418/j.issn.1001-165x.2019.02.011.
[12]	BRENNAN P A, DYLGJERI F, COLETTA R D, et al. Surgical tumour margins and their significance in oral squamous cell carcinoma[J]. J Oral Pathol Med, 2022, 51(4):311-314. doi:10.1111/jop.13276.
[13]	LI H, ZHANG Y, XU M, et al. Current trends of targeted therapy for oral squamous cell carcinoma[J]. J Cancer Res Clin Oncol, 2022, 148(9):2169-2186. doi:10.1007/s00432-022-04028-8.
[14]	JANIAK-KISZKA J, NOWACZEWSKA M, KAŹMIERCZAK W. Oral squamous cell carcinoma - clinical characteristics, treatment, and outcomes in a single institution retrospective cohort study[J]. Otolaryngol Pol, 2022, 76(3):12-17. doi:10.5604/01.3001.0015.7567.
[15]	MAITY R, CHATTERJEE M, BANERJEE A, et al. Gold nanoparticle-assisted enhancement in the anti-cancer properties of theaflavin against human ovarian cancer cells[J]. Mater Sci Eng C Mater Biol Appl, 2019, 104:109909. doi:10.1016/j.msec.2019.109909.
[16]	JUNEJA T, PANDYA M D, SHAH S. Molecular Landscape and computational screening of the natural inhibitors against HPV16 E6 oncoprotein[J]. Asian Pac J Cancer Prev, 2021, 22(8):2461-2469. doi:10.31557/APJCP.2021.22.8.2461.
[17]	SHI S, MA B, SUN F, et al. Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability[J]. J Biol Chem, 2021, 297(3):101016. doi:10.1016/j.jbc.2021.101016.
[18]	BUYUK B, JIN S, YE K. Epithelial-to-mesenchymal transition signaling pathways responsible for breast cancer metastasis[J]. Cell Mol Bioeng, 2022, 15(1):1-13. doi:10.1007/s12195-021-00694-9.
[19]	PENG J, WU H J, ZHANG H F, et al. miR-143-3p inhibits proliferation and invasion of hepatocellular carcinoma cells by regulating its target gene FGF1[J]. Clin Transl Oncol, 2021, 23(3):468-480. doi:10.1007/s12094-020-02440-5.
[20]	NOGUCHI S, HIRANO K, TANIMOTO N, et al. SLUG is upregulated and induces epithelial mesenchymal transition in canine oral squamous cell carcinoma[J]. Vet Comp Oncol, 2022, 20(1):134-141. doi:10.1111/vco.12755.

基因名称	引物序列（5′→3′）	产物大小/bp
Snail	上游：TCGGAAGCCTAACTACAGCGA	160
Snail	下游：AGATGAGCATTGGCAGCGAG	160
Slug	上游：TGTGACAAGGAATATGTGAGCC	123
Slug	下游：TGAGCCCTCAGATTTGACCTG	123
β-actin	上游：CTCGCCTTTGCCGATCC	145
β-actin	下游：GGGGTACTTCAGGGTGAGGA	145

基因名称	引物序列（5′→3′）	产物大小/bp
Snail	上游：TCGGAAGCCTAACTACAGCGA	160
Snail	下游：AGATGAGCATTGGCAGCGAG	160
Slug	上游：TGTGACAAGGAATATGTGAGCC	123
Slug	下游：TGAGCCCTCAGATTTGACCTG	123
β-actin	上游：CTCGCCTTTGCCGATCC	145
β-actin	下游：GGGGTACTTCAGGGTGAGGA	145

组别	mRNA		蛋白
组别	Snail	Slug	Snail	Slug
对照组	1.02±0.14	1.00±0.13	0.87±0.11	0.85±0.09
茶黄素低剂量组	0.57±0.05^a	0.63±0.07^a	0.47±0.08^a	0.46±0.07^a
茶黄素高剂量组	0.31±0.05^ab	0.29±0.04^ab	0.11±0.02^ab	0.09±0.01^ab
茶黄素高剂量+空载组	0.33±0.07	0.30±0.06	0.13±0.03	0.10±0.03
茶黄素高剂量+Snail过表达组	0.98±0.16^c	0.94±0.18^c	0.82±0.10^c	0.80±0.13^c
F	63.958^＊＊	57.763^＊＊	132.685^＊＊	129.660^＊＊

组别	mRNA		蛋白
组别	Snail	Slug	Snail	Slug
对照组	1.02±0.14	1.00±0.13	0.87±0.11	0.85±0.09
茶黄素低剂量组	0.57±0.05^a	0.63±0.07^a	0.47±0.08^a	0.46±0.07^a
茶黄素高剂量组	0.31±0.05^ab	0.29±0.04^ab	0.11±0.02^ab	0.09±0.01^ab
茶黄素高剂量+空载组	0.33±0.07	0.30±0.06	0.13±0.03	0.10±0.03
茶黄素高剂量+Snail过表达组	0.98±0.16^c	0.94±0.18^c	0.82±0.10^c	0.80±0.13^c
F	63.958^＊＊	57.763^＊＊	132.685^＊＊	129.660^＊＊

组别	存活率	凋亡率
对照组	100.00±14.26	2.71±0.64
茶黄素低剂量组	61.75±6.20^a	39.26±5.17^a
茶黄素高剂量组	34.02±5.32^ab	64.98±7.28^ab
茶黄素高剂量+空载组	31.46±4.93	65.17±8.13
茶黄素高剂量+Snail过表达组	93.13±15.41^c	5.05±1.28^c
F	57.918^＊＊	191.004^＊＊