Transcriptome Analysis of Theaflavin-3,3′-Digallate-Mediated Inhibitory Functions on Human Osteosarcoma HOS Cells
HE Tao1,2,3,4, CHEN Zhi-yu1,2,3, YANG Chao-hua1,2,3, WANG Lin-bang1,2,3, LI Qiao-chu1,2,3, ZHAN Fang-biao1,2,3, WANG Yang1,2,3, LIN Xiao-hong5, QUAN Zheng-xue1,2,3*
1. Chongqing Medical University, Chongqing 400016, China;
2. Department of Orthopaedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;
3. Orthopedic Laboratory of Chongqing Medical University, Chongqing 400016, China;
4. Department of Orthopedic Trauma, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing 401147, China;
5. Department of Ultrasound, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing 401147, China
OBJECTIVE To investigate the effects of theaflavin-3,3′-digallate (TF3) on proliferation and apoptosis of human osteosarcoma HOS cells and to explore its underlying mechanism through experiments including transcriptomics. METHODS Human osteosarcoma cells were treated with different concentrations of TF3 and 0.05% DMSO, respectively. Then, cell proliferation was detected by CCK-8 assay, crystal violet staining, colony formation assay and flow cytometry; cell apoptosis rate was detected by Hoechst33258 staining and flow cytometry. After treatment with of TF3 at different concentrations, the protein levels of the molecules related to proliferation and apoptosis of osteosarcoma HOS cells were determined by Western blot. The potential molecular mechanism of TF3 on HOS cells in vitro was revealed by transcriptomics and bioinformatic analysis. Real-time fluorescent quantitative PCR was performed in order to verify SCG2, EPHB1, and CLDN7 mRNA expression levels in cells based on results of transcriptome sequencing. RESULTS Compared with the control group, TF3 inhibited proliferation of osteosarcoma HOS cells and promoted apoptosis (P＜0.05) in a concentration-dependent manner. The expression levels of proliferation related proteins PCNA and Ki67 were all down-regulated by TF3 (P＜0. 01). TF3 decreased the expression of antiapoptotic protein Bcl-2 and apoptosis-related protein Caspase-3 (P＜0. 01). TF3 promoted the expression of apoptotic markers Bax, cleaved caspase-3 and cytochrome C (P＜0.01). Transcriptome analysis revealed 809 differential expressed genes(DEGs) after TF3 treatment in HOS cells, including 213 down-regulated and 596 up-regulated genes. The gene ontology (GO) analysis showed that the function of DEGs were mainly enriched in positive regulation of DNA replication, membrane, protein binding, etc. The Kyoto encyclopedia of genes and genome (KEGG) analysis showed that the function of DEGs were mainly enriched in apoptosis and tumor-related signaling pathways, including pathways in cancer, HIF-1 signaling pathway, TNF signaling pathway, etc. The expression of SCG2, EPHB1, and CLDN7 declined in comparison with the control group (P＜0.05). CONCLUSION TF3 inhibits the proliferation of osteosarcoma HOS cells, and also promote its apoptosis. Its molecular mechanism is potentially related to the regulation of multi-gene and multi-signaling pathway.
何涛, 陈治宇, 杨超华, 王霖邦, 李翘楚, 占方彪, 王旸, 林晓红, 权正学. 茶黄素-3,3′-双没食子酸酯对人骨肉瘤细胞的抑制作用及转录组学分析[J]. 中国药学杂志, 2022, 57(21): 1824-1833.
HE Tao, CHEN Zhi-yu, YANG Chao-hua, WANG Lin-bang, LI Qiao-chu, ZHAN Fang-biao, WANG Yang, LIN Xiao-hong, QUAN Zheng-xue. Transcriptome Analysis of Theaflavin-3,3′-Digallate-Mediated Inhibitory Functions on Human Osteosarcoma HOS Cells. Chinese Pharmaceutical Journal, 2022, 57(21): 1824-1833.
TA H T, DASS C R, CHOONG P F, et al. Osteosarcoma treatment: state of the art . Cancer Metastasis Rev, 2009, 28(1-2):247-263.
[2]
HATTINGER C M, FANELLI M, TAVANTI E, et al. Advances in emerging drugs for osteosarcoma . Expert Opin Emerg Drugs, 2015, 20(3):495-514.
[3]
O′NEILL E J, TERMINI D, ALBANO A, et al. Anti-Cancer Properties of Theaflavins . Molecules, 2021, 26(4):987. Doi: 10.3390/molecules26040987.
[4]
GAO Y, YIN J, TU Y, et al. Theaflavin-3,3′-digallate suppresses human ovarian carcinoma OVCAR-3 cells by regulating the checkpoint kinase 2 and p27 kip1 pathways . Molecules, 2019, 24(4):673. Doi: 10.3390/molecules24040673.
[5]
METZKER M L. Sequencing technologies - the next generation . Nat Rev Genet, 2010, 11(1):31-46.
[6]
SUPPLITT S, KARPINSKI P, SASIADEK M, et al. Current achievements and applications of transcriptomics in personalized cancer medicine . Int J Mol Sci, 2021, 22(3):1422. Doi: 10.3390/ijms22031422.
[7]
LIU M L, GAO Y, TIAN J S, et al. Transcriptomics study on anti-depressant effect of venlafaxine in treatment of CUMS-induced rats with stress . Chin Pharm J (中国药学杂志), 2021, 56(15):1220-1227.
[8]
SZKLARCZYK D, FRANCESCHINI A, WYDER S, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life . Nucleic Acids Res, 2015, 43(Database issue):D447-D452.
JEONG H, MASON S P, BARABÁSI A L, et al. Lethality and centrality in protein networks . Nature, 2001, 411(6833):41-42.
[11]
GILL J, AHLUWALIA M K, GELLER D, et al. New targets and approaches in osteosarcoma . Pharmacol Ther, 2013, 137(1):89-99.
[12]
ZHANG Y J, LIU X Z, ZENG L J, et al. Effect of multi-disciplinary cooperative pharmaceutical care of high dose methotrexate in patients with osteosarcoma . Chin Pharm J (中国药学杂志), 2021, 56(19):1612-1616.
[13]
ABDAL DAYEM A, CHOI H Y, YANG G M, et al. The anti-cancer effect of polyphenols against breast cancer and cancer stem cells: molecular mechanisms . Nutrients, 2016, 8(9):581. Doi: 10.3390/nu8090581.
[14]
ROBERTS E A H, CARTWRIGHT R A, OLDSCHOOL M M. The phenolic substances of manufactured tea. I. -Fractionation and paper chromatography of water-soluble substances . J Sci Food Agric, 1957, 8(2):72-80.
[15]
HE H F. Research progress on theaflavins: efficacy, formation, and preparation . Food Nutr Res, 2017, 61(1):1344521. Doi: 10.1080/16546628.2017.1344521.
[16]
HANAHAN D, WEINBERG R A. Hallmarks of cancer: the next generation . Cell, 2011, 144(5):646-674.
[17]
LI B, ZHOU P, XU K, et al. Metformin induces cell cycle arrest, apoptosis and autophagy through ROS/JNK signaling pathway in human osteosarcoma . Int J Biol Sci, 2020, 16(1):74-84.
[18]
ZHANG L L, YANG C M, HUANG Y R, et al. Inhibitory effect of cardamonin on human osteosarcoma cells and its underlying mechanism . Chin Pharm J (中国药学杂志), 2020, 55(18):1505-1511.
[19]
JIANG Z H, ZHOU X, LI R, et al. Whole transcriptome analysis with sequencing: methods, challenges and potential solutions . Cell Mol Life Sci, 2015, 72(18):3425-3439.
[20]
YAN Z X, LUO Q, ZHANG Y G, et al. A brief review on medicinal plants transcriptome research . Chin Pharm J (中国药学杂志), 2019, 54(7):513-520.
[21]
OUYANG Y R, LI H, BU J, et al. Hypoxia-inducible factor-1 expression predicts osteosarcoma patients′ survival: a meta-analysis . Int J Biol Markers, 2016, 31(3):e229-e234.
[22]
HE G P, PAN X Y, LIU X, et al. HIF-1α-Mediated mitophagy determines ZnO nanoparticle-induced human osteosarcoma cell death both in vitro and in vivo . ACS Appl Mater Interfaces, 2020, 12(43):48296-48309.
[23]
GASPARINI C, VECCHI BRUMATTI L, MONASTA L, et al. TRAIL-based therapeutic approaches for the treatment of pediatric malignancies . Curr Med Chem, 2013, 20(17):2254-2271.
[24]
ZHANG J, YU X H, YAN Y G, et al. PI3K/Akt signaling in osteosarcoma . Clin Chim Acta, 2015, 444: 182-192.
[25]
WU W, JING D, MENG Z, et al. FGD1 promotes tumor progression and regulates tumor immune response in osteosarcoma via inhibiting PTEN activity . Theranostics, 2020, 10(6):2859-2871.
[26]
WANG Z Y, WANG N, LIU P X, et al. AMPK and cancer . Exp Suppl, 2016, 107: 203-226.
[27]
WANG H, YIN J, HONG Y, et al. SCG2 is a prognostic biomarker associated with immune infiltration and macrophage polarization in colorectal cancer . Front Cell Dev Biol, 2022, 9: 795133. Doi: 10.3389/fcell.2021.795133.
[28]
WU Z, SHI J, SONG Y, et al. Claudin-7 (CLDN7) is overexpressed in gastric cancer and promotes gastric cancer cell proliferation, invasion and maintains mesenchymal state . Neoplasma, 2018, 65(3):349-359.
[29]
WANG L, PENG Q, SAI B, et al. Ligand-independent EphB1 signaling mediates TGF-β-activated CDH2 and promotes lung cancer cell invasion and migration . J Cancer, 2020, 11(14):4123-4131.
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