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Endocrine, Metabolic & Immune Disorders - Drug Targets

Author(s): Liehong Wang, Shengkun Zhang, Hua Cai, Qingling Qi, Chunhua Zhang, Ziyi Qi and CuiPing Huang*

DOI: 10.2174/1871530321666210622112753

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Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest, and Alleviates Angiogenic Property in Human Cervical Cancer Cells

Page: [510 - 517] Pages: 8

  • * (Excluding Mailing and Handling)

Abstract

Aims: The aim of the study was to assess the effect of blocking TLR9 signaling on the proliferation of cervical cancer cells and its angiogenic property.

Background: Toll-Like Receptors (TLRs) have been implicated for their crucial role in not only cervical cancer but also in other malignancies. TLR9 is expressed on an array of cells such as macrophages, dendritic cells, melanocytes, and keratinocytes and is reported to modulate oncogenesis along with tumorigenesis by augmenting NF-κB mediated inflammation within the tumor environment. TLR9 has also been reported to positively regulate oncogenesis within the cervix and as a marker to evaluate malignant remodeling of cervical squamous cells. Therefore, this study was designed to explore the functional relevance of blocking the TLR9signaling pathway in cervical cancer cells.

Objective: The objective of the current study was to investigate the effect of human TLR9 antagonist, ODN INH-18, on apoptosis and cell cycle regulation, and angiogenic property of human cervical cancer Caski cells.

Methods: MTT assay was performed to measure cell viability and flow cytometry analysis was performed to assess cell cycle arrest. Quantitative Real-Time PCR (qRT-PCR) analysis was performed to measure fold change in the gene expression of various markers of apoptosis, cell cycle regulation, and angiogenesis.

Results: The qRT-PCR results showed a higher expression level of TLR9 mRNA in Caski cervical cancer cells as compared to normal cervical keratinocytes. The apoptotic, angiogenic, and cell cycle regulatory factors were also deregulated in Caski cells in comparison to normal keratinocytes. The MTT assay demonstrated that treatment of TLR9 antagonist, ODN INH18, significantly reduced the proliferation of Caski cells in a dose-dependent manner. Treatment of ODN INH18 led to substantial cell cycle arrest in Caski cells at G0/G1 phase. Moreover, the qRT-PCR results demonstrated that ODN INH18 treatment led to suppressed mRNA expression of Bcl-2 and enhanced expression of Bax, signifying the induction of apoptosis in Caski cells. Moreover, the expression of cyclin D1, Cdk4, and Cdc25A was found to be reduced whereas expression of p27 was increased in ODN INH18-treated Caski cells; indicating G0/G1 phase arrest. Interestingly, expression of VEGF and VCAM-1 was found to be significantly inhibited in ODN INH18-treated Caski cells, substantiating alleviation of angiogenic property of cervical cancer cells.

Conclusion: The results of our study suggest that inhibiting TLR9 signaling might be an interesting therapeutic intervention for the treatment of cervical cancer.

Keywords: Toll-like receptor 9, cervical carcinoma, angiogenesis, apoptosis, cell cycle, human papilloma viruses.

Graphical Abstract

[1]
WHO Factsheet on cervical cancer, Available from: https://www.who.int/news-room/fact-sheets/detail/human-papillomavirus-(hpv)-and-cervical-cancer
[2]
Tripathi, R.; Rath, G.; Jawanjal, P.; Bharadwaj, M.; Mehrotra, R. Cyclin D1 protein affecting global women’s health by regulating HPV mediat-ed adenocarcinoma of the uterine cervix. Scientific reports, 2019, 9(1), 1-6.
[http://dx.doi.org/10.1038/s41598-019-41394-9]
[3]
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin., 2018, 68(6), 394-424.
[http://dx.doi.org/10.3322/caac.21492] [PMID: 30207593]
[4]
Minorics, R.; Bózsity, N.; Molnár, J.; Wölfling, J.; Mernyák, E.; Schneider, G.; Ocsovszki, I.; Zupkó, I. A molecular understanding of D-homoestrone-induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells. J. Cell. Mol. Med., 2015, 19(10), 2365-2374.
[http://dx.doi.org/10.1111/jcmm.12587] [PMID: 26228523]
[5]
Cho, H.; Chung, J.Y.; Song, K.H.; Noh, K.H.; Kim, B.W.; Chung, E.J.; Ylaya, K.; Kim, J.H.; Kim, T.W.; Hewitt, S.M.; Kim, J.H. Apoptosis inhibitor-5 overexpression is associated with tumor progression and poor prognosis in patients with cervical cancer. BMC Cancer, 2014, 14(1), 545.
[http://dx.doi.org/10.1186/1471-2407-14-545] [PMID: 25070070]
[6]
Senapati, R.; Senapati, N.N.; Dwibedi, B. Molecular mechanisms of HPV mediated neoplastic progression. Infect. Agent. Cancer, 2016, 11(1), 59.
[http://dx.doi.org/10.1186/s13027-016-0107-4] [PMID: 27933097]
[7]
Münger, K.; Baldwin, A.; Edwards, K.M.; Hayakawa, H.; Nguyen, C.L.; Owens, M.; Grace, M.; Huh, K. Mechanisms of human papilloma-virus-induced oncogenesis. J. Virol., 2004, 78(21), 11451-11460.
[http://dx.doi.org/10.1128/JVI.78.21.11451-11460.2004] [PMID: 15479788]
[8]
Yang, X.; Cheng, Y.; Li, C. The role of TLRs in cervical cancer with HPV infection: A review. Signal Transduct. Target. Ther., 2017, 2(1), 17055.
[http://dx.doi.org/10.1038/sigtrans.2017.55] [PMID: 29263932]
[9]
Tiwari, R.K.; Singh, S.; Gupta, C.L.; Bajpai, P. Microglial TLR9: Plausible novel target for therapeutic regime against glioblastoma multi-forme. Cell. Mol. Neurobiol., 2020, 20, 1-3.
[PMID: 32691190]
[10]
Hari, A.; Flach, T.L.; Shi, Y.; Mydlarski, P.R. Toll-like receptors: Role in dermatological disease. Mediators Inflamm., 2010, 2010, 437246.
[http://dx.doi.org/10.1155/2010/437246]
[11]
Jiang, D.S.; Wang, Y.W.; Jiang, J.; Li, S.M.; Liang, S.Z.; Fang, H.Y. MicroRNA-26a involved in toll-like receptor 9 mediated lung cancer growth and migration. Int. J. Mol. Med., 2014, 34(1), 307-312.
[http://dx.doi.org/10.3892/ijmm.2014.1764] [PMID: 24788552]
[12]
Di, J.M.; Zhou, J.; Zhou, X.L.; Gao, X.; Shao, C.Q.; Pang, J.; Sun, Q.P.; Zhang, Y.; Ruan, X.X. Cyclooxygenase-2 expression is associated with vascular endothelial growth factor-C and lymph node metastases in human prostate cancer. Arch. Med. Res., 2009, 40(4), 268-275.
[http://dx.doi.org/10.1016/j.arcmed.2009.03.002] [PMID: 19608016]
[13]
Melisi, D.; Frizziero, M.; Tamburrino, A.; Zanotto, M.; Carbone, C.; Piro, G.; Tortora, G. Toll-like receptor 9 agonists for cancer therapy. Biomedicines, 2014, 2(3), 211-228.
[http://dx.doi.org/10.3390/biomedicines2030211] [PMID: 28548068]
[14]
Schoenborn, J.R.; Wilson, C.B. Regulation of interferon-γ during innate and adaptive immune responses. Adv. Immunol., 2007, 96, 41-101.
[http://dx.doi.org/10.1016/S0065-2776(07)96002-2] [PMID: 17981204]
[15]
Urban-Wojciuk, Z.; Khan, M.M.; Oyler, B.L.; Fåhraeus, R.; Marek-Trzonkowska, N.; Nita-Lazar, A.; Hupp, T.R.; Goodlett, D.R. The role of TLRs in anti-cancer immunity and tumor rejection. Front. Immunol., 2019, 10, 2388.
[http://dx.doi.org/10.3389/fimmu.2019.02388] [PMID: 31695691]
[16]
Yu, L.; Wang, L.; Li, M.; Zhong, J.; Wang, Z.; Chen, S. Expression of toll-like receptor 4 is down-regulated during progression of cervical neoplasia. Cancer Immunol. Immunother., 2010, 59(7), 1021-1028.
[http://dx.doi.org/10.1007/s00262-010-0825-1] [PMID: 20177675]
[17]
Moradi-Marjaneh, R.; Hassanian, S.M.; Hasanzadeh, M.; Rezayi, M.; Maftouh, M.; Mehramiz, M.; Ferns, G.A.; Khazaei, M.; Avan, A. Therapeutic potential of toll-like receptors in treatment of gynecological cancers. IUBMB Life, 2019, 71(5), 549-564.
[http://dx.doi.org/10.1002/iub.2011] [PMID: 30729633]
[18]
Cannella, F.; Pierangeli, A.; Scagnolari, C.; Cacciotti, G.; Tranquilli, G.; Stentella, P.; Recine, N.; Antonelli, G. TLR9 is expressed in human papillomavirus-positive cervical cells and is overexpressed in persistent infections. Immunobiology, 2015, 220(3), 363-368.
[http://dx.doi.org/10.1016/j.imbio.2014.10.012] [PMID: 25454809]
[19]
Lim, E.J.; Park, D.W.; Lee, J.G.; Lee, C.H.; Bae, Y.S.; Hwang, Y.C.; Jeong, J.W.; Chin, B.R.; Baek, S.H. Toll-like receptor 9-mediated inhibi-tion of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression. Exp. Mol. Med., 2010, 42(10), 712-720.
[http://dx.doi.org/10.3858/emm.2010.42.10.070] [PMID: 20739833]
[20]
Belmont, L.; Rabbe, N.; Antoine, M.; Cathelin, D.; Guignabert, C.; Kurie, J.; Cadranel, J.; Wislez, M. Expression of TLR9 in tumor-infiltrating mononuclear cells enhances angiogenesis and is associated with a worse survival in lung cancer. Int. J. Cancer, 2014, 134(4), 765-777.
[http://dx.doi.org/10.1002/ijc.28413] [PMID: 23913633]
[21]
Crawford, R.A.; Caldwell, C.; Iles, R.K.; Lowe, D.; Shepherd, J.H.; Chard, T. Prognostic significance of the bcl-2 apoptotic family of pro-teins in primary and recurrent cervical cancer. Br. J. Cancer, 1998, 78(2), 210-214.
[http://dx.doi.org/10.1038/bjc.1998.466] [PMID: 9683295]
[22]
Rahman, S.F.A.; Muniandy, K.; Soo, Y.K.; Tiew, E.Y.H.; Tan, K.X.; Bates, T.E.; Kumaran, N.M. Co-inhibition of BCL-XL and MCL-1 with BCL-2 selective inhibitors A1331852 and S63845 enhances cytotoxicity of cervical cancer cell lines. Biochem. Biophys. Rep., 2020, 22, 100756.
[http://dx.doi.org/10.1016/j.bbrep.2020.100756] [PMID: 32346617]
[23]
Li, X.; Jiang, S.; Tapping, R.I. Toll-like receptor signaling in cell proliferation and survival. Cytokine, 2010, 49(1), 1-9.
[http://dx.doi.org/10.1016/j.cyto.2009.08.010] [PMID: 19775907]
[24]
Donjerkovic, D.; Scott, D.W. Regulation of the G1 phase of the mammalian cell cycle. Cell Res., 2000, 10(1), 1-16.
[http://dx.doi.org/10.1038/sj.cr.7290031] [PMID: 10765979]
[25]
Giacinti, C.; Giordano, A. RB and cell cycle progression. Oncogene, 2006, 25(38), 5220-5227.
[http://dx.doi.org/10.1038/sj.onc.1209615] [PMID: 16936740]
[26]
Hasan, U.A.; Trinchieri, G.; Vlach, J. Toll-like receptor signaling stimulates cell cycle entry and progression in fibroblasts. J. Biol. Chem., 2005, 280(21), 20620-20627.
[http://dx.doi.org/10.1074/jbc.M500877200] [PMID: 15788393]
[27]
McMahon, G. VEGF receptor signaling in tumor angiogenesis. Oncologist, 2000, 5(90001)5(Supp)(Suppl. 1), 3-10. http://dx.doi.org/10.1634/theoncologist.5-suppl_1-3
[PMID: 10804084]