Title:Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest,
and Alleviates Angiogenic Property in Human Cervical Cancer Cells
Volume: 22
Issue: 5
Author(s): Liehong Wang, Shengkun Zhang, Hua Cai, Qingling Qi, Chunhua Zhang, Ziyi Qi and CuiPing Huang*
Affiliation:
- Department of Obstetrics and Gynecology, The Second Hospital of Shandong University, 250033, China
Keywords:
Toll-like receptor 9, cervical carcinoma, angiogenesis, apoptosis, cell cycle, human papilloma viruses.
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.
