Toll-like receptor (TLR) gene expression and immunostimulatory effect of CpG oligonucleotides in hormone receptor positive cell line T47D and triple negative breast cancer cell line MDA-MB-468

Abstract

Background

We investigated the expression of TLR genes and the effects of CpG ODN in Estrogen Receptor positive, Progesterone Receptor positive breast cancer cell line (T47D) and a triple-negative breast cancer cell line (MDA-MB-468) followed by studying the immunostimulatory activity of CpG oligonucleotides in breast cancer cell lines T47D and MDA-MB-468.

Materials and Methods

We evaluated the expression pattern of TLR genes (TLR1 to TLR9) in T47D and MDA-MB-468 cells using Real-time qPCR analysis. The intracellular TLR9 protein expression was studied by flow cytometry. The effect of CpG ODN on cell viability was tested using MTT assay. The relative expression of pro-inflammatory (IL6 and TNFα) and anti- inflammatory/immunosuppressive cytokines genes (IL10 and TGF beta1) were examined by Real-time qPCR.

Results

We found that MDA-MB-468 cells expressed TLR2, TLR3, TLR6, TLR8, and TLR9 genes and T47D cells expressed TLR3, TRL5, TLR8, and TLR9 genes. Stimulation of TLR9 in vitro with CpG significantly reduced the cell viability of T47D and MDA-MB-468 cells. IL6 cytokine gene expression was significantly reduced in both CpG treated T47D cells and MDA-MB-468 cells. TNFα gene expression was significantly reduced after treatment with CpG in MDA-MB-468 cells but not in T47D cells. IL10 and TGFβ1 expression were downregulated in CpG treated T47D cells. Whereas, IL10 and TGFβ1 were elevated in CpG treated MDA-MB-468 cells.

Conclusion

Our in vitro finding gives preliminary evidence that triggering TLR9 using CpG ODN decreases the cell proliferation and alters the pro-inflammatory cytokines in favor of inhibition of hormone receptor positive breast cancer cells T47D and triple negative breast cancer cells MDA-MB-468.

Keywords:

• Breast cancer
• triple-negative breast cancer
• TLR
• CpG
• T47D
• MDA- MB-468
• apoptosis
• Immunotherapy

Acknowledgements

The authors express our sincere gratitude and acknowledge the funding received from Science and Engineering Research Board (SERB) under Fast Track scheme for Young Scientists, Government of India. We sincerely thank Dr. M.G.R. Educational and Research Institute (Deemed to be University) for providing the laboratory infrastructure and support for conducting the study. We thank Prof. Dr. Rama Vaidyanathan, Director – Dr. APJ Abdul Kalam Centre for Excellence in Innovation & Entrepreneurship for granting permission to use the Real-time qPCR system. We acknowledge the services provided by the flow cytometry facility at IIT-Madras. We acknowledge and thank Dr. Luke Elizabeth Hanna, Scientist ‘E’, Department of HIV-AIDS, ICMR-NIRT for permitting us to use the Real-time qPCR system.

Disclosure statement

Authors have no competing interests to declare.

Author contributions

NS conceived and designed the study and received funding for the study, involved in analysis and interpretation of data, drafting the article and revising it critically for important intellectual content and final approval of the version to be submitted. MR performed the experiments; involved in analysis and interpretation of data and drafted the article.

Additional information

Funding

Science and Engineering Research Board (SERB) is a National funding body under the Government of India.