Abstract
Purpose
MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection.
Methods
Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay.
Results
NCI-Н292Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3 at concentrations of 15 and 30 µM than uninfected NCI-Н292 cells (P < 0.05 and P < 0.001, respectively). Transcriptome analysis revealed differential expression of multiple genes involved in cancer progression and metastasis as well as epithelial–mesenchymal transition (EMT). Moreover, we have shown experimentally that NCI-Н292Myc.h cells were more capable of growing and dividing without binding to a substrate. The most likely mechanism explaining the observed changes was found to be TLR4- and IL-1b-mediated activation of NF-κB pathway.
Conclusions
Our results provide evidence that mycoplasma infection is an important factor modulating the effect of MDM2 inhibitors on cancer cells and is able to induce EMT-related changes.
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Acknowledgements
We thank Dr. Olga Timofeeva (Georgetown University Medical Center, USA) who kindly provided NCI-H292 cell line infected with M. hyorhinis (Н292Myc.h).
Funding
This work was supported by the Federal Targeted Program “Research and development on priority directions of science and technology in Russia, 2014–2020”, contract № 14.604.21.0101, unique identifier of the applied scientific project: RFMEFI60414X0101.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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432_2018_2658_MOESM7_ESM.jpg
Online fig. 1 Visualization of gene expression data by means of heatmap. Up-regulated genes are shown in red. Down-regulated genes are shown in blue. Н292_M1 and Н292_M2 – Н292 cells infected with M. hyorhinis; Н292_C1 and Н292_C2 – mycoplasma-free Н292 cells (JPG 1608 KB)
432_2018_2658_MOESM8_ESM.jpg
Online fig. 2 Results of a key node analysis. Summary network of signaling cascades activated in NCI-H292 cells infected with M. hyorhinis. Key node molecules and their complexes are shown in pink. Up-regulated transcription factors are shown in blue. Yellow frame indicates an increased level of expression of one or more molecules in a complex (JPG 2387 KB)
432_2018_2658_MOESM9_ESM.jpg
Online fig. 3 IL-1b-mediated positive feedback loop on a scheme generated by a key node analysis. Key node molecules and their complexes are shown in pink. Up-regulated transcription factors are shown in blue. Yellow frame indicates an increased level of expression of one or more molecules in a complex (JPG 463 KB)
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Boyarskikh, U.A., Shadrina, A.S., Smetanina, M.A. et al. Mycoplasma hyorhinis reduces sensitivity of human lung carcinoma cells to Nutlin-3 and promotes their malignant phenotype. J Cancer Res Clin Oncol 144, 1289–1300 (2018). https://doi.org/10.1007/s00432-018-2658-9
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DOI: https://doi.org/10.1007/s00432-018-2658-9