Effects Of TLR Agonist CBLB502 On A549 Cell Growth And Radiosensitivity

Objectives: To investigate the effects of TLR agonist CBLB502 on A549 cell growth and radiosensitivity, and to explore their molecular mechanisms.Method:1. The existence of TLR5-MyD88 signal pathway in A549 cell was confirmed through RT-PCR, Western-blot, and reporter gene analysis. Then the effects of CBLB502 on A549 cell growth and radiosensitivity were investigated through nude mouse transplantation tumor experiment. 2. DNA fragments bearing specific interfering sequences were ligated into pSicoR vectors, and lentivirus granules bearing different interfering sequences were prepared. HEK 293 cell was infected with lentivirus granules, and stable cell lines were constructed. Virus granules bearing effective interfering sequences were screened through RT-PCR and reportorgene analysis. 3. A549 cell was infected with eligible lentivirus granules, and stable cell lines were constructed. Infection efficiency was confirmed by RT-PCR and reporter gene analysis. The role of TLR5 and MyD88 molecules in CBLB502 regulated growth of lung cancer cells was investigated through nude mouse transplantation tumor experiment, and their molecular mechanisms were expored.Result:1. TLR5 mRNA expression was detected in A549 cell. Nuclear import of P65 protein was promoted and NF-κB reporter gene was efficiently activated by CBLB502 stimulation of A549 cell. Growth of A549 xenograft in mice was inhibited by CBLB502, but its radiosensitivity was not affected. 2. Lentivirus granules targeting TLR5 and MyD88 molecules were prepared, and effective interfering sequences were screened out utilizing HEK293 model. 3. TLR5 and MyD88 down regulated cell lines were independently constructed. These cell lines were subcutaneously transplanted into nude mice, and tumors were excised and weighted 22 days later. Tumor growth was accelerated in TLR5 and MyD88 down regulation group compared with pSicoR control group, with mean tumor weight 705.56±252.98 mg, 327.87±82.47 mg, and 152.86±72.99 mg in TLR5 down regulation group, MyD88 down regulation group, and pSicoR control group ,respectively. Further mechanistic exploration revealed down regulation of genes such as ENA-78, IL-8, MIP-3, etc, decreased neutrophil infiltration, with no impact on macrophage or vascular endothelial cell infiltration.Conclusion:1. TLR5 agonist CBLB502 can activate TLR5-MyD88-NF-κB signal pathway of lung cancer cell line A549 and inhibit the growth of A549 xenograft in mice, with no impact on radiosensitivity of A549 cell. 2. Growth inhibition of A549 cell by CBLB502 relies on TLR5 and MyD88 molecules, which relates to ENA-78, IL-8 and MIP-3 secretion and tumor cell killing by neutrophil.