Studies On Anticancer Activity And Mechanisms Of Novel Compounds Targeting Dual Inhibition Of TBK1and IKKi In Human Tongue Cancer

Objective:(1) To investigate TBK1and IKKi acting as the important role in human tongue cancer cells proliferation, and establish the theoretical basis of targeting dual inhibition of TBK1and IKKi for human tongue cancer therapy.(2) To evaluate anticancer activity of the compounds targeting dual inhibition of TBK1and IKKi.(3) To illustrate the compounds inducing the apoptosis of human tongue cancer cells and explore the related mechanism.(4) To observe the treatment effect of selected compound on human tongue cancer cells nude mice, investigate the anticancer effect and related mechanism, and provide proof of concept for further drug discovery efforts that may lead to novel strategies and efficient therapeutics for the treatment of human tongue cancer.Methods:(1) Western blotting analysis of the expression of p-IKKi, IKKi, p-TBK1, TBK1in human tongue cancer cells, SCC-9, SCC-15, and SCC-25, cell proliferation analysis of SCC-25cells transfected with TBK1or/and IKKi siRNAs.(2) Western blotting analysis of IRF-3and p-IRF-3(Ser396) in mouse macrophage RAW264.7cells treated with different concentrations of compounds for1h, followed by incubation with LPS (1μg/mL) for1h. Human tongue cancer cells were treated with different concentrations of indicated compounds for72h, and cell proliferation was analyzed by MTT assay.(3) Apoptotic cells in SCC-9cells treated with1.0μM200A for72h were identified by TUNEL staining (red). Cells were counterstained with DAPI (blue). Western blotting analysis of apoptotic protein was performed in SCC-9cells treated with the compounds. (4) Establishing SCC-9tumor growth in nude mouse model divided randomly into two groups. One group was administered ip with200A (100mg/kg, once a day), another group treated with vehicles. Tumor growth was monitored and measured every three days. Immunohistochemistry, Western blotting, RT-PCR analysis of the expression of VEGF and p-AKT were performed before and after200A treatment.Results:(1) p-IKKi, IKKi, p-TBK1, TBK1were expressed in the human tongue cancer cell lines, while the expression of p-IKKi was higher than p-TBK1. Knockdown of either IKKi or TBK1had only minor effects (P>0.05) on cell survival while knockdown of both TBK1and IKKi significantly (P<0.01) inhibited cell proliferation.(2) Western blotting analysis indicated that treatment with compounds targeting dual inhibition of TBK1and IKKi-SR8185,200A and200B reduced or completely blocked LPS-induced phosphorylation of IRJF-3, while200A displayed the best inhibition. MTT assay showed that these compounds displayed potent cytotoxic activities on tongue cancer cell lines with IC50values in low micromolar range (0.430-1.901μM). Among these compounds,200A presented highest anti-proliferative activity in all tested cancer cell lines with the lowest IC50.(3) Morphological study confirmed that compounds targeting dual inhibition of TBK1and IKKi could induce the apoptosis of human tongue cancer cells, the expressions of the pro-apoptotic proteins significantly increased in a time and dose dependent manner when human tongue cancer cells were exposed to the compounds. Meanwhile the expressions of Cyclin D1, p-AKT, and p-CYLD decreased.(4) Treatment of200A significantly suppressed the growth of SCC-9xenograft tumor in nude mice from day22(P＜0.05), from day31(P<0.01). It should be noted that there were no apparent signs of toxicity in nude mice treated with200A. After treatment, the tumor weight of200A-treated group mice was smaller than control group (P<0.05) with the tumor weight inhibition rate56.97%. Immunohistochemistry analysis showed that treatment with200A led to significant reduction of VEGF and p-AKT expression (P<0.01). Meanwhile RT-PCR and Western blotting confirmed it separately.Conclusion:(1) TBK1and IKKi are expressed and phosphorylated in human tongue cancer cell lines while the phosphorylation level of IKKi is higher than that of TBK1. Simultaneously targeting both TBK1and IKKi is necessary for efficient suppression of cancer cell growth, indicating that TBK1and IKKi may become molecular targets of new anticancer drugs.(2) Novel compounds targeting dual inhibition of TBK1and IKKi can be the candidates of anticancer drug because of strong inhibition activity on SCC-9, SCC-15and SCC-25human tongue cancer cells proliferation, especially200A shows the best.(3) Compounds targeting dual inhibition of TBK1and IKKi have the anticancer effect, which can induce the apoptosis of human tongue cancer cells in a time and dose dependent manner. The mechanism should be down-regulate the expression of Cyclin D1, p-AKT, p-CYLD. Besides, compound200A successfully suppress the growth of SCC-9xenograft tumor in nude mice without any apparent signs of toxicity, and the anticancer effect may be via their suppression of AKT activation and down-regulating the expression of VEGF, leading to impaired angiogenesis, and therefore the inhibition of tumor growth.