Multi-targeted Tyrosine Kinase Inhibitor ZD6474Through The NF-KappaB Pathway Inhibition Of IL-8Expression In Ovarian Cancer

Objective: IL-8, a pro-angiogenic factor, can promote tumor angiogenesis and has aclose relationship with tumor growth and metastasis, clinical study has found that highexpression of IL-8in ovarian cancer patients has poor prognosis. The nuclear factor-kappa B (Nuclearfactor-kappa B) is involved in cell proliferation, apoptosisregulation, immune response, inflammation, and tumor development and transfer ofimportant transcription factor. Important transcription factor for the expression of IL-8is also activated. In recent years, multi-targeted tyrosine kinase inhibitor has showed abroad-spectrum anti-tumor effect is powd close attention to in the fiels of concerntreatment and ZD6474is one them. ZD6474can inhibit VEGFR2and EGFR tyrosinekinase targets a broad-spectrum anti-angiogenic effect. In this study,ZD6474dealingwith overican cancer cell line SKOV3has control of IL-8and NF-kappaBpathway.An experimental basis for the development of new treatment strategies isprovided for ovarian cancer.Methods:1.MTT assay ZD6474proliferation of SKOV3cells.2. RT-PCR detect the expression of IL-8in SKOV3cells after ZD6474treatment atthe genetic level.3. ELISA detect ZD6474at the protein level of IL-8expression and regulation.4.Dualluciferase reporter system detects ZD6474IL-8expression and regulation ofNF-kappaB.Results:1.ZD6474inhibits the proliferation of SKOV3cells according to the experiment byusing different doses of ZD6474to treat the ovarian cancer cell line SKOV3. The resultsshowed that ZD6474significantly inhibited the proliferation of SKOV3and the ZD6474with concentration of2umol/L has reached the50%inhibition rate.2. The expression about whether ZD6474will affect IL-8in the gene level detection.Treat ovarian cancer cell line SKOV36h separately with0,0.1,1,2umol/L ZD6474to see the expression of real-time quantitative PCR (Real-Time PCR) to detect themRNA of IL-8. The results show that the ZD6474concentration of1mol/L cansignificantly inhibit IL-8mRNA expression (P <0.05).3. The expression and regulation of ZD6474detecting IL-8at the protein level. SKOV3cells was incubated overnight with different concentrations of ZD6474in the serumculture solution, and then was dealt with10ng/mL TNF-α8h. Finally ELISA detectsthe levels of IL-8in culture supernatant. The results are consistent with the Real-timePCR.4. By using the Dual Luciferase, whether that ZD6474has negative regulation of theIL-8is associated with NF-kappaB, respectively, upstream of the transcriptionalregulatory region of the wild-type IL-8gene deletion and NF-kappa B binding sites oftranscription regulatory region was cloned into the fluorescein enzymes upstream of thereporter gene construct p-IL-8-luc and p-IL-8(ΔNF-κB)-luc reporting system. Thetwo plasmids with PRL-TK-luc (internal reference) transfected SKOV3cells for24h after containing different concentrations of ZD6474serum-free medium overnight,and then was dealt with10ng/ml TNF-alpha6h, dual luciferase the results of the thedetection kit luciferase expression levels ZD6474inhibits the wild-type IL-8promoter(p-IL-8-luc) sequence mediated luciferase expression (P <0.05), while themissing NF-kappa B binding sites mutant IL-8start sub (P-IL-8((ΔNF-kappaB)-luc) luciferase expression mediated by the sequence had no inhibitory effect. Theresults show that ZD6474by NF-kappaB pathway has negative regulation of IL-8.And use p-NF-κB-luc luciferase reporter system to detect ZD6474active role for NF-kappaB.Results: ZD6474can effectively inhibit the activation of TNF-alpha-induced NF-kappa B.Conclusions: ZD6474inhibits the proliferation of ovarian cancer cell lines. Theinhibition may have connection with the inhibition of ZD6474on NF-kappa B, andthe expression meducal of IL-8. The study showed that ZD6474is expected to obtain a better therapeutic effect on high expression of IL-8ovarian cancer.