A Preliminary Study On The Regulation Of Angiogenesis In Hepatocellular Carcinoma By 2,4-Quinazoline Derivatives

1. The research background Hepatocellular carcinoma(HCC) is the most common primary malignant liver tumor and the third leading cause cancer‐related deaths worldwide. Although curative treatments such as surgical resection, liver transplantation, transcatheter arterial chemoembolization(TACE) and local ablation have improved the outcome in early‐stage HCC, most patients are not considered as candidates for these therapies because of an advanced tumor stage, inadequate liver function at the time of diagnosis and the lack of donor livers,etc. This limits the treatment to fewer options, such as target‐oriented chemotherapeutic methods. However, the survival rate has increased only a little over recent decades. Thus, a beteer pharmacological therapy is urgently needed for patients with advanced HCC.2. The research objective The present study was designed to investigate the effect of 2,4‐quinazoline derivatives of human hepatocellular carcinoma cells and human umbilical vein endothelial cell on the proliferation; on the adhesion ability; on the migration and invasion abilities; the angiogenesis of Chick embryo chorioallantoic membrane.Through researching the 2,4 quinazoline derivatives of endothelial cells and tumor cells, we analysis the inhibitory effects of the derivative on the angiogenesis.3. The research methods 1 Using MTT,we test the influence of 2,4‐quinazoline derivatives on the inhibition of the hepatocellular carcinoma cell(Hep G‐2,SMMC‐7721)and human umbilical vein endothelial cell(HUVEC)。 2 Using cell adhesion assays,we test the ability of the attachment of endothelial cells to type I collagen。 3 Using cell wounding assay,we evaluate 2,4‐quinazoline derivatives on the inhibition against migration. 4 Using CAM assay,we evaluate 2,4‐quinazoline derivatives’ anti‐angiogenesis activities 5 Using VEGFR2 kinase inhibition assay, we investigate the molecular mechanism of Quinazoline Derivatives‐induced inhibition on angiogenesis 6 To evaluate the expression of the VEGFm RNA,we use RT‐PCR assay. 7 Western blot analysis was performed to test the expression of Akt/m TOR/P70S6 K signaling.4. The research results1The cytotoxic activities of 2,4‐disubstituted quinazoline derivatives against hepatocellular carcinoma cells(Hep G‐2,SMMC‐7721)and Human Umbilical Vein Endothelial Cells(HUVEC) were determined by using the MTT assay. The results showed that most of 2,4‐disubstituted quinazoline derivatives had high cytotoxicity against hepatocellular carcinoma cell(Hep G‐2,SMMC‐7721)and poor cytotoxicity again Human Umbilical Vein Endothelial Cells. 2Most of the 2,4‐disubstituted quinazoline derivatives inhibited HUVEC adhesion at both 1h and 3h. Compound 11 d showed the most potent inhibition. 3Most of the 2,4‐disubstituted quinazoline derivatives show the inhibitory on migration when the concentration of these compounds was increased. Compound 11 d showed the best inhibitory activity 4Among all the CAMs treated with 2,4‐quinazoline derivatives,Compound 11 d excellently inhibited the angiogenesis of chick embryos. 5ELISA‐based tyrosine kinase assay was conducted to examine the effects of Quinazoline Derivatives on VEGF‐stimulated P‐VEGFR2. It was found that compound 11 d suppress kinase activity of VEGFR‐2 with an IC50 of 5.499 μM that is better than others. 6The m RNA level of VEGFA was determined by using RT‐ PCR, compound 11 d treatment changed the expression levels of VEGF in a dose‐dependent manner. 7Compound 11 d inhibites the activation of VEGF‐induced Akt/m TOR/P70S6 K signaling in HUVECs. 8 Compound 11 d induces HCC cell death via inhibition of Akt/m TOR/P70S6 K signaling.5.The research conclusion The results showed that most of 2,4‐ quinazoline derivatives possessed higher cytotoxicity and better inhibitive effect against the adhesion and migration. Among these derivatives, compound 11 d was the most potent agents that showed significant anti‐angiogenesis activities in chick embryo chorioallantoic membrane assay.Using a VEGFR2 kinase kit combined with colorimetric ELISA detection, optimization of the series led to the discovery of compound 11 d, a potent, bioavailable VEGFR2 inhibitor. In previous study, we identified the effect of compound 11 d on hepatocellular carcinoma growth and now we identified a mechanism of this action. Our data demonstrate that compound 11 d inhibits the biochemical function of AKT,m TOR and p70S6 K. Overall, the results indicate that the compound 11 d are promising anti‐HCC agents that deserve further development.