Study On The Anti-Tumor Activities And Mechanisms Of A Novel Multi-target Tyrosine Kinase Inhibitor, AL-8326

Objectives:To evaluate the anti-proliferation activity and the therapeutical effect on HL-60and SKOV3xenografled athymic mice of the novel anti-tumor multi-target tyrosine kinase inhibitor, AL-8326. To explore the mechanisms by which AL-8326induced cell cycle arrest, apoptosis, Aurora kinases activities inhibition and exerted tumor angiogenesis inhibition activity.Methods:1. Anti-tumor activity of AL-8326in vitro and in vivo:1) MTT assay and SRB assay were used to evaluate the cytotoxicity of AL-8326on16tumor cell lines and calculate the IC50value.2) HL-60, SKOV3, Bel-7402and Hela xenogratted athymic mice models were established to evaluate anti-tumor activity of AL-8326in vivo.2. The mechanism study of AL-8326:1) ELISA assay was used to exam whether AL-8326was an ATP-competitive inhibitor of tyrosine kinase.2) Western blotting assay was used to investigate the effect of AL-8326on the activation of Erkl/2and Akt when cells were exposed to hVEGF165or b-FGF.3) HL-60and SKOV3cells was treated with increasing concentrations of AL-8326, flow cytometry analysis was performed to determine cell apoptosis and autophagy by using Annexin-V/PI and AO staining; The expression of apoptosis-related proteins (cleaved-Caspase-3and cleaved-PARP) were conducted by Western blotting.4) HL-60, SKOV3and U937cells were treated with increasing concentrations of AL-8326, PI staining followed by flow cytometry analysis was performed to determine cell cycle distribution; Western blotting assay was conducted to validate the expression of the proteins related to cell cycle in HL-60, SKOV3and U937.5) Aurora-A/B/C kinases were treated with different ATP concentrations and increasing concentrations of AL-8326, the inhibition effect of AL-8326on Aurora-A/B/C kinases were analyzed.6) CAM assay was performed to test the antiangiogenic activity of AL-8326.7) HUVEC migration assay was used to evaluate the anti-invasion activity of AL-8326.Results:1. AL-8326showed broad anti-tumor spectrum in vitro and effective anti-tumor activity in vivo:1) Cytotoxicity assay demonstrated that AL-8326was a potential and high efficient anti-tumor compound with average IC50value of7.96±1.63μM in16tumor cell lines. To be specific, the IC50value of each cell line was:human ovarian carcinoma cell lines SKOV3(17.1±0.57μM), A2780(2.32±0.11μM), human promyelocytic leukemia cell line HL-60(4.84±0.45μM), T-lymphoblastic leukemia cell line Molt-4(4.85±1.43μM), human chronic myelogenous leukemia cell line K562(6.62±0.29μM), human histiocytic lymphoma cell lineU937(10.0±2.28μM), human breast carcinoma cell lines SKBR3(12.2±2.00μM), MDA-MB-231(7.77±0.02μM), human highly metastatic lung cancer cell line95-D (4.92±2.26μM)、human hepatoma cell lines BEL-7402(2.04±1.91μM), HepG2(8.29±2.32μM), SMMC-7721(8.51±1.61μM), human renal carcinoma786-O (10.1±3.97μM), coloncell line HCT-116(10.4μ3.18μM), human cervical carcinoma cell line Hela (11.2±2.71μM) andhuman gastric carcinoma cell line SGC7901(6.24±0.90μM). Meanwhile, the average IC50value of positive drug, Sunitinib in16tumor cell lines is6.13±1.72μM, which is a little lower than AL-8326.2) In HL-60xenografls model, the mice were sacrificed after AL-8326administration for14days. The tumor inhibition rate (%) of45mg/kg Sunitinib,45mg/kg AL-8326,15mg/kg AL-8326,5mg/kg AL-8326group were48.8%,84.2%,74.0%and47.9%, respectively, and the corresponding T/C (%) values were74.8%,24.1%,36.3%and64.8%, respectively; In SKOV3xenografts model, the mice were sacrificed after AL-8326administration for23days. The tumor inhibition rate (%) of45mg/kg Sunitinib,45mg/kg AL-8326,15mg/kg AL-8326,5mg/kg AL-8326group were72.7%,89.6%,80.4%and38.1%, respectively, and T/C (%) values were36.4%,12.9%,30.7%and72.0%, respectively; In Bel-7402xenografts model, the mice were sacrificed after AL-8326administration for27days. The tumor inhibition rate (%) of45mg/kg Sunitinib,45mg/kg AL-8326,15mg/kg AL-8326,5mg/kg AL-8326group were54.8%,74.7%,62.8%and47.5%, respectively, and T/C (%) values were49.4%,26.2%,35.9%and53.3%, respectively; In Hela xenografts model, the mice were sacrificed after AL-8326administration for19days. The tumor inhibition rate (%) of45mg/kg Sunitinib,45mg/kg AL-8326,15mg/kg AL-8326,5mg/kg AL-8326group were42.7%,76.9%,53.5%and27.9%, respectively, and T/C (%) values were45.2%,23.3%,38.9%and58.0%, respectively.2. AL-8326intervene the proliferation of tumor cells from induction of apoptosis, cell cycle arrest, inhibition of the activation of Aurora-B kinase and angiogenesis as well as cell migration:1) AL-8326is an ATP-competitive inhibitor:ELISA assay showed that, the inhibition of VEGFR-2by AL-8326was decreased in the presence of increasing concentrations of ATP. In order to achieve the same inhibition rate, increasing the concentrations of ATP resulted in an increase of AL-8326concentrations, suggesting that AL-8326is an ATP-competitive inhibitor.2) HUVECs were treated with0.001,0.01,0.1and1μM AL-8326as well as1and10μM Sunitinib followed with hVEGF165and b-FGF stimulation. The western blotting results showed an obvious inhibition of phosphorylation levels of Erkl/2and Akt based in an AL-8326dose-dependent manner. Furthermore, this effect under AL-8326treatment was much stronger than under same concentration of Sunitinib treatment.3) Al-8326exerts anti-tumor activity through induction of apoptosis and cell cycle arrest:HL-60and SKOV3cells were treated with5,10,15,20μM AL-8326and10nM Taxol respectively for24h. There were25.8%,41.9%,57.8%and78.5%apoptosis in serial concentrations of AL-8326treated HL-60cells; In SKOV3cells the apoptosis were31.1%,48.0%,52.4%and58.3%. Meanwhile, Taxol caused88.2%and57.0%cells apoptosis, respectively. When treated with AL-8326(2.5,5,10,15and20μM) for24h, Caspase-3activation (pro-Caspase-3was cleaved into its activated form, cleaved-Caspase-3) and the activation of cleavage of poly (ADP-ribose) polymerase (PARP) were observed in both HL-60and SKOV3cells;72h treatment of AL-8326could block the SKOV3cells in the G2/M phase in a dose-dependent manner, the G2/M phases were28.5%,67.4%and70.4%, respectively. In addition, AL-8326treatment induced the accumulation of cells with4N and8N DNA content of both HL-60and U937cells in a dose depend-manner (the percentage of polyploidy was87.4%,83.9%and18.3%in HL-60cells and13.7%,67.2%and66.4%in U937cells respectively, compared to0.84%or0.74%in control cells). In contrast, there were no obvious polyploidy appeared in Sunitinib and Taxol treated cells cells. Moreover, with2.5,5,10,15and20μM AL-8326treatment for24h, the expression of cdc25B, cyclin B and cdc2were decreased in both HL-60and SKOV3cells;4) AL-8326can selectively inhibit Aurora-B kinase activity in a dose-dependent manner; the IC50value was47nM. However, AL-8326had no obvious inhibition effect on Aurora-A, Aurora-C kinases activities (IC50>1,000nM). The results suggest that AL-8326has a selective inhibition effect on Aurora-B kinase activity in vitro.5) In Chick embryo chorioallantoic membrane (CAM) assay, AL-8326exhibited anti-angiogenesis activity in a dose-dependent manner;1.25,2.5and5μM Al-8326could obviously inhibit the migration of the HUVEC endothelia cells, and the effect of AL-8326on both angiogenesis and cell migration were more apparent than Sunitinib.Conclusion:AL-8326possessed broad spectrum and high anti-tumor activity in vitro, and exerted more effective anti-tumor activity than Sunitinib in vivo. AL-8326, as a multi-targeted molecule tyrosine kinase inhibitor, has a significant inhibition effect on the activities of VEGFR, FGFR, PDGFR and Ret tyrosine kinases. AL-8326is an ATP-competitive inhibitor of tyrosine kinase, down regulation of phosphorylation levels of Erk1/2and Akt, and exerted its anti-tumor activity by inducing cells apoptosis and the cell cycle arrest. Meanwhile, AL-8326can also exerted its anti-tumor activity by inhibiting the activity of Aurora-B kinases. Furthermore, AL-8326can inhibit the cancer angiogenesis and the migration ability of tumor cells. Taken together, AL-8326is a novel, high efficient multi-target small molecule tyrosine kinase inhibitor, fully possessed with the potential for further development into a new type of anti-cancer drugs, as well as the good prospects for clinical application.