Efficacy And Mechanism Study Of Multi-tyrosine Kinase Inhibitor AL3810 Against Thyroid Cancer

Thyroid cancer(TC)is the most prevalent endocrine neoplasia.Currently,treatment of thyroid cancer mainly include surgical resection,radioactive iodine(RAI)after surgery and inhibition of thyroid stimulating hormone(TSH).in the last decades,in spite that the research has facilitated significant progress in the development of more effective and novel treatment modalities for advanced thyroid cancer,many patients are refractory to RAI treatment,and alternative options for treatment remain limited.Therefore,it is important to look for new drugs and effective strategies to improve the treatment,the survival and their life quality.In our study,we systematically,in vitro and in vivo,evaluated the efficacy of AL3810,a novel and orally bioavailable small-molecule multiple tyrosine kinases inhibitor,against different kinds of thyroid cancer models.The results showed that the agent significantly inhibits thyroid cancer cells growth.AL3810 dose-dependently inhibited the proliferation of thyroid cancer cells TT,TPC-1 and SW579 after teatment for 72 or 120 hours,with mean IC50 values of 2.56 ?M,7.03 ?M and 590 nM respectively.And AL3810 potently inhibits growth of thyroid xenografts SW579 and TT in vivo.In SW579 xenografts model,the inhibition rate of AL3810 at a concentration of 5 mg/kg treating for three weeks reached 97.57%,same to the inhibition rate of Sorafenib 60 mg/kg.Encouragingly,the effect of AL3810 appeared to be sustained suppression in 2 weeks after drug withdrawal treated with AL3810 for 3 weeks.In the sanme time,we try to mimic therapy for advanced thyroid cancer,SW579 xenografts were allowed to grow to 2000 mm3 before the initiatial treatment with AL3810 daily,which markedly reduced tumor volume.Specifically,the mean tumor volume decreased to 300 mm3 after treatment with 10 mg/kg AL3810 for 5 weeks.Similarly,AL3810 substantially inhibited TT tumor growth with the inhibition rate of 66.15% at the dosage of 10 mg/kg treating for 28 days,which was significantly more effective than 60 mg/kg Sorafenib and 30 mg/kg XL184.Our previous data indicated that AL3810 is an angiogenesis inhibitor,therefore,the anti-angiogenesis efficacy of AL3810 was evaluated in the same human thyroid cancer xenograft models described above.To this end,tumor microvessels were immunohistochemically stained with an antibody against the endothelial cell marker CD34,which showed that treatment with AL3810 dramatically decreased the number of microvessels in the tumor compared with the control group: Specifically,the number of microvessels were decreased by 81.88% and 92.83% in SW579 xenograft tumors treated with AL3810 at the dosages of 5 mg/kg and 10 mg/kg,respectively,and 59.06%,63.78% and 81.10% in TT xenograft tumors treated with AL3810 at the dosages of 0.4,2 and 10 mg/kg respectively.Taken together with the antitumor activity of AL3810 described above,these results suggest that AL3810 inhibits thyroid cancer by inhibiting angiogenesis.Flow cytometry results showed that AL3810 arrested thyroid cancer cells in the G1 phase and induced apoptosis in a concentration-dependent manner,eventually led to cell death.After treatment with 0.5 ?M AL3810 for 48 hours,approximately 20% of SW579 cells underwent apoptosis.We further test the inhibition of AL3810 in thyroid cancer nude mice of subcutaneous tumor.Immunohistochemistry results,by testing apoptosis marker TUNEL,demonstrated that AL3810 dramatically induced apoptosis rate in TT and SW579 xenograft.In SW579 xenograft,which were administrated with AL3810 at the dosages of 0.4,2 and 10 mg/kg,the apoptosis rates were 4.66%,22.00% and 36.00% respectively.And in TT xenograft,the apoptosis rates were 6.66%,13.66% and 22.66% respectively.Again,confirm that AL3810 indeed caused apoptosis of thyroid cancer cell.In addition,the agent also could arrest cycle,treatment with 1 ?M AL3810 for 24 hours increased the percentage of SW579 cells in the G1 phase to 64.2%.We then detected the expression of proteins involved in the G1 phase after treating cells with AL3810 by Western Blot,which showed that AL3810 markedly up-regulated p21 and p27 protein and down-regulated CyclinD1,CDK2 and P-Rb,corresponding with the results of flow cytometry.Our previous results demonstrated that AL3810 could inhibit RET phosphorylation at the molecular level.Giving the important role of RET in the development of thyroid cancer,the antitumor activities of AL3810 were evaluated in RET gene fusion-driven thyroid cancer models to assess the therapeutic potential of AL3810 in thyroid cancer involving RET gene fusions in vivo and in vitro.At first,we choosed BaF3 cells transfected with RET.The results showed that AL3810 potently inhibit the proliferation of BaF3 transfected with RET in vitro,blocked cells in G1 phase,and induced cell apoptosis,while had no obvious effect for the growth of BaF3 parents cells.Western Blot results confirmed that the agent could inhibit the RET phosphorylation of BaF3-RET and thyroid cancer cells TT and TPC-1,in addition down-regulated the phosphorylation of downstream protein AKT,ERK1/2 and STAT3.The above results indicated that the agent can inhibit the RET signaling pathways both at the molecular and cellular levels.In conclusion,AL3810 showed promising antitumor activity against human thyroid cancer harboring RET alterations or independent RET.These datas suggest that AL3810 is an effective therapeutic agent in thyroid cancer,providing substantial experimental basis which deserving further clinical development.