The Mechanism And Effect Of Several Small Molecular Compounds On The Proliferation And Apoptosis Of Lung Cancer Cells

Lung cancer,which has high rate of morbidity and mortality,is one of the malignant tumors that is harmful to human's health and life.Due to late diagnosis or metastatic stages,the five years survival ratio of lung cancer is still less than 15 %.Traditional therapy includes operation,radiation therapy and chemical treatment.However,because of the limitations of traditional treatment and its serious side effects,the small molecular compounds targeting on the receptors of cell membrane have been widely studied recently.The inhibitors of small compound targeting on epidermal growth factor receptor(EGFR)are widely used.However,due to the long-term using,there are increasing cases showing the drug resistance.So we focus on another membrane protein called as vascular endothelial growth factor receptor(VEGFR).Now this kind of remedy is mainy used in the treatment of renal carcinoma,liver cancer and gastric carcinoma.And it is rarely in lung cancer.So we choose several kinds of VEGFR tyrosine kinase inhibitors to investigate the effect of inhibitors on the proliferation and apoptosis of non small cancer cells.This study might be hopeful to provide a new thought for lung cancer treatment.We investigated the effects of four kinds of small molecular compounds(Linifanib?Tivozanib?TSU-68 and Laetrile)on the proliferation of A549 and H358.Lung cancer cells were treated with different concentrations of compounds for 48 hours,and then observed under microscope.Then we used MTT assay to detect cell viability.The results showed that Linifanib and Tivozanib could inhibit cell viability in a dose-dependent manner.And we also observed that the cell number was decreased and cell morphology was changed after treatment.However,TSU-68 or Laetrile has no effect on cell viability.So in the following research we only focused on Linifanib and Tivozanib.The inhibition of cell viability are mainly from two aspects.One is cell apoptosis,the other is cell cycle arrest.Thus we detected cell cycle and apoptosis by flow cytometry analysis.The results showed that Linifanib could induce the cell apoptosis and cell cycle arrested in G2/M phase.Tivozanib could arrest cell cycle in G2/M phase.Cell cycle arrest in G2/M phase is mainly regulated by cyclin,cyclin-dependent kinase(CDK)and cyclin dependent kinase inhibitors(CKIs).CDK plays a positive role of regulation,while CKIs act as a negative control.The effect could be investigated by testing the mRNA expression of cell cycle regulators.So we tested the mRNA expression of P21 and Cyclin D1.We found that the mRNA expression of P21 has increased after Linifanib and Tivozanib treatment.While the mRNA expression of Cyclin D1 has not change significantly.The signal pathways in cells could be activated by the combination of the ligands and the receptors.And the biological processes,such as proliferation,differentiation and apoptosis,could be regulated by signal pathway.PI3K/AKT is an important signal pathway to regulate survival and proliferation.And it can be activated by VEGFR.So we tested the level of AKT phosphorylation.The result showed that Tivozanib could down-regulate the level of AKT phosphorylation and inhibit PI3K/AKT signal pathway.Next 16 HBE was used to investigate the effect of Linifanib or Tivozanib on the proliferation and apoptosis of normal cell.The results showed that the cell viability of 16 HBE treated with low concentration(2 ?M)of Linifanib was higher than that of lung cancer cells.However,the cell viability of 16 HBE treated with Linifanib at high concentration(5 ?M,10?M)or Tivozanib was decreased.The results indicate that low dosage of Linifanib in clinical therapy may cause less side effect.In summary,we found that Linifanib inhibitted the proliferation and apoptosis of A549 and H358.Linifanib induced G2/M cell cycle arrest by up-regulating the mRNA level of P21 and inhibiting the expression of cell cycle related protein.Tivozanib inhibitted the proliferation of A549 and H358.The effect of Tivozanib on the proliferation is caused by G2/M cell cycle arrest.This process is regulated by up-regulating the mRNA level of P21 and inhibiting PI3K/AKT signal pathway.And low dosage of Linifanib in clinical therapy may cause less side effect,which might be used to treat lung cancer.