Study On The Activity Of Pemetrexed Derivatives A12against Human Lung Cancer A549 Cells

Background:In malignant tumors,lung cancer is one of the most common cancers that endanger human life and health.Especially in China,its morbidity and mortality are at the first place.In addition to surgical procedures,chemotherapy is often used in the treatment of lung cancer.Pemetrexed?PMX?,which is used for clinical treatment of lung cancer,is commonly used in second line therapy and with other anticancer drugs for first-line treatment,which has good curative effect.However,the purity of domestic PMX is not high,and more and more adverse reactions have been gradually discovered.The transformation of its structure to reduce the occurrence of adverse reactions has gradually become a new direction of research.Objective:To investigate the inhibitory effect of PMX new derivatives on human lung cancer cells and its mechanism,in order to find out the better effective compounds for follow-up study.Methods:This study selected 12 kinds of novel PMX derivatives?A1-A12?and4 strains of human lung cancer cells?H322 cells,H1299 cells,A549 cells and SPC-A1cells?,firstly using MTT colorimetric method for antitumor activity screening in vitro experiments,the activity of 1 derivatives and 1 strains of good cell lines were obtained,and the detection of derivatives the inhibitory effect of the cell line in different time and different concentration conditions,and to observe the state of cell.Subsequently,in vivo antilung cancer activity research was carried out.BALB/c nude mice?SPF class?were selected as experimental animals.Tumor cells were cultured in vitro to the desired concentration and inoculated to the right axilla of nude mice.The number of tumor cells in each nude mice was 1 x 10⁶.The experiment setted up 3groups,6 nude mice in each group:?1?model group,for the Sodium Chloride Injection 0.9%;?2?PMX control group,for PMX?120 mg/Kg?;?3?Trial product group,for the derivatives?120 mg/Kg?,3 groups were administered intravenous injection,once evay 2 days.The volume of each group was 0.1 mL/10g,and the experiment lasted 21 days.The state of the nude mice was observed after each administration.The length and width of the transplanted tumor and the amount of food and body weight were measured every 3 days.The nude mice were killed after the administration,and the tumor was weighed and the tumor suppressor rate was calculated.At last,the study of anti lung cancer cells mechanism,mainly studies the derivatives on lung cancer cells?including vertical migration and migration ability?,cell cycle and apoptosis,respectively by Transwell assay and wound healing assay,PI staining assay and Annexin V-FITC/PI double staining experiments were performed to study.Results:?1?the MTT screening experimental results showed that although these compounds were the same as the PMX derivatives,but their effects on tumor cell effect were significantly different,the same kind of cells on the sensitivity of different derivatives were different,and the inhibitory effect of compound A12 on A549 cells was the most significant,IC₅₀0 is 1.26±0.15?mol/L,and this result had good repeatability.The compounds A12 and A549 cells were screened for further different action time MTT experiments and morphological observation.It was found that the survival rate of A549 cells was gradually decreased with the increase of action time and the increase of action concentration,and it was found that A12 could affect the morphology of A549 cells and make cell morphogenesis and shrinkage.?2?in vivo experiments confirmed that,compared with the model control group,PMX had no significant effect on the behavior,activity,body weight and food intake of nude mice compared with the model control group,while the nude mice giving PMX had a decrease in feeding intake.In the inhibition of tumor,the tumor inhibition rate of nude mice given to A12 was 41.73%,which was better than that of the PMX control group?32.77%?.It is indicated that the compound A12 has inhibitory activity on the growth of human lung cancer A549 cells transplanted in nude mice?P<0.01?.Under the same dosage,A12 had better antitumor effect than PMX,and the side effects were superior to those of the positive drug PMX.?3?The Transwell experimental results showed that after 8 hours A12 treatment,the number of A549 cells that migrated to the bottom of the Transwell compartment was less,the A12 compound has obvious effect on inhibiting migration of A549 cells?P<0.01?,and this inhibition is concentration dependent,this result was further confirmed by wound healing assay;PI staining results showed that the percentage of A12 can significantly increase the G2/M phase cells?P<0.01?,the upcoming A549 cell arrest in G2/M phase,so that it cannot enter G0 and copy the next cycle,then it is unable to perform its biological function;Annexin V-FITC/PI double staining results showed that A12 can significantly improve the early apoptosis rate of A549 cells?P<0.05?,late apoptosis and necrosis rate?P<0.01?and total apoptosis rate?P<0.05?.Conclusion:In the early stage of this study,a new PMX derivative A12 was screened.It was found that it had a good in vitro inhibitory activity on human lung cancer A549 cells,and it was also confirmed that could also inhibit the proliferation of A549 cells in vivo by nude mice A549 cell transplantation tumor model.Its mechanism is related to the migration of A549 cells,cell cycle and the process of apoptosis.