The Early Study On The Effect Of New Camptothecin Analogue FL118 On Human Non-Small Cell Lung Cancer

Lung cancer,known as “king of cancer”,is the one of the deadly cancer all over the world.Based on the pathological type,lung cancer is divided into small cell lung cancer and non-small cell lung cancer(NSCLC),of which non-small cell lung cancer accounts for more than 85% of all lung cancer cases.In the past decades,the incidence and mortality of non-small cell lung cancer have been increasing.At current stage,NSCLC has become the most deadly tumor in both China and the world.About 75 percent of patients with NSCLC have been diagnosed at the advanced stage due to lack of early signs.For patients with advanced or metastatic NSCLC who have lost the window for surgery,systemic chemotherapy and radiotherapy are the mainstream treatments.However,radiotherapy and chemotherapy resistance,as well as chemotherapeutic drugs-induced DNA damage and toxicity of liver and kidney hematopoietic system,greatly limit the effectiveness of current therapies lead to a low 5-year survival rate.Therefore,screening for anti-NSCLC drugs with great efficacy,few side effects,and low drug resistance has become a challenge in current clinical research.The researchers found a new camptothecin analogue,FL118,when screening anticancer drugs with the Survivin gene as a target and biomarker.Previous studies have shown that FL118 is a candidate drug with high efficacy,good tollerance,low drug resistance and broad anti-tumor spectrum.However,the effect of FL118 on NSCLC has not been clarified.This study aims to explore the effect of FL118 on non-small cell lung cancer,promote the further research and development of FL118,and provide new options for patients with non-small cell lung cancer.Objective: To study the effects of FL118 on the cell viability,apoptosis,invasion and metastatic potential of NSCLC A549 and H520 cells,and to provide an experimental basis for further study on the mechanism of FL118 against non-small cell lung cancer.Methods: CCK-8 assay was used to detect the effect of FL118 on the cell viability of A549 and H520 cells.The inverted phase contrast microscope were used to observe the morphological changes of the A549 and H520 cells.Western Blot,Hoechst-PI staining experiment and Annexin V-FITC/PI double staining followed by flow cytometry was used to detect the effect of FL118 on apoptosis of A549 and H520 cells.Transwell assay and wound scratch experiment were used to detect the effect of FL118 on the invasion and migration of A549 and H520 cells.Western Blot and Immunofluorescence and Western Blot were applied to detect the effects of FL118 on the expression levels of EMT marked proteins E-cadherin and N-cadherin and the transport of E-cadherin and N-cadherin in A549 and H520 cells.Results: Results from CCK-8 assay showed that A549 and H520 cells had no changes in cell viability after treatment with different concentrations of FL118(2 nM,5 nM,10 nM,20 nM and 50 nM)for24 h.When the incubation time was extended to 48 h,FL118 was able to decrease the cell viability of A549 and H520 cells in a dose-dependent manner with increasing concentrations.Morphological photographs showed that A549 and H520 cells were exposed to different concentrations of FL118(2 nM,5 nM,10 nM and 20 nM)for48 h,the cells gradually showed different degrees of apoptosis-like characteristics.The results of Western Blot showed that the expression levels of proapoptotic proteins Bax and cleaved-PARP increased with the increase of drug concentration,and the expression level of anti-apoptotic protein Bcl-2 decreased with the increase of drug concentration.The results of Hoechst/PI double staining and Annexin V/PI double staining followed by flow cytometry further indicated that FL118 can induce apoptosis in A549 and H520 cells,but the sensitivity of the two cells is different.In the Annexin V/PI double staining followed by flow cytometry experiment,the average apoptotic rate of A549 cells was 25% and 35% at concentration of 10 nM and 20 nM,respectively.Compared with the control group,the differences were significant.In H520 cells,when the concentration of FL118 was 10 nM,the mean apoptotic rate was 8%(p>0.05 vs.Control group).When the concentration of FL118 was 20 nM,the average apoptotic rate was 10%(compared to the control group,There was difference).(2)According to the CCK8 results for24 h,the optimal experimental dose(20 nM and 50 nM)was used to detect the effect of FL118 on the invasion and migration ability of A549 and H520 cells and EMT process.In Transwell and scratch experiment,FL118 significantly inhibited the invasion and migration of A549 and H520 cells with increasing drug concentration(compared with the control group,both have significant difference).Immunofluorescence and Western Blot assay showed that FL118 could significantly promote the expression level of E-cadherin and inhibit the expression level of N-cadherin,indicating that FL118 can significantly inhibit the EMT process of A549 and H520 cells.Conclusions: 1.FL118 significantly reduced the cell viability of A549 and H520 cells at 48 h,and was dose-dependent in a range of concentrations.2.FL118 can obviously induce apoptosis in A549 cells,but it has weak effect on apoptosis in H520 cells.The sensitivity difference of two cells in the induction of apoptosis was significant.3.FL118 can significantly inhibit the invasion and migration ability and EMT process of A549 and H520 cells at 24 h.