Small Molecule HDACi N3F Exerts Anti-proliferation Effect On Non-small Cell Lines And Researches On Its Mechanism

Lung cancer,also known as primary lung cancer,is characterized with high malignancy and poor clinical prognosis.Non-small cell lung cancer is one of the most common types of lung cancer,accounting for about 80 % of the total number of lung cancers,and the average 5-year survival rate is less than 15 %.Traditional treatment methods,such as surgery,may be effective in the treatment of non-small cell lung cancer.However,once lung cancer is found,it is basically mid-late and misses the best treatment period for surgery.With the traditional chemotherapy for lung cancer having the characteristics of drug resistance and obvious side effects,therefore,the development of new therapeutic drugs and new treatment strategies are crucial for enhancing the effectiveness of treating lung cancer and improving the survival rate of patients.Recent studies have found that the abnormal activity of histone deacetylase is closely related to the occurrence and development of various tumors.Histone deacetylase inhibitor(HDACi)is a new kind of inhibitor chemotherapy drugs,it has become a hot spot in the field of anti-tumor by inhibiting histone deacetylase to promote histone acetylation,promoting transcription and expression of related genes,inhibiting tumor cell proliferation,inducing apoptosis and autophagy,etc.Many HDACi have been already in clinical trial phase,and some have been applied to the clinical treatment of cancers,such as vorinostat(SAHA),which is the first HDACi to be supported by the U.S.FDA for the treatment of cutaneous T-cell lymphocyte tumors.N3F(N-(2-Chloro-5-(trifluoromethyl)phenyl)-N’-hydroxyoctanediamide)is a novel kind of HDACi obtained by structurally modifying from SAHA,which the structure of cap region is modified by replacing the phenyl No.2 position of SAHA with a chlorine atom and No.5 position with a trifluoromethyl group,so that N3 F can be better binding of HDACs,improve the defect of SAHA on poor selectivity and enhance the inhibitory activity on HDACs.In this study,the proliferation of small molecule HDACi N3 F against non-small cell lung cancer cell lines and the molecular mechanism of inducing lung cancer cell apoptosis were studied.Previous studies found that N3 F had significant inhibitory effect on proliferation of multiple lung cancer cells and tumor-bearing growth of subcutaneous lung cancer in nude mice,and could induce autophagy of lung cancer cells,with better effect than SAHA.Here,we will further explore its mechanism of inducing autophagy in lung cancer cells.CCK-8 and plate clone assays were used to detect the effects of N3 F on the proliferation of non-small lung cancer cell lines SPC-A-1 and A549.Cell scratch assay was used to detect the effect of N3 F on the migration of lung cancer cells.Hoechst 33258 staining and flow cytometry assays were used to detect N3F-induced apoptosis in lung cancer cells.The expression of HDAC3,apoptosis related proteins and autophagy related proteins were detected by Western blot.RGFP966,a specific inhibitor of HDAC3,was uesd to detect autophagy of lung cancer cells by Western blot.According to our research results,compared with the control group,N3 F group significantly inhibited SPC-A-1 and A549 cells proliferation,reduced the number of colony formation,and shortened cell migration distance,and significantly induced apoptosis and autophagy of lung cancer cells,significantly down-regulated HDAC3 expression,meanwhile,decreased apoptosis-related proteins Bcl-2 expression and up-regulated Bax expression significantly up-regulated acetyl transferase Tip60 and autophagy related proteins ULK1(Atg1)and Beclin-1(Atg6)expression in a dose-dependent matter(P<0.05).In conclusion,these data suggest that N3 F has significant antitumor activity,which may be due to the fact that N3 F induces apoptosis and autophagy through down-regulation of HDAC3,and the apoptotic pathway may be through the inhibition of Bcl-2 and up-regulation of Bax expression,while autophagy pathway may be through down-regulation of HDAC3,activation of Tip60,and further activation of ULK1 and Beclin-1.