Novel Histone Deacetylase Inhibitor N25 Exerts Anti-tumor Effects And Induces Autophagy In Human Glioma Cells By Inhibiting HDAC3

Glioma is both the most common and lethal primary brain tumor which occurs in adults and children.The median survival rate is only 13-16 months after standard therapy and more than 70% of glioblastoma patients die within two years of diagnosis.Therefore,development of novel and effective therapeutic drugs is very important for improving the effectiveness of glioma therapy and the survival of glioma patients.N25(Chemical name: Octanedioc acid(2,5-dimethoxy-phenyl)-amide hydroxy amide,patent number: ZL201310087797.9),a novel histone deacetylase Inhibitor,which was obtained through structural modification of SAHA.Cell autophagy,also known as type Ⅱ cell death,is the process which cells wrapped self-cytoplasmic protein or organelles into vesicles,then formed autolysosome after fusing with lysosome,and degraded their own components.Autophagy plays an important role in tumor,and has extremely complicated relationship with tumor which still unclear.To study the anti-tumor activity of N25 and clarify its molecular mechanism of inducing autophagy in glioma cells,we conducted a series of related experiments.First,we investigated its in vitro anti-proliferative effect by using CCK-8 method and evaluate its in vivo anticancer effect by establishing xenograft tumor model.Then,we detected whether N25 induces autophagy in glioma cells by transmission electron microscope.Moreover,we collected 20 glioma samples and 3 normal brain tissues,and analyzed the protein expression level of LC3,HDAC3,Tip60 in glioma samples by western blot.Finally,we analyzed the protein expression level of HDAC3,Tip60,ULK1(Atg1),and Beclin-1(Atg6)after treatment with N25 in glioma cells.and detected whether selective HDAC3 inhibitor RGFP966 induced autophagy in glioma cells.Our results showed that the anti-proliferative activity of N25 in glioma cells is slightly stronger than SAHA in vitro.The IC50 of N25 and SAHA were 2.55 μM and 3.58 μM respectively by calculating.The growth-inhibitory curves demonstrated that N25 suppressed U87-MG viability more significantly than SAHA at relatively low concentration in vitro.In xenograft tumor model,we found that N25 has significant antitumor activity in vivo and slightly stronger than SAHA,and the tumor inhibition rate of N25 is about 68% in the high dose group(96mg/kg).In the observation of transmission electron microscopy,we found that N25(5μM)could significantly induce the generation of gliama cells autophagosome.In the analysis of clinical glioma samples,we found that HDAC3 was significantly elevated and LC3 and Tip60 significantly decreased in glioma samples compared with normal brain tissues.After treatment of glioma cells with N25,HDAC3 was significantly inhibited,and LC3 and Tip60 were significantly increased.Nevertheless,we found N25 induced significantly the autophagy-related protein expression such as ULK1(Atg1),Beclin-1(Atg6)after treatment of glioma cells with N25.We also found that the selective HDAC3 inhibitor RGFP966 can also significantly induced autophagy in glioma cells.In conclusion,these data suggest that N25 has striking anti-tumor activity in part due to inhibition of HDAC3.Additionally,N25 may induce autophagy through a new pathway,which induces glioma cells autophagy through stimulating ULK1 and stimulating Beclin-1 after upregulating HAT Tip60 by down-regulating HDAC3.