Research On The Synthesis Of Hydroxamic Acids Histone Deacetylase Inhibitor N25and Its Anti-tumor Effect In Vitro

A large number of studies have demonstrated that the transformation of epigeneticplay a key role in the development of cancer. As an important part of epigenetics, themodification of histone acetylation has become a research hotspot. The state of histoneacetylation is regulated by histone acetyltransfer（HATs） and histone deacetylases（HDACs）,which leads to the change of chromatin structure and gene transcription, and further affectsto cell proliferation, cell cycle, apoptosis,and so on. Therefore, with the continuingdevelopment of HDACs-target study, histone deacetylase inhibitor （HDACi） is becoming apromising class of anticancer drug. Nowadays different structural types of HDACi havebeen synthesized and applied to anti-tumor experiments in vitro or clinical researches.Based on the structure-activity relationship of hydroxamic acids and HDACs, this researchaims to: synthesize N-（2,5-dimethoxyphenyl）-N’-hydroxyoctanediamide （N25） with thecompound of SAHA; make a research with glioma cell lines and lung cancer cell line as theobjectives to investigate its effect on HDAC and histone acetylation; evaluate the functionof N25in anti-tumor and discuss relevant machanism by applying pharmacodynamicmethod in vitro.Objective: This research aimed to obtain new compounds N25by modificating thestructure of SAHA, which was a hydroxamic acid HDACi. On the basis of examining theN25’ s antitumor activity in vitro and its effect on HDACs and histone acetylation, thefactor related to cell cycle and apoptosis inducing would be studied in the further. This paper aimed to explore the possibility of N25, as the ramification of SAHA, whether cantarget HDACs and change the level of histone acetylation, as well as to investigate thepossible mechanisms in antitumor effects.Methods:①Based on the structure-function relationshipof hydroxamic acids HDACiand HDACs, with the SAHA as lead compound, compound N25was obtained by using2,5-dimethoxyphenyl to take place of the phenyl in its surface-recognition cap group ofSAHA’ structure and referring to the synthesized path of SAHA. Hydroxamic acid groupswas identified by the color of ferric chloride solution. Infrared spectroscopy and NMR wererespectively applied in the identification of the main functional groups and structure. Themolecular weight of compound was detected by mass spectrometry.②In this paper, humanglioma U251, U87, T98G cells and human lung cancer H460, A549, H1299cells lines werethe main study subjects; the48h restrained effects of N25and SAHA on the above tumorcell lines（in7concentrations:0.5,1.0,2.0,4.0,8.0,16.0,32.0μMol/L） was investigatedwith CCK-8assay, and its median inhibitory concentration（IC50） was calculated, thenscreened out sensitive cell lines from various types of cancer cell lines which were used forthe follow-up experiments.③Western Blot method was applied to detecte the influence ofHDACs and histone acetylation in U251and H460cells which were N25treated for48hours.④Flow cytometry and PI single staining were performed to detecte the effect of N25on the cell cycle of U251and H460cells.⑤RT-PCR was used to studied N25’s effect onthe expression of cell cycle-related factor mRNA of U251and H460cell for48hours.⑥Flow cytometry and Annexin Ⅴ-FITC/PI assay were used to investigate inducedapoptosis effect in U251and H460cells with N25incubated for48hours.Results:①The synthetic compound was white powder, which showed amaranth aftertesting by the ferric chloride alcohol. Infrared spectroscopy had identified the majorfunctional groups such as carbonyl group, hydroxyl group, benzene ring, methoxy group,etc. Mass spectrometry’ result indicated that the molecular weight of sample was324.1774g/mol. The information of NMR reflected that those signals were hydrogen atoms ofN25.②N25and SAHA had showed a significant anti-proliferative effect and adose-dependent manner in three glioma cells and three lung cancer cells. N25’s half inhibition rate （IC50） on U251, U87, T98G glioma cells were respectively6.280±0.894（μMol/L）,11.332±0.505（μMol/L）,15.435±1.000（μMol/L）, however, SAHA’s IC50onthe three cells were6.322±1.271（μMol/L）,6.407±0.075（μMol/L）,10.035±2.004（μMol/L） respectively, which demonstrated that N25’s anti-proliferative effect inU251was slightly stronger than SAHA；but the sensibility of U87and T98G to SAHA wasstronger than N25. When N25was treated with H460, A549, H129lung cancer cells for48hours, the IC50were2.443±0.314（μMol/L）,3.463±0.841（μMol/L）,2.933±0.642（μMol/L） respectively; while SAHA’s IC50on them were5.163±0.973（μMol/L）,7.203±1.285（μMol/L）,6.869±1.132（μMol/L）respectively. It suggested N25had a stronger growth inhibition than SAHA in H460, A549and H1299cells, and N25had the most sensitive effect in killing H460.③N25couldsignificantly reduce the expression level of HDAC4protein and improve the overallacetylation levels of histone H3（K9+K14+K18+K23+K27） and single point level ofacetylation level H3-K9in U251and H460cells.④After U251cells were treated with N25for24hours, the ratio of G0/G1phase significantly declined （P0.001） and S phase wasalso slightly decreased,and the proportion of G2/M phase increased significantly （P0.001）.Thence the cell cycle arrested at the G2/M phase.When incubation time was extended to48h, the cell cycle was arrested in G0/G1phase, while the cell proportion of S and G2/Mphase were decreased（P0.01）. The large increase of APO implied that that N25couldinduce apoptosis in U251cells. After H460cells were treated by N25for48hours, cellcycle was arrested at the G2/M phase. The proportion of G0/G1and S-phase cellsdecreased, however, each dose group had a smaller impact on the APO.⑤N25raised theexpression ofP21^WAF1/CIP1gene respectively andP53gene showed a downregulatedexpression in U251and H460cells, which were similar with the result get from SAHA.⑥N25significantly induced apoptosis in U251and H460cells. In U251cells, there was asignificant difference between negative control group （1.1±0.3）, and the low-dose group（47.5±0.5）, medium dose group（64.2±7.1）, high-dose group（69.5±10.6）and SAHA（63.7±4.2）. They showed a significant difference （P <0.001） in U251cells.Apoptosis allsignificantly appeared in early, medium and late period, but apoptosis mainly appeared in the medium and late period. In the apoptosis of H460cell, compared with the negativecontrol group （0.2±0.1）, there was a significant deference among low-dose group（28.4±2.5）, medium dose group（32.0±4.9）, high-dose group（32.4±5.6） and SAHA（30.3±3.4）respectively. They also showed a significant difference （P <0.001）. The vastmajority of apoptotic cells were in the early of cell apoptosis（P <0.001）, but only a fewapoptosis appeared in the late period.Conclusions:①N25had been successfully synthesised which was the derivative ofSAHA.②N25had a significant growth inhibition effect on human glioma U251, U87,T98G cell lines and human lung cancer H460, A549, H1299cells. The anti-proliferativeeffects of N25were in a dose-dependent manner in vitro.③N25could significantly reducethe expression of HDAC4protein in U251and H460cells, and improved the level ofhistone H3acetylation. These revealed that N25could target HDAC4and would beregarded as a potential histone deacetylase inhibitor.④When N25treated U251for24hours or N25treated H460cells for48hours, the cell cycle were arrested at the G2/Mphase. When extended the incubation time to48hours in U251cells, cell cycle was blockedat the G0/G1phase. Moreover, the APO prompted that N25could induce apoptosis.⑤N25significantly increased the expression ofP21^WAF1/CIP1gene and reduced the level ofP53inU251and H460cells. The result suggested thatP21^WAF1/CIP1did not dependent on theregulation ofP53. On the other hand, the expression upregulation ofP21^WAF1/CIP1might berelated to cell cycle arrest.⑥N25significantly induced apoptosis in U251and H460cells.