| Background: As one of the most key enzymes, HDACs exert the function of regulating gene transcription. Mounting evidences point to a direct and close link between HDACs dysfunctions and cancers, therefore more and more HDACi are studied and exploited to treat various cancers by the way of promoting acetylation.According to different types of structures, HDACi are divided into hydroxamic acids,fatty acid salts, cyclic peptides, electrophilic ketone compounds, benzamides, etc. The action-mechanism concerning how to inhibit cell proliferation is relatively clear, for instance, induction of cell apoptosis and differentiation, arrest of cell cycle, induction of autophagy mediated cell death, inhibition formation of new blood vessels,prevention of migration and so on, thereby promoting cell death of tumor. With the increasing occurance of cancer patients and the accelerating pace of drug exploitation all over the world, SAHA as the first HDACi was applied for tumor therapy worldwide since had appeared on the market in Oct-2006, thus the current mainstream is concentrated on exploiting various HDACis and SAHA derivatives, especially the market of anti-cancer drugs had been enlarged with standard of living rapidly. The existing HDACis had inhibited some physiological HDACs with no selectivity so that caused extra side effects and toxicity, all results were attributed to their weak target-specific and sub-type selectivity. Due to environmental factors such as air pollution, the occurrence of lung cancer had already risen sharply for the past few years. In addition, the diagnosis and therapy treatment were faced with great difficulty.Meanwhile it has become hotspot and focus of global attention.Objectives: In this study, on the basis of analyzing the characteristics of SAHA matrix structure and atomic interaction, our research group modified the surface recognition domain and obtained a series of SAHA derivatives. Meanwhile, we optimized the synthesis pathways and finished the structure identifications. And then we implemented the pharmacological experiments in vivo and in vitro, HDAC inhibitory test and molecular docking stimulation in order to screen out derivatives with the most potent and /or lowest toxicity. In addition, we detected the phenomenon of autophagy which was induced by SAHA derivatives and probed into the molecular mechanism of autophagy and apoptosis mediated signal pathways. The scope of this study would pave the way for further research and exploition.Methods: With the optimization of synthetic route and solvents in the mixed anhydride method, several novel SAHA derivatives were synthesised respectively.After that, raw products were purified through the combination with CH2Cl2 solvent and slica column chromatography, and HPLC was applied to identify the purity. Four common methods of MS-ESI+、13C-NMR、1H-NMR and IR were used to identify the structures. In vitro biological evaluation, we designed seven various concentrations of compounds to examine their antiproliferation with lung cancer and normal cells by the CCK-8 method, then filtered out the most potent candidate. At the same time, we verified theoretically from the aspects of computer-aided molecular simulation and the binding forces of target HDAC8 which were determined by Auto Dock 6.0software. In addition, further various parameters of stronger compounds were measured(including: Free energy of binding, H-bond energy, Hydrogen bonding,Vander waals force). Besides we designed five various concentrations of compounds to detect the total HDAC inhibitory activity using ELISA method. In the same way,we investigated the mechanisms of apoptosis and autophagy molecular pathways,including Beclin-1, Caspase-3, Caspase-9 and Bcl-2 key proteins. With the help of transmission electron microscope(TEM), the autophagy phenomenon would be observed in lung cancer cells which was induced by derivatives N3 F. By adopting the method of xenoplastic transplantation nude mice, the derivatives were evaluated their anti-lung cancer effects and the toxic influence of heart, liver, spleen, lung and kidney important organs. Through the Bliss method for measuring the LD50 value of derivatives in kunming mice with intraperitoneal administration in order to investigate their toxic effects.Results: On the basis of optimal pathways and conditions in the mixed anhydride method, we had successfully synthesised compounds of Series A(N34, N4 I, N4 B,N24) and the yields(56.3%~72.5%) were improved, while completing the relevant structure identifications(MS-ESI+, 13C-NMR, 1H-NMR and IR) and purity identifications(>95%). In vitro study of lung cancer cells antiproliferation, all Series A compounds could show the better efficacy and less toxicity compared with positive SAHA. And therein N34 and N4 I were sensitive to SPC-A-1 cell line, the IC50 value of N34 was 0.9678μM which was equivalent to a quarter of SAHA and showed favorable structure-activity relationship. During screening of total HDAC inhibitory activity, the IC50 value of N34 in SPC-A-C cell line was 0.04379 μM which up to nanomolar concentration level. Additionally, the test of molecular docking bound with HDAC8, N34 excellently combined with amino acid resides which were located within crystal. The free energy, electrostatic energy and hydrogen bonding energy were stronger than positive SAHA. Researches on apoptosis and autophagy molecular signaling pathways, N34 could obviously up-regulate expression level of Beclin-1,Caspase-3, Caspase-9 and down-regulate the level of Bcl-2. In contrast with the inhibition of Caspase-apoptosis signaling pathways, we detected the increasing protein level of Beclin-1. In vivo study of Series B compounds(N3F, N2E), N3 F was the best candidate for antiproliferation against lung cancer cell SPC-A-1 and the fewest side effects to normal cells. The values of LD50 with intraperitoneal administration of N3 F and N2 E were 183.7501mg/kg(95% confidence interval118.3983mg/kg≤LD50≤285.1738 mg/kg) and 312.7871mg/kg(95% confidence interval 165.0207mg/kg≤LD50≤592.8696mg/kg). During in vivo efficacy evaluation,after fifteen-day intragastrical(i.g) administration of nude mce bearing human SPC-A-1 cell xenografts, also we monitored the tumor volume and weight, the body weight and organ distribution coefficient. The results showed that high and medium-doses of N3 F could exert the inhibitory effects on tumor growth and the inhibitory rates exceeded 50%. During the administration, each group of nude mice survived, the body weight had lost in some extent along with the inverse proportion doses. While the high-dose N3 F had less impact on losing weight. During the organ distribution coeffiient of heart, liver, spleen, lung and kidney, simply we observed the proportion of liver had increased which was representative of its side effects. By means of transmission electron microscope, we had caught sight of N3F-induced autophagic vacuoles which was representative of autophagy phenomenon.Conclusions : Based on SAHA matrix, we successfully obtained the Series A derivatives(N34, N4 I, N4 B, N24) through modifying the surface recognition area.Meanwhile optimizing reagents and conditions of mixed anhydride method together with elevated yield, it was shown that the optimized synthesis pathway was relatively perfect. Series A derivatives showed their favorable anti-proliferation activities against several lung cancer cells, of particular interest was SPC-A-1 cells treated with N34. The anti-proliferation and inhibitory activities were approximately four time and ten time compared with SAHA. Moreover, they displayed the lowest toxicity and correct structure-activity relationship. It was demonstrated that the binding activity with internal amino acid residues was obvious, as well as free energy, electrostatic energy and hydrogen bonding force compared with SAHA. Additionally, the related protein levels of autophagy and apoptosis were elevated distinctly. Particularlly, N3 F exerted inhibitory effects on several lung cancer cells and tumor-bearing nude mice notably, even induced the phenomenon of autophagy compared with SAHA. By the reason of their side effects, the liver was damaged and the body weight was lost to some extent, but the reaction was milder than SAHA. The values of LD50 with intraperitoneal administration of N3 F and N2 E were 183.7501mg/kg and312.7871mg/kg. This topic suggested that through modifying the structures of drug matrix, we can obtained the series of derivatives which could achieve the desired effect. |
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