Novel Histone Deacetylase Inhibitor Synthesized, Anti-tumor Function And Mechanism Researching In Vitro

Objection:The balance between histone acetyltransferase(HATs) and histone deacetylases (HDACs) regulates normal cell differentiation and metabolism activity. Disturbance of the balance between HATs and HDACs can lead to cancers. The research show that the novel histone deacetylase inhibitor(HDACI) of short-chain fatty acid medicines(butyric acid ,valpoic acid) can inhibit tumor cell growth and proliferation via various molecular mechnisms,including cell cycle arrest, induction of cell differentiation and apoptosis,as well as inhibition of angiogenesis.The novel short-chain fatty acid medicine 2,2,3,3-tetramethylcyclopropanecarbonylthiourea was synthesized to explore the inhibition effect about tumor cell and related protein expression.Methods:(1) Through the introduction of carbonylthiourea group, we synthesized some white crystallinity powders.(2)The powders structure was identificated by melting point measurement, infrared spectrum(IR),nuclear magnetic resonance(1HNMR) and mass spectrum(MS).(3)We observed that the different five 2,2,3,3-tetramethylcyclopropane carbonylthiourea administration concentration(0.0875, 0.175,0.35,0.70and1.40mmol/L)act on astrocytoma cell line U251 and lung adenocarcinoma cell line A549 after 24 hours by MTT colorimetry ,then IC50 were calculated.(4) HDAC3 and HDAC4,p21waf1/CIP1 protein expression in the cell lines U251 and A549 were identified by western blotting technology, after administrating 2,2,3,3-tetramethylcyclopropanecarbonylthiourea (1.00mmol/L,0.50mmol/L and 0.25mmol/L).Result:(1)Some white crystallinity powders were avaliable.(2) The powders were confirmed to be 2,2,3,3-tetramethylcyclpropanecarbonylthiourea through various testing index and atlas analysing.(3)For U251 glioma cell line,five administration concentration, corresponding inhibition ratios respectively were 18.56%,23.11%,24.48%,36.41%and 63.54% after 24h, positive medicine valproic acid (VPA),however, was 7.73%.The value of IC50 was 1.0287mmol/L. For A549 lung adenocarcinoma cell line, corresponding inhibition ratio respectively were 9.66%,15.93%,33.18%,84.32% and87.91%. The value of IC50 was 0.6235mmol/L.(4)The result of western blotting revealed in astrocytic cell line U251, the band of HDAC3 and HDAC4 protein is more clear in middle than high and low groups administration, positive group is more obvious. Control and reagent groups are most obvious, and the band of p21waf1/CIP1 protein are all conspicuous in high, middle and low administration groups. Positive administration group is less ambiguous, control and reagent groups are almost unexpression. In A549 cell line, the band of HDAC3 and HDAC4 protein are more obvious in control and reagent groups in contrast other groups. However, compare to high, positive, control and reagent groups, the band of p21waf1/CIP1 protein were more obvious in middle and low administration concentration. Moreover, The result revealed that molecular weight of HDAC3 , HDAC4 and p21waf1/CIP1 respectively are 50kDa, 130kDa and 21kDa.Conclusion:(1)2,2,3,3-tetramethylcyclopropanecarbonylthiourea were synthesised successfully.(2)The compound has sharply inhibited the growth and developed of two cancer cell lines, and display dose-dependent manner.(3)The electropherogram indicated that in U251 cell line,HDAC3 and HDAC4 expression are higher in middle group than high and low groups,positive drug created some effection,reagent and control groups displayed upregulation. The expression of p21waf1/CIP1 protein were all higher in high,middle and low groups administration than positive group,reagent and control groups are less dim. In A549 cell line, HDAC3 and HDAC4 expression are downregulation after drug effection. But the expression of p21waf1/CIP1 protein are lower in tumor cell,positive and DMSO reagent groups, and novel HDACI drug 2,2,3,3-tetramethylcyclopropanecarbonylthiourea administration, the expression showed upregulation trend. The above results pointed out 2,2,3,3-tetramethylcyclopropane carbonylthiourea inhibited cancer growth effection. The effection mechanism of the medicine is relation to HDACs downregulation, and p21waf1/CIP1 upregulation.