| Histone acetylation which is catalyzed by histone deacetylase (histonedeacetylases,HDACs) and histone acetyl transferase (histone acetyltransferases, HATs) isan important part of the epigenetics. HDACs and HATs maintain the balance of the histoneacetylation state,then gene transcription can conduct in a relatively stable and controllablerange. Once this balance is broken, such as upregulated HDACs expression or weakenedHATs activity will lead to low acetylation of histones. In this case,the low acetylatedhistones will combine with DNA tightly,which is followed by chromosome condensationand transcription inhibition.This abnormal gene transcription which is caused by theacetylation state changes plays an important role in the induction of tumorigenesis.Thestudy of HDACs-target drug-HDACi becomes a central issue and an important directionin anti-tumor areas.2,2,3,3-tetramethyl cyclopropane thiourea (TCCT) is a derivative ofVPA with its structure transformated,which is a short-chain fatty acids HDACi.Preliminarystudies found that TCCT could inhibit the expressions of class I of HDACs-HDAC3andclass II of HDACs-HDAC4in the U251and A549cells,so TCCT can be a novelHDACi.In this study,we researched the anti-tumor effect of TCCT by pharmacodynamicsassessment method in U251and A549cells in vitro, and has investigated its anti-tumoraction mechanism,so as to lay the foundation for developing the new HDACi kind ofanti-tumor medicine.Objective: The effects of a noval HDACi-TCCT on cell proliferation, the cell cyclefactors expression, cell cycle process and apoptosis were measured in U251and A549cells in this paper.Besides, the possible mechanism of TCCT anti-tumor effects wereinvestigated in this article.Methods: The human brain glioma U251and human lung adenocarcinoma A549cellswere cultured with RPMI1640and DMEM high glucose medium respectively in37°C,5%CO2incubator.There was10%fetal bovine serum or fetal calf serum in the RPMI1640andDMEM high glucose medium. Then the two cell lines were divided into five groups withdifferent-concentration TCCT, or the control groups.The two cell lines were treated with0.075mmol/L,0.150mmol/L,0.300mmol/L,0.600mmol/L,1.200mmol/L TCCT in theMTT assay,the solvent control and blank group were also established.The two cell lineswere treated with low dose of0.025mmol/L, middle dose of0.050mmol/L and high doseof1.000mmol/L TCCT respectively in other methods,VPA(1.000mmol/L) group and thesolvent control group were also established.○1MTT method was used to explore the effectsof TCCT on U251and A549cells proliferation (48h).○2RT-PCR was performed to detectthe effects of TCCT on cell cycle-related factor mRNA expression in U251and A549cells.○3PI single staining method was used to detect the effects of TCCT on U251andA549cell cycle.○4Annexin Ⅴ-FITC was applied to detect the effects of TCCT on U251and A549cells apoptosis.○5Western Blot was used to detect the effects of TCCT onhistones with specific sites acetylated and the cell cycle-related factor protein expression inU251cells.Results:○1MTT assay showed that the U251cell inhibition rates of0.075mmol/L,0.150mmol/L,0.300mmol/L,0.600mmol/L and1.200mmol/L concentra-tion groups were6.07%,17.86%32.68%,62.32%and84.29%respectively after TCCTtreatment for48h.Half of the U251cell inhibition rate(IC50)which was calculated by theSPSS Probit (probability distribution of the regression)was0.461±0.108mmol/L.The A549cell inhibition rates of different concentration groups were32.72%,49.85%,51.69%,61.77%and77.98%respectively after TCCT treatment for48h.Half of the A549cell inhibition rate(IC50)which was calculated by the SPSS Probit (probability distributionof the regression)was0.251±0.068mmol/L.These results showed that TCCT inhibited theproliferation of U251and A549cells with a dose-dependent manner.○2RT-PCR showedthat each treatment group of TCCT increased p21WAF1/CIP1mRNA expression whilereduced CyclinD1mRNA expression in U251cells compared with the solvent controlgroup.TCCT upregulated p21WAF1/CIP1mRNA expression weakly in A549cells,but it had almost no effects on the mRNA expression of CyclinD1.(?) The PI single staining demonstrated that the U251cell S phase percentages of low-dose group12.05±1.63%(p=0.264),middle-dose group19.40±2.55%(p=0.025) and high-dose group28.75±6.86%(p=0.003)were increased significantly compared with the solvent control group5.35±0.92%after TCCT treatment for24h.The U251cell S-phase percentage of VPA group (7.40±0.28%)(p=0.924)had few changes compared with the solvent control group5.35±0.92%.The U251cell S-phase percentages of the middle-dose group (p<0.05)and high-dose group (p<0.01)were increased significantly,but the VPA group (p>0.05)wasn’t increased significantly.The above indicated that the TCCT could induce U251cells arrest in S phase.The A549cell G2/M-phase percentage of all TCCT treatment groups were increased compared with the solvent control group.The A549cell G2/M-phase percentages of middle and high dose groups were increased significantly (p<0.01),and it was showed that the TCCT may induce the A549cells arrest in G2/M phase.4Annexin V-FITC assay indicated that the U251cell apoptosis rates of low-dose group36.85±1.20%(p<0.01),middle-dose group40.35±1.20%(p<0.01)and high-dose group47.70±1.56%(P<0.01)were all increased significantly compared with the solvent control group7.65±1.20%.The U251cell apoptosis rates of all TCCT treatment groups had statistically significance,which indicated that TCCT could significantly induce the U251cells apoptosis.The A549cell apoptosis rates of middle-dose group (6.05±0.64%, p<0.05)and high-dose group(8.05±0.49%, p<0.01) were increased significantly compared with the solvent control group3.00±0.99,while the A549cell apoptosis rate of low-dose group was not statistically significant.lt was showed that a certain dose of TCCT could induce A549cells apoptosis.5The result of western blot showed that none of the TCCT treatment groups could upregulate the expression of lysine9acetylated histone H3(Acetyl-Histone H3(Lys9))in U251cells,but it downregulated lysinel6acetylated histone H4(Acetyl-Histone H4(Lys16))in U251cells.And it was also showed that all TCCT treatment groups downregulated the protein expression of CyclinDl in U251cells.Conclusions:1The noval HDACi—TCCT can effectively inhibit the proliferation of U251and A549cells with a dose-dependent manner.TCCT can induce U251cells S phase arrest and apoptosis,and it can weakly induce A549cells in G2/M phase arrest.Besides,it can induced A549cells apoptosis under a certain dose.2The effects of TCCT on the expression of p21WAF1/CIP1and CyclinDl may contribute to the cell inhibition,cell cycle arrest and cell apoptosis in U251and A549cells. |
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