| In recent years, the mortality of esophageal cancer has increasedgradually and ranked front of the malignant tumors. The incidence ofesophageal cancer is high in our country. Cancer can be treated by surgery,chemotherapy, radiation therapy, immunotherapy, and monoclonal antibodytherapy recently. Chemotherapy plays a main role in the anti-tumor therapy.In this experiment, TE-1（the human esophageal squamous cell carcinoma cellline） was used to study the effects of N-[4-（4,6-dimethyl-2-pyrimidinyloxy）-3-methylphenyl]-N’-[2-（dimethylamino）] benzoyl urea （SUD）, a novelsynthesized small molecule compound, on cell growth, cell cycle, apoptosisand the proteins related to apoptosis.Part1The growth-inhibitory effect of SUD on TE-1cellsObjective: To study the effect of SUD on growth of TE-1cellsMethods: The effect of SUD on viability of TE-1cells was evaluated byMTT assay, growth curve assay and clonogenic assay. The morphologicalchanges of the TE-1cells were observed by an optical microscope.Results:（1） MTT assay showed that SUD decreased cell growth inconcentration-dependent and time-dependent manners. The growth-inhibitoryeffect of10-4mol/L SUD was stronger than that of positive control drug（P<0.01）.（2） The growth curves showed that SUD inhibited growth of TE-1cells （P<0.01）.（3） The clonogenic assay results indicated that the colonyformation rate of TE-1cells was18.00%in control group, which wasdecreased to0.38%,5.00%and7.22%by SUD10-4mol/L,10-5mol/L and10-6mol/L, respectively （P<0.01）.（4） Disappearance of junctions between cells,membrane asymmetry, and cell shrinkage phenomenon were observed underan optical microscope after the cells were treated with SUD. Conclusions:（1） SUD time-dependently and concentration-dependentlysuppressed the viability of TE-1cells. There was statistically significantdifference between SUD10-4mol/L group and5-Fu5×10-4mol/L group.（2）SUD could reduce colony formation of TE-1cells.（3） SUD causedmorphological changes of TE-1.Part2The effects of SUD on the cell cycle and apoptosis of TE-1cellsObjective: To study the effects of SUD on the cell cycle and apoptosisof TE-1cells.Methods: Cell-cycle distribution of TE-1cells was determined byfluorescence-activated cell sorting （FACS）. Apoptosis of TE-1was determinedby DAPI staining. Early apoptosis of TE-1was determined by AnnexinV-PIdouble staining. Western-blot was used to determine apoptosis-relatedproteins.Results:（1） Treatment of TE-1with SUD10-4mol/L led to an increaseof the S-phase cell population, which was accompanied by the reduction ofthe ratio of G1-cells. The number of cells in G1-phase increased and thenumber of cells in S-phase reduced in SUD10-5mol/L group. Treatment ofTE-1cells with SUD10-6mol/L increased G1-and G2/M-phase cellpopulation and decreased S-phase cell population. The apoptosis rate of TE-1cells was significantly enhanced by SUD （P<0.01）.（2） SUD could inducemorphological changes of TE-1cells such as cell nuclear pyknosis, chromatincondensation and formation of apoptotic bodies, which were more evident inhigh-concentration group.（3） After the cells were treated with SUD for48h,early apoptosis rate of the cells treated with SUD10-4mol/L,10-5mol/L and10-6mol/L was0.348±0.013,0.188±0.006and0.084±0.005, respectively,which was significantly higher （P<0.01） than that （0.010±0.002） in controlgroup.（4） SUD could significantly decrease apoptosis-inhibitory protein Bcl-2expression and raise expression of apoptosis-promoting proteins Bax and p53.SUD increased the ratio of Bax/Bcl-2in a concentration-dependent manner.The expression of caspase-3, caspase-9and PARP reduced and the expression of cleaved caspase-3, cleaved caspase-9and cleaved PARP raised in cellstreated with SUD for48h.Conclusions:（1） SUD10-4mol/L increased the population of TE-1cellsin S-phase. Treatment of TE-1cells with SUD10-5mol/L and10-6mol/Lincreased the population of cells in G1phase, and the G2/M-phase cells werealso increased by SUD10-6mol/L. Low concentrations of SUD inducedG1-phase arrest and the cells failed to enter S-phase.（2）. SUD inducedapoptosis of TE-1cells （3） The effect of SUD on apoptosis of TE-1cells couldbe associated with increasing expression of p53and Bax and reducingexpression of Bcl-2, followed by activation of caspase-3, caspase-9and PARP,suggesting that mitochondrial pathways were involved in SUD-inducedapoptosis of TE-1cells. |
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