| OBJECTIVE: First we investigated the protein, mRNA expression and methylation status of E-cad to evaluate its clinical value. Then, we detected the function of VPA, SAHA and DAC on the endometrial carcinoma(EC) cell lines RL-952 and HEC-1-B by tests in vivo and in vitro to understand the changes of cell cycle, apoptosis, growth inhibition effects, methylation status ,mRNA and protein expression of E-cad.METHODS: In the first part, we examined the expression of E-cad and its methylation status in clinical samples by immunohistochemical SP, RT-PCR and MSP methods. In the second part we studied the effects of DAC, VPA and SAHA on EC cell lines RL-52 and HEC-l-B by RT-PCR, MSP, TUNEL, MTT, Western-blot and FCM methods to observe the effects on cell growth , cell cycle, cell apoptosis , methylation status, mRNA and protein expression of E-cad. In the third part , the tumor growth inhibition effects , tumor apoptosis in situ and E-cad expression were observed in the nude mices with the xenografted implants of human EC HEC-l-B cell by immunohistochemical SP , RT-PCR TUNEL, TEM, HE stain and MSP methods. Results:1.E-cad and β -cat locates on cell membrane. From NE to CE , the E-cad expression decreased (P<0.05) . E-cad expression in stage I ~ II G1~G2 none and myometrial invasion ≤1/2 was higher than that in stageIII~IV, G3 and invasion >1/2 (P<0.05) . The expression of β -cat has the same trend with E-cad except that its expression in samples without lymph metastasis was higher than those with lymph metastasis (P<0.05) .2. The overall 5-year survival rate of positive expression group of E-cad/ β -cat was higher than that of negative expression group (P<0.05 ) .The overall 5-year survival rate of both positive expression group of E-cad and 3 -cat was higher than that ofnegative expression group or single positive expression group (P<0.05) .The rate of unmethylation group was higher than that of methylation group (P<0.05) . 3.The methylation rate of E-cad promoter was 0, 0 and 36.58% in NE, AHE and CE. Its methylation status was associated with clinical stage , myometrial invasion, lymph metastasis and grade (P<0.05) .4.E-cad promoter was in the status of methylation in HEC-l-B and RL-952 cell lines, DAC can change the status into demethylation, but VPA and SAHA can't. 5.The growth inhibition effects of DAC, VPA and SAHAwere dosage-dependent and time-dependent. DAC, VPA and SAHA can enhance AI and the ratio of Go/Gi in cell cycle. HEC-l-B cell line was more sensitive than RL-952 one in cell growth, inhibition of cell cycle and apoptosis.6. DAC, VPA and SAHA could up-regulate the mRNA and protein expression of E-cad and down-regulate the mRNA expression of bcl-2 in vitro. 7.The rate of successfully inoculation of subcutaneous xenograft was 85.71%, DAC and VPA are safe in the nude mices. After 6 weeks' therapy, the tumor volume of each treatment group was smaller than control group, the inhibition rate was higher than control group (p<0.05) .The volume of DAC group had significant difference with other treatment groups (/?<0.05) .Each VPA single treatment group had significant difference with each VPA combined treatment group (p<0.05) .There was no difference in VPA single treatment groups and VPA combined treatment groups (/?0.05) .8. The most part of dead cells was apoptosis cells. The AI of treatment groups had significant difference with that of control group(/><0.01). DAC group had significant difference with VPA combined treatment groups(/?<0.05 )and no difference with VPA single drug treatment group (/?0.05) . VPA combined treatment groups had significant difference with VPA 5mg treatment group (p<0.05 ) and no difference with VPA lOmg group (p>0.05) . There was no difference in VPA single treatment groups and VPA combined treatment groups (/?0.05 ) .9. DAC and VPA could up-regulate the mRNA and protein expression of E-cad anddown-regulate the mRNA expression of bcl-2 in vivo (p<0.05) .Conclusions:l.The down-expression of E-cad and the methylation of E-cad promoter were theearly events of EC. Its abnormal expression and methylation may play an importantrole in origin, development and prognosis of EC. The methylation of gene promoterwas an important regulation mechanism in E-cad expression.2.VPAn SAHA and DAC can inhibit EC cell lines RL-952 and HEC-l-B by the wayof enhancing AI ,the ratio of Go/Gi in cell cycle, up-regulating the expression ofE-cad and down-regulating the expression of bcl-2 in vitro.3.The rate of successfully inoculation of subcutaneous xenograft was 85.71%? DACand VPA could inhibit the growth of tumor significantly , especially joint treatment,without obvious side-effects. DAC and VPA could enhance the AI of tumor,up-regulate the mRNA and protein expression of E-cad and down-regulate themRNA expression of bcl-2 in vivo.
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