| Tumor multicellular spheroid is considered as an intermediate model to bridge the gap between monolayer and solid tumor,it is similar to human solid tumor subunit,and present resistance to many anticancer drugs[1].Ubiquitin-proteasome pathway(UPP)is vital to cell protein degradation,UPP Inhibitor itself can induce apoptosis,and can greatly augments apoptosis following chemotheraty and circuvent resistance of multidrug resistance cell[2,3].MG132 is one of UPP inhibitors,Whether MG132 can circuvent the multicellular drug resistance of MCS of cervical carcinoma HCE1 cell line and it's relevance to NF-κB,bcl-2 expression are still unknown.ObjectivesTo investigate wether DDP and the UPP inhibitor MG132 can induce human cevical carcinoma cell line HCE1 monolayer and MCS grow delay and apoptosis.To observe wether HCE1/MCS resistance to treatment with DDP and to prove wether MG132 can resensitive HCE1/MCS to treatment with DDP,and it's relationship with NF-κB,bcl-2 expression.Methods(一)To establish HCE1 multicellular spheroids model using liguid overlay technique and rotating culture technique,and to observe the growth and histological structure of HCE1/MCS,draw the growth curve.(二)HCE1 monolayer cells were cultured and treated with MG132(1.5umol/L,2.0umol/L,2.5 umol/L,5.0 umol/L,10.0 umol/L,20.0 umol/L)and DDP(5.0mg/L,10.0mg/L,15.0mg/L)for 48 hours respectively,Cell viability was measured by trypan blue exclusion assay,IC10 and IC50 was calculated respectively.(三)treatment and experiment:1.Groups:According to drugs,4 groups were established.Control group:Cells were treated with the same concentration of DMSO.MG132 group:Cells were treated with MG132 2.0umol/L.Cisplatin(DDP)group:Cells were treated with DDP6.0mg/L. MG132 and DDP group:cells were treated with DDP 6.0mg/L+MG132 2.0umol/L.1.HCE1/monolayer and HCE1/MCS were cultured and treated with 4 groups of drugs mentioned above for 48 hours,(1)cell growth were observed by light microscope.(2)cell viability were calculated by trypan blue exclusion assay.(3)cell cycle and apoptosis were detected by flowcymeter. 2.HCE1/monolayer and HCE1/MCS were cultured and treated with 4 groups of drugs mentioned above for 24 hours,(1)NF-κB(p65)were detected by western-blot assay.(2)bcl-2 expression were detected by immunohistochemistry.Results(一)establishment of HCE1 multicellular spheroid model:1.we observed the growth of HCE1 monolayer cells were in a good condation,after 24h in rotating culture,cells began to aggregated into multicellular spheroids,and becoming more and more compact and larger in size relay to culture time.2.histological structure of HCE1/MCS:central necrosis core and cell outgrowth around the MCS were observed when MCS adherented.Non-structure red-stain region was observed using HE stainning.3.Growth curve was drawed:be cultured till 10th days,the growth of HCE1/MCS turned into platform,the double time of HCE1/MCS cells is 4.43±0.47 days.(二)Inhibition rate of HCE1 to MG132 and DDP testing by trypan blue exclusion assay.HCE1/monolayer cells were cultured and treated with MG132 1.5umol/L,2.0umol/L,2.5umol/L,5umol/L,10umol/L,20umol/L for 48 hours,cell viability were:5.89%±0.48%,10.00%±0.33%,20.33%±1.21%,50.00%±1.76%, 61.67%±5.00%,78.56%±4.16%respectively,IC10 is 2.0umol/L.HCE1/monolayer cells were cultured and treated with DDP 5.0mg/L,10.0 mg/L,15.0 mg/L for 48 hours, cell viability were 39.22%±6.20%,80.34%±1.53%,93.89%±3.47%respectively. IC50 is 6.1mg/L.(三)treatment and experiment:1.After treated with 4 groups of drugs for 48 hours:(1)Morphology viewing:①control group:HCE1/monolayer cells growed in good condition,adherenced to irregular digonal corniform.HCE1/MCS were also in good condition,cells were round,adhenced to each other.②MG132 group:several cells shrinkage,turned round,and detached were observed in monolayer cells.Light Multicellular disaggregation,shrinkage,were observed in HCE1/MCS③DDP group: A lot of cells shrinkage,turned round,and detached were observed in monolayer cells. No more change were observed in HCE1/MCS.④MG132 and DDP group:The majority of cells shrinkage,turned round,and detached were observed in monolayer cells.Multicellular disaggregation,shrinkage,were observed in HCE1/MCS.(2)After treated with 4 groups of drugs for 48 hours:①control group:cell inhibition rate both were 1.00%±0.00%.②MG132 group: cell inhibition rate of HCE1/monolayer and HCE1/MCS were 11.67%±2.34%and 10.78%±1.17%respectively.No significant difference were observed.(P＞0.05)③DDP group:cell inhibition rate of HCE1/monolayer and HCE1/MCS were 45.00%±7.44%and 9.45%±5.98%respectively,significant difference were observed.(P＜0.05)④MG132 and DDP group:Cell inhibition rate of HCE1/monolayer were 92.67%±2.52%,91.33%±2.18%respectively.No significant difference were observed between them.(P＞0.05)(3)cell stage and apoptosis were detected by flowcyrneter:①control group:HCE1/MCS had a much higher G1/G0 stage cell(P＜0.05)and less S stage(P＜0.05)than HCE1/monolayer.There was no significant difference between G2/M stage cells between HCE1/MCS and HCE1/monolayer(P=0.21).②MG132 group:apoptosis and G2/M stage block were observed in both of HCE1/monolayer and HCE1/MCS.③DDP group:No apoptosis nor S stage block were observed of HCE1/MCS.④MG132 and DDP group: Apoptosis were observed in both of HCE1/monolayer and HCE1/MCS,apoptosis rate is 99.0%and 95.2%respetively.2.After treated with 4 groups of drugs for 24 hours(1)NF-κB expression were detected by western-blot assay:①HCE1/MCS cells expression higher NF-κB than HCE1/monolayer.(P＜0.05)②In multicelluar spheroids:(A)MG132 group:NF-κB expression is significantly lower than control group.(P＜0.05)(B)DDP group:NF-κB expression is significantly higher than control group.(P＞0.05)(C)MG132 and DDP group: NF-κB expression is significantly lower than control group.(P＜0.05),No significant change were observed after treated with MG132 and DDP+MG132.(2)bcl-2expression were detected by immunohistochemistry:①bcl-2 expression is stronger in HCE1/MCS than in HCE1/monolayer.②(A)MG132 group:bcl-2 expression is significantly lower than control group. (P＜0.05)(B)DDP group:bcl-2 expression is significantly higher than control group. (P＞0.05)(C)MG132 and DDP group:bcl-2 expression is significantly lower than control group.(P＜0.05),No significant change were observed after treated with MG132 and DDP+MG132(P＞0.05).Conclusions1.HCE1/MCS present natural resistance to DDP,maybe it can be a good model for study of tumor resistance to anticancer drugs.2.MG132 can induce inhibition and apoptosis of HCE1/MCS cells,and can partially reverse the natural resistance of HCE1/MCS to DDP.3.The natural resistance of HCE1/MCS maybe relation to the up-regulation of NF-κB,bcl-2 expression. |
PDF Full Text Request