The Experimental Research Of MG132 In The Activity Of Anti-cervical Cancer And Enhancement Of Sensitivity To Cisplatin Of Cervical Cancer Cells In Vivo And In Vitro

Cervical cancer is the one of the most common gynecological malignancy.Traditional therapy was based on surgery and radiotherapy. Neoadjuvant Chemotherapy is a new treatment rising in the past 10 years,which can effectively improve the quality of life and prognosis for the patient with locally advanced cervical cancer.However there are still about 20%of the patients having a drug-resistant phenomenon,to become a formidable problem in our clinical therapy.Ubiquitin-proteasome pathway plays a key role in the intracellular protein degradation.Research shows that proteasome inhibitors can not only induce the apoptosis of tumor cells but also increase the sensitivity of the chemotherapeutics and reverse the chemoresistance of drug-resistant cell lines.At present,the proteasome inhibitors have been used in multiple myeloma treatment and got satisfactory therapeutic efficacy.Given this important role the proteasome represents a novel target for chemotherapy with practical value.Nevertheless,the study of the proteasome inhibitor on the solid tumor is just at its starting stage and its mechanism of action is not yet clear,so it is of theoretical and clinical significance to investigate its function.MG132,a reversible proteasome inhibitor,has been used for the study of several tumors.To probe whether MG132 can enhance the sensitivity of cisplatin to cervical cancer cells and offer an experimental and theoretical basis on the possibility for the clinical application of proteasome inhibitor adjuvant therapy to cervical cancer this study which contains three parts:(1)The effect of MG132 on cell proliferation and apoptosis of human cervical Hela cells.(2)The observation whether MG132 could sensitize the Hela cells to cisplatin and inhibit the xenografts of Hela cells on the nude mice.(3)The research of MG132 on reversal of natural drug-resistance to cisplatin of HCE1/MCS Part OneObjectives:To investigate the effect of MG132 on cell proliferation and apoptosis of human cervical Hela cells in vitro and observe the influence of MG132 on the expression of p27.Methods:1 Detection of the cellular growth inhibition ratio by MTT assay.(1) HeLa cells were cultured and treated by MG132 with different concentrations for 24 hours respectively.Cellular viability and IC₅₀ were measured by MTT assay.(2)HeLa cells were cultured and treated by MG132 with different final concentration for 48 and 72 hours and the cellular growth inhibition ratio were detected by MTT assay respectively.2 The morphological features of the apoptosis of HeLa cells induced by MG132(1) we observed the morphological features of apoptotic cells induced by MG132 with inverted microscope.(2) Acridine orange combined with Ethidium bromide staining:Hela cells were treated with 10μmol/L MG132.Afterwards they were collected 48 hours later and observed after acridine orange combined with EB staining by fluorescence microscope.(3) Transmission electron microscope was used to observe the morphological alteration of Hela cells treated with 10μmol/L MG132 for 48 hours.3 The biochemistry feature of HeLa cells treated with MG132: After treated with MG132 10μmol/L for 48 h,Hela cells were collected and DNA was abstracted for gel electrophorosis.4 Analysis of apoptosis rate and cell cycle of HeLa cells through flow cytometry been exposed to MG132.(1) HeLa cells were cultured and treated by MG132 with disdinct concentrations for 48h,the apoptotic rate of HeLa cells were analyzed by flow cytometry after PI staining respectively.(2)Treated with MG132 10μmol/1 for 24,48 and 72 hours,the apoptotic rate and cell cycle distribution were examined through flow cytometry after PI staining.5 Immunocytochemistry was explored to detect the expression of p27 protein of different groups in which HeLa cells treated by MG132 with various final concentrations for 24 hours respectively.Results:1 After treated with MG-132,the growth of Hela cells were inhibited obviously.There were statistically significant diffferences of the proliferative inhibition among different experimental groups (F=3569.301,P＜0.001),and there were also statistically significant differences of the proliferative inhibition rates for different duration of treatment(F=6155.339,P＜0.001).There was an interaction between different concentrations and different duration by MG132(F=272.722, P＜0.001).2 Hela cells became round,small,cell shrinkage,membrane blebbing and so on after being exposed to MG132.3 Acridine orange combine with Ethidium bromide staining showed that most of the cells in control group appeared fluorescent green uniform.But in experement group,most cells were yellow-green fluorescence.4 DNA ladder exist in experiment group.5 The typical ultrastructure of apoptosis cell was found by electron microscope after the cells treated with MG132:cell shrinkage,nucleus pycnosis,chromatin margination and apoptotic body can be seen.6 Quantification of the apoptosis effect by flow cytometric analysis showed that it was in a good correspondence with the loss in cell viability induced by MG132.There were statistically significant differences among experimental groups with different density of MG132 (F=1182.745,P＜0.001);Meanwhile,treated with MG132 10μmol/l for 24,48 and 72 hours,the apoptotic rate of HeLa cells advanced obviously, and there were significant differences among experimental groups (F=357.090,P＜0.001) Cell cycle arrested at G2/M phase when the duration of treatment prolonged.(F=49.206,P=0.001).7 In contrast to control group,The p27 protein expression level obvious up-regulated in MG132 group.(P＜0.05).Conclusions:1 These observations suggested that the proteasome inhibitor MG132 could actively suppress the proliferation and promote apoptosis of human cervical carcinoma cell line HeLa cells in a dose and time-dependent manner. 2 The effect of apoptosis induced by MG132 in cervical cancer cells were proposed to be followed by p27-overexpressing. Part TwoObjective 1 To observe whether proteasome inhibitor MG132 can enhance the sensitivity of cisplatin to HeLa cell and detect the expression of GST-Ï€and NF-κB.2 To observe the therapeutic efficacy of proteasome inhibitor MG132 combined with cisplatin on the xenografts of Hela cells on the nude mice.Methods 1 Inverted microscope was used to observe the morphological features and take photos of HeLa cells:HeLa cells were divided into four groups:the first group was just cultured by culture medium for 24h;the second group was treated by cisplatin2.5μg/ml for 24hours;the third group was treated by MG132 5μmol/l for 24hours;the forth group was treated by MG132 5μmol/l combined with cisplatin 2.5μg/ml for 24hours.2 MTT assay was used to detect the growth inhibition rate of HeLa cells:The first control group were only treated by culture medium; cisplatin group were treated by different concentrations of cisplatin for 24,48,72 hours respectively;the second control group were treated by DMSO which have the same concentrations with MG132 group;MG132 group were treated by MG132 5μmol/1 for 24,48 hours;MG132 combined with cisplatin group were treated by different concentrations of cisplatin respectively combined with MG132 5μmol/1 for 24 and 48 hours.3 PI staining flow cytometry was used to confirmed the apoptotic rates and cell cycles of HeLa cells:HeLa cells were divided into four groups:the first group was just treated by DMSO for 48hours;the second group was treated by cisplatin2.5μg/ml and DMSO for 48hours;the third group was treated by MG132 5μmol/l for 48hours;the forth group was treated by cisplatin2.5μg/ml combined with MG132 5μmol/l for 48hours.4 Western blotting was used to detect the expression of p65 of HeLa cells:HeLa cells were divided into four groups:the first group was just cultured by culture medium;the second group was treated by MG132 5μmol/l for 48hours;the third group was treated by cisplatin2.5μg/ml for 48hours;the forth group was treated by MG132 5μmol/l combined with cisplatin2.5μg/ml for 48hours.5 Strep tavidin-peroxidase conjugated method(SP) of immunocytochemistry was used to detect the expression of GST-Ï€in HeLa cells:HeLa cells were divided into three groups:the first group was just cultured by DMSO for 24hours;the second group was treated by cisplatin2.5μg/ml and DMSO for 24hours;the third group was treated by MG132 5μmol/l combined with cisplatin2.5μg/ml for 24hours.6 Establishment of the xenografts model of Hela cells on the nude mice. (1) Cells were cultured to logarithm phrase and digested with EDTA and then adjusted density of cells to 4×10⁷/ml.(2) Inoculability in nude mice:We inoculated the cells on the right anterior limbs of the nude mice by hypodermic injection under the sterile condition and observed the growth state of xenografts on the nude mice.7 The grouping and drug treatment of the bearing cancer mice.When the volum of the xenografts reached 40-70mm³,we divided the nude mice into four stochastic groups.There were 5 mice in every group and managed by the way there in after.1) control group:0.2ml 0.9%saline,i.p,2)MG132 group:MG132 5mg/kg,0.2ml,i.p,3)cisplatin group:cisplatin 3mg/kg,0.2ml i.p,4) MG132 with cisplatin group: MG132 5mg/kg,0.2ml and cisplatin 3mg/kg,0.2ml i.p.8 Inspection of xenograft's growth and detection of the correlated index.(1)Measurement of xenograft and draw growth curve:to mark the day starting treated as the first day,then measure the diameter of the xenograft per trid,calculate the volume and draw growth curve.(2) Calculation of tumor inhibiting rate and inhibiting rate of the weight of xenograft:we measured the weight of the 22th-day mice xenograft,calculated tumor inhibiting rate and inhibiting rate of the weight of xenograft and compared the differences of the volum and weight of xenograft in every group.(3) Inspection of pathology and histology:some of xenografts were fixed by 10%formaldehyde.Then they were dehydrated and embed. After slicing up and HE stain.They were observed by light microscope.Results 1 Morphological features was observed by inverted microscope:compared with culture medium group and cisplatin group, we observed that the cells in the MG132 combined with cisplatin group have changed a lot,most cells turned round and small and had balloon inside.2 MTT assay:there statistical significant diffferences of the proliferative inhibition rates among the groups with different concentration of cisplatin(P＜0.001) and there were also statistically significant differences of the proliferative inhibition rates among cisplatin groups with different durations(P＜0.001).The effect of different concentrations and different durations of cisplatin on cell growth was interactive(P＜0.001).There were also statistically significant diffferences of the proliferative inhibition rates among the cisplatin, MG132,MG132 combined with cisplatin groups(P＜0.001).3 Flow cytometric analysis:the MG132 combined with cisplatin group induced apoptosis of HeLa cells more effectively than the control,cisplatin and MG132 groups,and there were statistically significant differences among experiment groups(P＜0.001).Cell cycle arrested at S phase significantly.4 Western blotting:compared with the control group,the expression of p65 in cisplatin group was obviously up-regulated (P＜0.05).Compared with the control group,expression of p65 in MG132 and th MG132 combined with cisplatin groups was obviously down-regulated(P＜0.05).But there was no statistically significant differences between MG132 and MG132 combined with cisplatin groups. (p＞0.05).5 Immunocytochemistry:compared with the control group and the cisplatin group,the expression of GST-Ï€showed a clear decrease among the MG132 combined with cisplatin groups(P＜0.001)。6 Comparison of the weight and volum of xenograft:when the nude mice were sacrificed,the weight of the xenografts of every group are:control group:0.86±0.12g,MG132 group:0.74±0.13g,cisplatin group:0.49±0.08g,the MG132 with cisplatin group:0.27±0.04g.The volume of the xenografts of every group are:control group:1004±239.83mm³,MG132 group:863±192.52mm³,cisplatin group:551±143.60mm³,the MG132 with cisplatin group were 236±71.64mm³ (P＜0.001).The analysis of variance was used to examine the differences between the four groups on the volume and the weight of the xenografts on the nude mice.There were statistically significant differences.(p＜0.05)7 Tumor inhibiting rate and inhibiting rate of the weight of xenograf:comparing with the control group,the tumor inhibiting rates of MG132 group,cisplatin group and the MG132 with cisplatin group were 15%,48%,81%,the inhibiting rates of the weight of the xenografts were 9%,43%,69%respectively.Conclusion 1.Proteasome inhibitor MG132 can enhance the sensitivity of cisplatin to HeLa cell in vitro.2.The effect of enhancement of MG132 on sensitivity of cisplatin to Hela cells was proposed to be followed by the down-regulation of p65 and GST-Ï€3.MG132 can inhibit the xenografts of Hela cells on the nude mice and can increase the inhibition effect of cisplatin to them. Part ThreeObjectives To investigate the effect of the UPP inhibitor MG132 and cisplatin on proliferation and apoptosis of human cervical carcinoma cell line HCE1 monolayer and MCS and to compare the difference between the two cell culture models.To observe whether HCE1/MCS is resist to the treatment of cisplatin and to probe if MG132 can reverse the HCE1/MCS resistance to cispaltin,as well as the relationship between the effect and the expression of NF-κB,bcl-2.Methods 1 An HCE1 multicellular spheroids model was establish using liquid overlay technique and rotating culture technique.To observe the growth and histological structure of HCE1/MCS and draw the growth curve.2 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 cispaltin(5.0ug/mL,10.0ug/mL,15.0ug/mL) for 48 hours respectively and cell viability was detected by trypan blue exclusion assay with IC₁₀ and IC₅₀ calculated respectively.3 Drug treatment and experiment:(1).Grouping:according to the combinations of 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 cisplatin (6.0ug/mL);MG132 and cisplatin group:cells were treated with cisplatin 6.0ug/mL + MG132 2.0umol/L.(2).HCE1/monolayer and HCE1/MCS were cultured and treated with the 4 groups of drugs mentioned above for 48 hours,1) cell growth was observed by light microscope.2) cell viability were measured by trypan blue exclusion assay.3) cell cycle and apoptosis were detected by flow cytometer.(3).HCE1/monolayer and HCE1/MCS were cultured and treated with 4 groups of drugs mentioned above for 24 hours,1) The expression of NF-κB(p65) was detected by western-blot assay.2) bcl-2 expression was detected by immunohistochemistry.Results 1 Establishment of HCE1 multicellular spheroid model:(1). Growth state of HCE1 monolayer cells:in a good condition and after rotating for 24h,cells began to aggregate into multicellular spheroids and became more and more compact and larger in size with the culture time prolonged.(2).Histological structure of HCE1/MCS:central necrosis core and cell outgrowth around the MCS were observed when MCS got adherence.Non-structure red-stain region was observed using HE stain. (3).Growth curve of HCE1/MCS:the growth of HCE1/MCS turned into platform after 10 days.The double time of HCE1/MCS cells was 4.43±0.47 days. 2 Growth inhibition rate of HCE1 induced by MG132 and cisplatin tested by trypan blue exclusion assay.(1)HCE1/monolayer cells were cultured and treated with MG132 1.5umol/L,2.0umol/L,2.5 umol/L,5 umol/L,10 umol/L,20 umol/L for 48 hours,growth inhibition rates 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 was 2.0umol/L.(2)HCE1/monolayer cells were cultured and treated with DDP 5.0ug/mL,10.0 ug/mL,15.0 ug/mL for 48 hours,cell viability were 39.22%±6.20%,80.34%±1.53%,93.89%±3.47%respectively.IC50 is 6.1mg/L.3 Drug treatment and experiment:(1).After treated with 4 groups of drugs for 48 hours.1)Morphology viewing:â‘ control group:HCE1/monolayer cells grew in good condition, adherent to irregular diagonal corniform.HCE1/MCS were also in good condition,cells were round,adhered to each other.â‘¡MG132 group: several cells shrinkage,turned round,and detached which were observed in monolayer cells.A few of multicellular spheroids' disaggregation and shrinkage were observed in HCE1/MCSâ‘¢Cisplatin group:A lot of cells shrinkage,turned round,and detached which were observed in monolayer cells.No more change were observed in HCE1/MCS.â‘£MG132 and cisplatin group:The majority of cells shrinkage,turned round and detachment were observed in monolayer cells.Disaggregation and shrinkage were observed in HCE1/MCS.2) After treated with 4 groups of drugs for 48 hours:â‘ control group:cell inhibition rate were 1.00%±0.00%both in monolayer and MCS groups.â‘¡MG132 group:cell inhibition rate of HCE1/monolayer and HCE1/MCS were 11.67%±2.34%and 10.78%±1.17%respectively.There was no significant difference between the tow groups(P＞0.05)â‘¢Cisplatin group:cell inhibition rate of HCE1/monolayer and HCE1/MCS were 45.00%±7.44%and 9.45%±5.98%respectively.There were significant differences.(P＜0.05)â‘£MG132 and cisplatin group:Cell inhibition rate of HCE1/monolayer were 92.67%±2.52%,91.33%±2.18%respectively.There was no significant difference between them.(P＞0.05)3) Cell cycle and apoptosis in experiment groups were detected by flow cytometer.â‘ control 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 in G2/M stage cells between HCE1/MCS and HCE1/monolayer(P=0.21).â‘¡MG132 group:apoptosis and G2/M stage block were observed in both HCE1/monolayer and HCE1/MCS.â‘¢Cisplatin group:neither apoptosis nor S stage block were observed of HCE1/MCS.â‘£MG132 and DDP group:Apoptosis were observed in both HCE1/monolayer and HCE1/MCS,with apoptosis rate of 99.0%and 95.2%respectively.(2).After treated with 4 groups of drugs for 24 hours.1) NF-κB expression was detected by western-blot assay:â‘ HCE1/MCS cells expressed higher NF-κB than HCE1/monolayer.(P＜0.05)â‘¡In multicelluar spheroids:(A) MG132 group:NF-κB expression was significantly lower that of the control group.(P＜0.05)(B) Cisplatin group:NF-κB expression was significantly higher than that of the control group.(P＞0.05)(C) MG132 and cisplatin group:NF-κB expression was significantly lower than that of the control group.(P＜0.05),there was no significant change after treated with MG132 and cisplatin+ MG132.2) bcl-2expression was analysed by immunohistochemistry:â‘ bcl-2 expression was stronger in HCE1/MCS than in HCE1/monolayer.â‘¡(A) MG132 group:bcl-2 expression was significantly lower than that of the control group.(P＜0.05)(B) DDP group:bcl-2 expression was significantly higher than that of control group.(P＞0.05)(C) MG132 and cisplatin group:bcl-2 expression was significantly lower than that of the control group.(P＜0.05),and there was no significant difference between the groups exposed by MG132 and cisplatin + MG132(P＞0.05).Conclusions 1 HCE1/MCS present natural resistance to cisplatin and it may become a good model for the study of tumor chemoresistance. 2.MG132 can induce inhibition and apoptosis of HCE1/MCS cells, up-regulate the sensitivity to cisplatin of HCE1 cells and partially reverse the natural resistance of HCE1/MCS to cisplatin.3.The partially reversion of the natural resistance to cisplatin induced by MG132 in HCE1/MCS were proposed to be in relation to the up-regulation of NF-κB,bcl-2 expression.