Differential Protein Expression In Cervical Cancer And The Role Of Chemotherapeutics In Treatment Of Hela Cells

Background:Cervical cancer is one of the most common cancers of women in the world and on the top of gynecology cancers in our country.The morbidity of cervical cancer is increased with the age,which increased significantly after 40 years old.In the past 30 years,the morbidity and mortality of cervical cancer were decreased all over the world.But cervical cancer is still the main cause of women died of cancer.Early diagnosis in cervical cancer is the most efficient method in improving prognostic. Examination method,such as cervix smear,is still lack of sufficient sensitivity. SCCA and CEA,serum tumor markers,are used in prospective detection of cervical cancer.SCCA increase significantly when tumor recur or progress,while has no marked change in earlier period.In a word,there are no ideal tumor markers to discovery cervical cancer early or to be used as monitoring index in follow-up visit. It is very urgent to find a sensitiveness and effective method in early diagnosis of cervical cancer.Protein array technique is used widely in clinic.It is very important to diagnose and screen disease,judge the progression,prognosis and therapeutic effect.Proteome provided a new platform for cervical cancer research,which analyze quantitatively and compare change of proteins category and quantity in normal and process sample. It is helpful to illuminate the regulation network among proteins and hoped to find the key molecule controlling the progress of cervical cancer.It brings new idea and pathway to diagnosis and medicine research of cervical cancer.Therapeutic regimen of cervical cancer is decided with clinical stage,age, general state of health and tumor correlation factor.The earlier stage(Ⅰ～ⅡA stage) is treated by operation.Patients who are not suitable for operation can be treated with radiotherapy,which has the same therapeutic efficiency with operation.Part advanced stage(â…¡B～ⅢB stage)is treated by radiotherapy and chemotherapy together. Advanced stage(â…£stage),tumor metastasis and recurrence,is treated by chemotherapy plus radiotherapy.For few years,chemotherapy of cervical cancer is changed from the alleviative treatment to an important ingredient of cervical cancer combined therapy because of the progress of combination chemotherapy based on DDP.Traditional active drug in treatment of cervical cancer include cisplatin(DDP),carboplatin(CBP),fluorouracil(5-Fu),cyclophosphamide(CTX),ifosfamide (IFO),adriamycin(ADM),amethopterin(MTX),bleomycin(BLM),mitomycin (MMC)and so on.New drugs such as paclitaxel(Taxol),gemcitabine(GEM),irinotecan(CPT-11),vinorelbine(NVB),topotecan(TPT)are the active drug in treatment of cervical cancer.DDP is the most competent cytotoxic drug in the treatment of recurrence and metastatic cervical cancer.Combination chemotherapy increases the therapeutic effect.Including BIP,PF,TP,CPT-11+DDP and TPT +DDP.In the American NCCN guideline of latest edition,DDP,combination of DDP+ Taxol and combination of DDP+CPT-11 are the first-line chemotherapy of recurrence cervical cancer.Paclitaxel liposome,the similar drug with paclitaxel,is used in treatment of nonsmall-cell lung cancer,breast cancer and ovarian cancer. Paclitaxel liposome has good therapeutic effect in clinic.But it is not proved that combination chemotherapy can improve obviously the survival time and quality of patients compared with simple chemotherapy.It needs to approach new drug and administration route to elevate the chemotherapy efficacy and prolong survival time of patients.Objectives:1.To search the differentially expressed proteins and set up diagnostic model in earlier period and advanced stage of cervical cancer.2.To investigate the inhibitory proliferation effect and apoptosis induction of Taxo1,Liposome single or combined with DDP on Hela human cervical carcinoma cells.Methods:1.Identify the differentially expressed proteins between earlier period and advanced stage in the serum of patients with cervical cancer.(1)Sample collection:44 cervical carcinoma patients were divided into two groups,26 patients in earlier period and 18 patients in advanced stage. Collect the venous blood of all patients.Centrifuge blood at 4℃and take supernatant,which was preserved in frigidaire at-70℃.(2)Serum protein mass peak were detected by SELDI-TOF-MS and weak canonic chips.Fetch array data and draw protein mass spectra figure.The protein fingerprints were obtained,two diagnostic models was set up,and bioinformatic analysis was performed to identify the differentially expressed proteins in the serum of the patients.(3)The density of SCCA in serum was detected by ELISA method.Control group included the normal value of SCCA;treatment group included the level of SCCA which surpass 3 times of normal value.The peak value of protein mass spectra in control group and treatment group were detected.2.Inhibitory effect of chemotherapeutics on Hela human cervical carcinoma cells in vitro(1)Hela human cervical carcinoma cells were cultured in RPMI 1640 containing 10%fetal calf serum.Exponentially growing cells were chosen for experiment. Hela cells were divided into 6 groups:control group,DDP-treated group, Taxol-treated group,Liposome-treated group,DDP+Taxol combination treatment group and DDP+Liposome combination treatment group.MTT assay included cells treated with DDP(2.5,5,10,20μg/ml),Taxol(4.5,9,18,36μg/ml),Liposome(4.5,9,18,36μg/ml)alone and combination DDP+Taxol(2.5+4.5,5+9,10+18)(μg/ml)and combination DDP+Liposome (2.5+4.5,5+9,10+18)(μg/ml)for 24,48 and 72 hours respectively.Apoptosis analysis included cells treated with DDP(10μg/ml),Taxol(18μg/ml)and Liposome(10μg/ml)alone and combination DDP+Taxol(10+18)μg/ml and combination DDP+Liposome(10+18)μg/ml.(2)Group as the above mentioned,the inhibitory of Hela cells were detected by MTT assay after treatment.Calculate the inhibitory rates of cells according OD value.Interaction between DDP and Taxol,DDP and Liposome were assessed using the q value,where q＞1.15,0.85≤q≤1.15,q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.(3)Group as the above mentioned,collected cells and analyzed apoptosis by Annexin V-FITC/PI and flow cytometry.Calculate the inhibitory rates of apoptosis.(4)All datas were analysised by SPSS 13.0 statistical softwire.Experimental data were expressed as mean±S.D.The inhibitory rates were analyzed by univariate analysis of variance.The groups with same dose and same time were analyzed by independent-samples t test.The others data were analyzed by One-way ANOVA.The method of comparision within different time and groups was LSD multiple comparison test if homogeneity of variance,and Dunnett's T3 test if heterogeneity of variance..P-Values were considered to be significant at＜0.05(2-sided)Results:1.Differentially expressed proteins in earlier stage and advanced stage of cervical cancer(1)In the range of 1002～18369 in relative molecular mass,79 differentially expressed proteins were detected in the serum of cervical cancer patients in two groups and 43 mass spectra peak value had significant difference (P＜0.05),among which 11 proteins with mass/charge ratio of 11523.83,11679.13,5841.076,7971.04,16109.71,15932.17,11480.07,5902.02,15861.78,18368.15 and 8141.23,respectively,showed lower expression in the serum of cervical cancer patients in earlier period,P＜0.001.(2)Two diagnostic models for cervical cancer were generated to discriminate earlier period and advanced stage,including 11679.13(M/Z)protein with the correct classification of 93.18%(41/44)and 5902.02(M/Z)protein with the correct classification of 81.81%(36/44).(3)Divide the 44 serum sample into two groups according to the value of SCCA. Control group included 22 patients with normal value of SCCA.Treatment group included 16 patients with the level of SCCA which surpass 3 times of normal value.The peak value of protein mass spectra in control group and treatment group were detected.The result indicated:in the range of 1002～ 18369 in relative molecular mass,78 differentially expressed proteins were detected in two groups and 43 mass spectra peak value had significant difference(P＜0.05),among which 11 proteins with mass/charge ratio of 11523.83,11679.13,5841.076,7971.04,16109.71,15932.17,11480.07,5902.02,15861.78,18368.15 and 8141.23,respectively,showed lower expression in control group.These differentially expressed proteins were the same with which in earlier period and advanced stage.2.The inhibitory effect of chemotherapeutics on Hela cells in vitro(1)The MTT result indicated:DDP,Taxol and Liposome alone inhibited cell proliferation time-and dose-dependently(P＜0.00l).With the increase of dose and the lasting of the time,inhibition ratio of DDP increased accordingly。The platform stage didn't appear when DDP(20μg/ml)had affected cells for 72 hours.The inhibition of Taxol didn't increase accordingly with the change of time when the dose of Taxol achieved 36μg/ml.In a short time,the inhibition of Liposome didn't increase accordingly with the increasing of dose.(2)Combination groups inhibited cell proliferation time-and dose-dependently (P＜0.001).Combination group(DDP 2.5μg/ml+Taxol 4.5μg/ml)had a different inhibition in 24h and 72h.Combination group(DDP 5μg/ml+ Taxol 9μg/ml)had the best inhibition in 48h.Combination group(DDP 5μg/ml+ Liposome 9μg/ml)had the same inhibition in 48h and 72h.So did combination group(DDP 10μg/ml+Liposome 18μg/ml).All in all,the inhibition of combination between DDP and Liposome didn't increase after 48h and medium dosage.(3)Calculate and analysis the value of IC₅₀every group.The result indicated: the value of IC₅₀diminished with the drugs worker longer,there was no significant difference in combination between DDP and Taxol and combination between DDP and Liposome in 24h.There was no significant difference in DDP,combination between DDP and Taxol,combination between DDP and Liposome in 48h.There was a significant difference in every group at 72h..(4)q Value of DDP combined with Taxol showed:Coadministration of DDP with Taxol showed synergism and additive effect of all concentration levels at 24, 48,and 72h(0.85≤q≤1.15).Combination group(DDP 2.5μg/ml+ Taxol 4.5μg/ml)had the max q value(2.19)at 48h.Combination group(DDP 2.5μg/ml+ Liposome 4.5μg/ml)had the max q value(1.90)at 48h.(5)Single and combination group induced Hela cells apoptosis.Cells apoptosis was detected after treated with single and combination group(P＜0.001). There was a significant difference in single and combination group.The rate of apoptosis in combination between DDP and Taxol was higher than in combination between DDP and Liposome.Conclusion:1.Differential protein expression existed in earlier period and advanced stage in cervical carcinoma.Two diagnostic models for cervical cancer were generated using software,which was used to reference of early diagnosis and stage in clinic.Serum SCCA at the level of 3 times of normal value was considered has a relationship with advanced stage.Differential protein expression existed in both group which SCCA at normal value and group which SCCA at the level of 3 times of normal value.These differential protein were the same with which in earlier period and advanced stage.2.DDP,Taxol and Liposome alone inhibited cell proliferation time-and dose-dependently.DDP has synergism effect with Taxol and Liposome.The single and combination could promote Hela cells apoptosis.