Proteomic Analysis Of Cervical Cancer Pre- And Post-neoadjuvant Chemotherapy With Paclitaxel And Cisplatin

Objective:To establish 2-DE patterns of cervical cancer pre-and post-neoadjvant chemotherapy with paclitaxel and cisplatin in sensitive cases and to find canditate proteins correlating to TP for improving effects of cervical cancer neoadjuvant chemotherapy with TP or finding new anti-cancer drugs.Methods:1. Six cervical cancer patients were selected which have the same characterization:①locally advanced cauliflower-type before chemotherapy;②neoadjuvant chemotherapy with paclitaxel plus cisplatin:all patients had once chemotherapy, paclitaxel 175mg/m2, cisplatin 60mg/m2;③Evaluation of efficacy of chemotherapy in 10 days were partial remission(PR), tumor size decreased 50% or more;④Radical hysterectomy and pelvic lymph node dissection were carried out about 15 days after chemotherapy;⑤Pathological diagnosis after operation were moderately differentiated squamous cell carcinoma. Samples were immediately conserved in -80℃after collection pre- and post- neoadjvant chemotherapy. Protein samples extracted from tissues were isolated by 2-D electrophoresis. The images obtained by Coomassie brilliant blue staining and scanning images were analyzed with PD-quest 7.0 software to identify differentially expressed protein spots between the two groups.2.According to the proteins classification and the degree of difficulties for buying antibodies, four differentially expressed proteins were selected for verification. By Western blot methods the expressions of four different proteins were observed pre-and post-chemotherapy in the six cases of cervical cancer tissue to detect whether the results are consistent with the proteomics. The protein were extracted from the cervical cancer tissues as proteomics study did. The same amount of sample were put to PAGE gel after measuring protein concentration. Then SDS-PAGE electrophoresis, PVDF transfer membrane, nonfat dry milk closed, and antibody incubation, HRP-ECL luminescence color, Fuser film, scanning and analysis were made.GAPDH as an internal reference.Results:1.The high-resolution, good reproducible 2-DE images were established.598±64 and 673±79 protein spots were found respectively pre- and post- chemotherapy, the average match rate between groups were 85.4%. Differentially expressed proteins between the two groups points were 102. Thirteen points significantly differentially expressed were selected for MS analysis. All candidate proteins were identified successfully. Compared with pre- chemotherapy, the expression of 11 proteins were downregulated and 2 proteins were upregulated in the post- chemotherapy group. Downregulated proteins include:actinin al,lamin Bl,eukaryotic translation elongation factor 1γ, enolase 1, annexin 1, epithelial cell marker protein 1, aldolase A, heat shock protein 27, pyruvate kinase, keratinⅡ-type and heat shock protein 70; Upregulated proteins include:apolipoprotein A1 and annexin V. All these proteins can be categorized into three groups:①Cytoskeletal proteins:actininα1, lamin B1, keratin typeⅡ, annexinⅠ, annexinⅤ;②Glycolytic enzymes:enolase 1, aldolase A, pyruvate kinase;③Others:eukaryotic translation elongation factor 1γ, epithelial cell marker protein 1, HSP27, HSP70 and apolipoprotein Al.2. The expression of HSP27, HSP70, enolase 1, aldolase A were conformed by Western blot method and results showed that the four proteins in post- chemotherapy group have lower expression than pre- chemotherapy group. The results were consistent with the proteomics study.Conclusion:1. The establishment of two-dimensional electrophoresis images for pre- and post- neoadjuvant chemotherapy with paclitaxel and cisplatin of cervical cancer show that the TP can lead to changes in the protein expression profile of cervical cancer.2. TP in chemotherapy of cervical cancer may play its roles by regulating cytoskeletal proteins, HSP, glycolytic enzymes and other proteins.3. Western blot results showed that the expression of HSP70, HSP27, enolase 1, aldolase A in post-chemotherapy group were lower than the pre-chemotherapy group and it was consistent with proteomics results. More study of these proteins may be expected to find new anticancer drug targets or to improve the efficacy of TP by down-regulating them.