Effect Of Aurora Kinase A On Cervical Squamous Cell Carcinoma And Its Study Of Mechanism

Objective: Definitive radiation therapy(RT)(with or without cisplatin-based chemotherapy)is one of the most effective treatments for cervical squamous cell carcinoma(CSCC),but efficacy is limited due to resistance.In the present study,we investigated the relationship between the expression of Aurora kinase A(Aurora-A,AURKA)and response to RT in patients with CSCC.To investigate the effect of MLN8237,DPP and docetaxel on CSCC cell lines(HCC94,SiHa)in vitro and with the purpose of understanding the toxicity of MLN8237 to normal cells to detect the effect of MLN8237 on cervical immortalized cell line H8.To further explore the mechanism of Aurora A in vitro on CSCC and the mechanism of sensitization to chemotherapy and RT.Methods:1)The expression of Aurora A in biopsy specimens of untreated primary tumors in 129 Uyghur patients with CSCC was investigated immunohistochemically.Primary treatment in these patients was definitive radical RT,which consisted of pelvic RT plus brachytherapy(total point A dose:70–85 Gy)(with or without cisplatin-based chemotherapy).The prognostic value of tumoral Aurora-A expression and patients’ clinical outcomes were evaluated.2)The HCC94 and Siha cells were treated with MLN8237,DPP,docetaxel uM,respectivly.3)The H8 cells were treated with MLN8237.4)The HCC94 and Siha cells were treated with vary combination of the three drug with the MLN8237,cisplatin and with docetaxel groups.5)The HCC94 and SiHa cells were treated with the combination with various drugs of 1/2 IC50(48h)and IC50(48h);The inhibitory rate of HCC94,Si Ha and H8 cells was analyzed with MTT.6)The cells were treated with cisplatin,docetaxel and their combination groups(MLN8237,DPP,docetaxel,and the control group.7)The cells were incubated with the three kinds of drug according to the above concentration for 24 hours,respectively;and the cells also were incubated with vary dose of combination of the vary two out of three drugs,and with the smaller dose of combination of the three drug;and then the cells were exposed to X-ray irradiation(IR)(0,2,4,8,10Gy).Coloies and survival fraction(SF)of cells were detected by clonogenic assay.The survival curve was gained by single hit multi-target model,and the extrapolated values,extrapolation value(N),mean lethal dose(D0),quasi-threshold dose(Dq),and sensitivity enhancement ratio(SER)were also calculated by the single hit multi-target model.8)The HCC94 and SiHa cells were treated with MLN8237 1uM,2uM for 24 h,set up control,migration ability was detected by scratch assay.The HCC94 and SiHa cells were treated with MLN8237,Cell cycle was detected by Flow cytometry;Apoptosis of cells was measured by Annexin V-FITC.The HCC94 and SiHa cells were treated with MLN8237,set up control group,Aurora kinase A,β-action mRNA was detected by RT-PCR,Aurora kinase A protein,(T288)phosphorylated Aurora kinase A proteinprotein and β-action protein were detected by western blot.Results: 1)Aurora-A expression was significantly associated with lymph node metastasis(P<0.001),large tumor size(P<0.001),low hemoglobin(Hb)level(P=0.011)and recurrence(P<0.001),but not other clinicopathological factors.Definitive RT was unfavorable in patients with high Aurora-A expression(P?<?0.001).In 129 enrolled patients,lymph node metastasis,large tumor size,low Hb level,and AURKA overexpression were prognostic factors for both recurrent free survival(RFS)and overall survival(OS)in univariate analysis.However,only high Aurora A expression was an adverse independent risk factor for both RFS(hazard ratio,3.953;95% CI,1.473-10.638;P = 0.006)and OS(hazard ratio 9.091;95%CI 2.597-32.258;P<0.001)in multivariate analyses.2)MLN8237 was capable of inhibiting HCC94 and Siha cells growth comparing with control group(P<0.05);MLN8237 appears more inhibiting HCC94 and Siha cells grouth effect attending by the increasing dose and time.DPP,docetaxel also showed the same trend.3)Compared with the control,The concentration of MLN8237 up to 80 uM did not cause H8 cell activity decreased(P> 0.05),even up to 320 uM only lead to a small amount of toxicity(cell survival rate at 85%).3)The combined groups inhibited the cells grouth obversely than single drug groups after 24 hours(P <0.05).Inhibition rate increased gradually with MLN8237 increasing dose combined with DDP/docetaxel.4)The survival rate of HCC94 and SiHa cells were only 15.859 ± 4.657% and 18.678 ± 5.387% with 1/2 IC50(48h)of three drugs,respectively.The survival rate of HCC94 and SiHa cells were only 6.957 ± 5.846%,9.561 ± 6.241% with IC50(48h)of three drugs,respectively.5)And the results were further confirmed in the plate clone formation experiments.6)Both the HCC94 cells and the SiHa cells survival fraction were obviously lower after treatment with the combination of MLN8237 and radiation than that of radiation alone.The cell survival curve of combination groups were to the left compared with radiotherapy groups.The front one was relatively flat and the "shoulder area" is not obvious.7)MLN8237 significantly inhibited cell migration,compared with the control group.MLN8237 groups obviously increase the G2/M phase and polyploid cell percentage,while G0/G1 + S phase ratio significantly decreased(P < 0.05),compared with the control group;Compared with the control group,the apoptotic cells in MLN8237 groups increased significantly(P < 0.05),and with the increase of time and dose,the apoptosis was more obvious.8)Aurora kinase A mRNA and protein expression between MLN8237 groups and the control group was not statistically significant.(T288)phosphorylation Aurora kinase A protein expression significantly suppressed,even disappear.Conclusion:1)Aurora A may serve as a predictive biomarker of radiation response and a therapeutic target to reverse radiation therapy resistance.2)DPP,docetaxel and MLN8237 respectively inhibited the proliferation of HCC94 and SiHa cell lines,and they showed a time and concentration dependent in a certain range.3)The cell line H8 had no obvious toxic effect.4)In clinical applications,for patients who cannot tolerate high-dose cisplatin or docetaxel,we should add appropriate dose of MLN8237,and reduce appropriate dose of cisplatin or docetaxel,in order to achieve the same or even better therapeutic effect without increasing side effects.For patients who can tolerate high-dose cisplatin or docetaxel,We should try to increase the dose of the drug in the tolerance range,in order to give full play to anti-tumor effect.5)Appropriately increasing the dose of MLN8237 can enhance the radiosensitization effect.6)In vitro,the sensitization mechanism of MLN8237 in the treatment of chemotherapy and radiation may be achieved by inhibiting the migration ability of tumor cells,inducing G2/M cell cycle arrest and polyploid formation,inducing apoptosis.7)MLN8237 did not inhibit the transcription level,nor did it affect the translation level,but changed the Aurora kinase A spatial conformation and thus affects its activity.