Research On The Growth Inhibition And The Chemosensitivity Of Cervical Cancer Cell By Inhibiting Aurora Kinase B

Introduction and ObjectivesCervical cancer is one of the most common malignancies of women. It severely threatens women's health. Radiation and surgery therapy have been used for patients with advanced or recurrent cancer, but many patients have already lost the chance of operation in fact, with the basic and clinical research advances rapidly, platinum-based neoadjuvant chemotherapy (NACT) has greatly improved the prognosis of these patients,as well as the cases with larger lesions.But because of the toxicity to normal tissue and the acquired resistance, it severely restricts the effectiveness and application of platinum-chemotherapy. Therefore, looking for some drugs with low toxicity, relatively selective for tumor cells and synergistic effect for chemotherapy seems extremely important. It is the key to improve the survival of cervical cancer patients, especially for those advanced patients.Aurora kinase family of mammalian proteins are evolutionarily conservative at the structure and function, they can be divided into three kinds:Aurora-A, Aurora-B, Aurora-C according to their location in cells.Aurora-B is primarily located in the centromere region of the chromosome in the early stage of mitosis, and then transferred to the microtubule of the spindle equatorial plane in the late stage of mitosis.When the spindle elongates and the cytokinesis occurs, Aurora-B will aggregate in the fissures of the cell cortex and the center of the spindle.In finally, the protein will concentrate in the intermediate filament, binding to another three chromosomal passenger proteins INCENP, survivin and borealin to form a complex. This complex, playing an important role in proper arrangement and separation of chromosomes, has the function to ensure Aurora-B can precisely locate and activate before and during the mitosis period.Aurora-B is essential for the phosphorylation of histone H3 serine-10 in mitosis, which is very important for chromosome condensation.When histone H3 phosphorylation is inhibited, the chromosome condensation is inhibited, and cell mitosis is impeded. So,histone H3 phosphorylation can be used as a direct downstream target of Aurora-B to measure the effect of Aurora-B kinase inhibition.Studies found that the Aurora-A and Aurora-B were over-expressed in lung cancer, prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, bladder cancer,carcinoma of esophagus.Enhancing their expression can cause cell genome instability, mitotic errors and cell malignant transformation; suppressing their expression can inhibit cell proliferation and promote apoptosis.Aurora kinase family therefore has become a potentially valuable anti-tumor therapeutic target.There are a lot of Aurora kinase inhibitors previously reported, one of which is ZM447439.ZM447439 is an ATP competitive inhibitor, which can inhibit the microtubule inaction caused by the abnormal kinetochore separation,prevent chromosomes alignment and separation,inhibiting spindle checkpoint,inhibit cytokinesis.Cells treated with ZM447439 exit mitosis without dividing, the cell cycle is arrested, polyploidy increased, cell size increased, proliferation decreased, apoptosis increased, etc. Through RNA interference and induced mutation techniques, Girdler et al. proved that the inhibition of Aurora-B by ZM447439 can cause the changes of tumor cell morphology and biological behavior.Aurora-B kinase is only expressed in the proliferating cells, most of human normal cells are in a steady state, so the inhibitor of Aurora-B kinase on the tumor cells have a strong selective. First, this paper tested the higher express of Aurora-B kinase in cervical cancer tissue, then combined ZM447439 with cisplatin in cervical cancer cells, investigated the effect of ZM447439 on cell and the synergy effect with cDDP. The study of these questions at home and abroad is now rarely reported. Through this study, we provide new ideas for target therapy and clinical chemotherapy for cervical cancer.Materials and MethodsPart I:Detected Aurora-B protein in different tissues of the cervix by immunohistochemistry and then analyzed the relationship between Aurora B protein and clinical parameters of cervical cancer.Part II:The effect of ZM447439 on proliferation of SiHa cells was tested by MTT; the changes of cell cycle and apoptosis were detected by flow cytometry; cell morphology was observed with microscope and electron microscope; Aurora B and H3-P protein was detected by Western blot.Part III:The changes of cell proliferation and cell cycle and apoptosis under ZM447439 and cisplatin were respectively tested using MTT, flow cytometry. HPV16E6,P53,BCL-2, VEGF protein expression were detected by Western blot.ResultsPart I:The rate of Aurora-B expression is highest in cervical cancer and has no significant correlation to clinical stage, lymph node metastasis and age(P> 0.05). Aurora-B protein expression has significant correlation to cancer cell differentiation and pathological type (P<0.05).Part II:ZM447439 can reduce the number of SiHa cell, increase the volume of cell, and lead to apoptosis in a dose-and time-dependent manner. The results of Flow cytometry showed G2/M arrest and the increase of early apoptosis by ZM447439. Protein blot investegated that ZM447439 inhibited Aurora B and H3-P expression.Part III:MTT results showed that cisplatin inhibited the growth of SiHa cells in the form of time and dose-dependent. Combined with cisplatin, ZM447439 significantly enhanced the effect of inhibiting proliferation and promoting apoptosis of cisplatin, and caused significant arrest of S phase, and inhibited HPV16E6, BCL-2, VEGF protein expression, and enhanced the expression of P53 protein. ConclusionsPartâ… :Aurora B provides a new target for the treatment of cervical cancer. The role of Aurora B in the pathogenesis of cervical squamous cell carcinoma and adenocarcinoma may be different.Aurora B expression has relation to the degree of maturity of cervical cancer cells.Partâ…¡:Inhibiting the expression of Aurora B and H3 phosphorylation can inhibit the proliferation of cervical cancer SiHa cells, promote apoptosis,cause cell arrest at mitosis.Partâ…¢:The inhibition of Aurora B significantly enhanced the chemosensitivity of cisplatin for cervical cancer SiHa cells, significantly induced apoptosis,showed synergistic effect. The mechanism may be related to the inhibition HPV16E6,BCL-2 expression, and the recovering of P53 function.