A Study On The Relationship Between β-catenin Expression And Cisplatin Resistance In Human Ovarian Cancer Cells

Ovarian cancer, one of the most malignant tumors in ovarian carcinomas, has a high morbidity second to cervical cancers, but has a top mortality. Ovarian cancers are not usually detected at the early stage due to their concealed onset, easy metastasis and poor prognosis. Such characteristics of epithelial ovarian cancers make it difficult to get the basic improvement and contribute to 30% ~ 40% of 5-year survival rate for the patients. In recent years, comprehensive therapy based on surgery with adjuvant radiation and chemo has been regarded as the main cure methods for ovarian cancers. Ovarian cancer cells reduced sensitivity to platinum may cause tumor recurrence, metastasis, and increasing mortality. Therefore, discussing the relevant drug-resistant mechanisms of ovarian cancers and finding molecular targets or effective drug-resistant reversal agents, will become undoubtedly important to provide a new method for tumor gene therapy.Classical Wnt signaling pathway, plays an important regulatory role in signal transduction. And beta-catenin is the key molecule of Wnt signaling pathways, which participates in molecular adhesion, growth, cell differentiation, damage repair, neoplasia,and metastasis.Beta-catenin(β-cat) is an important adhesive molecule in cells adhesion ,and the signal transduction of Wnt pathway. Disfunction of each member of Wnt pathway can lead to beta-catenin change. When it accumulates in cytoplasm to some extent then goes into nuclear where downstream proto-oncogenes activated byβ-catenin and express related proteins. It is of great significance for the occurrence, development and prognosis in a variety of malignant tumors.There are some evidences thatβ-catenin plays a role in drug resistance.On one hand, nuclear incrementalβ-catenin provoke DNA-binding proteins family T cells factor/lymphocytes enhanced factor (Tcf/Lef) activated, and thus the downstream target genes such as MDR1, c-myc, cyclinD1 ,etc, may express that could cause cancer cells resistent to drug sensitivity. On the other hand,β-catenin gene can be adjusted by apoptosis-related important factors,whch affect chemoradiotherapy resistance by influencing cells growth and apoptosis.This study adopts the immunochemical method to detectβ-catenin expression in ovarian cancer A2780 cells that are sensitive to cisplatin and its counterpart ,cisplatin resistant CP70 cells. analysis the relationship betweenβ-catenin expression and platinum resistance in ovarian cancers. SiRNA and small molecule inhibitors were applied to inhibitβ-catenin expression to observe the influence of chemo sensitivity withβ-catenin gene silence in ovarian cancer cells. This paper discusses the relationship betweenβ-catenin and cisplatin resistance in ovarian cancer cells, and provide valuable experimental basis for platinum resistance reversal of ovarian cancer.【Objectives】1. To investigate the correlation ofβ-catenin gene and cisplatin resistance in ovarian cancer cells.2. To verify the influence of cisplatin onβ-catenin expression in ovarian cancer cells.3. To observe the impacts ofβ-catenin gene silence on cisplatin resistance of ovarian cancer cells transfected byβ-catenin siRNA.【Methods】1. Immunocytochemistry,immunofluorescence were used to detect theβ-catenin protein expression and location in cisplatin sensitive A2780 cells and resistant CP70 cells of ovarian cancers.2. Western blot assay was used to detectβ-catenin expression in A2780 cells and CP70 cells.3. The MTT method was to testify the sensitivity to cisplatin of A2780 cells and CP70 cells.4. Western blot assay was used to observe the changes ofβ-catenin expression in A2780 and CP70 cells when treated by different doses of cisplatin.5.β-catenin siRNA instantaneously transfected into cisplatin resistant CP70 cells, real time-RT PCR tested the mRNA level of intracellularβ-catenin. Western blot assay used to detect the protein expression as well.6. Immunocytochemistry,immunofluorescence were used to prove theβ-catenin protein expression in CP70 cells afterβ-catenin siRNA transfecting.7. MTT method was adopted to investigate the influence ofβ-catenin siRNA instantaneously transfection on cisplatin sensitivity of the resistant cells.8. TUNNEL was used to detect the cell apoptosis induced by cisplatin afterβ-catenin siRNA transfection.【Results】1. The location ofβ-catenin of both CP70 cells and A2780 cells are in nucleu and cytoplasm. In A2780,β-catenin mainly expresses in cytoplasm,but in CP70 ,β-catenin expresses mainly in nucleu and cytoplasm.2.β-catenin protein expression level is higher in CP70 cell than that in A2780 cell. They reached significantly statistical difference(P<0.01).3. The 50% inhibitory concentration(IC50) value of cisplatin in A2780 and CP70 cells was 14.3μM and 74.0μM,the resistance index was 5.17.4.β-catenin protein changing trends varied in both cells.The result of Western blot showed that expression ofβ-catenin increased then decreased in CP70 cell lines pretreated by DDP with increasing dosage,but declined in A2780 cells which was a passive corretation with DDP dosage.5.β-catenin silenced by instantaneously transfecting siRNA into CP70 ovarian cancer cell for 48h,β-catenin mRNA level in CP70 was decreased conspicuously,as well as the results ofβ-catenin expression tested by western blot.6. Immunocytochemistry and immunofluorescence showed thatβ-catenin protein level in CP70 was decreased conspicuously afterβ-catenin siRNA instantaneously transfection.7. Cisplatin resistant CP70 cell instantaneously transfected withβ-catenin siRNA enhanced its sensitity to cisplatin.The 50% inhibitory concentration(IC50) value of cisplatin in CP70 cells was from 74μM before siRNA interference to 24.59μM after siRNA interference.he drug resistance reversal fold(RF) was 3.01,which reached the significant statistical difference(P<0.05).8. Afterβ-catenin siRNA transfection,CP70 cells apoptosis inducted by cisplatin was increased. Apoptostic index ofβ-catenin siRNA transfected CP70 cells was(43.1 + 4.2)%,2.14 times as the control group treated only by DDP.They had significantly statistical difference(P<0.01).【Conclusion】1. The expression ofβ-catenin and sub-cellular localizationin in cisplatin sensitive cells and resistant cells had different characteristics.β-catenin protein expression in cisplatin resistant CP70 cells is higher than that in sensitive A2780 cells.It is showed that the expression and sub-cellular localization ofβ-catenin had relevance with cisplatin resistance in ovarian cancer.2.β-catenin expression differently changed in A2780 and CP70 with different concentration of cisplatin exposure.β-catenin level differed in the two cells suggested thatβ-catenin plays an important role in cisplatin resistance in ovarian cancer cells.3. To some extent,inhibitingβ-catenin expression greatly enhances apoptosis induced by cisplatin and cisplatin chemotherapy sensitivity.The mechanism may be related to its apoptotic regulation.β-catenin may become a effective molecular target for platinum- resistant reversl of ovarian cancer.