CAJ | 학술논문

目的：研究细胞外基质中的一种主要蛋白：纤维连接蛋白（F N ）介导的人膀胱癌细胞（T24）对化疗药物丝裂霉素（MMC）的耐药现象，并阐明其发生的信号转导机制。方法 T24细胞与FN黏附后予以MMC处理及磷脂酰肌醇3‐激酶（PI3‐K）特异性抑制剂LY294002处理，分别检测其细胞凋亡及细胞周期情况。分别采用比色法、免疫荧光、蛋白印迹法及流式细胞术等技术检测Caspase‐8、Caspase‐9、凋亡诱导因子（AIF）3种凋亡通路的激活情况。蛋白印迹法检测细胞周期G1／S期转换的关键分子糖原合成激酶3β（GSK‐3β）、Cyclin D1的表达水平。同时利用蛋白印迹法检测PI3‐K／Akt信号通路的激活情况。结果 FN与 T24细胞黏附后显著抑制了MMC诱发的细胞凋亡，且这种作用是通过抑制Caspase‐9及AIF两种凋亡通路而非Caspase‐8凋亡通路实现的。同时，FN通过失活GSK‐3β而稳定了Cyclin D1的表达水平，从而拮抗了MMC诱导的 T24细胞G1／S期停滞。FN介导的对 T24细胞的保护作用是 PI3‐K／Akt依赖的，可以被LY294002阻断。
목적：연구세포외기질중적일충주요단백：섬유련접단백（F N ）개도적인방광암세포（T24）대화료약물사렬매소（MMC）적내약현상，병천명기발생적신호전도궤제。방법 T24세포여FN점부후여이MMC처리급린지선기순3‐격매（PI3‐K）특이성억제제LY294002처리，분별검측기세포조망급세포주기정황。분별채용비색법、면역형광、단백인적법급류식세포술등기술검측Caspase‐8、Caspase‐9、조망유도인자（AIF）3충조망통로적격활정황。단백인적법검측세포주기G1／S기전환적관건분자당원합성격매3β（GSK‐3β）、Cyclin D1적표체수평。동시이용단백인적법검측PI3‐K／Akt신호통로적격활정황。결과 FN여 T24세포점부후현저억제료MMC유발적세포조망，차저충작용시통과억제Caspase‐9급AIF량충조망통로이비Caspase‐8조망통로실현적。동시，FN통과실활GSK‐3β이은정료Cyclin D1적표체수평，종이길항료MMC유도적 T24세포G1／S기정체。FN개도적대 T24세포적보호작용시 PI3‐K／Akt의뢰적，가이피LY294002조단。
Objective To investigate that cell adhesion to fibronectin (FN) ,a major protein of extracellular matrix ,induces drug resistance in human bladder cancer cells ,and to study fibronectin mediated survival signaling pathway in bladder cancer chemotherapy resistance in vitro . Methods T24 cells (human bladder cancer cell lines) were precoated with fibronectin ,and treated with Mito‐mycin C (MMC) and the specific PI3‐K inhibitor LY294002 ,and the apoptosis and cell cycles were analyzed respectively .The activity of Caspase‐8 ,‐9 and apoptosis‐inducing factor (AIF) apoptosis pathways were assessed using colorimetric assay ,immunofluorescence ,western blot and flow cy‐tometry .The expression of glycogen synthase kinase‐3β(GSK‐3β) and Cyclin D1 ,as the key regula‐tor of G1/S phase transition ,were determined by western blot .The expression of PI3‐K ,Akt , phospho‐Akt andβ1‐integrin were also examined by western blot . Results Apoptosis induced by MMC was significantly resisted by FN adhesion in T24 cells ,and this effect was through inhibition of Caspase‐9 and AIF apoptosis pathways but not Caspase‐8 pathway .FN antagonized MMC‐induced G1/S‐phase arrest by inactivating GSK‐3βto stabilize Cyclin D1 expression in T24 cells .Furthermore ,FN‐medi‐ated protection of T24 cells was dependent on activity of PI3‐K/Akt signaling pathway ,and the protection could be abolished by the PI3‐K inhibitor LY294002 . Conclusions FN‐mediated PI3‐K/Akt activation protects T24 cells from MMC‐induced cell death through inhibition of both Caspase‐9 and AIF medi‐ated apoptosis and GSK‐3β/Cyclin D1 involved G1/S‐phase arrest .