Abstract：
Cervical cancer is the fourth leading cause of cancer deaths in women globally. Combining gene therapy with chemo- and radiotherapy may improve cervical cancer treatment outcomes. This study evaluated the effects of Annexin A5(ANXA5) overexpression alongside 5-fluorouracil (5-FU) and irradiation on the viability of CaSki cervical squamous cell carcinoma (SCC) cells. pAdenoVator-CMV-ANXA5-IRES-GFP-plasmid and mock plasmid were transfected into CaSki cells using calcium-phosphate. Seventy-two hours post-transfection, GFP expression was quantified by fluorescence microscopy and flow cytometry to evaluate transfection efficiency. ANXA5 overexpression was confirmed via qPCR. Twenty-four hours post-transfection, cells received a single dose of 8 Gy and were treated with 1 and 2 µg/ml of 5-FU (IC50 = 2.783 µg/ml). Cell viability, apoptosis, cell cycle stage, and Bcl-2 and Bax gene expression were assessed via MTT, annexin V/7-AAD, PI staining, and qPCR assays, respectively. ANXA5 was overexpressed 31.5-fold compared to control (p < 0.0001). MTT assays showed ANXA5 overexpression dose-dependently reduced CaSki cell viability (p < 0.001). IC50 of 5-FU was reduced from 2.783 μg/mL to 1.794 μg/mL when combined with ANXA5 overexpression. Additive effects on cell death were observed for ANXA5 plus 5-FU or irradiation versus ANXA5 alone. Apoptosis assays indicated combinatorial treatment increased CaSki cell apoptosis over ANXA5 alone. Cell cycle analysis revealed ANXA5 arrested cell cycle at G1/S phases; the percentage of cells in the S phase further rose with combination treatment. Finally, combination therapy significantly decreased Bcl-2 expression and increased Bax versus control (p < 0.001). Altogether, ANXA5 overexpression alongside 5-FU and irradiation may improve cervical squamous cell carcinoma (SCC) treatment efficacy. Further, in vivo investigations are warranted to confirm these in vitro results.
摘要：
宫颈癌是全球女性癌症死亡的第四大原因。将基因治疗与化疗和放疗相结合可以改善宫颈癌的治疗效果。这项研究评估了膜联蛋白A5（ANXA5）过表达以及5-氟尿嘧啶（5-FU）和辐照对CaSki宫颈鳞状细胞癌（SCC）细胞活力的影响。使用磷酸钙将pAdenoVator-CMV-ANXA5-IRES-GFP-质粒和模拟质粒转染到CaSki细胞中。转染后72小时，通过荧光显微镜和流式细胞术定量GFP表达以评估转染效率。通过qPCR确认ANXA5过表达。转染后24小时，细胞接受单剂量8Gy,用1和2µg/ml5-FU处理(IC50=2.783µg/ml).细胞活力，凋亡，细胞周期阶段，并通过MTT评估Bcl-2和Bax基因的表达，附件五/7-AAD，PI染色,和qPCR分析，分别。与对照相比,ANXA5过表达31.5倍(p<0.0001)。MTT测定显示ANXA5过表达剂量依赖性地降低CaSki细胞活力(p<0.001)。当与ANXA5过表达组合时,5-FU的IC50从2.783μg/mL降低至1.794μg/mL。对于ANXA5加5-FU或照射与单独的ANXA5观察到对细胞死亡的加合作用。凋亡测定表明,与单独的ANXA5相比,组合处理增加了CaSki细胞凋亡。细胞周期分析显示，ANXA5将细胞周期阻滞在G1/S期；S期细胞的百分比随着组合处理而进一步上升。最后,与对照组相比,联合治疗显著降低Bcl-2表达,增加Bax(p<0.001).总之,ANXA5过表达与5-FU和放疗一起可能会提高宫颈鳞状细胞癌（SCC）的治疗效果。Further,有必要进行体内研究以证实这些体外结果。
