National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China.
Radiotherapy Department, Tianjin Medical University General Hospital, Tianjin, China.
Thorac Cancer. 2020 May;11(5):1271-1279. doi: 10.1111/1759-7714.13393. Epub 2020 Mar 10.
Radioresistance in tumors limits the curative effect of the radiotherapy. Mimetic compounds of second mitochondria-derived activator of caspase (Smac) are potential new tumor radiation-sensitizing drugs because they can increase radiation-induced tumor cell apoptosis. Here, we observed the radiosensitization effect of a new Smac mimetic Antennapedia protein (ANTP)-SmacN7 fusion peptide in A549 cells and investigated the underlying mechanisms behind the effects of this protein on tumor cells.
The ANTP-SmacN7 fusion peptide was synthesized and linked with fluorescein isothiocyanate to observe the protein's ability to penetrate cells. A549 cells were divided into the control, radiation-only, ANTP-SmacN7-only and ANTP-SmacN7 + radiation groups. The cells were exposed to 0, 2, 4 and 6 Gy, with 20 μmol/L of ANTP-SmacN7. The radiation-sensitizing effects of the ANTP-SmacN7 fusion proteins were observed via clonogenic assay. Apoptosis was detected using flow cytometry. A comet assay was used to assess DNA damage. The levels and degrees of cytochrome-c, PARP, H2AX, caspase-8, caspase-3, and caspase-9 activation were detected via western blot assay. The radiation sensitization of the fusion peptide, expression of γ-H2AX and C-PARP were compared after adding the caspase inhibitor, Z-VAD.
ANTP-SmacN7 fusion proteins entered the cells and promoted A549 cell radiosensitization. Treatment with ANTP-SmacN7 + radiation significantly reduced the A549 cell clone-forming rate, increased the cytochrome-c, cleaved caspase-8, cleaved caspase-3 and cleaved caspase-9 expression levels, promoted caspase activation, and increased the rate of radiation-induced apoptosis. The ANTP-SmacN7 fusion peptide significantly increased radiation-induced double-stranded DNA rupture in the A549 cells and increased DNA damage. Adding Z-VAD reduced the fusion peptide's proapoptotic effect but not the level of double-stranded DNA breakage.
The ANTP-SmacN7 fusion peptide exerted a remarkable radiosensitization effect on A549 cells. This protein may reduce tumor cell radioresistance by inducing caspase activation and may be a potential new Smac mimetic that can be applied in radiosensitization therapy.
肿瘤的放射抵抗性限制了放射治疗的疗效。模仿第二线粒体衍生的半胱天冬酶激活剂（Smac）的模拟化合物是潜在的新型肿瘤放射增敏药物，因为它们可以增加放射诱导的肿瘤细胞凋亡。在这里，我们观察了一种新的 Smac 模拟物 Antennapedia 蛋白（ANTP）-SmacN7 融合肽在 A549 细胞中的放射增敏作用，并研究了该蛋白对肿瘤细胞作用的潜在机制。
合成 ANTP-SmacN7 融合肽并与异硫氰酸荧光素偶联，以观察该蛋白穿透细胞的能力。将 A549 细胞分为对照组、单纯照射组、ANTP-SmacN7 单纯处理组和 ANTP-SmacN7+照射组。将细胞暴露于 0、2、4 和 6 Gy 以及 20 μmol/L 的 ANTP-SmacN7 下。通过集落形成实验观察 ANTP-SmacN7 融合蛋白的放射增敏作用。通过流式细胞术检测细胞凋亡。彗星试验用于评估 DNA 损伤。通过 Western blot 检测细胞色素-c、PARP、H2AX、caspase-8、caspase-3 和 caspase-9 的激活水平和程度。在添加半胱天冬酶抑制剂 Z-VAD 后，比较融合肽的放射增敏作用、γ-H2AX 和 C-PARP 的表达。
ANTP-SmacN7 融合蛋白进入细胞并促进 A549 细胞放射增敏。用 ANTP-SmacN7+照射处理可显著降低 A549 细胞集落形成率，增加细胞色素-c、裂解 caspase-8、裂解 caspase-3 和裂解 caspase-9 的表达水平，促进半胱天冬酶激活，增加放射诱导的细胞凋亡率。ANTP-SmacN7 融合肽显著增加 A549 细胞中放射诱导的双链 DNA 断裂，并增加 DNA 损伤。添加 Z-VAD 可降低融合肽的促凋亡作用，但不降低双链 DNA 断裂水平。
ANTP-SmacN7 融合肽对 A549 细胞具有显著的放射增敏作用。该蛋白可能通过诱导半胱天冬酶激活来降低肿瘤细胞的放射抵抗性，是一种潜在的新型 Smac 模拟物，可应用于放射增敏治疗。
