Hyperthermia and cisplatin combination therapy promotes caspase-8 accumulation and activation to enhance apoptosis and pyroptosis in cancer cells

Hyperthermia can play a synergistic role with chemotherapy in combination therapy. Although the association between caspase activation, apoptosis, and pyroptosis have been published for both cisplatin (CDDP) and hyperthermia therapies independently, the interactions between these molecular pathways in combination therapy are unknown. The present study aimed to investigate the possible interactions between caspase 8 activation, apoptosis, and pyroptosis in combination therapy. Cells were treated with CDDP (15 µg/ml), followed by hyperthermia at optimized temperature (42.5 °C) in water-bath. After combination therapy, cell viability was analyzed by CCK-8, and cell death was analyzed by Annexin-V-FITC/PI and caspases activation. Immuno-staining and co-immuno-precipitation were used to examine the interaction between p62 and caspase-8. Pyroptosis was investigated by western blotting and transmission electron microscopy. E3 ligase Cullin 3 was knockdown by siRNA. In addition, caspase-8 activation was modulated by CRISPR-Cas9 gene-editing or pharmacological inhibition. Combination therapy promoted K63-linked polyubiquitination of caspase-8 and cellular accumulation of caspase-8. In turn, polyubiquitinated caspase-8 interacted with p62 and led to the activation of caspase-3. Knockdown of the E3 ligase Cullin 3 by siRNA reduced caspase-8 polyubiquitination and activation. In addition, combination therapy induced release of the pore-forming N-terminus from gasdermins and promoted pyroptosis along with caspase-8 accumulation and activation. Knockdown of caspase-8 by CRISPR/Cas9 based gene editing reduced the sensitivity of tumor cells to apoptosis and pyroptosis. Our study presented a novel mechanism in which hyperthermia synergized with chemotherapy in promoting apoptosis and pyroptosis in a caspase-8 dependent manner.

热疗与化疗联合应用时可发挥协同增效作用。尽管已有研究分别报道了顺铂（cisplatin, CDDP）或热疗单独应用时，半胱天冬酶激活、细胞凋亡与细胞焦亡之间的关联，但二者联合治疗时这些分子通路间的相互作用仍未明确。本研究旨在探究联合治疗中半胱天冬酶-8（caspase-8）激活、细胞凋亡与细胞焦亡之间的潜在相互作用。实验中，细胞先经15 µg/ml的顺铂处理，随后置于恒温水浴箱中于优化后的42.5 °C条件下进行热疗。联合治疗后，采用CCK-8法检测细胞活力，通过膜联蛋白V-FITC/碘化丙啶（Annexin-V-FITC/PI）双染色及半胱天冬酶活性检测分析细胞死亡情况。采用免疫染色与免疫共沉淀技术检测p62与半胱天冬酶-8之间的相互作用。通过蛋白质印迹（Western blotting）与透射电子显微镜观察细胞焦亡情况。采用小干扰RNA（siRNA）敲低E3泛素连接酶Cullin 3的表达。此外，通过成簇规律间隔短回文重复序列相关蛋白9（CRISPR-Cas9）基因编辑或药物抑制手段调控半胱天冬酶-8的激活。联合治疗可促进半胱天冬酶-8发生K63位连接的多泛素化修饰，并增加细胞内半胱天冬酶-8的积累。随后，泛素化修饰的半胱天冬酶-8可与p62结合，并介导半胱天冬酶-3的激活。通过siRNA敲低E3泛素连接酶Cullin 3的表达，可降低半胱天冬酶-8的多泛素化水平与激活活性。此外，联合治疗可诱导Gasdermin家族蛋白的孔形成N端结构域释放，并伴随半胱天冬酶-8的积累与激活，进而促进细胞焦亡。基于CRISPR/Cas9的基因编辑技术敲低半胱天冬酶-8的表达，可降低肿瘤细胞对细胞凋亡与细胞焦亡的敏感性。本研究揭示了一种全新的机制：热疗与化疗通过半胱天冬酶-8依赖的方式协同促进细胞凋亡与细胞焦亡。