It is well accepted that the immune system efficiently contributes to positive outcomes of chemotherapeutic cancer treatment by activating immunogenic cell death (ICD). However, only a limited number of ICD-inducing compounds are well characterized at present; therefore, identification of novel ICD inducers is urgently needed for cancer drug discovery, and the need is becoming increasingly urgent. Herein, we assessed the antitumour activity of bullatacin by MTS assay and apoptosis assay. ICD biomarkers, such as calreticulin (CRT), high-mobility group protein B1 (HMGB-1), heat shock protein (HSP)70, HSP90 and ATP, were assessed by Western blotting, ELISA and flow cytometry. Western blot and qPCR assays were performed to explore the underlying mechanisms of bullatacin-induced ICD. Flow cytometry was used to detect macrophage phagocytosis. First, bullatacin induced apoptosis in both SW480 cells and HT-29 cells in a time-dependent manner at 10 nM, as assessed by flow cytometry. Moreover, Western blot and flow cytometry assays showed that CRT and HSP90 (biomarkers of early ICD) significantly accumulated on the cell membrane surface after approximately 6 h of treatment with bullatacin. In addition, ELISAs and Western blot assays showed that the second set of hallmarks required for ICD (HMGB1, HSP70 and HSP90) were released in the conditioned media of both SW480 and HT-29 cells after 36 h of treatment. Furthermore, qPCR and Western blot assays indicated that bullatacin triggered ICD via activation of the endoplasmic reticulum stress (ERS) signalling pathway. Finally, bullatacin promoted macrophage phagocytosis. This study documents that bullatacin, a novel ICD inducer, triggers immunogenic tumour cell death by activating ERS even at a relatively low concentration in vitro.

中文翻译：

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Bullatacin 通过激活内质网伴侣蛋白触发结肠癌细胞的免疫原性细胞死亡

众所周知，免疫系统通过激活免疫原性细胞死亡 (ICD) 有效地促进化疗癌症治疗的积极结果。然而，目前只有有限数量的 ICD 诱导化合物得到很好的表征。因此，癌症药物发现迫切需要鉴定新型ICD诱导剂，并且需求变得越来越迫切。在此，我们通过 MTS 测定和细胞凋亡测定评估了布拉他星的抗肿瘤活性。通过蛋白质印迹、ELISA 和流式细胞术评估 ICD 生物标志物，例如钙网蛋白 (CRT)、高迁移率组蛋白 B1 (HMGB-1)、热休克蛋白 (HSP)70、HSP90 和 ATP。进行蛋白质印迹和 qPCR 测定以探索布拉他星诱导的 ICD 的潜在机制。流式细胞术用于检测巨噬细胞吞噬作用。第一的，通过流式细胞术评估，bulatacin 在 10 nM 以时间依赖性方式诱导 SW480 细胞和 HT-29 细胞凋亡。此外，蛋白质印迹和流式细胞术分析表明，在用布拉他星处理约 6 小时后，CRT 和 HSP90（早期 ICD 的生物标志物）在细胞膜表面显着积累。此外，ELISA 和蛋白质印迹分析表明，ICD 所需的第二组标志（HMGB1、HSP70 和 HSP90）在处理 36 小时后在 SW480 和 HT-29 细胞的条件培养基中释放。此外，qPCR 和蛋白质印迹分析表明布拉他星通过激活内质网应激 (ERS) 信号通路触发 ICD。最后，布拉他星促进巨噬细胞吞噬作用。该研究记录了一种新型 ICD 诱导剂 Bullatacin，