Background

Mitoxantrone (MTX) is a synthetic compound used as a second line chemotherapeutic drug for prostate cancer. It has been reported to trigger immunogenic cell death (ICD) in animal model studies, but the underlying mechanism is not fully understood yet, especially not in prostate cancer cells.

Methods

ICD was determined by assessing the release of damage-associated molecular patterns (DAMPs) in the prostate cancer-derived cell lines LNCaP, 22RV1 and PC-3. Short hairpin RNAs (shRNAs) were used to knock down target gene expression. Phagocytosis was assessed using a dual labeling technology in dendric cells co-cultured with cancer cells. The PERK gene promoter was cloned for dual luciferase assays. Chromatin immunoprecipitation (ChIP) was used to determine p53 protein-DNA binding activity. Immunocompetent mice and murine RM-1 prostate cancer cells were used for vaccination experiments.

Results

MTX treatment induced typical characteristics of DAMP release, including increased cell surface exposure of calreticulin (CALR), and extracellular release of ATP and high mobility group box-1 (HMGB1) protein. MTX also enhanced phagocytosis by dendritic cells. Moreover, MTX treatment increased eukaryotic initiation factor 2α (eIF2α) S51 phosphorylation, which was reduced when PERK and GCN2 were silenced using shRNAs. In addition, PERK or GCN2 silencing significantly reduced MTX-induced release of DAMPs in vitro and anti-tumor immunity in vivo. MTX treatment also resulted in dendritic cell activation in mice, which was attenuated when PERK or GCN2 were silenced in cancer cells used for vaccination. Further analysis revealed that PERK and GCN2 expression was enhanced by MTX treatment, of which PERK, but not GCN2, was enhanced via a p53-dependent mechanism.

Conclusion

MTX triggers ICD by activating eIF2α via PERK/GCN2 upregulation in prostate cancer cells. MTX-induced PERK expression upregulation depends on the p53 pathway, while that of GCN2 requires further investigation.

中文翻译：

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米托蒽醌通过 p53 依赖性 PERK 表达触发免疫原性前列腺癌细胞死亡。

背景

米托蒽醌 (MTX) 是一种合成化合物，用作前列腺癌的二线化疗药物。据报道，在动物模型研究中它会引发免疫原性细胞死亡 (ICD)，但其潜在机制尚未完全了解，尤其是在前列腺癌细胞中。

方法

ICD 是通过评估前列腺癌衍生细胞系 LNCaP、22RV1 和 PC-3 中损伤相关分子模式 (DAMP) 的释放来确定的。短发夹 RNA (shRNA) 用于敲低靶基因表达。在与癌细胞共培养的树突细胞中使用双重标记技术评估吞噬作用。克隆PERK基因启动子用于双荧光素酶测定。染色质免疫沉淀 (ChIP) 用于确定 p53 蛋白-DNA 结合活性。免疫活性小鼠和小鼠 RM-1 前列腺癌细胞用于疫苗接种实验。

结果

MTX 处理诱导 DAMP 释放的典型特征，包括钙网蛋白 (CALR) 的细胞表面暴露增加，以及 ATP 和高迁移率族蛋白 1 (HMGB1) 蛋白的细胞外释放。MTX 还增强了树突状细胞的吞噬作用。此外，MTX 处理增加了真核起始因子 2α (eIF2α) S51 磷酸化，当使用 shRNA 沉默 PERK 和 GCN2 时磷酸化减少。此外，PERK 或 GCN2 沉默显着降低了 MTX 诱导的体外 DAMP 释放和体内抗肿瘤免疫。MTX 治疗还导致小鼠树突状细胞激活，当用于疫苗接种的癌细胞中 PERK 或 GCN2 沉默时，树突状细胞激活减弱。进一步分析显示，MTX 处理增强了 PERK 和 GCN2 的表达，其中 PERK 而不是 GCN2，

结论

MTX 通过前列腺癌细胞中的 PERK/GCN2 上调激活 eIF2α 来触发 ICD。MTX 诱导的 PERK 表达上调取决于 p53 通路，而 GCN2 的上调需要进一步研究。