半胱氨酸耗竭使前列腺癌细胞对增强 DNA 损伤的药物和免疫检查点抑制剂敏感。
Cysteine depletion sensitizes prostate cancer cells to agents that enhance DNA damage and to immune checkpoint inhibition.
机构信息
Division of Pharmacology and Toxicology and Dell Pediatric Research Institute, The University of Texas at Austin, 1400 Barbara Jordan Blvd, Austin, TX, 78723, USA.
Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, 78712, USA.
出版信息
J Exp Clin Cancer Res. 2023 May 11;42(1):119. doi: 10.1186/s13046-023-02677-2.
BACKGROUND
Prostate Cancer (PCa) represents one of the most commonly diagnosed neoplasms in men and is associated with significant morbidity and mortality. Therapy resistance and significant side effects of current treatment strategies indicate the need for more effective agents to treat both androgen-dependent and androgen-independent PCa. In earlier studies, we demonstrated that depletion of L-cysteine/cystine with an engineered human enzyme, Cyst(e)inase, increased intracellular ROS levels and inhibited PCa growth in vitro and in vivo. The current study was conducted to further explore the mechanisms and potential combinatorial approaches with Cyst(e)inase for treatment of PCa.
METHODS
DNA single strand breaks and clustered oxidative DNA damage were evaluated by alkaline comet assay and pulsed field gel electrophoresis, respectively. Neutral comet assay and immunofluorescence staining was used to measure DNA double strand breaks. Cell survival and reactive oxygen species level were measured by crystal violet assay and DCFDA staining, respectively. Western blot was used to determine protein expression. FACS analyses were preformed for immune cell phenotyping. Allograft and xenograft tumor models were used for assessing effects on tumor growth.
RESULTS
PCa cells treated with Cyst(e)inase lead to DNA single and double strand breaks resulted from clustered oxidative DNA damage (SSBs and DSBs). Cyst(e)inase in combination with Auranofin, a thioredoxin reductase inhibitor, further increased intracellular ROS and DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo. A combination of Cyst(e)inase with a PARP inhibitor (Olaparib) also increased DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo without additional ROS induction. Knockdown of BRCA2 in PCa cells increased DSBs and enhanced sensitivity to Cyst(e)inase. Finally, Cyst(e)inase treatment altered tumor immune infiltrates and PD-L1 expression and sensitized PCa cells to anti-PD-L1 treatment.
CONCLUSIONS
The current results demonstrate the importance of oxidative DNA damage either alone or in combination for Cyst(e)inase-induced anticancer activity. Furthermore, cysteine/cystine depletion alters the tumor immune landscape favoring enhanced immune checkpoint inhibition targeting PD-L1. Thus, combinatorial approaches with Cyst(e)inase could lead to novel therapeutic strategies for PCa.
背景
前列腺癌（PCa）是男性最常见的肿瘤之一，与显著的发病率和死亡率相关。治疗耐药性和当前治疗策略的显著副作用表明，需要更有效的药物来治疗雄激素依赖性和雄激素非依赖性 PCa。在早期研究中，我们证明了用工程化的人类酶胱氨酸酶耗竭 L-半胱氨酸/胱氨酸可增加细胞内 ROS 水平并抑制体外和体内 PCa 的生长。本研究旨在进一步探讨胱氨酸酶治疗 PCa 的机制和潜在组合方法。
方法
通过碱性彗星试验和脉冲场凝胶电泳分别评估 DNA 单链断裂和簇状氧化 DNA 损伤。中性彗星试验和免疫荧光染色用于测量 DNA 双链断裂。通过结晶紫试验和 DCFDA 染色分别测量细胞存活和活性氧水平。Western blot 用于测定蛋白质表达。流式细胞术分析用于免疫细胞表型分析。同种异体移植和异种移植肿瘤模型用于评估对肿瘤生长的影响。
结果
用胱氨酸酶处理的 PCa 细胞导致由簇状氧化 DNA 损伤引起的 DNA 单链和双链断裂（SSBs 和 DSBs）。胱氨酸酶与硫氧还蛋白还原酶抑制剂 Auranofin 联合使用，进一步增加了细胞内 ROS 和 DNA DSBs，并协同抑制了体外和体内的 PCa 细胞生长。胱氨酸酶与 PARP 抑制剂（奥拉帕利）的联合使用也增加了 DNA DSBs，并协同抑制了体外和体内的 PCa 细胞生长，而没有额外的 ROS 诱导。PCa 细胞中 BRCA2 的敲低增加了 DSBs，并增强了对胱氨酸酶的敏感性。最后，胱氨酸酶处理改变了肿瘤免疫浸润和 PD-L1 表达，并使 PCa 细胞对抗 PD-L1 治疗敏感。
结论
目前的结果表明，氧化 DNA 损伤无论是单独作用还是联合作用，对于胱氨酸酶诱导的抗癌活性都很重要。此外，半胱氨酸/胱氨酸耗竭改变了肿瘤免疫景观，有利于增强针对 PD-L1 的免疫检查点抑制。因此，胱氨酸酶的组合方法可能为 PCa 带来新的治疗策略。
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