Albert Einstein College of Medicine, Bronx, New York.
Montefiore Medical Center, Bronx, New York.
Clin Cancer Res. 2021 Feb 1;27(3):865-876. doi: 10.1158/1078-0432.CCR-20-2385. Epub 2020 Nov 9.
To explore the effects of pelareorep on autophagy in multiple models of colorectal cancer, including patient-derived peripheral blood mononuclear cells (PBMCs).
HCT116 [KRAS mutant (mut)] and Hke3 [ wild-type (WT)] cells were treated with pelareorep (multiplicity of infection, 5) and harvested at 6 and 9 hours. LC3 A/B expression was determined by immunofluorescence and flow cytometry; five autophagic proteins were analyzed by Western blotting. The expression of 88 autophagy genes was determined by qRT-PCR. Syngeneic mouse models, CT26/Balb-C ( mut) and MC38/C57B6 ( WT), were developed and treated with pelareorep (10 × 10 plaque-forming unit/day) intraperitoneally. Protein and RNA were extracted from harvested tumor tissues. PBMCs from five experimental and three control patients were sampled at 0 (pre) and 48 hours, and on days 8 and 15. The gene expression normalized to "pre" was determined using 2 method.
Pelareorep induced significant upregulation of LC3 A/B in HCT116 as compared with Hke3 cells by immunofluorescence (3.24 × and 8.67 ×), flow cytometry (2.37 × and 2.58 ×), and autophagosome formation (2.02 × and 1.57 ×), at 6 and 9 hours, respectively; all < 0.05. Western blot analysis showed an increase in LC3 A/B (2.38 × and 6.82 ×) and Beclin1 (1.17 × and 1.24 ×) at 6 and 9 hours, ATG5 (2.4 ×) and P-62 (1.52 ×) at 6 hours, and VPS-34 (1.39 ×) at 9 hours (all < 0.05). Induction of 13 transcripts in cell lines (>4 ×; 6 and 9 hours; < 0.05), 12 transcripts in CT26 (qRT-PCR), and 14 transcripts in human PBMCs ( < 0.05) was observed. , and expression was upregulated in all three model systems.
Pelareorep hijacks host autophagic machinery in -mut conditions to augment its propagation and preferential oncolysis of the cancer cells.
探索 pelareorep 在包括源自患者外周血单核细胞（PBMC）的结直肠癌多种模型中的自噬作用。
用 pelareorep（感染复数，5）处理 HCT116[KRAS 突变（mut）]和 Hke3[野生型（WT）]细胞，并在 6 小时和 9 小时收获细胞。通过免疫荧光和流式细胞术测定 LC3 A/B 的表达；通过 Western blot 分析 5 种自噬蛋白。通过 qRT-PCR 测定 88 种自噬基因的表达。建立并经 pelareorep（10×10 噬菌斑形成单位/天）腹腔内给药的同基因小鼠模型 CT26/Balb-C（mut）和 MC38/C57B6（WT）。从收获的肿瘤组织中提取蛋白质和 RNA。在 0（预处理）和 48 小时以及第 8 天和第 15 天，从 5 名实验患者和 3 名对照患者中采集 PBMC。使用 2-∆∆CT 方法确定相对于“预处理”的基因表达。
与 Hke3 细胞相比，pelareorep 在 HCT116 中诱导 LC3 A/B 的免疫荧光（3.24×和 8.67×）、流式细胞术（2.37×和 2.58×）和自噬体形成（2.02×和 1.57×）分别显著上调，均 < 0.05。Western blot 分析显示 LC3 A/B（2.38×和 6.82×）和 Beclin1（1.17×和 1.24×）在 6 小时和 9 小时、ATG5（2.4×）和 P-62（1.52×）在 6 小时以及 VPS-34（1.39×）在 9 小时增加（均 < 0.05）。在细胞系中观察到 13 个转录物的诱导（>4×；6 小时和 9 小时；均 < 0.05）、CT26 中的 12 个转录物（qRT-PCR）和人 PBMCs 中的 14 个转录物（均 < 0.05）。在所有三个模型系统中， 、 和 的表达均上调。
Pelareorep 在 -mut 条件下劫持宿主自噬机制，以增强其增殖和对癌细胞的优先溶瘤作用。
