Tinzaparin is an anticoagulant and antiangiogenic drug with inhibitory properties against tumor growth. VEGF stimulates angiogenesis, while an association between reactive oxygen species (ROS) and angiogenesis is involved in tumor progression. The present study aimed to investigate the effect of tinzaparin on VL30 retrotransposition‑positive mouse HC11 mammary stem‑like epithelial cells, previously reported to be associated with induced mammosphere/cancer stem cell (CSC) generation and tumorigenesis. Under 24 h serum starvation, 15.2% nominal retrotransposition frequency was increased to 29%. Additionally, while treatment with 3‑12 ng/ml VEGF further induced retrotransposition frequency in a dose‑dependent manner (up to 40.3%), pre‑incubation with tinzaparin (2 IU/ml) for 0.5‑4 h reduced this frequency to 18.3% in a time‑dependent manner, confirmed by analogous results in NIH3T3 fibroblasts. Treatment with 10‑40 pg/ml glucose oxidase (GO) for 24 h induced HC11 cell retrotransposition in a dose‑dependent manner (up to 82.5%), while a 3 h pre‑incubation with tinzaparin (1 or 2 IU/ml) elicited a 13.5 or 25.5% reduction in retrotransposition, respectively. Regarding tumorigenic VL30 retrotransposition‑positive HC11 cells, treatment with 2 IU/ml tinzaparin for 5 days reduced proliferation rate in a time‑dependent manner (up to ~55%), and after 3 weeks, disaggregated soft agar‑formed foci, as well as low‑adherent mammospheres, producing single mesenchymal‑like cells with a ~50% reduced retrotransposition. With respect to the VL30 retrotransposition mechanism: While 12 ng/ml VEGF increased the level of VL30 and endogenous reverse transcriptase (enRT) transcripts ~1.41‑ and ~1.16‑fold, respectively, subsequent tinzaparin treatment reduced both endogenous/ROS‑ and VEGF‑induced levels 1.15‑ and 0.40‑fold (VL30) and 0.60‑ and 0.52‑fold (enRT), respectively. To the best of our knowledge, these data demonstrate for the first time, the novel inhibition activity of tinzaparin against ROS‑ and VEGF‑induced VL30 retrotransposition, and the proliferation and/or aggregation of mouse HC11 mammosphere/tumor‑initiating CSCs, thus contributing to the inhibition of VL30 retrotransposition‑induced primary tumor growth.

中文翻译：

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Tinzaparin 抑制 HC11 小鼠祖乳腺细胞中氧化应激和/或 VEGF 诱导的 VL30 逆转录转座：癌症干细胞增殖抑制与乳腺球解聚之间的关联。

Tinzaparin 是一种抗凝剂和抗血管生成药物，具有抑制肿瘤生长的特性。VEGF 刺激血管生成，而活性氧 (ROS) 和血管生成之间的关联与肿瘤进展有关。本研究旨在研究亭扎肝素对 VL30 逆转录转座阳性小鼠 HC11 乳腺干样上皮细胞的影响，先前报道该细胞与诱导的乳腺球/癌症干细胞 (CSC) 生成和肿瘤发生有关。在 24 小时血清饥饿下，15.2% 的标称逆转录转座频率增加到 29%。此外，虽然用 3-12 ng/ml VEGF 处理以剂量依赖性方式进一步诱导逆转录转座频率（高达 40.3%），但与亭扎肝素（2 IU/ml）预孵育 0.5-4 小时将该频率降低至 18.3 % 以时间相关的方式，NIH3T3 成纤维细胞中的类似结果证实了这一点。用 10-40 pg/ml 葡萄糖氧化酶 (GO) 处理 24 小时以剂量依赖性方式（高达 82.5%）诱导 HC11 细胞逆转录，同时与亭扎肝素（1 或 2 IU/ml）预孵育 3 小时分别导致逆转录转座减少 13.5% 或 25.5%。关于致瘤性 VL30 逆转录转座阳性 HC11 细胞，用 2 IU/ml 亭扎肝素处理 5 天以时间依赖性方式降低增殖率（高达约 55%），并且在 3 周后，分解的软琼脂形成病灶，以及作为低粘附性乳腺球，产生单个间充质样细胞，逆转录转座减少约 50%。关于 VL30 逆转录转座机制：虽然 12 ng/ml VEGF 增加了 VL30 和内源性逆转录酶 (enRT) 转录物的水平~1.41 倍和~1.16 倍，随后的亭扎肝素治疗分别使内源性/ROS 和 VEGF 诱导的水平分别降低了 1.15 倍和 0.40 倍（VL30）以及 0.60 倍和 0.52 倍（enRT）。据我们所知，这些数据首次证明亭扎肝素对 ROS 和 VEGF 诱导的 VL30 逆转录转座的新抑制活性，以及​​小鼠 HC11 乳腺/肿瘤起始 CSC 的增殖和/或聚集，从而有助于VL30 逆转录转座诱导的原发性肿瘤生长的抑制作用。