Suares Alejandra, Tapia Cinthya, González-Pardo Verónica
Instituto de Ciencias Biológicas y Biomédicas del Sur (INBIOSUR), Departamento de Biología Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)-CONICET, San Juan 670, 8000, Bahía Blanca, Argentina.
IFIBYNE - Instituto de Fisiología, Biología Molecular y Neurociencias (UBA-CONICET), Ciudad Universitaria, 1428, Ciudad Autónoma de Buenos Aires, Argentina.
Heliyon. 2019 Aug 27;5(8):e02367. doi: 10.1016/j.heliyon.2019.e02367. eCollection 2019 Aug.
The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. We have previously shown that 1α,25(OH)D or its less-calcemic analog TX 527 inhibits the proliferation of endothelial cells expressing vGPCR, NF-κB activity and induces apoptosis in a VDR dependent manner. In this work, we further explored whether 1α,25(OH)D or TX 527 regulates PI3K/Akt/mTOR axis and induces autophagy as part of its antineoplastic mechanism of action. Proliferation assays indicated that vGPCR cell number decreased in presence of LY294002 (PI3K/Akt inhibitor) likewise 1α,25(OH)D or TX 527 (10 nM, 48 h). Also, Akt phosphorylation was found decreased in dose (0.1-100 nM) and time response studies (12-72 h) after both compounds treatments. In addition, decreased phosphorylated Akt was significantly observed in the nucleus. Moreover, regulation of Akt phosphorylation was NF-κB and VDR dependent. TNFAIP3/A20, an ubiquitin-editing enzyme, a direct NF-κB target gene and a negative regulator of Beclin-1, was down-regulated whereas Beclin-1 was up-regulated after 10 nM of 1α,25(OH)D or TX 527 treatment. Decrement in Akt phosphorylation was accompanied by a reduced mTOR phosphorylation and an increase in the autophagy marker LC3-II. Since increment in autophagosomes not always indicates increment in autophagy activity, we used Chloroquine (CQ, 1 μM), an inhibitor of autophagy flow, to confirm autophagy after both VDR agonists treatment. In conclusion, VDR agonists, 1α,25(OH)D or TX 527, inhibited PI3K/Akt/mTOR axis and induced autophagy in endothelial cells expressing vGPCR by a VDR-dependent mechanism.
卡波西肉瘤相关疱疹病毒G蛋白偶联受体（KSHV/vGPCR）是卡波西肉瘤发病机制中的关键分子。我们之前已经表明，1α,25(OH)D或其低钙类似物TX 527以VDR依赖的方式抑制表达vGPCR的内皮细胞增殖、NF-κB活性并诱导细胞凋亡。在这项研究中，我们进一步探讨了1α,25(OH)D或TX 527是否调节PI3K/Akt/mTOR轴并诱导自噬，作为其抗肿瘤作用机制的一部分。增殖试验表明，在存在LY294002（PI3K/Akt抑制剂）的情况下，vGPCR细胞数量减少，同样在1α,25(OH)D或TX 527（10 nM，48小时）存在时也是如此。此外，在两种化合物处理后的剂量（0.1 - 100 nM）和时间反应研究（12 - 72小时）中发现Akt磷酸化降低。另外，在细胞核中显著观察到磷酸化Akt减少。而且，Akt磷酸化的调节是NF-κB和VDR依赖的。TNFAIP3/A20，一种泛素编辑酶，是NF-κB的直接靶基因和Beclin-1的负调节因子，在10 nM的1α,25(OH)D或TX 527处理后被下调，而Beclin-1被上调。Akt磷酸化的减少伴随着mTOR磷酸化的降低和自噬标志物LC^3-II的增加。由于自噬体的增加并不总是表明自噬活性增加，我们使用氯喹（CQ，1 μM），一种自噬流抑制剂，来确认两种VDR激动剂处理后的自噬。总之，VDR激动剂1α,25(OH)D或TX 527通过VDR依赖的机制抑制PI3K/Akt/mTOR轴并在内皮细胞中诱导自噬。
