Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Gyeongsang 52727, Republic of Korea.
Int J Oncol. 2018 Nov;53(5):1953-1966. doi: 10.3892/ijo.2018.4552. Epub 2018 Sep 4.
In the tumor microenvironment, extracellular nucleotides are released and accumulate, and can activate the P2Y2 receptor (P2Y2R), which regulates various responses in tumor cells, resulting in tumor progression and metastasis. Moreover, the inflammasome has recently been reported to be associated with tumor progression. However, the role of P2Y2R in inflammasome activation in breast cancer cells is not yet well defined. Therefore, in this study, we investigated the role of P2Y2R in inflammasome-mediated tumor progression in breast cancer using breast cancer cells and radiotherapy-resistant (RT‑R) breast cancer cells. We established RT‑R-breast cancer cells (RT‑R‑MDA‑MB‑231, RT‑R‑MCF‑7, and RT‑R-T47D cells) by repeated irradiation (2 Gy each, 25 times) in a previous study. In this study, we found that the RT‑R breast cancer cells exhibited an increased release of adenosine triphosphate (ATP) and P2Y2R activity. In particular, the RT‑R‑MDA‑MB‑231 cells derived from highly metastatic MDA‑MB‑231 cells, exhibited a markedly increased ATP release, which was potentiated by tumor necrosis factor (TNF)-α. The MDA‑MB‑231 cells exhibited inflammasome activation, as measured by caspase‑1 activity and interleukin (IL)-1β secretion following treatment with TNF‑α and ATP; these effects were enhanced in the RT‑R‑MDA‑MB‑231 cells. However, the increased caspase‑1 activities and IL‑1β secretion levels induced in response to treatment with TNF‑α or ATP were significantly reduced by P2Y2R knockdown or the presence of apyrase in both the MDA‑MB‑231 and RT‑R‑MDA‑MB‑231 cells, suggesting the involvement of ATP-activated P2Y2R in inflammasome activation. In addition, TNF‑α and ATP increased the invasive and colony-forming ability of the MDA‑MB‑231 and RT‑R‑MDA‑MB‑231 cells, and these effects were caspase‑1-dependent. Moreover, matrix metalloproteinase (MMP)-9 activity was modulated by caspase-1, in a P2Y2R-dependent manner in the MDA‑MB‑231 and RT‑R‑MDA‑MB‑231 cells. Finally, nude mice injected with the RT‑R‑MDA‑MB‑231-EV cells (transfected with the empty vector) exhibited increased tumor growth, and higher levels of MMP-9 in their tumors and IL‑1β levels in their serum compared with the mice injected with the RT‑R‑MDA‑MB‑231-P2Y2R shRNA cells (transfected with P2Y2R shRNA). On the whole, the findings of this study suggest that extracellular ATP promotes tumor progression in RT‑R-breast cancer cells and breast cancer cells by modulating invasion and associated molecules through the P2Y2R-inflammasome activation pathway.
在肿瘤微环境中，细胞外核苷酸被释放并积累，并可以激活 P2Y2 受体（P2Y2R），调节肿瘤细胞中的各种反应，导致肿瘤进展和转移。此外，炎症小体最近被报道与肿瘤进展有关。然而，P2Y2R 在乳腺癌细胞中炎症小体激活中的作用尚未得到很好的定义。因此，在这项研究中，我们使用乳腺癌细胞和放射抗性（RT-R）乳腺癌细胞研究了 P2Y2R 在炎症小体介导的乳腺癌进展中的作用。我们通过在之前的研究中重复照射（每次 2 Gy，共 25 次）建立了 RT-R-乳腺癌细胞（RT-R-MDA-MB-231、RT-R-MCF-7 和 RT-R-T47D 细胞）。在这项研究中，我们发现 RT-R 乳腺癌细胞表现出三磷酸腺苷（ATP）释放增加和 P2Y2R 活性增强。特别是，源自高转移性 MDA-MB-231 细胞的 RT-R-MDA-MB-231 细胞表现出明显增加的 ATP 释放，这一释放被肿瘤坏死因子（TNF）-α增强。MDA-MB-231 细胞表现出炎症小体激活，如 TNF-α和 ATP 处理后 caspase-1 活性和白细胞介素（IL）-1β分泌所测量的那样；这些效应在 RT-R-MDA-MB-231 细胞中增强。然而，用 P2Y2R 敲低或在 MDA-MB-231 和 RT-R-MDA-MB-231 细胞中存在 Apyrase 处理后，TNF-α或 ATP 处理诱导的 caspase-1 活性和 IL-1β分泌水平显著降低，表明 ATP 激活的 P2Y2R 参与炎症小体激活。此外，TNF-α和 ATP 增加了 MDA-MB-231 和 RT-R-MDA-MB-231 细胞的侵袭和集落形成能力，这些作用依赖于 caspase-1。此外，MMP-9 活性在 MDA-MB-231 和 RT-R-MDA-MB-231 细胞中受到 caspase-1 的调节，这种调节依赖于 P2Y2R。最后，用 RT-R-MDA-MB-231-EV 细胞（转染空载体）注射的裸鼠表现出肿瘤生长增加，并且其肿瘤中的 MMP-9 水平和血清中的 IL-1β水平均高于用 RT-R-MDA-MB-231-P2Y2R shRNA 细胞（转染 P2Y2R shRNA）注射的小鼠。总的来说，这项研究的结果表明，细胞外 ATP 通过调节侵袭和相关分子，通过 P2Y2R-炎症小体激活途径促进 RT-R-乳腺癌细胞和乳腺癌细胞的肿瘤进展。
