Knigin Adi, Avniel-Polak Shani, Leibowitz Gil, Oleinikov Kira, Gross David J, Grozinsky-Glasberg Simona
Neuroendocrine Tumor Unit, ENETS Center of Excellence, Endocrinology and Metabolism Department, Hadassah Medical Organization and Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
Cancers (Basel). 2021 Dec 16;13(24):6327. doi: 10.3390/cancers13246327.
(1) Background: Neuroendocrine neoplasms of the lung (LNENs, lung carcinoids) are often diagnosed at an advanced stage when they are not surgically curable, and treatment options are limited. One of the approved options for treating inoperable tumors is everolimus-an mTOR inhibitor (mTORi). Activation of mTOR, among many other effects, inhibits autophagy, which is a cell survival mechanism in general, and in tumor cells in particular. Everolimus may paradoxically encourage cancer cell survival. In practice, the drug inhibits tumor development. Chloroquine (CQ) is a known antimalarial compound that inhibits autophagy. Our research is focused on the hypothesis that autophagy plays a key role in the development of tumor resistance to mTORi, and that the addition of autophagy inhibitors to mTORi exerts a synergistic effect on suppressing tumor cell proliferation. We have recently demonstrated that the combination of CQ with different mTORi increases their potency compared with mTORi alone in both in vitro and in vivo models of pancreatic NENs. In this study, we examined the effects of CQ and mTORi on in vitro and in vivo LNEN models. Aims: Testing the effects of CQ together with mTORi on cell proliferation, apoptosis, and autophagy in in vitro and in vivo LNEN models. (2) Methods: The NCI-H727 LNEN cells were treated with CQ ± mTORi. Cells' viability and proliferation were measured using XTT and Ki-67 FACS staining. The effects of the treatments on the mTOR pathway and autophagy were examined using Western blotting. Cytotoxicity was measured using a cytotoxicity kit; apoptosis was measured by PI FACS staining and Western blotting. We further established an LNEN subcutaneous murine xenograft model and evaluated the effects of the drugs on tumor growth. (3) Results: CQ alone suppressed LNEN cells' viability and proliferation and increased their cytotoxicity and apoptosis; these effects were augmented when CQ was added to an mTORi. We also showed the possible mechanisms for these results: on the one hand we could see a decrease in P62 levels and the absence of LC3-II (both inversely related to autophagy) following treatment with the mTORi, and on the other hand we could demonstrate an increase in their levels when CQ was added. The effect was less apparent in the murine xenograft model. (4) Conclusions: By inhibiting autophagy and inducing apoptosis, CQ suppresses tumor cell growth in LNENs. CQ potentiates mTORi effects, implying that further studies are needed in order to elucidate its possible role in tumor inhibition in patients with LNENs.
(1) 背景：肺神经内分泌肿瘤（LNENs，肺类癌）通常在无法通过手术治愈的晚期阶段被诊断出来，治疗选择有限。治疗不可切除肿瘤的获批方案之一是依维莫司——一种mTOR抑制剂（mTORi）。mTOR的激活除了产生许多其他作用外，还会抑制自噬，而自噬一般是一种细胞存活机制，对肿瘤细胞而言尤其如此。依维莫司可能反而会促进癌细胞存活。实际上，该药物会抑制肿瘤发展。氯喹（CQ）是一种已知的抑制自噬的抗疟化合物。我们的研究聚焦于这样一个假设：自噬在肿瘤对mTORi产生耐药性的过程中起关键作用，并且在mTORi中添加自噬抑制剂对抑制肿瘤细胞增殖具有协同作用。我们最近已证明，在胰腺神经内分泌肿瘤的体外和体内模型中，CQ与不同的mTORi联合使用比单独使用mTORi能增强其效力。在本研究中，我们检测了CQ和mTORi对体外和体内LNEN模型的影响。目的：检测CQ与mTORi联合使用对体外和体内LNEN模型中细胞增殖、凋亡和自噬的影响。(2) 方法：用CQ ± mTORi处理NCI - H727 LNEN细胞。使用XTT和Ki - 67流式细胞术染色检测细胞活力和增殖。通过蛋白质免疫印迹法检测处理对mTOR通路和自噬的影响。使用细胞毒性试剂盒测量细胞毒性；通过PI流式细胞术染色和蛋白质免疫印迹法检测凋亡。我们进一步建立了LNEN皮下小鼠异种移植模型，并评估了药物对肿瘤生长的影响。(3) 结果：单独使用CQ可抑制LNEN细胞的活力和增殖，并增加其细胞毒性和凋亡；当CQ添加到mTORi中时，这些作用会增强。我们还展示了这些结果的可能机制：一方面，在用mTORi处理后，我们可以看到P62水平降低且LC3 - II缺失（两者均与自噬呈负相关），另一方面，当添加CQ时，我们可以证明它们的水平升高。在小鼠异种移植模型中，这种效果不太明显。(4) 结论：通过抑制自噬和诱导凋亡，CQ可抑制LNENs中的肿瘤细胞生长。CQ增强了mTORi的作用，这意味着需要进一步研究以阐明其在LNENs患者肿瘤抑制中的可能作用。
