Brokowska Joanna, Herman-Antosiewicz Anna, Hać Aleksandra
Department of Medical Biology and Genetics, Faculty of Biology, University of Gdansk, Wita Stwosza 59, Gdansk, 80-308, Poland.
Department of Molecular Biology, Faculty of Biology, University of Gdansk, Gdansk, Poland.
Eur J Nutr. 2024 Dec 16;64(1):46. doi: 10.1007/s00394-024-03539-z.
Autophagy is a degradation process whose activation underlies beneficial effects of caloric restriction. Isothiocyanates (ITCs) induce autophagy in cancer cells, however, their impact on primary cells remains insufficiently explored, particularly in non-epithelial cells. The aim of this study was to investigate whether ITCs induce autophagy in primary (non-immortalized) mesenchymal cells and if so, to determine the molecular mechanism underlying its activation and consequences on cell functioning.
Primary human dermal fibroblasts (HDFa) and prostate cancer cells (PC3) as well as two ITCs, sulforaphane and phenethyl isothiocyanate, were applied. Cell viability was measured by the MTT test, protein synthesis - by H-leucine incorporation, and protein level - by immunoblotting. A number of mutant huntingtin (mHtt) aggregates was assessed by fluorescence microscopy.
Both ITCs efficiently induced autophagy in fibroblasts which coincided with suppression of mTORC1 - a negative autophagy regulator - and protein synthesis arrest. A dephosphorylation of mTORC1 substrate, S6K1, and ribosomal S6 protein was preceded by activation of AMPK, an inhibitor of mTORC1 and autophagy activator. A similar response was observed in phenethyl isothiocyanate-treated prostate cancer cells. We also showed that ITCs-induced autophagy and/or translation block do not affect cells viability and can protect cells against an accumulation of mHtt aggregates - a main cause of Huntington's disease.
Our study showed that ITCs induce autophagy and inhibit protein synthesis in both primary mesenchymal and cancer cells via modulation of the AMPK-mTORC1-S6K1 pathway. Moreover, it suggests that ITCs might have a potential in developing therapeutics for Huntington's disease.
自噬是一种降解过程，其激活是热量限制产生有益作用的基础。异硫氰酸盐（ITCs）可诱导癌细胞发生自噬，然而，它们对原代细胞的影响仍未得到充分研究，尤其是对非上皮细胞的影响。本研究的目的是调查ITCs是否能诱导原代（未永生化的）间充质细胞发生自噬，如果可以，确定其激活的分子机制以及对细胞功能的影响。
使用原代人皮肤成纤维细胞（HDFa）、前列腺癌细胞（PC3）以及两种ITCs，即萝卜硫素和苯乙基异硫氰酸盐。通过MTT试验测定细胞活力，通过H-亮氨酸掺入法测定蛋白质合成，通过免疫印迹法测定蛋白质水平。通过荧光显微镜评估突变型亨廷顿蛋白（mHtt）聚集体的数量。
两种ITCs均能有效诱导成纤维细胞发生自噬，这与抑制mTORC1（一种自噬负调节因子）和蛋白质合成停滞相一致。mTORC1底物S6K1和核糖体S6蛋白的去磷酸化之前是AMPK的激活，AMPK是mTORC1的抑制剂和自噬激活剂。在苯乙基异硫氰酸盐处理的前列腺癌细胞中也观察到了类似的反应。我们还表明，ITCs诱导的自噬和/或翻译阻滞不会影响细胞活力，并且可以保护细胞免受mHtt聚集体（亨廷顿舞蹈病的主要病因）的积累。
我们的研究表明，ITCs通过调节AMPK-mTORC1-S6K1途径诱导原代间充质细胞和癌细胞发生自噬并抑制蛋白质合成。此外，这表明ITCs在开发亨廷顿舞蹈病治疗药物方面可能具有潜力。
