Division of Bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 406-772, Korea.
Int J Mol Sci. 2019 Jan 14;20(2):318. doi: 10.3390/ijms20020318.
Autophagy is a natural physiological process, and it induces the lysosomal degradation of intracellular components in response to environmental stresses, including nutrient starvation. Although an adequate autophagy level helps in cell survival, excessive autophagy triggered by stress such as starvation leads to autophagy-mediated apoptosis. Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, including monoclonal antibodies. However, apoptosis induced by high stress levels, including nutrient deficiency, is a major problem in cell cultures grown in bioreactors, which should be overcome. Therefore, it is necessary to develop a method for suppressing excessive autophagy and for maintaining an appropriate autophagy level in cells. Therefore, we investigated the effect of silkworm storage protein 1 (SP1), an antiapoptotic protein, on autophagy-mediated apoptosis. SP1-expressing CHO cells were generated to assess the effect and molecular mechanism of SP1 in suppressing autophagy. These cells were cultured under starvation conditions by treatment with Earle's balanced salt solution (EBSS) to induce autophagy. We observed that SP1 significantly inhibited autophagy-mediated apoptosis by suppressing caspase-3 activation and reactive oxygen species generation. In addition, SP1 suppressed EBSS-induced conversion of LC3-I to LC3-II and the expression of autophagy-related protein 7. Notably, basal Beclin-1 level was significantly low in the SP1-expressing cells, indicating that SP1 regulated upstream events in the autophagy pathway. Together, these findings suggest that SP1 offers a new strategy for overcoming severe autophagy-mediated apoptosis in mammalian cells, and it can be used widely in biopharmaceutical production.
自噬是一种自然的生理过程，它会在环境压力（包括营养饥饿）下诱导溶酶体降解细胞内成分。虽然适当的自噬水平有助于细胞存活，但过度的自噬，如由饥饿引起的自噬，会导致自噬介导的细胞凋亡。中国仓鼠卵巢（CHO）细胞广泛用于生产生物制药，包括单克隆抗体。然而，在生物反应器中培养的细胞中，由高应激水平（包括营养缺乏）引起的凋亡是一个主要问题，需要加以克服。因此，有必要开发一种方法来抑制过度的自噬，并在细胞中维持适当的自噬水平。因此，我们研究了蚕蛹存储蛋白 1（SP1）作为一种抗凋亡蛋白对自噬介导的细胞凋亡的影响。生成表达 SP1 的 CHO 细胞以评估 SP1 抑制自噬的作用和分子机制。通过用 Earle 的平衡盐溶液（EBSS）处理这些细胞来诱导自噬，使这些细胞在营养饥饿条件下培养。我们观察到 SP1 通过抑制半胱氨酸蛋白酶 3（caspase-3）的激活和活性氧（ROS）的产生，显著抑制自噬介导的细胞凋亡。此外，SP1 抑制了 EBSS 诱导的 LC3-I 向 LC3-II 的转化和自噬相关蛋白 7 的表达。值得注意的是，表达 SP1 的细胞中的基础 Beclin-1 水平显著降低，表明 SP1 调节自噬途径中的上游事件。综上所述，这些发现表明 SP1 为克服哺乳动物细胞中严重的自噬介导的细胞凋亡提供了一种新策略，并且可以广泛应用于生物制药生产。
