Urocortin Inhibits Starvation-induced Autophagy In Human Breast Cancer Cells

Corticotropin- releasing factor (CRF) family peptides are composed of CRF, Urocortin (Ucn), Urocortin2 (Ucn2) and Urocortin3 (Ucn3). CRF family peptides are widely distributed in the central nervous system and the periphery system. They can regulate migration, proliferation and apoptosis of many types of cells through the two subtypes receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2), which belong to G-protein-coupled receptors. Ucn is a 41 amino acids polypeptide. The research suggest Ucn inhibits Beclinl-mediated autophagic cell death in cardiac myocytes exposed to ischaemia/reperfusion injury. However, the effect of Ucn on autophagy remains unclear in human breast cancer cells.The level of basic autophagic activity for cells is low in normal conditions. Therefore, autophagy was induced in cells by starvation in Earle’s balanced salt solution (EBSS). Then, the autophagic activity was measured by the formation of autophagosomes under transmission electron microscopy (TEM), we observed the formation of double-membrane autophagic vacuoles in cells treated with EBSS, which was indicative of autophagy induction. Besides, microtubule-associated protein 1 light chain 3 (LC3) and P62, the important proteins of autophagosome formation, were determined to assess the level of autophagic activity in cells. The experiments were carried out with human breast cancer cell lines (MDA-MB-231 and MCF-7 cells).The purpose of this research is to investigate the contribution and relevant mechanisms of Ucn on starvation-induced autophagy in breast cancer cells. As shown by the results of Western blot analysis and lentivirus GFP-LC3 infection, treatment of Ucn enhanced the level of P62, while Ucn significantly decreased LC3B-Ⅱ/LC3B-Ⅰ ratio and the number of GFP-LC3 dot formation. To ascertain whether CRFRs were involved in the regulation of autophagy, Astressin (Ast), a nonspecific CRFRs antagonist was used in the experiment. The findings revealed that Ast abrogated the effect of Ucn on starvation-induced autophagy in both MDA-MB-231 and MCF-7 cells, indicating that Ucn had an influence on autophagy through CRF receptors.Subsequently, we further investigated the mechanism of Ucn-mediated decreased ratio of LC3B-Ⅱ/LC3B-Ⅰ. Since change in level of LC3B-Ⅱ could be caused by either autophagosome formation or degradation in lysosome, it was necessary to clarify that the decreased ratio of LC3B-Ⅱ/LC3B-Ⅰ induced by Ucn was due to the decreased autophagosome formation or the increased autolysosome degradation. Blockade of autophagosome-lysosome fusion by a vacuolar H+ATPase inhibitor, BafAl, significantly augmented the number of GFP-LC3 dots. However, Ucn apparently diminished the number of GFP-LC3 dots mediated by BafAl. Taken together, these results suggested that Ucn reduced LC3B-Ⅱ/LC3B-Ⅰ ratio through inhibiting autophagosome formation. As mammalian target of rapamycin (mTOR) kinase is an important regulator of autophagy, we further investigated the influence of Ucn on the phosphorylation level of mTOR in breast cancer cells. The results showed that Ucn had no effect on the phosphorylation level of mTOR under starvation conditions, suggesting that mTOR signaling pathway may be not involved in the inhibition of starvation-induced autophagy by Ucn.In conclusion, Ucn inhibited starvation-induced autophagy through suppressing autophagosome formation. Meanwhile, the effect of Ucn depended on CRF receptors. In addition, mTOR signaling pathway may don’t participate in the inhibitory effect of Ucn on starvation-induced autophagy in human breast cancer cells.