Bioinformatic searches of genome databases revealed that the number of autophagy-related genes (ATG) is considerably lower in trypanosomes than in higher eukaryotes and even in yeast. This raises the question of whether autophagy in this protozoan parasite is more primitive and represents a rudimentary paradigm due to its early branching off the evolutionary tree. We here present the crystal structure of TbATG8B. This molecule (MW 13.7 kDa) belongs to the ubiquitin-like proteins showing the typical ubiquitin fold and strong sequence homology to LC3, the human homologue. Due to its characteristic folding, it should readily bind to TbATG4.1 for being processed. This presumption was tested by molecular modeling approaches, docking TbATG8B to a homology model of TbATG4.1. Although exchanges of several amino acids are evident from sequence comparisons, the overall structure seems very much alike and the necessary catalytic triad (C-D-H) is well conserved in TbATG4.1. Thus membrane formation during appearance of the autophagic bodies seems very similar in trypanosomes and their higher eukaryotic counterpart.

We recently reported that carnosol induces ROS-dependent autophagy and apoptosis in breast cancer cells. We also reported that carnosol inhibits breast cancer cell migration, invasion, and in ovo tumor growth, as well as targets STAT3, PCAF, and p300 to proteasome degradation. Here, we investigated the molecular mechanisms underlying its anti-malignant activity in breast cancer. We report that carnosol induces a ROS-dependent type I and type II programmed cell death (PCD-I or PCD-II, respectively), which occurred independently of each other. Indeed, chemical inhibition of autophagy had no effect on the induction of apoptosis, evident by the absence of cleaved PARP. Electron microscopy revealed that carnosol-treated cells exhibited enlarged endoplasmic reticulum, characteristic of ER stress. Markers of the three unfolded protein response pathways (PERK, IRE-1 α, and ATF6), namely ATF4, CHOP, phospho-IRE-1α, XBP1S, and cleaved ATF6 were upregulated in a ROS-dependent manner. In addition, carnosol induced a ROS-dependent activation of p38MAPK, increased the overall level of protein polyubiquitination, and targeted mTOR protein to proteasome degradation. Interestingly, inhibition of p38MAPK, by SB202190 and 203580, reduced cell death, selectively blocked the induction of IRE-1α and ATF6 UPR sensors and inhibited autophagy. In addition, inhibition of p38 reduced the carnosol-induced polyubiquitination and rescued mTOR, PCAF, and STAT3 from proteasomal degradation. Importantly, activation of PERK sensors and induction of apoptosis occurred independently of p38 activation. Taken together, our results suggest that ROS-dependent induced-ER stress contributes to carnosol-induced apoptotic and autophagic cell death in breast cancer cells, and further confirm that carnosol is a promising agent for breast cancer therapy.

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Breast cancer is the leading cause of cancer-related deaths among women. Among breast cancer types, triple negative breast cancer (TNBC) is the most aggressive, and is resistant to hormonal and chemotherapeutic treatments. As such, alternative approaches that may provide some benefit in fighting this debilitating pathology are critically needed; hence the utilization of herbal medicine. Origanum syriacum L., one of the most regularly consumed plants in the Mediterranean region, exhibits antiproliferative effect on several cancer cell lines. However, whether this herb modulates the malignant phenotype of TNBC remains poorly investigated. Here, we show that in MDA-MB-231, a TNBC cell line, Origanum syriacum L. aqueous extract (OSE) inhibited cellular viability, induced autophagy determined by the accumulation of lipidized LC3 II, and triggered apoptosis. We also show that OSE significantly promoted homotypic cell-cell adhesion while it decreased cellular migration, adhesion to fibronectin, and invasion of MDA-MB-231 cells. This was supported by decreased activity of focal adhesion kinase (FAK), reduced α2 integrin expression, and downregulation of secreted PgE2, MMP2 and MMP-9, in OSE-treated cells. Finally, we also show that OSE significantly inhibited angiogenesis and downregulated the level of nitric oxide (NO) production. Our findings demonstrate the ability of OSE to attenuate the malignant phenotype of the MDA-MB-231 cells, thus presenting Origanum syriacum L. as a promising potential source for therapeutic compounds for TNBC.

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