D-Threonine methyl ester

Photodynamic therapy (PDT), utilizes a photochemical reaction between photosensitizer and light to cause cancer death by generating reactive oxygen species (ROS). X-box binding protein 1 (XBP1), a downstream product of the IRE1α-XBP1 pathway, regulates diverse target genes, including various proto-oncogenes and its overexpression was closely related to the occurrence and progression of malignant tumors. The present study was performed to explore the role of XBP1 in human osteosarcoma HOS cells treated with pyropheophorbide-α methyl ester (MPPα)-mediated photodynamic therapy (PDT) (MPPα-PDT) and its potential mechanisms. The protein IRE1α and XBP1 increased with a time-dependent manner after MPPα-PDT treated, which indicated that MPPα-PDT induced the activation of the IRE1α-XBP1 pathway in HOS cells. Besides, MPPα-PDT treated alone or combined with XBP1 knockdown could both restrain the cell viability, but the latter one has more notable effect, which indicated that XBP1 knockdown may enhance the cell inhibitory effect by MPPα-PDT. Simultaneously, the apoptotic rate measured by flow cytometry (FCM) was increased surprisedly and the expression of apoptosis proteins was increased when knockdown XBP1 under the MPPα-PDT. In addition, antioxidant-related proteins such as the Catalase and SOD1 protein levels decreased, while the intracellular ROS content increased in HOS cells when knockdown XBP1 under the MPPα-PDT. These results suggested that the mechanism of XBP1 mediating resistance in HOS cells might be related to the expression of antioxidant molecules. In summary, this study found that the IRE1α-XBP1 pathway was activated in HOS cells after MPPα-PDT treated, and furthermore, XBP1 knockdown could decrease HOS cell viability through apoptosis and enhance the anti-tumor effect of MPPα-PDT remarkably in the meantime, which related to the regulation of oxidation-antioxidant system.

Background: Arctium lappa L. roots are very popular cultivated vegetables, which possesses various pharmacological activities. Our previous studies have demonstrated that Arctium lappa L. roots exerted protective effects against H2O2, glutamate and N-methyl-D-aspartic acid (NMDA)-induced neuronal injury in vitro. However, whether Arctium lappa L. roots could prevent against cerebral ischemia and the underlying mechanism remain unclear. Purpose: The objective of the present study was to investigate the neuroprotective effects of ethyl acetate extract of Arctium lappa L. roots (EAL) and the active ingredient 4,5-O-dicaffeoyl-1-O-[4-malic acid methyl ester]-quinic acid (DCMQA) in EAL against cerebral ischemia and explore the underlying mechanism. Study design: The neuroprotective effects of EAL and DCMQA were investigated in rats with permanent middle cerebral artery occlusion (MCAO) and in oxygen glucose deprivation/reoxygenation (OGD/R)-stimulated SH-SY5Y cells, respectively. Methods: The infarct volume, brain edema and neurological deficits were measured following MCAO. TUNEL and Nissl staining were performed to detect neuronal loss and apoptosis of neurons in rat brains. Cell survival was measured by MTT and LDH assay. In addition, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels were determined by DCFH-DA and JC-1 fluorescent probe, respectively. Hoechst 33342 staining and Annexin V-FITC/PI double staining were performed to evaluate neuronal apoptosis. The expression levels of proteins were evaluated by western blot. Results: EAL reduced brain infarct volume, ameliorated brain edema and improved neurological deficits in MCAO rats. In addition, EAL inhibited oxidative stress and inflammatory responses following MCAO. Besides, active compound DCMQA alleviated cytotoxicity as well as inhibited over-production of intracellular ROS and loss of MMP induced by OGD/R in SH-SY5Y cells. Moreover, EAL and DCMQA inhibited apoptosis by decreasing the expressions of pro-apoptotic proteins including bax, cytochrome c and cleaved caspase-3 while promoting the bcl-2 expression in MCAO rats and OGD/R-stimulated neurons, respectively. In addition, DCMQA suppressed the production of autophagosomes and down-regulated expression of Beclin 1 and LC3. Furthermore, inhibiting AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway contributed to DCMQA-mediated suppression of autophagy induced by OGD/R. Conclusion: Our findings demonstrate that Arctium lappa L. roots protect against cerebral ischemia through inhibiting apoptosis and AMPK/mTOR-mediated autophagy in vitro and in vivo, providing a theoretical basis for the development of CQAs in Arctium lappa L. roots as neuroprotective drugs for the prevention and treatment of ischemic stroke.

Lysosomal damage activates AMPK, a regulator of macroautophagy/autophagy and metabolism, and elicits a strong ubiquitination response. Here we show that the cytosolic lectin LGALS9 detects lysosomal membrane breach by binding to lumenal glycoepitopes, and directs both the ubiquitination response and AMPK activation. Proteomic analyses have revealed increased LGALS9 association with lysosomes, and concomitant changes in LGALS9 interactions with its newly identified partners that control ubiquitination-deubiquitination processes. An LGALS9-inetractor, deubiquitinase USP9X, dissociates from damaged lysosomes upon recognition of lumenal glycans by LGALS9. USP9X's departure from lysosomes promotes K63 ubiquitination and stimulation of MAP3K7/TAK1, an upstream kinase and activator of AMPK hitherto orphaned for a precise physiological function. Ubiquitin-activated MAP3K7/TAK1 controls AMPK specifically during lysosomal injury, caused by a spectrum of membrane-damaging or -permeabilizing agents, including silica crystals, the intracellular pathogen Mycobacterium tuberculosis, TNFSF10/TRAIL signaling, and the anti-diabetes drugs metformin. The LGALS9-ubiquitin system activating AMPK represents a novel signal transduction system contributing to various physiological outputs that are under the control of AMPK, including autophagy, MTOR, lysosomal maintenance and biogenesis, immunity, defense against microbes, and metabolic reprograming. Abbreviations: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; CAMKK2: calcium/calmodulin dependent protein kinase kinase 2; DUB: deubiquitinase; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; STK11/LKB1: serine/threonine kinase 11; TNFSF10/TRAIL: TNF superfamily member 10; USP9X: ubiquitin specific peptidase 9 X-linked.