Stress-induced cellular defense machinery has a critical role in mediating cancer drug resistance, and targeting stress-related signaling has become a novel strategy to improve chemosensitivity. Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with potent anticancer bioactivities in multiple malignancies, whereas its underlying mechanisms remain unclear. Here in, we found that BA has synergistic effects with taxol to induce breast cancer cells G2/M checkpoint arrest and apoptosis induction, but had little cytotoxicity effects on normal mammary epithelial cells. Drug affinity responsive target stability (DARTS) strategy further identified glucose-regulated protein 78 (GRP78) as the direct interacting target of BA. BA administration significantly elevated GRP78-mediated endoplasmic reticulum (ER) stress and resulted in the activation of protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor 2a/CCAAT/enhancer-binding protein homologous protein apoptotic pathway. GRP78 silencing or ER stress inhibitor salubrinal administration was revealed to abolish the anticancer effects of BA, indicating the critical role of GRP78 in mediating the bioactivity of BA. Molecular docking and coimmunoprecipitation assay further demonstrated that BA might competitively bind with ATPase domain of GRP78 to interrupt its interaction with ER stress sensor PERK, thereby initiating the downstream apoptosis cascade. In vivo breast cancer xenografts finally validated the chemosensitizing effects of BA and its biofunction in activating GRP78 to trigger ER stress-mediated apoptosis. Taken together, our study not only uncovers GRP78 as a novel target underlying the chemosensitizing effects of BA, but also highlights GRP78-based targeting strategy as a promising approach to improve breast cancer prognosis.

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Abstract. Ligustrazine, also known as 2,3,5,6-tetramethylpyrazine (TMP), one of the major active compounds of Ligusticum wallichii Franchat., has been shown to reduce neuroinflammation and protect neurons during cerebral ischemia/reperfusion injury. However, whether it reduces blood-brain barrier (BBB) permeability during ischemic stroke is unclear. The aim of the present study was to investigate the role that TMP plays in protecting the BBB integrity in ischemia/reperfusion injury and to investigate the relevant mechanisms involved. Rats received an intraperitoneal injection of 20 mg/kg TMP 15 min before the onset of ischemia, which was induced by middle cerebral artery occlusion. Infarct volume, neurological score, brain edema, BBB permeability and tight junction protein impairment were observed. The results showed that TMP reduced the neurological score and levels of brain infarction and edema. In addition, TMP significantly decreased BBB permeability and prevented the impairment of occludin and claudin-5, two tight junction protein components of the BBB, in rat brains with ischemia/reperfusion injury. In addition, the expression and activity of matrix metalloproteinases, enzymes responsible for the degradation of the extracellular matrix and tight junctions, were reduced in the rat brains by TMP treatment. These results combined suggest that TMP reduces BBB permeability as well as neuronal damage in focal cerebral ischemia/reperfusion injury in rats.