ABSTRACT
Although Salviamiltiorrhiza has been reported to have anti-cancer mechanisms, such as caspase activation, cell cycle arrest, an anti-angiogenesis effect, and Bcl-2 family regulation, its underlying mechanism of endoplasmic reticulum (ER) stress-mediated apoptosis has never been demonstrated. Thus, in this current study, ER stress-related apoptosis via miR-216b of the ethanol extract of Salviamiltiorrhiza (SM) is elucidated for the first time. SM treatment inhibited the viability of U266 and U937 cells in a concentration-dependent manner. However, SM-exposed Raw264.7 cells were intact compared to U266 or U937 cells. Treatment with SM significantly elevated the generation of reactive oxygen species (ROS). The anti-proliferative effect of SM was reversed by pretreatment with the ROS scavenger, N-acetyl-l-cysteine (NAC), compared to cells treated only with SM. Also, SM treatment increased the ER stress by elevation of phosphorylated activating transcription factor 4 (p-ATF4), phosphorylated eukaryotic Initiation Factor 2 (p-eIF2), and phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK) expression. Caspase-3 and Poly (ADP-ribose) polymerase (PARP) were cleaved and CCAAT-enhancer-binding protein homologous protein (CHOP) was activated by SM treatment. PARP cleavage and CHOP activation were attenuated by NAC pretreatment. Furthermore, SM increased the tumor suppressor, miR-216b, and suppressed its target, c-Jun. miR-216b inhibitor attenuated the apoptotic effect of SM. Taken together, SM treatment induced apoptosis through regulation of miR-216b and ROS/ER stress pathways. SM could be a potential drug for treatment of multiple myeloma and myeloid leukemia.
Subject(s)
Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Endoplasmic Reticulum Stress/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Hematologic Neoplasms/drug therapy , MicroRNAs/genetics , Salvia miltiorrhiza/chemistry , Apoptosis , Cell Line, Tumor , Cell Proliferation/drug effects , Hematologic Neoplasms/genetics , Hematologic Neoplasms/metabolism , Hematologic Neoplasms/pathology , Humans , Oxidative Stress/drug effects , Plant Extracts/chemistry , Plant Extracts/pharmacology , Reactive Oxygen Species/metabolism , U937 CellsABSTRACT
Maladaptive repair after AKI may lead to progressive fibrosis and decline in kidney function. Sphingosine 1-phosphate has an important role in kidney injury and pleiotropic effects in fibrosis. We investigated the involvement of sphingosine kinase 1 and 2 (SphK1 and SphK2), which phosphorylate sphingosine to produce sphingosine 1-phosphate, in kidney fibrosis induced by folic acid (FA) or unilateral ischemia-reperfusion injury. Analysis of Masson trichrome staining and fibrotic marker protein and mRNA expression 14 days after AKI revealed that wild-type (WT) and Sphk1-/- mice exhibited more kidney fibrosis than Sphk2-/- mice. Furthermore, kidneys of FA-treated WT and Sphk1-/- mice had greater immune cell infiltration and expression of fibrotic and inflammatory markers than kidneys of FA-treated Sphk2-/- mice. In contrast, kidneys of Sphk2-/- mice exhibited greater expression of Ifng and IFN-γ-responsive genes (Cxcl9 and Cxcl10) than kidneys of WT or Sphk1-/- mice did at this time point. Splenic T cells from untreated Sphk2-/- mice were hyperproliferative and produced more IFN-γ than did those of WT or Sphk1-/- mice. IFN-γ blocking antibody administered to Sphk2-/- mice or deletion of Ifng (Sphk2-/-Ifng-/- mice) blocked the protective effect of SphK2 deficiency in fibrosis. Moreover, adoptive transfer of Sphk2-/- (but not Sphk2-/-Ifng-/- ) CD4 T cells into WT mice blocked FA-induced fibrosis. Finally, a selective SphK2 inhibitor blocked FA-induced kidney fibrosis in WT mice. These studies demonstrate that SphK2 inhibition may serve as a novel therapeutic approach for attenuating kidney fibrosis.
