St. John's Wort Suppresses Growth in Triple-Negative Breast Cancer Cell Line MDA-MB-231 by Inducing Prodeath Autophagy and Apoptosis.

The rational regulation of programmed cell death by means of autophagy and apoptosis has been considered a potential treatment strategy for cancer. We demonstrated the inhibitory effect of St. John's Wort (SJW) on growth in the triple-negative breast cancer (TNBC) cell line and xenografted mice and its target mechanism concerning autophagic and apoptotic cell death. SJW ethanol extract (SJWE) inhibited proliferation in a dose-dependent manner. SJWE treatment dramatically increased autophagy flux and apoptosis compared with the control. The autophagy inhibitor, 3-methyladenine (3-MA), reversed the SJWE-induced inhibition of cell proliferation and regulation of autophagy and apoptosis, indicating that SJWE induced apoptosis through prodeath autophagy. Furthermore, SJWE inhibited tumor growth and induced autophagy and apoptosis in the tumor of MDA-MB-231 xenografted athymic nude mice. Our results indicate that SJWE might have great potential as a new anticancer therapy for triple-negative breast cancer by inducing prodeath autophagy and apoptosis.

St. John's Wort Regulates Proliferation and Apoptosis in MCF-7 Human Breast Cancer Cells by Inhibiting AMPK/mTOR and Activating the Mitochondrial Pathway.

St. John's Wort (SJW) has been used as an estrogen agonist in the systems affected by menopause. Also, hypericin, a bioactive compound of SJW, has been used as a photosensitizer in photodynamic therapy. In the present study, we investigate the anti-proliferative and pro-apoptotic effects of SJW to demonstrate the chemo-preventive effect in human breast cancer cells. MCF-7 cells were cultured with DMSO or various concentrations of SJW ethanol extract (SJWE). Cell viability, proliferation, apoptosis, the expression of proteins involved in cell growth and apoptosis, and caspase-3/7 activity were examined. SJWE dose-dependently suppressed cell growth and induced apoptosis of MCF-7 cells. Mechanistically, SJWE enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and decreased the expression of p-mammalian target of rapamycin (p-mTOR) and p-eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Also, SJWE inhibited the phosphorylation of protein kinase B (Akt) and showed increases in the expression of pro-apoptotic proteins Bax and Bad with decreases in the expression of anti-apoptotic proteins including B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), and p-Bcl-2-associated death promoter (p-Bad). SJWE at 50 µg/mL showed markedly enhanced caspase-7 activation. Taken together, our results provide evidence that SJWE shows anti-proliferative and pro-apoptotic effects via inhibition of AMPK/mTOR and activation of a mitochondrial pathway. Therefore, SJWE can be used as a chemo-preventive agent without photo-activation.

Neuroprotection by valproic Acid in mouse models of permanent and transient focal cerebral ischemia.

Valproic acid (VPA) is a well-known anti-epileptic and mood stabilizing drug. A growing number of reports demonstrate that VPA is neuroprotective against various insults. Despite intensive efforts to develop new therapeutics for stroke over the past two decades, all treatments have thus far failed to show clinical effect because of treatment-limiting side effects of the drugs. Therefore, a safety-validated drug like VPA would be an attractive candidate if it has neuroprotective effects against ischemic insults. The present study was undertaken to examine whether pre- and post-insult treatments with VPA protect against brain infarct and neurological deficits in mouse transient (tMCAO) and permanent middle cerebral artery occlusion (pMCAO) models. In the tMCAO (2 hr MCAO and 22 hr reperfusion) model, intraperitoneal injection of VPA (300 mg/kg, i.p.) 30 min prior to MCAO significantly reduced the infarct size and the neurological deficit. VPA treatment immediately after reperfusion significantly reduced the infarct size. The administration of VPA at 4 hr after reperfusion failed to reduce the infarct size and the neurological deficit. In the pMCAO model, treatment with VPA (300 mg/kg, i.p.) 30 min prior to MCAO significantly attenuated the infarct size, but did not affect the neurological deficit. Western blot analysis of acetylated H3 and H4 protein levels in extracts from the ischemic cortical area showed that treatment with VPA increased the expression of acetylated H3 and H4 at 2 hrs after MCAO. These results demonstrated that treatment with VPA prior to ischemia attenuated ischemic brain damage in both mice tMCAO and pMCAO models and treatment with VPA immediately after reperfusion reduced the infarct area in the tMCAO model. VPA could therefore be evaluated for clinical use in stroke patients.

Inhibition of matrix metalloproteinase-9 attenuated neural progenitor cell migration after photothrombotic ischemia.

Recent studies have shown that neuroblasts migrate from the subventricular zone (SVZ) into the injured area after ischemic brain insults. However, it is not well understood which mechanism mediates this ectopic migration and which types of cells migrate into the damaged region from the SVZ. The present study was designed to investigate the characteristics of the migration of nestin-positive neural stem cells toward the region of ischemic injury after focal cortical ischemia. Nestin-eGFP transgenic mice were used to effectively model the migration of SVZ cells. Photothrombotic ischemia was induced by injection of rose bengal (30 mg/kg) and exposure to cold light. Migration of nestin-positive cells was examined using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and bromodeoxyuridine (BrdU) labeling. The number of nestin-positive cells was increased significantly in the peri-infarct area at 5 and 7 days after photothrombosis. A subset of nestin-positive cells was co-labeled with DiI or BrdU. Some of the nestin-positive cells co-expressed doublecortin (DCX) and only a few nestin-positive cells co-labeled with anti-epidermal growth factor receptor (EGFr) antibody. However, no nestin-positive cells were immunoreactive for glial fibrillary acidic protein (GFAP). The inhibition of matrix metalloproteinases (MMPs) using the MMP inhibitor, FN-439, decreased nestin-positive cells in the peri-infarct region at 7 days after photothrombosis. Although MMP-9 was not co-expressed in the nestin-positive cells in the peri-infarct cortex, MMP-9 did co-localize with GFAP-positive astrocytes. These results suggest that nestin-positive neural progenitor cells migrate into the peri-infarct cortex after photothrombotic ischemia and that MMP-9 is involved in the migration.

System L-amino acid transporters are differently expressed in rat astrocyte and C6 glioma cells.

The system L-amino acid transporter is a major nutrient transport system that is responsible for Na+-independent transport of neutral amino acids including several essential amino acids. We have compared and examined the expressions and functions of the system L-amino acid transporters in both rat astrocyte cultures and C6 glioma cells. The rat astrocyte cultures expressed the l-type amino acid transporter 2 (LAT2) with its subunit 4F2hc, whereas the l-type amino acid transporter 1 (LAT1) was not expressed in these cells. The C6 glioma cells expressed LAT1 but not LAT2 with 4F2hc. The [14C]l-leucine uptakes by the rat astrocyte cultures and C6 glioma cells were Na+-independent and were completely inhibited by the system l selective inhibitor, BCH. These results suggest that the transport of neutral amino acids including several essential amino acids into rat astrocyte cultures and C6 glioma cells are for the most part mediated by LAT2 and LAT1, respectively. Therefore, the rat astrocyte cultures and C6 glioma cells are excellent tools for examining the properties of LAT2 and LAT1, respectively. Moreover, the specific inhibition of LAT1 in cancer cells might be a new rationale for anti-cancer therapy.