Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.

The anti-excitotoxic efficacy of the pineal hormone melatonin was investigated in kainate-injured brains of rats. Kainate (a glutamate-receptor agonist, 2.5 nmol in 1 microl) was directly injected to unilateral striatum. Melatonin (10 mg/kg) was administrated intraperitoneally 1 h before and 1, 3, and 5 h after intrastriatal kainate injection in adult Sprague-Dawley rats. Three days after kainate injection, a significant neuronal damage was found, as determined by Nissl staining and the TUNEL method, not only in the injected striatum, but also in the ipsilateral neighboring cortex. The kainate-induced cortical apoptotic neuronal death was significantly attenuated by treatment with melatonin compared with the vehicle control group. However, no detectable changes were observed in the contralateral side of the brain in either vehicle- or melatonin-treated rats. Moreover, the biochemical results indicated that kainate can indeed induce oxidative stress, such as a decrease in the content of total glutathione (GSH), oxidized glutathione (GSSG), and an increase in the ratio of GSSG/GSH in the striatum and cortex compared with the contralateral brain regions. In the kainate-injected striatum, melatonin did not reduce the oxidative stress, but in the neighborhood of injected area-cortex, kainate-induced oxidative stress was significantly reduced by melatonin. Enhancement of glutathione-peroxidase activity was induced by intrastriatal kainate injection, not only in the cortical area of control and melatonin-treated rats, but also in striatum of control rats. However, a large elevation was found in the melatonin-treated cortex. Taking the morphological and biochemical data together, the present results suggest that melatonin functions as an antioxidant by upregulating the glutathione antioxidative defense system, thereby reducing neuronal death caused by excitotoxicity and preventing the kainate-induced damage from spreading to adjacent brain regions.

To determine the roles of different members of the family of B cell lymphoma protooncogene (Bcl-2) in relation to neurotoxin-induced neuronal degeneration, the pattern of the expression of a number of molecules of the Bcl-2 family was studied immunocytochemically in the retinas of C57BL/6J mice after intraperitoneal (IP) injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Three days to 12 weeks after MPTP treatment, a detectable reduction of tyrosine hydroxylase immunoreactivity in the amacrine cells was observed, with an increase of Bcl-2 expression in the Müller glial cells, and a de novo expression of Bad and Bax in the retinal ganglion cells, optic nerve fibers and plexiform layers. In contrast, a slight decrease of Bcl-x(L) immunoreactivity in the retinal ganglion cells was observed, whereas Bcl-x(S/L) immunoreactivity was increased slightly in the retinas of MPTP-treated mice compared with that of the controls. In animals that received MPTP injection, an increase in immunostaining of GFAP, glutamine synthetase, and Mac-1 (CD11b) in astrocytes, Müller cells, and microglia was invariably observed, indicating an activation or dysfunction of retinal glial cells. These findings are consistent with the current view that glial dysfunction is important in mediating the cytotoxic effect of a variety of neurotoxic molecules, including MPTP, and that different members of Bcl-2 family may have different roles as far as neuronal degeneration or neuroprotection is concerned.