Cocaine potentiates an inflammatory response in C6 astroglia-like cells.

Cocaine is a highly addictive drug that mediates its effect through altering dopamine metabolism in the central nervous system (CNS), resulting in a feeling of euphoria. Owing to its high lipophilicity, cocaine easily crosses the blood brain barrier of the CNS and reaches various domains of the brain, where it can trigger cellular damage. Cocaine-induced CNS damage may arise due to increased levels of free radicals and nitric oxide (NO) in immunecompetent astroglial cells. In the present study, the potential ability of cocaine to exacerbate the production of inflammatory products, primarily superoxide free radicals (O2 -), hydrogen peroxide (H2O2) and NO/nitrite (NO2 -) was examined in rat C6 astroglia-like cells challenged with lipopolysaccharide (LPS), a bacterial endotoxin, and interferon gamma (IFNγ), a pro-inflammatory cytokine. Furthermore, the role of cocaine in increasing the expression of hypoxia inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) in cells was also determined. First, the viability of the cells was assessed when treated with cocaine (0.5-7 mM) for 24 and 48 h. The results showed that cocaine toxicity was both time and dose-dependent. In subsequent studies, cells were challenged with or without LPS and IFNγ, followed by co-treatment with cocaine (1-4 mM) for 24 h. Cocaine treatment did not increase O2 - or H2O2 production in the challenged or unchallenged cells. Similarly, cocaine treatment did not increase NO/NO2 - production in the unchallenged cells; however, NO/NO2 - levels in the challenged cells was increased 40-50-fold upon cocaine treatment compared with the corresponding unchallenged group. The HIF-1α and VEGF levels were significantly increased in the challenged cells at higher cocaine doses compared with the unchallenged cells. Since high concentrations of NO are associated with inflammation, the high levels of NO production observed in the present study suggested that cocaine may have potentiated the inflammatory response in the challenged C6 astroglia-like cells.

Identification of cytotoxic markers in methamphetamine treated rat C6 astroglia-like cells.

Methamphetamine (METH) is a powerfully addictive psychostimulant that has a pronounced effect on the central nervous system (CNS). The present study aimed to assess METH toxicity in differentiated C6 astroglia-like cells through biochemical and toxicity markers with acute (1 h) and chronic (48 h) treatments. In the absence of external stimulants, cellular differentiation of neuronal morphology was achieved through reduced serum (2.5%) in the medium. The cells displayed branched neurite-like processes with extensive intercellular connections. Results indicated that acute METH treatment neither altered the cell morphology nor killed the cells, which echoed with lack of consequence on reactive oxygen species (ROS), nitric oxide (NO) or inhibition of any cell cycle phases except induction of cytoplasmic vacuoles. On the other hand, chronic treatment at 1 mM or above destroyed the neurite-like processors and decreased the cell viability that paralleled with increased levels of ROS, lipid peroxidation and lactate, depletion in glutathione (GSH) level and inhibition at G0/G1 phase of cell cycle, leading to apoptosis. Pre-treatment of cells with N-acetyl cysteine (NAC, 2.5 mM for 1 h) followed by METH co-treatment for 48 h rescued the cells completely from toxicity by decreasing ROS through increased GSH. Our results provide evidence that increased ROS and GSH depletion underlie the cytotoxic effects of METH in the cells. Since loss in neurite connections and intracellular changes can lead to psychiatric illnesses in drug users, the evidence that we show in our study suggests that these are also contributing factors for psychiatric-illnesses in METH addicts.

Milk thistle seed extract protects rat C6 astroglial cells from acute cocaine toxicity.

Cocaine is a powerful addictive drug, widely abused in most Western countries. It easily reaches various domains within and outside of the central nervous system (CNS), and triggers varying levels of cellular toxicity. No pharmacological treatment is available to alleviate cocaine-induced toxicity in the cells without side-effects. Here, we discerned the role of milk thistle (MT) seed extract against cocaine toxicity. First, we investigated acute cytotoxicity induced by treatment with 2, 3 and 4 mM cocaine for 1 h in astroglial, liver and kidney cells in vitro, and then in living shrimp larvae in vivo. We showed that astroglial cells are more sensitive to cocaine than liver, kidney cells or larvae. Cocaine exposure disrupted the general architecture of astroglial cells, induced vacuolation, decreased cell viability, and depleted the glutathione (GSH) level. These changes may represent the underlying pathology of cocaine in the astrocytes. By contrast, MT pretreatment (200 µg/ml) for 30 min sustained the cell morphological features and increased both cell viability and the GSH level. Besides its protective effects, the MT extract was revealed to be non-toxic to astroglial cells, and displayed high free-radical scavenging activity. The results from this study suggest that enhanced GSH level underlies cell protection, and indicate that compounds that promote GSH synthesis in the cells may be beneficial against cocaine toxicity.

Autoxidation of gallic acid induces ROS-dependent death in human prostate cancer LNCaP cells.

BACKGROUND: Prostate cancer is the second most common cause of mortality. Gallic acid (GA) is a natural polyphenol, and we tested its in-vitro cytotoxicity after 24 h in prostate cancer LNCaP cells. MATERIALS AND METHODS: GA autoxidation was measured fluorimetrically for H(2)O(2), and O(2)(•-) radicals by chemiluminescence. Intracellular reactive oxygen species (ROS) levels were detected with 2',7'-dichlorodihydrofluorescein diacetate. Cytotoxicity was evaluated by crystal-violet, while apoptosis and mitochondrial membrane potential were determined by flow cytometry. Cytochrome c release was detected by enzyme-linked immunosorbent assay, and caspase-8, -9 and -3 activities were measured calorimetrically. RESULTS: GA autoxidation produced significant levels of H(2)O(2) and O2.-. Increased intracellular ROS levels with GA were reduced by N-acetyl-L-cysteine (NAC) and L-glutathione (GSH). Cells were protected against GA cytotoxicity when pretreated with increasing levels of superoxide dismutase/catalase mixture, NAC, or GSH for 3 h. The number of apoptotic cells increased with GA dose. GA caused mitochondrial potential loss, cytochrome c release, and activation of caspases 3, 8 and 9. CONCLUSION: The ROS-dependent apoptotic mechanism of GA kills malignant cells effectively; it is likely that GA could be a good anticancer agent.

Differential cytotoxicity of triphala and its phenolic constituent gallic acid on human prostate cancer LNCap and normal cells.

BACKGROUND: Prostate cancer is one of the most commonly diagnosed solid malignancies among US men. We identified gallic acid (GA) as a major bioactive cytotoxic constituent of a polyherbal Ayurvedic formulation - triphala (TPL). Both TPL and GA were evaluated on (AR)(+) LNCaP prostate cancer and normal epithelial cells. MATERIALS AND METHODS: Total polyphenols in TPL were determined using Folin and Ciocalteu method, followed by GA quantitation by high performance liquid chromatography. Cell toxicity was evaluated by crystal violet after 24, 48, 72 and 96 h. RESULTS: TPL contains 40% unidentified polyphenolic acids, of which 2.4% comprised GA. GA induced severe morphological alterations and was about 3-fold more cytotoxic towards cancer cells than TPL. This activity increased further in the presence of dihydrotestosterone. GA toxicity on normal cells was low at 72 h. Combination of GA with flutamide caused higher toxicity to cancer cells than either of the compounds alone. CONCLUSION: GA appears to have promising anticancer activity.