Antioxidant effect of curcumin and its analogues in PC12 cells Università degli Studi di Sassari Antioxidant effect of curcumin and its analogues in PC12 cells MELIS S1, SERRA PA1, PIRISINU M1, DELOGU G2, FABBRI D2, DETTORI MA2, MIGHELI R1 1 Department of Clinical and Experimental Medicine, University of Sassari, Italy 2 Institute of Biomolecular Chemistry, CNR of Sassari, Italy Scuola di Dottorato in Scienze Biomediche – Indirizzo Neuroscienze - XXIX ciclo INTRODUCTION Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic pathway (1). Several studies have shown that oxidative stress plays a key role in the dopaminergic neurodegeneration. The PC12 cell line, originated from rat pheochromocytoma, a benign tumor of the adrenal medulla, release dopamine and other catecholamines as dopaminergic neurons (2). Identification of the biochemical pathways involved in neuronal cell death due to oxidative stress and apoptosis could contribute to the development of drugs for the treatment of certain neurodegenerative disorders, including PD. In the last years, natural antioxidants and their derivatives gained increased interest for their antioxidant properties (3). Curcumin is a potent antioxidant compound, derived from the curry spice turmeric. This molecule is obtained by solvent extraction from the dried and ground rhizome of Curcuma Longa Linn (Zingiberaceae) (4) (Fig. 1). Curcumin acts such as a free radical scavenger and antioxidant, inhibiting lipid peroxidation. The potential neuroprotective effects of curcumin and its derivatives (Fig. 2) have been evaluated in this study on PC12 cells against H2O2 damage. Fig. 1: Curcuma longa. Fig. 2 curcumin 13DF81 AD687 MATERIALS AND METHODS Cell culture Experiments were performed on PC12 cells during their exponential phase of growth. Cells were maintained at 37°C in 100 mm plastic tissue culture dishes in atmosphere of 5% CO2/95% air in Dulbecco’s modified Eagle’s medium (DMEM/F12) supplemented with 10% horse serum, 5% fetal calf serum and 1% penicillin/streptomycin. At the start of experiment, PC12 were seeded at 1x105 cells/well in 24-well plates (time 0) for 24 h. PC12 cells were preincubated for 20’ with curcumin and its derivatives then exposed to H2O2 (100µM) for 24 h. § * A B C Cell viability Cells were processed with the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay after 24 h of incubation. Viable cells convert the soluble dye MTT to insoluble (in aqueous medium) blue formazan crystals. In brief, 1 mg of MTT [200 µl of a 5 mg/ml stock solution in phosphate-buffered saline (PBS)] was added per milliliter of medium, and the cultures were allowed to incubate at 37° C for 4 h. The MTT was removed and cells were rinsed with PBS and centrifuged at 4,000 rpm for 15 min. Thereafter, the supernatant was discarded, and the pellet was dissolved in 2 ml of isopropanol; after centrifugation at 4,000 rpm for 5 min, the color was read at 578 nm using a microplate reader. Cell viability was expressed as a percentage of untreated cells, which served as the negative control group and was designated as 100%; the results were expressed as a percentage compared to the negative control. All assays were performed using samples in duplicate. Fluorescence Microscopy PC12 cells were seeded on gelatin (1mg/ml in sterile milliQ water), coated glasse coverslips at 105 cells/ml and cultured overnight. Cells were treated with 13DF81 at 10 µM for 24 h. Afterwards, cells were rinsed two times with PBS and stained with DAPI (5 µg/ml in PBS) for 15 min, protecting them from light. Cells were then rinsed once time with PBS and sealed on microscope slides. Samples were analized by fluorescence microscopy. RESULTS As shown in figure 3, curcumin and its natural analogs used in increasing concentrations (from 0,1 to 40 µM) did not show any toxic effect on PC12 cell (dark green bars of each group). Viability decreased up to 58% after H2O2 100 µM treatment (red bars of each graph). As shown in orange bars of each group, curcumin and 13DF81 were able to protect against hydrogen peroxide. In particular, in cells pretreated with 5 µM of curcumin viability increased up to 68% of the control value (panel A). A range from 1 to 40 µM of 13DF81 treatments showed interesting protection; between these concentrations, 13DF81 at 10 µM treatments restored viability up to 80% of the control value (panel C). On the other hand, AD687 did not show any protection in the tested concentrations (panel B). As shown in fluorescence micrographs (Fig. 4), the number of apoptotic nuclei (white arrows) decreased in cells received 10 µM pretreatments of 13DF81 20 min before H2O2 100 µM (figure 4, panel D). Fig.4: Fluorescence micrographs of PC12 cell nuclei. A: control; B: 13DF81 10 µM; C: H2O2 100 µM; D: 13DF81 10 µM + H2O2 100 µM. Cells were stained with the DNA specific fluorescent dye DAPI (3 g/ml)). A B A B C D C D * Fig.3: Panel A: Curcumin; panel B: AD687; panel C: 13DF81. Green bars: viability of controls; red bars: viability after H2O2 (100 µM) exposure; dark green bars: viability after increasing concentration of curcumin and its derivatives; orange: both treatments. (*p<0.05 vs H2O2) (§p<0.05 vs control) CONCLUSIONS The preliminary results showed a protective action of the natural compound curcumin and its natural-like derivatives against H2O2-induced oxidative stress on PC12 cells. In particular, viability was protected by all used concentrations of 13DF81, for this reason further studies will be required to investigate its antioxidant properties. 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