Effect of cocaine (COC) (1 mM, 6 hours) on DNA fragmentation in PC12 Cells. A flow cytometric analysis was conducted after the cells had been incubated for 6 and 24 hours with cocaine. Results are expressed as mean + S.E.M. from three individual experiments. * p < 0.05 vs PBS (one-way ANOVA followed by Newman-Keuls test).

Schematic representation of neurotoxic action of cocaine in PC12 cells. The treatment of PC 12 cells with cocaine can alter the Bax/Bcl-2 ratio, reducing the Bcl2 levels, which could lead to activation of caspase 3 and the trigger of cell death process, seen after 24 hours of treatment. On the other hand, to protect the cell from cocaine-toxic effects activation of NF-κB occurs through D1 receptors. The activation of this transcription factor could lead to transcription of anti-apoptotic genes, such as BDNF that tries to mimitaze the process of cell death.

Effect of PDTC, Sodium Salicilate and SCH23390 on cocaine-Induced toxicity in PC12 Cells (A and C: LDH release assay; B and D: MTT reduction assay). PC12 cells were pre-treated for 20 min with various concentrations of PDTC, Sodium Salicilate or SCH23390 prior to cocaine (1 mM, 6 hours) treatment. Cell viability was estimated by MTT reduction (570 nm) and LDH release (490 nm) assays. Results are expressed as mean + S.E.M. from three individual experiments. * p < 0.05 vs PBS and # p < 0.05 vs COC (cocaine) (one-way ANOVA followed by Newman-Keuls test)

Influence of SCH23390 on NF-κB activation induced by cocaine in PC12 cells. Cells treated with SCH23390 (10, 50, 100 μM) 20 min prior to cocaine (COC) (1 mM) or PBS treatments were incubated for 6 hours and EMSA assay was performed with the nuclear extract. Panel A represents autoradiography of the EMSA assay. The position of specific NF-κB/DNA binding complex p50/p65 is indicated. Bottom graphic (panel B) represents densitometric analysis of the p50/p65 bands presented in the top panel. Results are expressed as mean + S.E.M. from three individual experiments. * p < 0.05 vs PBS and # p < 0.05 vs COC (one-way ANOVA followed by Newman-Keuls test).

(A) Cocaine (COC) exposure increased α-spectrin (240 kDa) cleavage and (E) increased cleavage of caspase 3 in PC12 cells. Cell lysates were extracted from PBS and cocaine (1 mM, 6 hours) treated PC12 cells. Cell lysate protein content (30 μg) was loaded and run on a SDS-polyacrylamide gel and submitted to Immunoblot assay. The densitometric analysis (% of control) of the 150,120 kDa (cleaved product of spectrin) and caspase-3 cleaved product (17 kDa) band complexes represented in A and D are shown on B, C and F respectably. β-Actin was used as internal control. (D) COC exposure raised caspase-3 activity and expression in PC12 cells. Cells were treated with 1 mM of COC for 6 hours. Cell lysates were analyzed for caspase-3 activity, using the caspase-3 substrate (Ac-DEVD-AMC). Results are expressed as mean + S.E.M. from three individual experiments; * p < 0.05 vs PBS (Student's t-test).

(A) Cocaine (COC) exposure decreased Bcl-2 protein expression in PC12 cells. Cell lysates were extracted from PBS and COC (1 mM, 6 hours) treated PC12 cells. Cell lysate protein content (30 μg) was loaded and run on a SDS-polyacrylamide gel and submitted to Immunoblot assay. The densitometric analysis (% of control) of the Bax and Bcl-2 band complexes represented in (A) are shown (B and C). β-Actin was used as internal control. (D) Effect of incubation with COC (1 mM) for 6 or 24 hours on levels of mRNA for Bax, Bcl-2 and BDNF in PC12 cells. Cocaine increased BDNF mRNA levels after 6 hours (G) and decreased Bcl-2 mRNA levels after 6 and 24 hours of cocaine treatment (F) as indicated by RT-PCR assays. No changes were observed for Bax mRNA levels after both 6 and 24 hours of cocaine treatment (E). Results are expressed as mean + S.E.M. from three individual experiments. * p < 0.05 vs PBS (one-way ANOVA followed by Newman-Keuls test).

PC12 cells were treated with 1 mM cocaine (COC) or PBS for 6 hours. Nuclear extract (5 μg) was submitted to EMSA assay. (A) Positions of protein/DNA complexes observed are indicated by numbers. Localization of free probe is also indicated. (B) Competition studies in the absence or presence of unlabeled specific (NF-κB consensus sequence 2-fold molar in excess) or non-specific oligonucleotide (TFIID consensus sequence, 2-fold molar in excess), as indicated. (C) Supershift assays incubated in the absence or presence of 2 μL of antibodies (1:10 dilution) against p50, p65, c-Rel and p52 subunits, as indicated. Antibodies were added 20 min prior to addition of the radiolabeled NF-κB consensus oligonucleotide. The position of specific NF-κB/DNA binding complex p50/p65 (band 1) is indicated. NS represents no specific binding (bands 2 and 3). (D) Concentration response curve and time-course of NF-κB activation induced by COC in PC12 cells. Nuclear proteins (5 μg) were extracted from cells treated with different concentrations of cocaine: 0.5, 0.25,0.5, 0.75, 1, 2 mM or PBS for 6 hours (E) Different time points of NF-κB activation: 1, 3, 6 and 8 hours with 1 mM of COC (panel B) or (F) 24 hours with 1 and 2 mM COC or PBS. The position of specific NF-κB/DNA binding complex (p50/p65 complex) is indicated. (G) Representative immunoblot expression of IκB-α in PC12 cells after incubation with COC (1 mM) or PBS for 6 hours. The densitometric analysis (% of control) of the IκB-α band complex represented in (G) is shown (H). β-Actin was used as internal control. Results are expressed as mean + S.E.M. from three individual experiments. * p < 0.05 vs PBS (Student's t-test).