Fig. 5.
(A–D) Western blot analysis of caspase activation in different Jurkat cell clones after treatment with lidocaine. (A) Jurkat wild-type cells, Jurkat cells overexpressing the B-cell lymphoma 2 protein (Bcl-2), and (B) cells deficient of either caspase 9, (C) caspase 8, or (D) Fas-associated protein with death domain (FADD) were treated with 1.25 μm staurosporine (STS) or the indicated concentrations of lidocaine. After 12 h, cell lysates were prepared and subjected to Western blot analysis using antibodies against caspase 8, caspase 9, Bcl-2, or the cleaved active form of caspase 3 (act. caspase-3). Analysis of actin expression served as a loading control. Caspase-3 activation was detected after treatment with 3 mm (≈ 0.08%) and 6 mm (≈ 0.16%) lidocaine in Jurkat wild-type cells, but not in Bcl-2–overexpressing (A) or in caspase 9–deficient cells (B). In contrast, caspase-3 activation was not compromised in Jurkat cells deficient of caspase 8 (C) or FADD (D) as compared with wild-type cells. Treatment with 10 mm (≈ 0.27%) lidocaine led to no detectable caspases-3 activation, indicating necrotic cell death. (E) Lidocaine treatment triggers the mitochondrial release of cytochromecrelease. Parental Jurkat cells were treated with 1.25 μm STS or the indicated concentrations of lidocaine. After 12 h, mitochondrial and cytosolic fractions were prepared and analyzed for cytochromeccontent using Western blot analysis. Detection of the translocase of outer mitochondrial membrane 20 (Tom 20) confirmed the purity of the mitochondrial and cytosolic fractions. Release of mitochondrial cytochromecinto the cytosol was strongly pronounced after treatment with 3 and 6 mm, but less evident at 10 mm lidocaine.