IL-1beta acts in synergy with endogenous IL-1beta in A375-S2 human melanoma cell apoptosis through mitochondrial pathway

Abstract

Interleukin-1beta (IL-1beta) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1beta-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1beta did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1beta (pro-IL-1beta) and endogenous IL-1beta production were involved in the apoptotic process. IL-1beta enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1beta induces endogenous IL-1beta production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.

Fig. 1

Inhibitory effects of four cytokines on A375-S2 cells. A375-S2 cells were incubated with IL-1α, IL-1β, TNF and IL-6 at various doses for 72 hr. The data are presented as mean shown only a part here of the results for three independent experiments.

Fig. 2

IL-1β-induced apoptosis in A375-S2 cells. Morphologic changes of A375-S2 cell nuclei were observed by fluorescence microscopy. (A) medium control. (B) After treatment with 1 nM IL-1β for 72 hr, the cells were stained with Hoechst 33258 using ×400 manification. Arrows indicate condensed and fragmented nuclei. (C) Internucleosomal DNA fragmentation induced in A375-S2 cells by IL-1β. The cells were treated with 1 nM IL-1β for 0, 24, 48 and 72 hr (lane 2, 3, 4 and 5; M: molecular weight marker).

Fig. 3

Effects of IL-1β on the activation of caspase-1, -3, -8 and -9 in A375-S2 cells. The cells were treated with 1 nM IL-1β for 0, 12, 24, 48 and 72 hr. The data are presented as mean±S.D. of the results for three independent experiments.

Fig. 4

Expression of PARP and ICAD in IL-1β-treated A375-S2 cells. The cells were treated with 1 nM IL-1β for 0, 12, 24, 48 and 72 hr. Cell lysates were separated by 12% SDS-PAGE electrophoresis, and PARP and ICAD proteins were detected by Western blot analysis.

Fig. 5

IL-1β-induced production of IL-1β by A375-S2 cells. (A) The cells were treated with 1 nM IL-1β for 0, 12, 24, 48 and 72 hr. The cell lysates were separated by 12% SDS-PAGE electrophoresis, and pro-IL-1β protein bands was detected by Western blot analysis. (B) A375-S2 cells (1.25, 0.63, 0.32, 0.16, 0.08×10⁶ cells/mL) were treated with 0.005, 0.05, 0.5, 5 nM IL-1β for 24 hr.

Fig. 6

IL-1β-induced mitochrondria-dependent apoptotic cell death. A375-S2 cells were treated with 1 nM IL-1β for 0, 12, 24, 48 and 72 hr. The cell lysates were separated by 12% SDS-PAGE electrophoresis, and the proteins were detected by Western blot analysis.