Bcl-2 is a negative regulator of interleukin-1[beta] secretion in murine macrophages in pharmacological-induced apoptosis

BACKGROUND AND PURPOSE Cucurbitacin R, a natural anti-inflammatory product, has been shown to exhibit activity against both adjuvant-induced arthritis and delayed-type hypersensitivity reactions induced by various agents. Previous studies have demonstrated that the effects of cucurbitacin R stem from its inhibition of both cytokine production and lymphocyte proliferation. EXPERIMENTAL APPROACHES Effects of cucurbitacin R were investigated on lipopolysaccharide-stimulated RAW 264.7 cells. Cell cycle evolution was analysed by flow cytometry, detection of apoptosis by DNA ladder, Bcl-2, p21, p53, Bax, cleaved caspase-1 (p10), caspase-9, and caspase-3, cleaved caspase (p17) and interleukin-1[beta] detection was followed by Western blot analysis and mRNA expression with quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR). KEY RESULTS Cucurbitacin R was found to induce apoptosis in lipopolysaccharide-stimulated RAW 264.7 macrophages through the inhibition of Bcl-2 expression, which regulates pro-inflammatory caspase-1 activation and interleukin-1[beta] release. Also, cucurbitacin R arrested the cell cycle in the G2/M phase and increased the subG0 population in lipopolysaccharide-stimulated RAW 264.7 macrophages. Moreover, it increased the expression of proteins p53 and p21, down-regulated the expression of Bcl-2, activated the activity of caspase-1 and augmented the production of interleukin-1[beta]. Finally, the transfection of RAW 264.7 macrophages with a Bcl-2 expression plasmid produced the inhibition of apoptosis and caspase-1 activation/interleukin-1[beta] release induced by cucurbitacin R in RAW 264.7 cells. CONCLUSIONS AND IMPLICATIONS Taken together, these results point to a new apoptotic process in which interleukin-1[beta] release is directly regulated by Bcl-2 status; this contributes to the evidence that apoptotic processes do not induce inflammation. [PUBLICATION ABSTRACT]