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


METHODS
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-1beta detection was followed by Western blot analysis and mRNA expression with quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR).


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-1beta release. Also, cucurbitacin R arrested the cell cycle in the G(2)/M phase and increased the subG(0) 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-1beta. Finally, the transfection of RAW 264.7 macrophages with a Bcl-2 expression plasmid produced the inhibition of apoptosis and caspase-1 activation/interleukin-1beta release induced by cucurbitacin R in RAW 264.7 cells.


CONCLUSIONS
Taken together, these results point to a new apoptotic process in which interleukin-1beta release is directly regulated by Bcl-2 status; this contributes to the evidence that apoptotic processes do not induce inflammation.
Escandell
J M
JM
Departament de Farmacologia, Facultat de Farmàcia, Universitat de València, Burjassot, Spain.
Recio
M C
MC
Giner
R M
RM
Máñez
S
S
Ríos
J L
JL
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D015153:Blotting, Western; D020170:Caspase 1 / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D005434:Flow Cytometry; D053583:Interleukin-1beta / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014315:Triterpenes / Q000493:pharmacokinetics*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D014315:Triterpenes; C517916:cucurbitacin R; D053148:Caspase 3; D020170:Caspase 1