Characterization of cytokine response induced by ingenol 3-angelate in WEHI-231 cells

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Ingenol 3-angelate (I3A) is one of the active ingredients in Euphorbia peplus, which has been used in traditional medicine against skin conditions including cancer, and has now entered a Phase I clinical trial for the treatment of actinic keratosis. We previously reported (Kedei et al., Cancer Res. 2004, 64(9): 3243-55) that I3A is a potent ligand for PKC and induces a unique, biphasic pattern of induction of interleukin (IL)-6 compared with the typical phorbol ester, phorbol 12-myristate 13-acetate (PMA). In the present study we have characterized in detail the cytokine response to I3A treatment in WEHI-231 cells. Using the Lincoplex mouse cytokine array, the only other cytokine induced by I3A or PMA in the WEHI-231 cells was TNF alpha. I3A and PMA induced TNF alpha secretion with an EC₅₀ of 5.48 + 0.74 and 2.00 + 0.14 nM, respectively. The pattern of TNF secretion was monophasic. Cytokine secretion is generally regulated at the transcriptional level, so we tested the effect of I3A and PMA on IL-6 and TNF alpha RNA levels by RT-PCR. I3A and PMA increased the level of TNF-alpha RNA with an EC₅₀ of 6.92 + 0.99 nM and 1.34 + 0.16 nM, respectively. The induction of IL-6 RNA, similarly to the protein levels, was biphasic. The promoter region of the IL-6 gene contains binding sites for multiple transcription factors, including NF-IL6, NFΚB and AP-1. We tested the activation of NF-IL6 and different members of the AP-1 and NFΚB families in the nuclear extracts of WEHI-231 cells treated with I3A or PMA using the TransAM Elisa kit. We could not detect major changes in NF-IL6 levels, but I3A and PMA were very potent in inducing activation of cFos, JunB and JunD of the AP-1 family (at 4 hours EC₅₀ values were 0.77 + 0.15 nM, 1.4 nM and 0.5 nM for I3A, and 0.44 + 0.15 nM, 1.13 nM, and 0.45 nM for PMA, respectively). The response of the NFΚB family members to I3A or PMA treatment was more complicated and changed with time. Up to 6 hours, p65 and, to a lesser extent, cRel showed a biphasic pattern of activation, namely lower drug concentrations (0.3-10 nM I3A) decreased and higher drug concentrations (30-300 nM I3A) increased or did not effect the DNA binding. After treatment for 18 hours with I3A or PMA, p65 showed a monophasic decrease in DNA binding. RelB was activated by I3A and PMA with an EC₅₀ of 4.49 + 0.89 and 0.65 + 0.101, respectively. Our results suggest that multiple AP-1 and NFΚB family members are activated by I3A and PMA and that p65 could be a negative regulator of IL-6 secretion. Its biphasic decrease in DNA binding after I3A treatment in combination with the changes in the activation of other factors could result in the observed biphasic IL-6 secretion pattern.