A sesquiterpene quinone, 5-Epi-smenospongine, promotes TNF-α production in LPS-stimulated RAW 264.7 Cells.

Eight sesquiterpene quinones: ilimaquinone (1), smenospongidine (3), smenospongiarine (5), smenospongine (7), and their corresponding 5-epimers 2, 4, 6, and 8, isolated from the Palauan marine sponge Hippospongia sp., were examined regarding their effects on TNF-α production in LPS-stimulated RAW 264.7 cells. 5-Epi-smenospongine (8) promoted the production of TNF-α to a level three times greater than the control at 10 micron, but compounds 1-7 did not show apparent activity. The results suggest that the cis-decaline ring and a primary amine in the benzoquinone ring are necessary for activity. This is the first study to report the modulation of TNF-α production by a sesquiterpene quinone.


Test Compounds
Sesquiterpene quinones 1-8 were isolated as described in the previous paper [10]. The structures of 1-8 are shown in Fig. 1.

Detection of murine TNF-α by ELISA
TNF-α concentrations of the culture supernatants under control and various test conditions were measured by ELISA using a combination of monoclonal and polyclonal antibodies. All samples were assayed at least in duplicate. Data are presented as the mean ± SE of three independent experiments.

Determination of cell proliferation
Cell proliferation was evaluated by enumerating the viable cells using the MTT formazan production method [16]. RAW264.7 cells (1 x 10 6 cells/mL) were treated with LPS (with or without test compounds) and then transferred to 96-well microtiter plates. After incubation for 24 h, 20 μL of MTT reagent (5 mg/mL in PBS) was added to each well and further incubated for 3 h. The production of formazan was assessed by measuring optical density (OD 570 nm). Data are shown as the values relative (%) to each PMA-stimulated optical density.

Results and Discussion
RAW 264.7 cells are known to produce TNF-α in response to the addition of LPS, and this system is used to detect the modulating activities of compounds on TNF-α production.
5-Epi-smenospongine (8) stimulated the production of TNF-α to a level three times greater than the control, but compounds 1-7 did not show any apparent activity (Fig. 2).
Compounds 3, 5, and 7 exhibited stronger activity on the promotion of IL-8 production than the corresponding 5-epimers 4, 6, and 8 in PMA-stimulated HL-60 cells [10], which showed the contribution of the trans-decaline ring in enhancing activity. The basic functional group at C-18 increased IL-8 production, since 3-8 exhibited stronger activities than 1 and 2 [10]. It was therefore revealed that the production of TNF-α induced by 8 involves a different mechanism from that involved in IL-8 production.
In this study, we revealed the production of TNF-α production in LPS-stimulated RAW 264.7 cells by one of the eight sesquiterpene quinones, 5-epi-smenospongine (8). This is the first report on the modulation of TNF-α production by sesquiterpene quinones.
From the comparison of structures 1-8, it is suggested that the cis-decaline ring and a primary amine in the benzoquinone ring are necessary for the activity of 8.
As we previously reported, compounds 3 to 8 induced the production of IL-8 by PMA-stimulated HL-60 cells [10]. In this study, we investigated their actions on TNF-α produced by LPS-stimulated mouse macrophage cell lines. Compound 8 induced TNF-α production. However, there was no influence of the other compounds. This may have been associated with the different actions of these compounds related to variations in receptors, signal molecules, and transcription factors in the production process of IL-8 and TNF-α as inflammatory cytokines. Therefore, the results of our experiment suggest that individual inflammatory cytokines can be controlled; these compounds should be further developed in the future.