Hirotsugu Ogura

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-α-mediated activation of NF-κB and caspase-8

The transcription factor nuclear factor kappaB (NF-kappaB) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-alpha-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-alpha-mediated activation of NF-kappaB and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in a dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-alpha-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-alpha-mediated activation of NF-kappaB and caspase-8.

Odoroside A and ouabain inhibit Na+/K+-ATPase and prevent NF-κB-inducible protein expression by blocking Na+-dependent amino acid transport

Inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin-1 (IL-1), trigger the activation of transcription factor NF-kappaB that induces the expression of a variety of genes, including intercellular adhesion molecule (ICAM)-1. Odoroside A [3beta-O-(beta-D-diginosyl)-14-hydroxy-5beta,14beta-card-20(22)-enolide] was found to inhibit the cell-surface expression of ICAM-1 induced by TNF-alpha and IL-1 at comparable concentrations in human lung carcinoma A549 cells. In this study, the molecular mechanism underlying the inhibition of TNF-alpha-induced cell-surface ICAM-1 expression by odoroside A together with the specific Na(+)/K(+)-ATPase inhibitor ouabain was further investigated. Odoroside A and ouabain neither prevented IkappaBalpha degradation nor NF-kappaB translocation to the nucleus upon TNF-alpha stimulation. While odoroside A and ouabain had no inhibitory effect on the induction of ICAM-1 mRNA, they inhibited the TNF-alpha-induced ICAM-1 expression at the protein level. Consistent with these results, odoroside A and ouabain potently reduced de novo protein synthesis, largely due to its ability to block Na(+)-dependent transport of amino acids across the plasma membrane, but not to interfering with the translation machinery. As a direct molecular target, odoroside A was found to inhibit the ATP-hydrolyzing activity of Na(+)/K(+)-ATPase as potently as ouabain. These results clearly demonstrate that odoroside A and ouabain prevent NF-kappaB-inducible protein expression by blocking the Na(+)-dependent amino acid transport.

ERK and p38 MAP kinase are involved in downregulation of cell surface TNF receptor 1 induced by acetoxycycloheximide

Tumor necrosis factor (TNF)-alpha activates the nuclear factor kappaB (NF-kappaB) signaling pathway. The protein synthesis inhibitor cycloheximide (CHX) and its structural derivative acetoxycycloheximide (Ac-CHX) have been recently shown to block the TNF-alpha-induced activation of NF-kappaB via ectodomain shedding of TNF receptor 1 (TNF-R1) in human lung carcinoma A549 cells. In this study, we show that ERK and p38 MAP kinase are involved in the downregulation of cell surface TNF-R1 upon exposure to Ac-CHX and the subsequent inhibition of TNF-alpha-induced NF-kappaB activation. Ac-CHX was capable of promoting the sustained activation of ERK, JNK, and p38 MAP kinase. Treatment with the MEK inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the JNK inhibitor SP600125, reversed the diminished expression of cell surface TNF-R1 as well as the blockade of TNF-alpha-induced IkappaBalpha degradation in Ac-CHX-treated cells. Our results indicate that Ac-CHX triggers the downregulation of cell surface TNF-R1 via the activation of ERK and p38 MAP kinase, thereby preventing activation of the NF-kappaB signaling pathway by TNF-alpha.

Eudesmane-type sesquiterpene lactones inhibit multiple steps in the NF-κB signaling pathway induced by inflammatory cytokines

Inflammatory cytokines, such as interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), induce the intracellular signaling pathway leading to the activation of nuclear factor κB (NF-κB). A series of eudesmane-type sesquiterpene lactones possessing an α-methylene γ-lactone group and/or an α-bromo ketone group were synthesized and evaluated for their inhibitory effects on the NF-κB-dependent gene expression and signaling pathway. Our present study reveals that eudesmane-type α-methylene γ-lactones and α-bromo ketones inhibit multiple steps in the NF-κB signaling pathway induced by IL-1α and TNF-α.

Polar cardenolide monoglycosides from stems and twigs of Nerium oleander and their biological activities

Twelve polar cardenolide monoglycosides, 1, 2, 4–13, and oleagenin (3) were isolated from the methanol extract of stems and twigs of Nerium oleander. Among these, oleagenin (3) and cardenolide monoglycosides named cardenolide B-1 (1) and cardenolide B-2 (2) were isolated from natural sources for the first time. The in vitro antiinflammatory activity of compounds 1–13 was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). Compounds 4–7 were active at an IC50 value of less than 0.4 μM. The cytotoxic activity of compounds 1–13 was evaluated against three human cell lines: normal human fibroblast cells (WI-38), malignant tumor cells derived from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 4, 6, and 7 were active toward these three cell lines at IC50 values of less than 0.7 μM, and compounds 5 and 8 were active toward the cell lines at IC50 values of less than 1.5 μM. The multidrug resistance (MDR) cancer-reversal activity of compounds 1–13 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compound 1 and 12 showed significant effects on calcein accumulation.

The biological activities of cardenolide triglycosides from stems, twigs, and leaves of Nerium oleander

Sixteen cardenolide triglycosides (1–16) were isolated from stems, twigs, and leaves of Nerium oleander. Among them, 3β-O-(4-O-gentiobiosyl-d-diginosyl)-7β,8-epoxy-14-hydroxy-5β,14β-card-20(22)-enolide, named cardenolide B-3 (16), was isolated from natural sources for the first time. The in vitro anti-inflammatory activities of compounds 1–16 were examined on the basis of inhibitory activity against the induction of intercellular adhesion molecule-1 (ICAM-1). Compounds 1–5 were active at an IC50 value of less than 7 μM. The cytotoxic activity of isolated compounds was evaluated against three human cell lines: normal human fibroblast cells (W-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 1–5 were active toward WI-38 cells; compounds 1, 3, and 5 were active toward VA-13 cells; and compounds 1–5 were active toward HepG2 cells at IC50 values of less than 10 μM. The multidrug-resistant (MDR) cancer-reversal activity of compounds 1–16 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 13 and 14 showed significant effects on calcein accumulation.