Effects of C. Crepidioides in nude mice inoculated with S-180 cells
To explore the effect of C. crepidioides on tumor growth in nude mice transplanted with murine S-180, we treated tumor-bearing mice with 5 g/kg of C. crepidioides extract. The mean tumor volume was significantly lower than that of control mice after 14-day treatment (P = 0.0433, Figure 1A). However, the difference in tumor volume after 21- and 27-days treatment, compared with control, was less conspicuous. There was no significant difference in body weight gain from day 0 to day 28 between the control and C. crepidioides-treated groups (data not shown), and mice treated with C. crepidioides appeared generally healthy during the same period. On the other hand, treatment of S-180 cells with C. crepidioides for 29 days produced strong H&E staining for apoptosis (Figure 1B). These results suggest that C. crepidioides has antitumor in vivo effect.
Supernatant from
C. crepidioides
-stimulated RAW264.7 cells but not
C. crepidioides
alone causes cell growth inhibition of S-180 cells
To study the mechanism of the antitumor effect of C. crepidioides, we determined the effects of C. crepidioides on cell growth of S-180 cells in vitro. Cell growth was assessed by the WST-8 assay. Incubation with C. crepidioides alone at concentrations up to 500 μg/ml for 72 h did not affect cell growth (Figure 1C). Activation of macrophages by agents such as bacterial LPS stimulates their growth inhibitory effects on a wide variety of tumor cells [4]. Based on this property, we investigated the effects of macrophages on S-180 cells. RAW264.7 cells were incubated with various concentrations of C. crepidioides. C. crepidioides did not have any growth inhibitory activity on RAW264.7 cells at concentrations up to 500 μg/ml for 24 h, but a mild inhibitory effect was noted at 500 μg/ml and incubation for 48 and 72 h (Figure 1D).
S-180 cells were also cultured with supernatants from RAW264.7 cells that had been stimulated by various concentrations of C. crepidioides for 72 h. The supernatants suppressed cell growth of S-180 cells in a C. crepidioides dose-dependent manner (Figure 2A), while C. crepidioides-containing control medium had no growth inhibitory activity on S-180 cells, similar to the data shown in Figure 1C (Figure 2B).
C. Crepidioides induces NO production and expression of iNOS mRNA
Previous studies demonstrated the crucial role of NO in the tumoricidal activity of murine macrophages [14–16]. The addition of exogenous NO donor, NOR3, to the culture medium of S-180 cells inhibited cell growth (data not shown). Therefore, we examined the effects of C. crepidioides on NO production by RAW264.7 cells in vitro. NO production by stimulated RAW264.7 cells was assessed by measuring nitrite in the culture medium. C. crepidioides stimulated the production of nitrite from RAW264.7 cells in time- and dose-dependent manners (Figure 2C and D). In in vivo experiments, mice orally administered C. crepidioides had significantly high serum nitrite and nitrate levels compared with the control (Figure 3).
What is the mechanism of C. crepidioides-stimulated NO production? NO is synthesized by NOS-catalyzed conversion of L-arginine to L-citrulline. Whereas the activity of neuronal and endothelial NOS is mainly regulated post-translationally by cytoplasmic calcium levels or by phosphorylation by various protein kinases, iNOS is primarily regulated at the transcriptional level [17]. Next, we determined whether C. crepidioides-induced NO production from RAW264.7 cells was catalyzed by iNOS. The addition of 500 μg/ml C. crepidioides to RAW264.7 cells resulted in the expression of iNOS mRNA from 1 h after treatment (Figure 4A). In another setting, incubation with C. crepidioides for 3 h at 15.6 μg/ml induced iNOS mRNA expression in RAW264.7 cells (Figure 4B). The main antioxidant isolated from C. crepidioides is isochlorogenic acid [3]. Isochlorogenic acid stimulated iNOS expression in a manner similar to that by C. crepidioides (Figure 4C and D). These results suggest that the C. crepidioides-induced increase in NO production by RAW264.7 cells is mediated by the induction of iNOS expression, and that isochlorogenic acid seems to contribute to at least part to this augmented production of NO.
NF-κB sites are necessary for C. Crepidioides inducibility of iNOS promoter
To assess the effect of C. crepidioides on iNOS promoter activity, RAW264.7 cells were transfected with a murine iNOS-luciferase promoter/reporter construct and then incubated with various concentrations of C. crepidioides. The cells were lysed, and luciferase activity was measured. C. crepidioides increased the expression of luciferase from the iNOS promoter in a dose-dependent manner (Figure 5A). The expression of the iNOS gene in macrophages is regulated mainly at the transcriptional level, particularly by NF-κB [18–21]. The murine iNOS promoter contains two putative NF-κB binding sites, one upstream (GGGATTTTCC, −971 to −962 bp, designated NF-κB2) and one downstream (GGGACTCTCC, −85 to −76 bp, designated NF-κB1). To test the relative contribution of the NF-κB binding sites to the C. crepidioides-mediated activation of iNOS, we introduced a deletion into each or both sites. A single deletion of the κB2 site markedly inhibited C. crepidioides-mediated promoter activation, whereas a single deletion of the κB1 site resulted in moderate activation. On the other hand, double deletion completely abolished the C. crepidioides-mediated promoter activation (Figure 5B).
C. Crepidioides induces binding of NF-κB family proteins to two NF-κB sites
We next characterized the nuclear proteins in C. crepidioides-treated RAW264.7 cells that bind to sequences from the iNOS promoter in an NF-κB-dependent manner. EMSA was performed using two probes; miNOS κB1 and κB2, oligonucleotides consisting of the NF-κB1 and NF-κB2 elements. C. crepidioides induced a time-dependent appearance of nuclear proteins that bound to both probes in RAW264.7 cells (Figure 6A). In both probes, the addition of excess unlabeled κB1 and κB2 oligonucleotides to the binding reaction completely abolished the formation of inducible DNA-protein complexes (Figure 6B, lanes 2 and 9). In contrast, the formation of these DNA-protein complexes was not blocked by the addition of excess of unrelated oligonucleotide AP-1 (Figure 6B, lanes 3 and 10). To identify the NF-κB family members that bind to the NF-κB motifs of the murine iNOS gene promoter, the binding reactions were preincubated with antibodies specific to p50, p65, c-Rel and p52. The anti-p50 and anti-p65 antibodies induced the supershifted bands and reduced the intensity of complexes κB1 and κB2 (Figure 6B, lanes 4, 5, 11 and 12). The c-Rel antibody also supershifted complexes κB1 and κB2 (Figure 6B, lanes 6 and 13). These results indicate that the complexes κB1 and κB2 correspond to p50/p65/c-Rel.
NF-κB signal is essential for C. Crepidioides-induced iNOS expression
Does C. crepidioides-mediated upregulation of iNOS gene expression involve signal transduction components in NF-κB activation? Activation of NF-κB requires the phosphorylation of two conserved serine residues of NF-κB inhibitory subunit, IκBα (Ser32 and Ser36) within the N-terminal domain [22]. Phosphorylation leads to the ubiquitination and 26 S proteasome-mediated degradation of IκBα, thereby releasing NF-κB from the complex and its translocation to the nucleus and activation of various genes [22]. Next, we determined the role of IκBα phosphorylation and degradation in C. crepidioides-induced NF-κB translocation and activation by Western blot analysis using antibodies against phosphorylated and total IκBα. Treatment of RAW264.7 cells with C. crepidioides resulted in phosphorylation and degradation of IκBα within 30 min (Figure 6C).
Next, RAW264.7 cells were transfected with the luciferase reporter plasmid regulated by NF-κB elements (κB-LUC; [11]) and then incubated with C. crepidioides (Figure 6D). The results showed that C. crepidioides induced NF-κB activation. To further confirm the involvement of IκBα phosphorylation and degradation, we transfected the cells with transdominant mutant of IκBα in which two critical serine residues required for inducer-mediated phosphorylation were deleted [6]. Overexpression of mutant IκBα inhibited the C. crepidioides-induced iNOS promoter activation (Figure 6D), suggesting the involvement of IκBα phosphorylation and degradation in C. crepidioides-induced iNOS expression.
IκBβ also contains an N-terminal regulatory region required for stimulus-induced degradation, a key step in NF-κB activation [22]. Overexpression of mutant IκBβ in which two critical serine residues required for inducer-mediated phosphorylation were deleted [7], also inhibited the C. crepidioides-induced iNOS promoter activation (Figure 6D). These results suggest the involvement of IκBβ phosphorylation and degradation in C. crepidioides-induced iNOS expression.
The IKK complex consists of two catalytic subunits IKKα and IKKβ, and the regulatory subunit IKKγ [22]. In the NF-κB activation pathway, IKK complex can induce serine phosphorylation of IκBα and IκBβ [22]. Previous studies suggested the involvement of serine/threonine kinase NIK in phosphorylation and activation of the IKK complex [23]. To determine the roles of NIK, IKKα, IKKβ and IKKγ in C. crepidioides-induced iNOS expression in RAW264.7 cells, plasmids encoding dominant-negative mutants of NIK, IKKα, IKKβ and IKKγ were used. As shown in Figure 6D, transfection with dominant–negative mutants of NIK, IKKα, IKKβ and IKKγ significantly attenuated C. crepidioides-induced iNOS expression. Taken together, these findings demonstrate that C. crepidioides induces iNOS expression via activation of NF-κB mediated through the NIK/IKK pathway.
Isochlorogenic acid activates NF-κB
To determine the role of active compounds on C. crepidioides-induced iNOS expression, we examined whether isochlorogenic acid activates NF-κB. Treatment of RAW264.7 cells with isochlorogenic acid resulted in phosphorylation of IκBα (Figure 7A) and induction of nuclear proteins that bound to both miNOS κB1 and κB2 probes (Figure 7B). These results suggest that isochlorogenic acid in C. crepidioides seems to contribute at least in part to the induction of iNOS expression through IκBα phosphorylation and subsequent NF-κB activation.
Effects of NF-κB inhibitors on C. Crepidioides-induced iNOS expression and NO production
Because activation of the iNOS promoter by C. crepidioides required the activation of NF-κB, we blocked NF-κB activation with Bay 11-7082, an inhibitor of IκBα phosphorylation [24] or LLnL, a proteasome inhibitor [25]. Bay 11-7082 and LLnL reduced C. crepidioides-induced iNOS promoter activity (Figure 8A). In addition, Bay 11-7082 and LLnL diminished C. crepidioides-induced iNOS mRNA expression and NO production in RAW264.7 cells (Figure 8B and C).