Antitumor activity and macrophage nitric oxide producing action of medicinal herb, Crassocephalum crepidioides
© Tomimori et al.; licensee BioMed Central Ltd. 2012
Received: 8 February 2012
Accepted: 21 June 2012
Published: 21 June 2012
Crassocephalum crepidioides, a plant distributed in Okinawa Islands, is known in folk medicine; however, its anticancer activity has not been investigated. The aim of this study was to determine the in vitro and in vivo antitumor activities of C. crepidioides on murine Sarcoma 180 (S-180) and related molecular mechanisms.
The antitumor effect of C. crepidioides was evaluated in S-180-cell-bearing mice. Cell growth was assessed using a colorimetric assay. Nitrite and nitrate levels were measured by colorimetry. The expression levels of inducible NO synthase (iNOS) in murine RAW264.7 macrophages was assessed by reverse transcriptase-polymerase chain reaction. Activation of iNOS promoter was detected by reporter gene. Activation of nuclear factor-κB (NF-κB) was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling was analyzed using inhibitors of NF-κB and dominant-negative mutants, and Western blot analysis.
C. crepidioides extract delayed tumor growth in S-180-bearing mice. However, it did not inhibit S-180 cell growth in vitro. Supernatant of cultured C. crepidioides-stimulated RAW264.7 macrophages was cytotoxic to S-180 cells. This cytotoxicity was associated with nitric oxide (NO) production. NF-κB signaling pathway was crucial for the transcriptional activation of iNOS gene. Isochlorogenic acid, a component of C. crepidioides, induced NF-κB activation and iNOS expression.
The results highlight the oncolytic and immunopotentiation properties of C. crepidioides mediated through NF-κB-induced release of NO from macrophages.
Plants contain several classes of phytochemicals that have antioxidative, antimutagenic and anticarcinogenic effects. The edible plant, Crassocephalum crepidioides S. MOORE (Japanese name; Benibanaborogiku), is wildly distributed in the Okinawa Islands and used in folk medicine for the treatment of acute hepatitis, fever and edema. Compounds with antimalarial activity and strong antimutagenicity had been isolated previously from C. crepidioides[1, 2]. Other preparations from C. crepidioides have been described to have antioxidant and hepatoprotective properties . However, the anticancer activity of C. crepidioides has not been investigated.
This is the first study that examines the antitumor effects of C. crepidioides extract. Although this extract is effective in inhibiting the in vivo growth of implanted Sarcoma-180 (S-180) cells, it did not inhibit the growth of the same cells in vitro. Activation of macrophages by agents such as bacterial lipopolysaccharide (LPS) stimulates their growth inhibitory effects against a wide variety of tumor cells . In the present study, we report that C. crepidioides extract can induce the production of nitric oxide (NO), a major mediator of the tumoricidal activity of murine macrophages. In addition, serum nitrite and nitrate levels were significantly elevated in mice administered C. crepidioides extract compared with levels in the control group. Specifically, we characterized the mechanisms of the actions of C. crepidioides extract on inducible NO synthase (iNOS) promoter in murine macrophages. The antitumor efficacy of the extract was based on immunopotentiation, mediated, at least in part, by isochlorogenic acid.
Fresh C. crepidioides was harvested in Subtropical Field Science Center of the University of the Ryukyus, Okinawa, Japan, and air-dried. Dried C. crepidioides (50 g) was extracted twice with 500 ml of boiling water for 30 min and the supernatant was decanted. After filtration, the combined supernatants were evaporated in vacuum and finally lyophilized to the powder. The extract obtained was used as an original extract, and dissolved with pure water when necessary. Isochlorogenic acid was purified using the procedure described previously with some modifications . The extract dissolved in pure water was applied to a HP-20 (Mitsubishi Chemical, Tokyo, Japan) column eluting water and increasing amount of methanol (MeOH) to yield 70% MeOH fraction. After passing the fraction through C18 Sep-Pak cartridge (Waters, Millford, MA, USA), the final purification of the fraction was carried out by a Toyopearl HW-40 C (Tosho, Tokyo, Japan) column with 50% MeOH as an eluent. The 50% MeOH fraction contained 94% of isochlorogenic acid by absorption at 320 nm, following separation by reversed phase HPLC on C18 column (Nomura Chemical, Seto, Japan). C. crepidioides was dissolved in Dulbecco’s modified Eagle’s medium to a final concentration of 20 mg/ml.
Antibodies to nuclear factor-κB (NF-κB) subunits p65, p50, c-Rel and p52 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to actin was purchased from NeoMarkers (Fremont, CA, USA). Antibodies to IκBα and phospho-IκBα (Ser32 and Ser36) were obtained from Cell Signaling Technology (Beverly, MA, USA). N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL) and Bay 11-7082 were purchased from Sigma-Aldrich (St Louis, MO, USA) and Calbiochem (La Jolla, CA, USA), respectively.
In vivo therapeutic effect of C. Crepidioides
Four-week-old female BALB/c strain athymic nu/nu mice were obtained from Ryukyu Biotec Co. (Urasoe, Japan). They were engrafted with 2 × 105 S-180 cells by subcutaneous injection in the back region. Treatment was initiated on the day of cell inoculation. C. crepidioides was dissolved in distilled water at a concentration of 333 mg/ml, and 5 g/kg body weight of C. crepidioides was administered by oral gavage every day for 29 days. Tumor size was monitored once a week. All mice were sacrificed on day 28 and the tumors dissected out immediately. Tumors were fixed for paraffin embedding and tissue sectioning, and evaluated histologically using hematoxylin and eosin (H&E). This experiment was performed according to the guidelines for Animal Experimentation of the University of the Ryukyus and approved by the Animal Care and Use Committee of the same University.
The mouse sarcoma cell line S-180 and macrophage cell line RAW264.7 were cultured in Eagle’s Minimum Essential Medium and Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, respectively.
Assays for cell growth
S-180 and RAW264.7 cells were seeded on 96-well plates and cultured for 24 h. C. crepidioides was added at various concentrations and incubated for 24, 48 and 72 h. In addition, the culture media were removed and replaced by supernatants from C. crepidioides-stimulated RAW264.7 cells. The growth of cells was evaluated by measuring the mitochondrial-dependent conversion of the water-soluble tetrazolium (WST)-8 (Nacalai Tesque, Kyoto, Japan) to a colored formazan product . After 24, 48 and 72 h of culture, WST-8 (5 μl) was added to each well containing cultured cells in the last 4 h of incubation. Absorbance at 450 nm was measured using an automated microplate reader. The cell growth in untreated control cultures was considered 100%, and the growth of each treated group was compared relative to this value.
Measurement of NO
Nitrite (NO2−), the stable end product of NO was measured in the supernatants by the colorimetric assay. Briefly, the medium was removed from individual wells and treated with Griess reagent (1% sulphanilamide and 0.1% naphtylethylene diamine dihydrochloride in 2% H3PO4) for 10 min at room temperature. The optical density of the samples was obtained using an automated microplate reader at 550 nm. A standard curve using a standard solution of NaNO2 in culture medium was employed to calculate the nitrite concentration. The levels of nitrite (NO2−) and nitrate (NO3−) anions derived from NO in murine sera were measured by an ENO-20 NO analyzer (EiCOM, Kyoto, Japan).
Western blot analysis
Cells were lysed and equal amounts of protein (20 μg) were subjected to electrophoresis on sodium dodecyl sulphate-polyacrylamide gels followed by transfer onto a polyvinylidene difluoride membrane and probing with the specific antibodies. The bands were visualized with an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA). The reported results were obtained from at least two independent experiments with a similar pattern.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total cellular RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 1 μg total cellular RNA using an RNA-PCR kit (Takara Bio Inc., Otsu, Japan) with random primers. The primers used were 5’-TCATTGTACTCTGAGGGCTGACACA-3’ (forward) and 5’-GCCTTCAACACCAAGGTTGTCTGCA-3’ (reverse) for murine iNOS, and 5’-GTGGGGCGCCCCAGGCACCA-3’ (forward) and 5’-CTCCTTAATGTCACGCACGATTTC-3’ (reverse) or β-actin. The length of RT-PCR was 25 cycles for iNOS and 28 cycles for β-actin. The PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining.
Transfection and luciferase assay
The IκBαΔN- and IκBβΔN-dominant-negative mutants are IκBα and IκBβ deletion mutants lacking the N-terminal 36 and 23 amino acids, respectively [6, 7]. The dominant-negative mutants of IκB kinase (IKK)α, IKKα (K44M), IKKβ, IKKβ (K44A), IKKγ, IKKγ (1-305) and NF-κB-inducing kinase (NIK), NIK (KK429/430AA) have been described previously [8, 9]. pGL3 iNOS plasmid was generated by inserting the murine iNOS promoter region (−1588 to +161 bp surrounding the transcription start site) into the pGL3-basic vector (Promega, Madison, WI, USA) . Three internal deletion mutants, pGL3 iNOS κB2−, pGL3 iNOS κB1− and pGL3 iNOS κB1/κB2−, were constructed by deletion of two NF-κB sites defined as the κB1 (−85 to −76) and κB2 (−971 to −962). For reporter assays, an NF-κB site-dependent luciferase vector, κB-LUC  was also used. RAW264.7 cells were plated and transfected with the appropriate reporter and effector plasmids using Lipofectamine reagent (Invitrogen). After 18-20 h, C. crepidioides was added and incubated for 6 h. The cells were lysed in reporter lysis buffer (Promega). Lysates were assayed for reporter gene activity with the dual-luciferase assay system (Promega). Luciferase activities were normalized relative to the Renilla luciferase activity from phRL-TK.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were obtained as described by Antalis and Godbolt  with modifications, and EMSA was performed as described previously . The probes used were prepared by annealing the sense and antisense synthetic oligonucleotides; a κB1 site from the murine iNOS gene (5’-tcgaCCAACTGGGGACTCTCC CTTTGGGAA-3’), a κB2 site from the murine iNOS gene (5’-tcgaTGCTAGGGGGATTTTCC CTCTCTCTG-3’) and an AP-1 element of the interleukin (IL)-8 gene (5’-gatcGTGATGACTCA GGTT-3’). The above underlined sequences represent the NF-κB or AP-1 binding site. The reported results were obtained from at least two independent experiments with a similar pattern.
Data are expressed as mean ± SD. Differences between groups were assessed for statistical significance by the Mann-Whitney’s U-test. A P value < 0.05 denoted the presence of a statistically significant difference.
Effects of C. Crepidioides in nude mice inoculated with S-180 cells
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 . 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).
C. Crepidioides induces NO production and expression of iNOS mRNA
NF-κB sites are necessary for C. Crepidioides inducibility of iNOS promoter
C. Crepidioides induces binding of NF-κB family proteins to two NF-κB sites
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 . 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 . 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; ) 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 . 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 . Overexpression of mutant IκBβ in which two critical serine residues required for inducer-mediated phosphorylation were deleted , 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 . In the NF-κB activation pathway, IKK complex can induce serine phosphorylation of IκBα and IκBβ . Previous studies suggested the involvement of serine/threonine kinase NIK in phosphorylation and activation of the IKK complex . 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
Effects of NF-κB inhibitors on C. Crepidioides-induced iNOS expression and NO production
Alternative therapeutic tools obtained from plants to fight cancer have attracted great interest. This is the first study to report the antitumor effect of C. crepidioides extract in S-180 cell-bearing mice. The results demonstrated the effectiveness of C. crepidioides in inhibiting the growth of implanted S-180 cells, although this effect was not mirrored in the in vitro studies. Activated macrophages are important in host defense against tumors, including tumor cytotoxicity. NO appears to be a major mediator of macrophage tumoricidal activity [14–16]. NO secreted by activated macrophages inactivates iron-containing enzymes critical to the viability of tumor cells . The present study was designed to determine whether C. crepidioides extract activated macrophages to the tumoricidal phenotype.
The results demonstrated that C. crepidioides extract alone did not inhibit S-180 cell growth in vitro. However, the supernatant from C. crepidioides-treated RAW264.7 cells inhibited S-180 cell growth. This antitumor activity was associated with NO production in activated RAW264.7 cells, and administration of C. crepidioides in mice induced a significant increase in serum nitrite and nitrate levels. Thus, NO seems a significant component of the pathway responsible for tumor regression. In this context, it should be emphasized that this experimental approach only allows the study of the effects mediated by soluble mediators. Other interactions involving direct contact between S-180 cells and neighboring macrophages will have additional effects on S-180 cells, modulating the proliferation and apoptosis of S-180 cells.
Another part of the present study investigated the molecular mechanism of C. crepidioides-induced NO production. NO formation is catalyzed by iNOS from L-arginine. Macrophage iNOS is not expressed in resting cells and differs from the constitutive neuronal and endothelial NOS . Extensive studies of the transcriptional control of iNOS expression in murine macrophages have stressed the importance of binding of NF-κB [18–21], IRF-1 , Stat1  and Oct-1 [29, 30] to their recognition sequences on the iNOS promoter region for activation of iNOS transcription by interferon γ, LPS, IL-6 and Taxol. We showed here that C. crepidioides rapidly activated NF-κB. Moreover, we confirmed the essential role of NF-κB in C. crepidioides-induced iNOS promoter activity using deletion mutant forms of the two NF-κB recognition sites in the iNOS promoter, termed NF-κB1 and NF-κB2. Nuclear protein complexes that bind specifically to NF-κB1 and NF-κB2 after treatment of RAW264.7 cells with C. crepidioides contained p50/p65/c-Rel. The components of the NF-κB complexes formed in response to LPS consisted primarily of p50, with very low levels of p65 and c-Rel subunits [18, 21]. These results support the view that LPS and C. crepidioides extract activate NF-κB through the same signaling pathway. Because various phytochemicals have been shown to suppress LPS-induced iNOS expression , we examined the effects of C. crepidioides extract on LPS-induced iNOS mRNA expression and NO production. However, C. crepidioides failed to suppress both events induced by LPS (data not shown).
Bay 11-7082 and LLnL are relatively specific inhibitors of NF-κB activation [24, 25]. Both agents blocked the promoter activity of iNOS, the expression of iNOS mRNA, and the production of nitrite, indicating the likely involvement of NF-κB in the induction of not just iNOS-driven reporter constructs but also the iNOS gene itself in C. crepidioides-treated macrophages. We also confirmed the important role of NF-κB and the upstream target of C. crepidioides by showing that overexpression of dominant-negative potent inhibitors of NF-κB activation (NIK, IKKs and IκBs) inhibited C. crepidioides-induced activation of iNOS promoter.
Compounds with antioxidant activities have been isolated previously from C. crepidioides, including isochlorogenic acid and the flavonoids quercetin and kaempferol. However, the latter two have anti-inflammatory properties and are known to inhibit LPS-induced iNOS expression and NO production [32, 33]. For this reason, isochlorogenic acid was selected in this study. Results indicated that isochlorogenic acid induces NF-κB activation and subsequent iNOS induction, and thus contributes, at least in part, to the tumoricidal effects of C. crepidioides.
Considered together, our results indicated that C. crepidioides causes regression of murine S-180 tumor and that this effect is mediated by activated macrophages through NO production. These results suggest that C. crepidioides is an interesting plant for the development of novel anticancer agents. However, further studies are needed to determine the effects of C. crepidioides extract and isochlorogenic acid on tumor growth in patients with malignant tumors.
We reported in the present study the suppressive effects of C. crepidioides extract on tumor growth in S-180 model through the induction of NO production. Also, the extract stimulated macrophages and induced NO production via NF-κB signaling pathway. NO production may play an important role in the antitumor activity of C. crepidioides on S-180 cells. The results suggest that C. crepidioides is a potentially useful chemopreventive and chemotherapeutic agent, thus justifying further investigation of other possible beneficial biological properties.
We thank Drs. Kouji Wada and Yoko Aniya for the generous supply of C. crepidioides extract and isochlorogenic acid, and for assistance with the preparation of the manuscript. We acknowledge also Drs. Tamara K. Nowling, Dean W. Ballard, Romas Geleziunas and Kuan-Teh Jeang for providing pGL3 iNOS plasmid; for the expression vectors for IκBα- and IκBβ-dominant-negative mutants; for NIK-, IKKα- and IKKβ-dominant-negative mutants; and for IKKγ-dominant-negative mutant. We also thank Dr. Shigeki Sawada for conducting the pathological examination.
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