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
−), 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’-tcgaCCAACTGGGGACTCTCCCTTTGGGAA-3’), a κB2 site from the murine iNOS gene (5’-tcgaTGCTAGGGGGATTTTCCCTCTCTCTG-3’) and an AP-1 element of the interleukin (IL)-8 gene (5’-gatcGTGATGACTCAGGTT-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.