2021 年 44 巻 5 号 p. 686-690
Although more than 400 species of Cordyceps s.l. have been identified, most have not been well explored regarding their potential for medicinal use. In this study, the profiles of constituents of ten different species of Ophiocordyceps, which is an unexplored species of Cordyceps, were analyzed and their anti-tumor effects were further examined. Although all Ophiocordyceps samples exhibited similar peak patterns, Ophiocordyceps gracilioides (O. grac) had a distinct constituent profile from the other samples. Furthermore, O. grac was the most active in suppressing the transcriptional activities of both nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription (STAT)3, and the production of interleukin (IL)-6 from breast cancer cells. This study demonstrated that O. grac is a relatively unexplored Cordyceps s.l. that may have medicinal potential to inhibit the NFκB-STAT3-IL-6 inflammatory pathway in cancer.
Cordyceps s.l. are parasitic ascomycete fungi in traditional medicine. Among many Cordyceps s.l., Ophiocordyceps sinensis or Cordyceps militaris, known as “To-chu-ka-so” in Japan, have been extensively examined regarding their medicinal use.1–3) Several biological activities and pharmacological effects of Cordyceps s.l. have been reported, including liver function improvement,4) anti-cancer,5) anti-oxidation,6) anti-aging,7) and anti-diabetic activities.8) It was also suggested that Cordyceps can be used to support conventional cancer therapies, such as surgery, radiation therapy, and chemotherapy to reduce their side effects.9,10) Although several bioactive ingredients of Cordyceps s.l. are known,11) cordycepin, a derivative of the nucleoside adenosine, is the major constituent of C. militaris underlying its medicinal effects.12) Regarding its anti-cancer activity, Cordyceps s.l. induces apoptosis or cell cycle arrest in cancer cells, and reduces metastasis in experimental animal models.13,14) In addition, inhibition of the nuclear factor-kappa B (NF-κB) signaling pathway or the down-regulation of tumor-promoting inflammatory cytokine expression in cancer cells may be the molecular mechanism of the anti-cancer effects of Cordyceps s.l.15,16)
Inflammatory genes are the key players in tumor progression and metastasis, and are known to be controlled by transcription factors such as signal transducer and NF-κB or activator of transcription 3 (STAT3).17,18) As the inflammatory cytokine interleukin (IL)-6 is a potent activator of STAT3 and the main target gene of NF-κB,19) the NFκB-STAT3-IL-6 inflammatory pathway is considered an attractive pharmacological target to control the pathogenesis of many types of tumors.
Although more than 400 species of Cordyceps s.l. have been identified, most have not been well explored regarding their potential for medicinal use. In this study, the profiles of constituents of ten different species of Ophiocordyceps, which is an unexplored species of Cordyceps s.l., were analyzed using total ion chromatography and their anti-tumor effects were examined further by targeting the NFκB-STAT3-IL-6 inflammatory pathway.
Ophiocordyceps samples were originally collected from across Japan and isolated by the surface sterilization method20) or ejected ascospores. Based on the arrangement of the internal transcribed spacer using DNA sequencing and/or morphological features,21) 10 different species of Ophiocordyceps were identified (Table 1). To prepare the samples of Ophiocordyceps, the mycelium were incubated in a Sabouraud glucose liquid medium at 23 °C in a dark room. Thereafter, the cultures were subjected to filtration and lyophilized. The lyophilized cultures were used for the subsequent experiments by dissolving in relevant solvents.
| Sample No. | Scientific name | Region of collection in Japan |
|---|---|---|
| 1 | Ophiocordyceps cf. elongatistromata | Tomakomai, Hokkaido |
| 2 | Ophiocordyceps entomorrhiza | Toyama, Toyama |
| 3 | Ophiocordyceps gracilioides | Toyama, Toyama |
| 4 | Ophiocordyceps heteropoda | Toyama, Toyama |
| 5 | Ophiocordyceps neovolkiana | Nantan, Kyoto |
| 6 | Ophiocordyceps nikkoensis | Tomakomai, Hokkaido |
| 7 | Ophiocordyceps nutans | Toyama, Toyama |
| 8 | Ophiocordyceps sobolifela | Kanazawa, Ishikawa |
| 9 | Ophiocordyceps sphecocephala | Toyama, Toyama |
| 10 | Ophiocordyceps tricentri | Toyama, Toyama |
The HPLC analysis was performed on an Agilent 1260 Infinity II Series (Agilent, Waldbronn, Germany) consisting of a binary pump (G7112B), a vialsampler (G7129A), and a 1290 photodiode array detector (G7117B). Chromatography was carried out on a TSK-gel ODS-80Ts (4.6 × 150 mm, 5 µm) column (Tosoh Co., Tokyo, Japan). The following gradient was used: mobile phase: water (solvent system A) and methanol (solvent system B) in a gradient mode (B from 0 to 90% in 60 min), with a flow rate of 0.5 mL/min.
CellsMurine mammary carcinoma 4T1 cells were obtained from ATC C (VA, U.S.A.) and the 4T1 luciferase reporter-expressing cell lines (4T1-NFκB-luc2 and 4T1-STAT3-luc2) were established previously.22–24) Cells were maintained in RPMI 1640 (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences, Tokyo, Japan), 0.2% (w/v) NaHCO3, 1 mM L-glutamine (Life Technologies, Gaithersburg, MD, U.S.A.), and antibiotics (100 units/mL of penicillin and 100 mg/mL of streptomycin; Meiji Seika Pharma Co., Ltd., Tokyo, Japan). All cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
Luciferase Reporter Assay4T1-NFκB-luc2 cells and 4T1-STAT3-luc2 cells were seeded at 1 × 104 cells per well in RPMI 1640 containing 10% FBS in 96-well plates (Ref 655090; Greiner Bio-One, Kremsmünster, Austria) and incubated overnight. Cells were further incubated with or without samples for another 24 h, and then D-luciferin was added at a final concentration of 150 µg/mL for 30 min. The luciferase activity was measured using the imaging system (IVIS Lumina II; Caliper Life Sciences, Hopkinton, MA, U.S.A.). The viability of cells was determined using the Cell Counting Kit-8 (CCK-8) assay kit (Dojindo Co., Kumamoto, Japan) according to the manufacturer’s instructions. The absorbance at 450 nm and reference at 620 nm were measured using a Sunrise plate reader (Tecan, Grödig, Austria).
Western BlottingCells (1 × 106 cells per well) were seeded and grown for 24 h in 6-well plates. After treatment, the cells were rinsed in cold phosphate-buffered saline (PBS), scraped, and lysed in whole-cell lysis buffer (25 mmol/L N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.7, 300 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 20 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonylfluoride, 1 mmol/L dithiothreitol, 10 mg/mL aprotinin, and 10 mg/mL leupeptin). Cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Merck Milipore, Darmstadt, Germany). The membranes were treated with Block Ace (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) for at least 4 h, and probed with the indicated primary antibodies overnight, followed by horseradish peroxidase-conjugated secondary antibodies (DAKO, Glostrup, Denmark). Bands were visualized using Pierce™ enhanced chemiluminescence (ECL) Western blotting Substrate (Thermo Scientific, Waltham, MA, U.S.A.). Primary antibodies used (at a dilution of 1 : 1000) were specific to STAT3 (79D7, #4904), p-STAT3 (Tyr705) (Y705, #9131), p-STAT3 (Ser727) (S727, #9134), p65 (L8F6, #6956), p-p65, (Ser536) (93H1, #3033) (Cell Signaling Technology, Beverly, MA, U.S.A.), and β-actin (C4, sc-47778, Santa Cruz, CA, U.S.A.).
Measurement of IL-6 Production4T1 cells (2 × 105 cells per well) were plated on 24-well plates and allowed to adhere overnight. Cells were treated with Ophiocordyceps gracilioides (O. grac) for 24 h and the supernatant was collected. IL-6 standards in different concentrations were diluted in 1% bovine serum albumin (BSA) in PBS, and supernatants were diluted 1 : 10 in media. Secreted IL-6 levels in culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA) MAX™ Standard Set Mouse IL-6 (BioLegend, San Diego, CA, U.S.A.).
Statistical AnalysisData were analyzed using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, U.S.A.). Significantl differences between groups were analyzed by one-way ANOVA with Dunnett’s test. A p-value less than 0.05 was considered to indicate a significant difference. Data are expressed as the mean ± standard deviation (S.D.) of three independent experiments.
In order to profile the constituents of 10 different Ophiocordyceps (Table 1), each sample was subjected to total ion chromatography (TIC) on HPLC. Although all Ophiocordyceps samples exhibited similar peak patterns from 0 to 12 min, sample number 3 (Ophiocordyceps gracilioides) had a distinct peak at a retention time of 39.4 min from the other samples (Fig. 1A). As a reference, a peak of cordycepin (at a retention time of 14.8 min determined using standard compounds) is shown in Fig. 1B.
All samples were dissolved in methanol to 5 mg/mL, and different chromatography patterns of several species of Ophiocordyceps were identified using HPLC (A). Detection on TIC and diode array detector (DAD) chromatography of cordycepin was at 254 nm (B).
To examine the anti-tumor effects of 10 different Ophiocordyceps, we first investigated their effects on the transcriptional activity of NF-κB and STAT3 using 4T1 luciferase reporter cell lines. By measuring both luciferase activity and cell viability in 4T1-NFκB-luc2 and 4T1-STAT3-luc2 cells, we compared the relative inhibitory activity of Ophiocordyceps on both NF-κB and STAT3. As shown in Fig. 2, sample number 3 inhibited both NF-κB and STAT3 activity stronger than other tested samples. As sample number 3, O. grac, had the most significant cytotoxic effects on 4T1 cells, we further examined its anti-tumor effects of O. grac.
4T1-NFκB-luc2 and 4T1-STAT3-luc2 cells were plated at a density 1 × 104 cells/well in 96-well plates and treated with or without 10 species of Ophiocordyceps (500 µg/mL) for 24 h. D-Luciferin (150 µg/mL) was added and incubated for another 30 min, and the luminescence was measured. The cell viability of the culture was determined using CCK-8 reagent. The relative activity of NF-κB and STAT3 was calculated by dividing the luminescence by the cell viability in each culture. Results are presented as the mean ± standard deviation (S.D.) (n = 3). Significance was assessed by one-way ANOVA with Dunnett’s test. * p < 0.05 versus control.
As shown in Fig. 3, O. grac inhibited the transcriptional activities of NF-κB (Fig. 3A) and STAT3 (Fig. 3B) in a dose-dependent manner in 4T1 breast cancer cells even at a dose where not significantly inhibit cell viability (at 62.5 µg/mL, Fig. 3C). In addition to the inhibition of NF-κB and STAT3 transcriptional activities, O. grac inhibited the phosphorylation of p65 and STAT3 (Figs. 4A, B). This suggests that O. grac inhibit the activation of NF-κB and STAT3 in 4T1 cells.
4T1-NFκB-luc2 cells (A) or 4T1-STAT3-luc2 cells (B) were treated with the indicated dose of O. grac for 24 h. D-Luciferin (150 µg/mL) was added and incubated for another 30 min, and the luminescence was measured. The cell viability of the culture was determined using CCK-8 reagent (C). The relative activity of NF-κB and STAT3 was calculated by dividing the luminescence by the cell viability in each culture. Results are presented as the mean ± S.D. (n = 3). Significance was assessed by one-way ANOVA with Dunnett’s test. * p < 0.05 versus control (0 µg/mL).
4T1 cells (1 × 106 cells/well) were seeded in 6-well plates and treated with the indicated dose of O. grac for 24 h, and equal amounts of protein in cell lysates were analyzed by Western blotting (A). The β-actin protein levels were used to confirm that equal amounts of protein were subjected to electrophoresis. The band intensities of phosphorylated proteins were assessed relative to non-phosphorylated proteins using ImageJ software (B).
We next evaluated the consequence of NF-κB and STAT3 inhibition by O. grac by measuring IL-6 production by 4T1 cells, which is known to be regulated by NF-κB and STAT3. As shown in Fig. 5, IL-6 production by 4T1 cells was significantly inhibited by O. grac in a dose-dependent manner, suggesting potential anti-tumor effects of O. grac through inhibition of the NFκB-STAT3-IL-6 inflammatory pathway.
4T1 cells (2 × 105 cells/well) were seeded in 24-well plates and treated with the indicated dose of O. grac. After 24 h, culture supernatants were collected and cytokine IL-6 was quantified using the ELISA kit according to the manufacturer’s instructions. The data are presented as the mean ± S.D. (n = 3). Significance was assessed by one-way ANOVA with Dunnett’s test. * p < 0.05
In this study, we examined 10 different species of Ophiocordyceps regarding their anti-tumor activity by targeting NF-κB and STAT3 activities in 4T1 breast cancer cells. Among these, O. grac was the most active in inhibiting the NFκB-STAT3-IL-6 inflammatory pathway.
Cordyceps s.l. has been used in traditional medicine in East Asian countries such as Japan, China, and Korea.25,26) O. sinensis and C. militaris are the best-known and widely used species of Cordyceps s.l. with medicinal activities.27,28) Cordycepin is one of the most representative active components of Cordyceps s.l., and exerts anti-tumor effects by inducing apoptosis through multiple pathways,12) including the inhibition of NF-κB phosphorylation.29) Regarding the potential role of cordycepin in the anti-tumor activity of O. grac, there was no peak of cordycepin (at a retention time of 14.8 min determined using standard compounds) observed in HPLC analysis of O. grac; therefore we concluded that O. grac has unique active components. Ophiocordyceps is genus of Ophiocordycipitaceae,30) and Cordyceps gracilioides (syn. Ophiocordyceps gracilioides) was first described by Kobayasi in 1941,31) segregated into Ophiocordyceps gracilioides in 2007 by Sung et al.,32) and recently distinguished to Paraisaria by Mongkolsamrit in 2019 based on multigene phylogenies.33) Therefore, O. grac belongs to the Paraisaria clade of Ophiocordyceps, which attacks the larval stages of Coleoptera (Elateridae). The sample of O. grac used in this study exhibited morphological characteristics of single stroma, globose, fertile head, and immersed perithecia, consistent with the previously reported features of this species.33,34) Contrary to Ophiocordyceps sinensis or Cordyceps militaris, the medicinal use of Ophiocordyceps has not been extensively examined, although there is one report of the anti-tumor effects of Ophiocordyceps sobolifela (same species as sample No. 8 in this study).35) Our study demonstrated that O. grac is the most active in inhibiting the NFκB-STAT3-IL-6 inflammatory pathway; therefore, further exploration of the use of O. grac is expected for cancer treatment.
This study was supported by a Grant from the Frontier Research Program of Toyama Prefecture. M-K.S. was supported by a scholarship from the Rotary Yoneyama Memorial Foundation.
The authors declare no conflict of interest.
