Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo

Hepatocellular carcinoma (HCC), the major form of primary liver cancer, is a common cause of cancer-related death worldwide. Signal transducer and activator of transcription 3 (STAT3) signaling is constantly activated in HCC and has been proposed as a chemotherapeutic target for HCC. Antrodia camphorata (AC), a medicinal mushroom unique to Taiwan, is traditionally used for treating HCC. Whereas natural AC is scarce, cultured AC mycelia are becoming alternatives. In this study, we investigated the anti-HCC effects of the ethyl acetate fraction of an ethanolic extract of AC mycelia (EEAC), particularly exploring the involvement of STAT3 signaling in these effects. We found that EEAC reduced cell viability, induced apoptosis, and retarded migration and invasion in cultured HepG2 and SMMC-7721 cells. Immunoblotting results showed that EEAC downregulated protein levels of phosphorylated and total STAT3 and JAK2 (an upstream kinase of STAT3) in HCC cells. Real-time PCR analyses showed that STAT3, but not JAK2, mRNA levels were decreased by EEAC. EEAC also lowered the protein level of nuclear STAT3, decreased the transcriptional activity of STAT3, and downregulated protein levels of STAT3-targeted molecules, including anti-apoptotic proteins Bcl-xL and Bcl-2, and invasion-related proteins MMP-2 and MMP-9. Over-activation of STAT3 in HCC cells diminished the cytotoxic effects of EEAC. In SMMC-7721 cell-bearing mice, EEAC (100 mg/kg, i.g. for 18 days) significantly inhibited tumor growth. Consistent with our in vitro data, EEAC induced apoptosis and suppressed JAK2/STAT3 activation/phosphorylation in the tumors. Taken together, EEAC exerts anti-HCC effects both in vitro and in vivo; and inhibition of STAT3 signaling is, at least in part, responsible for these effects. We did not observe significant toxicity of EEAC in normal human liver-derived cells, nude mice and rats. Our results provide a pharmacological basis for developing EEAC as a safe and effective agent for HCC management.

specific for aflatoxins B 1 , B 2 , G 1 and G 2 . Passed 15 mL of the test solution through the column at a flow rate of about 6 mL/min. Wash the column twice with 10 mL H 2 O, followed by passing 2-3 mL of air. Next, 1.0 mL methanol was used as the eluting solution. Elute the column with 1.0 mL methanol at a flow rate of 1-2 mL/min. Collect all eluates. Transferred accurately 1.0 mL of elutes in a 2-mL volumetric flask and made up to the mark with distilled water for assay (diluted elutes). The concentrations of aflatoxins were determined by HPLC analysis. The chromatographic procedure was carried out using a mixture of methanol and distilled water (45+55) as the mobile phase and a flow rate of 0.8 mL/min. 0.05% iodine solution as post-column derivatization reagent, flow rate at 0.2 mL/min, reaction temperature at 70 ℃ and reaction time at 1 min were set as a post-column reactor system. 100 µL of a mixture of aflatoxins standard solution (benzene and acetonitrile 98:2, v/v) or diluted elutes were analyzed by HPLC. Water was used as control. Aflatoxin B1, B2, G1 and G2 were not detected in these mycelia.

Acute oral Toxicity Study
In acute oral toxicity study, 24 rats were randomly divided into two groups, 12 for each (6 females and 6 males). Rats were intragastrically administrated with a single dose of 5000 mg/kg body weight EEAC on day 1 and then observed for next 14 days. The signs of toxic effects and/or mortality were observed carefully every 1 h after administration on the first day, followed by daily observation. Body weights were recorded every week.

Repeated Dose 28-day Oral Toxicity Study
In repeated dose 28-day oral toxicity study, forty SD rats were randomly divided into four groups containing 10 rats for each (5 females and 5 males): control group, 250 mg/kg group, 500 mg/kg group and 1000 mg/kg group. And then EEAC was administrated to rats by gavage once a day for 30 consecutive days.

Measurement of Organ Indexes
SD Rats were sacrificed after blood sample were obtained and then gross observation was performed to analyze the macroscopic external features of organs, including heart, liver, spleen, lung, kidney, testis, ovary and uterus. These organs were carefully removed and then weighted individually. Organ index was calculated as follows: (organ weight/ body weight) ×100.

Supplementary Figure S2 Cytotoxic effects of EEAC on human HCC cells and normal human liver-derived (MIHA) cells.
Human HCC cells and MIHA cells were treated with various concentrations of EEAC for 24 h and cell viability was measured by the MTT assay. Data were shown as mean ± SD from three independent experiments.

Supplementary Figure S3 Effects of (25S)-Antcin H on the viability of HepG2 cells.
HepG2 cell were treated with indicated concentrations of (25S)-Antcin H for 48 h. Cell viability was assessed by MTT assays. Data were shown as mean ± SD from three independent experiments. **P<0.01 vs. the control.