Anti-Rad51 and anti-excision repair cross-complementation group 1 (ERCC1) antibodies were purchased from Abcam (Cambridge, MA, USA). Cyclin B1, cyclin A2, cyclin D1, cleaved-PARP (Asp214), phospho-p53 (Ser15), phospho-cdc2 (Tyr15), LC3A/B, sequestosome 1 (SQSTM1)/p62 and cyclin-dependent kinase 2 (CDK2) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-cyclin-dependent kinase 4 (CDK4) and anti-cyclin-dependent kinase 6 (CDK6) were obtained from Bethyl (Montgomery, TX, USA). Cyclin E, Bcl-2, Bcl-x_L, Ku70, Ku80, anti-p53, survivin, DNA-PKcs, glyceraldegyde-3-phosphate dehydrogenase (GAPDH), horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-Histone H2AX (γ-H2AX, Ser139) was provided by Millipore (Billerica, MA, USA). Alexa Fluor^® 488 goat anti-mouse IgG (H+L) and Alexa Fluor 594 goat anti-rabbit IgG (H+L) secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Roswell Park Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS), and antibiotics (penicillin and streptomycin) were obtained from Welgene (Daegu, South Korea).

The combined mixture of seven mushrooms and P. ginseng root extracts (also called Amex7) used in this study was obtained from Han Kook Shin Yak Pharmaceutical Co., Ltd. In the first step, P. ginseng root was extracted with purified water at 80–100°C for 2–3 h. The water extract from P. ginseng root was filtered through a 150-µm pore size, and then evaporated in a vacuum below 60°C. In the second step, the extract of P. ginseng was mixed with extract powders of Phellinus linteus, Lentinula edodes, Trametes versicolor, Cordyceps militaris, Sparassis crispa, Hericium erinaceum, and Grifola frondosa at 200 rpm for 20 min. The mixture was filtered through a 5-µm pore size and subsequently sterilized at 98°C for 10 min. Fig. 1 shows the preparation process of Amex7 and Table I lists the combined ratio of several compounds in Amex7.

HT-29 human colorectal cancer cells were obtained from the Korean Cell Line Bank (Seoul, South Korea). HT-29 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin and incubated in an atmosphere of 5% CO₂ at 37°C.

Amex7, a combined mixture of several medicinal mushrooms and P. ginseng extracts, was kindly provided by Han Kook Shin Yak Co., Ltd. For in vivo experiments, Amex7 was freshly mixed with drinking water at 1.25, 6.25, and 12.5 ml/kg. For in vitro experiments, Amex7 was suspended at a concentration of 1, 2, 4, 8, and 16% in culture medium.

Balb/c nude mice (5-week-old males) were obtained from Nara Biotech Co. (Seoul, South Korea) and maintained in a laminar airflow cabinet under specific pathogen-free conditions. Mice xenografted with HT-29 cells were obtained by subcutaneous inoculation of 3×10⁶ HT-29 cells into the right hind leg. After tumor implantation, we monitored the condition of the animals twice daily and prepared analgesics to minimize animal suffering. When the tumor attained a volume of approximately 180–200 mm³, the mice were randomly divided into four groups (n=10): a) control, b) 1.25 ml/kg, c) 6.25 ml/kg, and d) 12.5 ml/kg. The tumor volume (V) was calculated using the standard formula: V (mm³) = π/6 × (shortest diameter)² × (longest diameter). Mice were euthanized by CO₂ inhalation when the average tumor volume of the control group reached 2,000 mm³.

Cells were seeded in 60-mm culture dishes at 5×10⁴ per well and cultured overnight. Then, the cells were treated with serial concentrations of Amex7 (0, 1, 2, 4, 8, and 16%) for 6, 12, and 24 h. At the indicated time points, cells were washed with phosphate buffered saline (PBS), harvested, and immediately stained with 0.4% trypan blue. Viable cells were quantified using a hemocytometer under an inverted microscope. Cell viability of control group was set at 100% and the effects of Amex7 on cell viability were expressed relative to the control group.

Cells were seeded at a density of 3×10⁶ cells/dish in a 100π dish and incubated for 24 h. The cells were treated with 4% Amex7 and incubated for a further 6, 12, and 24 h. The cells were observed by light microscopy (×200) (Motic AE31 microscope, Ted Pella Inc., Redding, CA, USA).

Cells were treated with 4% Amex7, incubated for 6, 12, and 24 h, harvested, stained with propidium iodide (1 mg/ml) according to the manufacturer's protocol, and then analyzed using a FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). A minimum of 10,000 cells were counted for each sample and data analysis was performed using the CellQuest software.

The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and the proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes blots were blocked with 5% (v/v) skim milk in PBS with 0.1% Tween-20, incubated with the indicated primary (1:1,000 dilution) and secondary antibodies (1:1,000 dilution), developed using enhanced chemiluminescence (ECL) immunoblotting substrate (Pierce, Rockford, IL, USA), and imaged with the ImageQuant LAS-4000 mini (GE Healthcare, Fairfield, CT, USA). The signal intensity of the bands was measured with the Multi Gauge image analysis software (Fujifilm, Tokyo, Japan).

HT-29 cells were seeded on coverslips and grown overnight. After treatment with 4% Amex7 for 6, 12, and 24 h, the cells were washed and fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed again, permeabilized in PBS/0.4% Triton X-100 for 10 min at room temperature, and blocked in PBS/4% FBS for 1 h at room temperature. The coverslips were stained with primary anti-γ-H2AX (1:500 dilution) antibody, incubated overnight at 4°C, and then stained with Alexa Fluor 488 Goat anti-Mouse IgG (H+L) secondary antibody and DAPI for 1 h at room temperature. The coverslips were washed and mounted in VectaMount™ mounting medium (Vector Laboratory, Inc., Burlingame, CA, USA).

All animal protocols and studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korean Institute of Radiological and Medical Sciences (KIRAMS 2015-0058).

Results from the in vivo study are expressed as the mean ± standard error of the mean (SEM) and those from the in vitro study were plotted as the mean ± standard deviation (SD). The statistical analysis was performed using a Student's t-test and one-way ANOVA followed by Tukey's HSD test using the statistical package for the social sciences (SPSS) software (version 23.0; Chicago, IL, USA). The level of statistical significance was set at a p<0.05.

We evaluated the in vivo anticancer effect of Amex7 at various concentrations (1.25, 6.25, and 12.5 ml/kg) in mice xenografted with HT-29 cells. When the mean tumor volume in the control group reached approximately 2,000 mm³, the tumor volume in the mice treated with Amex7 at 1.25, 6.25, and 12.5 ml/kg was 46.7±1.5, 46.4±1.2 and 43.9±1.8% smaller, respectively, than the control (Fig. 2A). A significant concentration-independent anticancer effect (p<0.001) of Amex7 was observed in the mouse model. Moreover, there were no marked changes in body weight between the control and Amex7 treatment groups during the administration period of Amex7 (Fig. 2B).

To assess the anti-proliferative effect of Amex7 on human colorectal cancer cells, we exposed HT-29 cells to serial concentrations of Amex7 (0, 1, 2, 4, 8, and 16%) for 6, 12, and 24 h. By using cell count assay, Amex7 treatment resulted in 99, 93, 73, 45 and 1% cell viability for 6 h; 79, 77, 52, 4 and 0% cell viability for 12 h; and 73, 52, 29, 0 and 0% cell viability for 24 h at 1, 2, 4, 8, and 16%, respectively (Fig. 3A). Furthermore, Amex7 changed cell morphology and increased cell aggregations and cellular debris compared with the control cells in time-dependent manner (Fig. 3B). Our data showed that Amex7 induces concentration- and time-dependent inhibition of HT-29 human colorectal cancer cell lines.

To determine whether the anti-proliferative effect of Amex7 was associated with regulation of the cell cycle, we used flow cytometry to analyze the cell cycle distribution of HT-29 cells in the presence of Amex7. Fig. 4A and B show that Amex7 significantly decreased the proportion of G0/G1 phase cells (p<0.001) and accumulation of G2/M phase cells compared with the control (p<0.001). Furthermore, Amex7 markedly increased the proportion of sub-G1 phase cells at 24 h, but not at 6 and 12 h after treatment of Amex7 compared with the control. These results suggested that Amex7 inhibited cell proliferation by inducing G2/M phase arrest and apoptosis.

To further confirm the reduction of cells in the G0/G1 phase and the increase of cells in the G2/M phase, the expression levels of cell cycle regulatory proteins such as cyclin A2, cyclin B1, cyclin D1, cyclin E, phospho-cdc2 (Tyr15), CDK2, CDK4, and CDK6 were examined by immunoblotting. At 6, 12, and 24 h after Amex7 treatment, the expression levels of cyclin D1, cyclin E, and CDK4, which are involved in G1/S progression, were significantly decreased (p<0.01) and CDK6 protein level was slightly decreased. Moreover, Amex7 markedly increased cyclin A2 and cyclin B1 protein levels, which are required for G2/M progression (p<0.05), but did not change the expression level of phospho-cdc2 (Tyr15) (Fig. 4C and D). In summary, we showed that Amex7 regulated cell cycle progression by significantly reducing G1/S phase-related proteins and increasing G2/M phase-related proteins.

In order to identify the induction of apoptosis by Amex7, we observed the expression of p53, phospho-p53 (Ser15), Bcl-2, Bcl-x_L, survivin, and cleaved-PARP (Asp214) by immunoblotting. At 12 and 24 h after treatment of Amex7, cleaved-PARP protein (Asp214) was increased and anti-apoptotic proteins, including Bcl-2, Bcl-x_L, and survivin, were decreased. In addition, Amex7 treatment for 12, 24 h reduced total p53 protein level, whereas phosphorylated p53 at Ser15 (Fig. 5A and B). Next, to evaluate the induction of autophagy by Amex7, we showed the conversion of the autophagosome protein LC3A/B-I to LC3A/B-II and the expression of SQTM1/p62 using immunoblotting. Starting 12 h after treatment of Amex7, LC3A/B-II and SQTM1/p62 were consistently induced (Fig. 5C and D). These results suggested that Amex7 induced apoptosis and autophagosome formation in human colorectal cancer cells.

To investigate the effect of Amex7 on the kinetics of DNA damage and repair, we examined the immunofluorescent staining for γ-H2AX: a marker for DNA strand breaks (DSBs) and stained nuclear DNA with DAPI. The images were analyzed using two (green/blue) fluorescence channels. At 24 h after treatment of Amex7, we observed an increased number of γ-H2AX foci compared with the control group (p<0.05) (Fig. 6A and B). Next, to evaluate whether Amex7 could affect the DNA repair pathway, we examined the expression of homologous recombination (HR) repair proteins and non-homologous end joining (NHEJ) repair proteins using immunoblotting. As shown in Fig. 6C and D, Amex7 treatment for 12 and 24 h significantly decreased HR repair proteins, including Rad51 and ERCC1, in HT-29 cells (p<0.05). However, Amex7 did not affect NHEJ repair proteins such as the catalytic subunit of DNA-PKcs, Ku70, and Ku80. These results showed that Amex7 enhanced anticancer effects by increasing DNA damage and reducing HR repair proteins.