Khz (fusion of
The BEAS-2B (normal immortalized), 1799 (non-transformed), 1198 (transformed, non-tumorigenic) and 1170-I (tumorigenic) cell lines comprise an
Khz (Brain Group Co., Ltd., Seoul, Korea) was extracted first in powder form (1 kg) using clean water (8.5 liters), at 115°C for 60 min extracts at a pressure of 1.8–2 kW, followed by 60 min maturing at a hydraulic pressure of 5 KW in order to separate Khz and debris. Subsequently, clean water (7.5 liters) was added to the residual water of the first extraction, and extraction was performed at 115°C, for 60 min at a pressure of, followed by 60 min maturation and hydraulic crossroad gathering. Finally, the extracts from the first extract and the secondary extraction steps were mixed and boiled at 100°C for 5 min.
An MTT assay was used to determine the rate of cell survival. The MCF-7 cells were seeded into 96-well plates at a density of 2,000 cells per well, and 10/100
The cells treated with Khz were washed twice in cold phosphate-buffered saline (PBS) and were stained with annexin V-fluorescein isothiocyanate (FITC) (cat. no. A13199, Invitrogen Life Technologies, Carlsbad, CA, USA) and propidium iodide (PI; 5
The levels of cytoplasmic reactive oxygen species (ROS) in the cells were estimated using the oxidation-sensitive fluorescent dye, 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA; Invitrogen Life Technologies). For the DCF staining, the cells were loaded with H2DCF-DA (100 nM) for 1 h at 37°C, and were then washed once with PBS, ROS levels were analyzed following Khz treatment using a flow cytometer (FACSCalibur; BD Biosciences). N-acetyl cysteine was purchased from Sigma-Aldrich.
The Ca2+ levels in the MCF-7 cells were determined by staining with fluo-4 AM. The cells, which had been treated with Khz for different durations (0, 0.5, 1 and 2 h), harvested and washed twice with PBS, and then resuspended in fluo-4 AM, followed by incubated incubation at 37°C for 30 min. The changes in Ca2+ concentration were analyzed using flow cytometry. Ethylene glycol tetraacetic acid (EGTA) was purchased from Sigma-Aldrich.
The cells were lysed in extraction buffer (Sigma-Aldrich) containing 31.25 mM Tris-HCl (pH 6.8), 1% sodium dodecyl sulfate (SDS), 10% glycerol and 2.5% mercaptoethanol, and the whole-cell lysates were subjected to separation using 10% SDS-polyacrylamide gel (Sigma-Aldrich) electrophoresis. The size-fractionated proteins on the gel were then transferred onto a nitrocellulose membrane (GE Healthcare Life Sciences), and the membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween 20 (Sigma-Aldrich), and was incubated with the following primary antibodies: Goat polyclonal anti-caspase-7 (cat. no. sc-22179), anti-caspase-8 (cat. no. sc-6136) and anti-caspase-9 (cat. no. sc-22182), purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Following washing once with PBS, the membrane was incubated with a horseradish peroxidase-conjugated mouse anti-goat IgG secondary antibody (cat. no. sc-2354; Santa Cruz Biotechnology, Inc.). The protein band of interest was detected using enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Shanghai, China) (
Cell viability was expressed as the mean ± standard deviation. Statistical comparisons were made between the control and treatment groups using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Khz is the fusion of G.
To assess whether Khz induces apoptosis, the MCF-7 cells were double stained with annexin V-FITC and PI and analyzed using flow cytometry, which is the most sensitive and specific assessment for apoptotic cells in suspension culture. PI was used to differentiate between the apoptotic cells, which maintained plasma membrane integrity (annexin V+/PI−) and those exhibiting loss-of-membrane integrity (annexin V+/PI+). Following Khz treatment at different concentrations for 24 h, the majority of the apoptotic cells were observed in the upper right quadrant (annexin V+/PI+), indicating that almost all of the apoptotic cells were in the late stages of apoptosis (data not shown). Therefore, to detect cells in early apoptosis, the durations of treatment were reduced. Treatment with 1/2 Khz for 1 h resulted in the presence of early apoptotic cells in the lower right quadrant (annexin V+/PI−) (
An
Subsequently, the present study analyzed the activation of caspases following Khz treatment to determine whether Khz-induced apoptosis was caspase-dependent. The levels of cleaved caspases 7, 8 and 9 in the MCF-7 cells increased following Khz treatment (
As oxidative stress is involved in apoptosis induced by a variety of stresses, the production of ROS following Khz treatment was analyzed in the present study. As shown in
Assessment of the effect of Khz on intracellular calcium levels revealed that Khz evoked an increase in [Ca2+]
In a previous study, it was demonstrated that tanshinone IIA induces apoptosis in A549 human lung cancer cells through the induction of ROS and by decreasing mitochondrial membrane potential (
The pro-apoptotic and cytotoxic effects of Khz demonstrated in the present study suggest it has potential as a chemotherapeutic agent for the treatment of human breast cancer. Further investigations of the effects of Khz, including
This study was supported by Mr. Young Lye Chae, chief executive officer, at Brain Group Co., Ltd., Pharmacology and Drug Development, Korean Institute of Science and Management Career College (Seoul, Korea; grant no. BRG815-1386).
Chemical structure of (A) ganoderic acid T. and (B) compound isolated from the sclerotia of
Khz induces apoptosis in transformed cells. (A) Shape of fused fruiting bodies of
Effect of Khz on the viability of MCF-7 cells, measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (A) MCF-7 cells were incubated with Khz for the indicated time-periods. (B) MCF-7 cells were treated with the indicated concentrations of Khz for 48 h. Each point represents the mean of triplicate samples ± standard deviation. *P<0.05, **P<0.01.
Detection of apoptosis using flow cytometry following annexin V-fluorescein isothiocyanate/PI staining. Lower left quadrant, viable cells (annexin V−/PI−); lower right quadrant, early apoptotic cells (annexin V+/PI−); upper right quadrant, late apoptotic cells (annexin V+/PI+); upper left quadrant, necrotic cells (annexin V−/PI+). MCF-7 cells were treated with a 1:2 dilution of Khz, and apoptosis was analyzed after 0.5, 1, and 2 h by flow cytometry. The cells were exposed to 1/2 Khz for 0.5, 1 and 2 h. The numbers in the panels represent the percentage of annexin V+/PI− and annexin V+/PI+ cells. Data are representative of three independent experiments. PI, propidium iodide.
Detection of apoptosis in the transformed cells. BEAS-2B, 1799, 1198 and 1170-I cells were treated with 1/2 Khz for 1 h. Apoptosis was analyzed using propidium iodide staining followed by flow cytometric analysis. The histograms reveal two distinct sub-populations, characterized by low (M1) and high (M2) fluorescence, respectively, corresponding to cells with low and high apoptosis staining.
Protein expression levels of caspase 7, 8 and 9 in MCF-7 cells following exposure to Khz. (A) MCF-7 cells were treated with 1/2 Khz, and caspase activation was analyzed using immunoblotting. (B) Cells were pretreated with the pan-caspase inhibitor z-VAD (20
Effect of Khz on the production of ROS in MCF-7 cells. (A) MCF-7 cells were treated with Khz (1/2) for different time-periods (0, 0.5, 1 and 2 h) and the production of ROS was evaluated using flow cytometry. Increasing the duration of incubation with Khz led to increased levels of ROS in the MCF-7 cells. (B) Cells were pretreated with NAC (1 mM) for 1 h and treated with 1/2 Khz, following which ROS levels were analyzed. The histograms reveal two distinct sub-populations, characterized by low (M1) and high (M2) fluorescence, respectively, corresponding to cells with low and high ROS staining. ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; H2DCF-DA, 20,70-dichlorodihydrofluorescein diacetate.
Effect of Khz on the production of Ca2+ in MCF-7 cells. (A) MCF-7 cells were treated with Khz (1/2) for different time-periods (0, 0.5, 1 and 2 h) and intracellular Ca2+ concentrations were measured. The Ca2+ concentrations were significantly different between the Khz-treated group and the control group. Khz treatment increased the concentration of Ca2+ in the MCF-7 cells in a time-dependent manner. (B) Cells were pretreated with EGTA (10