Traditional Chinese medicine (TCM) has been increasingly applied in both preventing and treating a variety of cancers in the last decades, attributing to its fewer side effects as compared with chemotherapy drugs. Hellebrigenin, a component of Chanpi from the skin of
The incidence of colorectal cancer (CRC) is ranked the third in terms of morbidity and the fifth in terms of mortality of malignancies in China, where new colorectal cancer cases had reached over 0.8 million and 0.4 million deaths occurred in 2018 (
Traditional Chinese medicine (TCM) has been increasingly applied in preventing and treating a variety of cancers in the last decades because of its characteristics of diverse components, multiple targets, low toxicity, and a few side effects, overall regulation, and synergistic effects (
Bufadienolides are the primary bioactive components of Chanpi, including approximately one hundred monomers, most of which have potent anti-cancer activity and have been drawn attention as novel candidates in cancer therapy (
In the present study, the effect and mechanism of hellebrigenin on human colorectal cancer cell lines HCT116 and HT29were investigated. We showed that exposure of CRC cell lines to hellebrigenin led to cell death with morphological and biochemical characteristic changes of apoptosis. Further study demonstrated that reactive oxygen species (ROS) were upstream of hellebrigenin-mediated apoptosis, and the apoptosis was triggered through the intrinsic pathway in CRC cells. These findings suggest that hellebrigenin may be a potential chemotherapeutic agent for colorectal cancer treatment.
Human colorectal cancer cell lines HCT116 and HT29 cells were purchased from Genechem Co., Ltd., (Shanghai, China). Hellebrigenin was prepared from water extract of Chanpi and dissolved in DMSO at a stock concentration of 10 mM. Fetal bovine serum was purchased from Gibco (USA). Sulforhodamine B (SRB) was purchased from Sigma (USA). FITC-Annexin V Apoptosis Detection Kit I was purchased from Becton Dickinson Biosciences (USA). Reactive Oxygen Species (ROS) Assay Kit and Mitochondrial Membrane Potential Assay Kit were purchased from Beyotime (China). Antibodies against cleaved caspase-9 (#7237), cleaved caspase-3 (#9664), BAX (#272), BCL-2 (#3498), Cytochrome C (#11940) and VDAC (#4661) were purchased from Cell Signaling Technology (USA). Antibodies against PCNA (EM111201) and β-actin (R1207-1) were obtained from Huabio (China).
The human colorectal cancer cell lines HCT116 and HT29 were cultured in RPMI-1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) at 37°C in a humidified cell culture incubator containing 5% CO2.
Cells were seeded into 96-well plates at a density of 5 × 103cells per well and cultured for 24 h to allow adherence. Next, cells were incubated with a series of concentrations of hellebrigenin. After incubated for 24, 48 and72 h, cells were performed SRB colorimetry (
Cells were seeded into 6-well plates at a density of 500 cells per well and cultured for 24 h. Next, cells were pretreated with 100 to 400 nM of hellebrigenin for 48 h and then cultured with fresh cell culture medium for 2 weeks. Then, cells were washed with phosphate buffer saline (PBS) followed by fixed with methanol for 1 h and stained with Giemsa solution (Solarbio, Beijing, China). Finally, colonies of cells were counted to evaluate cell proliferation.
Cells were plated into a 6-well plate at a density of 1 × 105cells per well and cultured for 24 h. Then cells were incubated with 100 to 400 nM of hellebrigenin for 48 h. The cells were washed and collected with cold PBS and resuspended in 500 μL binding buffer (0.01 M Hepes-NaOH (pH7.4), 0.14 M NaCl, 2.5 mM CaCl2). Later, cells reacted with 5 μL FITC-Annexin-V and 5 μL PI at room temperature in the dark for 15 min. Finally, the stained samples were analyzed by flow cytometerC6 plus (BD, USA).
Cells were plated into a 6-well plate at a density of 1 × 105cells per well and cultured for 24 h. After incubated with 100 to 400 nM of hellebrigenin for 48 h, cells were stained with Hoechst 33342 (Beyotime, Shanghai, China) for 30 min in the dark. Cells were photographed by fluorescence microscope (Olympus, Tokyo, Japan) in which the bright blue of condensed or fragmented nuclei in apoptotic cells were observed.
Cells were plated into a 6-well plate at a density of 1 × 105 cells per well and cultured for 24 h, followed by treating with 100 to 400 nM of hellebrigenin for 48 h. Cells were then washed by PBS and cultured in fresh RPMI-1640 medium with 10 μM of 5,5,6,6’-tetrachloro-1,1,3,3’-tetraethyl-imidacarbocyanine (JC-1) for 20 min in the dark. Afterward, cells were washed by JC-1 staining buffer and added fresh RPMI-1640 medium without fetal bovine serum to be photographed by fluorescence microscope.
Cells were plated into a 6-well plate at a density of 1 × 105 cells per well and cultured for 24 h followed by treating with 100 to 400 nM of hellebrigenin for 48 h. Cells then were washed by PBS and incubated in fresh RPMI-1640 medium with 10 μM Dichloro-dihydro-fluoresceindiacetate (DCFH-DA) probe for 30 min in dark. Later, cells washed with ice-cold PBS and added fresh RPMI-1640 medium without fetal bovine serum to be examined by fluorescence microscope.
Mitochondria of cells were prepared using Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China). Briefly, cells were collected from culture dishes and suspended in ice-cold PBS. Cells were then precipitated by centrifugation and treated with mitochondrial separation reagent for 15 min in ice. Later, the cell suspension was milled in a glass homogenizer and centrifugated at 600
HCT116 cells were suspended in PBS at a concentration of 1 × 107 per µL and injected subcutaneously into the right forelimb of male BALB/c nude mouse (N = 10) at 4 weeks of age (Beijing Huahengkang Biological Technology, Beijing, China). Mice were maintained in specific pathogen-free conditions. When the mean volume of the tumors reached approximately 70 mm3, the mice were randomized to either the vehicle control group (N = 5) or hellebrigenin treated group (N = 5). Hellebrigenin was dissolved in normal saline and intraperitoneally injected into mice of the hellebrigenin-treated group at a dose of 2.5 mg/kg for 12 continuous days. Mice of the vehicle control group were treated with the same volume of normal saline and DMSO. Volumes of tumors were measured and calculated every day by the following formula: Volume = 1/2 (length × width2). Then tumor neoplasm was harvested 12 days after compound administration.
Tumor tissues were fixed with formalin, embedded in paraffin, and sectioned. The slices were incubated with primary antibody against PCNA (1:200) overnight at 4°C, and HRP-secondary antibody for 2 h at room temperature and then subjected to 3,3’-diaminobenzidine chromogen development. Samples were photographed by microscope (Olympus, Tokyo, Japan).
Total protein was extracted from cells or tumor tissue by RIPA lysis buffer with proteinase inhibitor PMSF (Beyotime, China) and phosphatase inhibitor (Sigma-Aldrich, USA). Protein samples were quantified, separated by SDS-PAGE in 12% gel, and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% BSA for 2 h at room temperature and probed with primary antibodies at 4°C overnight. Then the membranes were incubated with HRP-conjugated anti-rabbit antibody for 1 h at room temperature. Protein bands were visualized by ChemiDoc Imaging System (Bio-Rad, USA).
All experiments were performed at least three times independently. Statistical analyses were performed using SPSS 17.0 (IBM, Armonk, New York, USA). Statistical significance was determined by ANOVA analysis, and a two-tailed value of
To investigate the effect of hellebrigenin on the proliferation of human CRC cells, two human CRC cell lines, HCT116 and HT29, were exposed to various concentration of hellebrigenin (0, 15.62, 31.25, 62.5, 125, 250, and 500 nM) for 24, 48, and 72 h, respectively. The cell viability detected by SRB assay showed that hellebrigenin significantly suppressed the growth of HCT116 and HT29 cells (
Colonies outgrew from single cells after cells treated with various doses of hellebrigenin (C and D). The data showed three independent experiments (*
In order to identify whether the proliferation inhibition by hellebrigenin was associated with apoptosis, we detected apoptotic cells by FITC-Annexin-V and PI staining. Results showed that the proportion of apoptotic cells obviously increased following treatment with 400 nM hellebrigenin for HCT116 and HT29, respectively (
Mitochondrial apoptosis-associated protein BAX, BCL-2, cleaved caspase-3, and cleaved caspase-9 detected after cells treated with hellebrigenin for 48 h (D). Nuclei of cells stained by Hoechst 33342 after cells treated with hellebrigenin for 48 h (E, the scale bar is 50 μm). Data of three independent experiments performed one-way ANOVA analysis (*
Mitochondria play an important role in regulating apoptosis, and exhaustion of MMP is a symbolic event in the early stage of intrinsic apoptosis. Subsequently, cytochrome C is released from mitochondria to the cytoplasm, and apoptosis progresses. We examined the change of MMP by fluorescent probe JC-1 with a microscope. MMP significantly declined after treated with 400 nM hellebrigenin for 48 h, evidenced by enhanced green fluorescence representing strengthened permeating of JC-1 monomer in mitochondria (
An unstable state of ROS is a precipitating factor of apoptosis, and usually, excessive ROS production causes oxidative damage to cells resulting in cell death. In order to find out whether ROS was involved in apoptosis induced by hellebrigenin, we detected the level of ROS in CRC cells by fluorescent probe DCFH-DA. Results showed that the production of ROS elevated, evidenced by green fluorescence increase when cells were treated with 200 and 400 nM hellebrigenin. It revealed that hellebrigenin promoted toxic levels of ROS production to trigger apoptosis in CRC cells (
To investigate the antitumor activity of hellebrigenin
Hellebrigenin is a natural product found in the toad skin secretions and plants of Urginea, including Helleborus and Kalanchoe genera (
ROS is a set of short-lived molecules with unpaired electrons, including superoxide, hydroxyl, hydrogen peroxide, and hypochlorous acid. Endogenous ROS is byproducts of oxygen consumption and is mainly generated in mitochondria electron transport chain and NADPH oxidases complex (
Although mitochondrion is one of the main ROS generators, increased production of ROS in turn causes damage to the mitochondrial membrane resulting in the release and translocation of cytochrome C from mitochondria to cytosol and then triggering caspase-independent or caspase-dependent apoptosis (