Cell death can occur by apoptosis [also known as programmed cell death (PCD) I] or by necrosis and autophagy (which is known as PCD II) (
The stems of
Antibodies against Beclin 1 (cat. no. 3495), light chain (LC) 3 (cat. no. 12741), B-cell lymphoma (Bcl)-2 (cat. no. 2872) and Bcl-2-associated X (Bax; cat. no. 2772), Akt (cat. no. 4691), Akt-Ser308 (cat. no. 9275), mechanistic target of rapamycin (mTOR; cat. no. 2983), mTOR-Ser2448 (cat. no. 5536), p70 ribosomal protein S6 kinase (p70S6K; cat. no. 2708) and p70S6K-Thr389 (cat. no. 9234) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The enhanced chemiluminescence (ECL) kit was purchased from GE Healthcare Life Sciences (Chalfont, UK). DMSO and MTT were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Other reagents were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).
The human colorectal cancer cell line HT-29 was acquired from the Cell Bank of Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). HT-29 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and maintained at 37°C in a humidified incubator in an atmosphere of 5% CO2.
Cell viability was measured by MTT assay. HT-29 cells were grown until logarithmic phase, and then seeded in a 96-well plate at a density of 1×104 cells/well overnight prior to treatment. The cells were then incubated with different concentrations of COE (0, 20, 40, 80, 160 and 320 mg/l) in triplicate. After 24, 48 and 72 h of incubation, the cells were incubated with medium containing MTT for 4 h, and the formazan crystals were dissolved with 150 µl DMSO. The plates were incubated on an agitator for 15 min at room temperature, and the absorbance was measured at 490 nm with a microplate reader. The drug concentration at which the cell viability was reduced to 50% [defined as the half maximal inhibitory concentration (IC50)] by 24 h of treatment was then determined. The effects of COE on cell viability were also determined in the absence or presence of the autophagy inhibitor 3-methyladenine (3-MA) (5 mM; Sigma-Aldrich; Merck Millipore) 3 h prior to COE treatment or the autophagy inducer rapamycin (100 nM; Sigma-Aldrich; Merck Millipore) 1 h prior to COE treatment to investigate the effects of COE-induced autophagy on the viability of HT-29 cells.
Apoptosis was detected with the Annexin V-FITC/PI Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). In brief, after 24 h of treatment with different concentrations of COE, cells were washed twice with ice-cold PBS and re-suspended in 500 µl binding buffer. A total of 5 µl annexin V-FITC and 5 µl PI were added to the cell suspension and then incubated for 15 min at room temperature in the dark. FITC and PI fluorescence was analyzed in a FACSort flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with CellQuest Pro software (BD Biosciences). The results were generated from three independent experiments.
Following treatment with COE, total RNA was extracted from the cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) under RNase-free conditions, and RT was performed in a 20-µl reaction with 200 ng total RNA using the Two-Step RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). RT-qPCR was performed on a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) in Axygen® 96-well reaction plates (Thermo Fisher Scientific, Inc.). PCR was performed under the following conditions: Initiation step of 95°C for 10 min, denaturation step of 95°C for 15 sec, and the annealing and extension step at 60°C for 60 sec for 40 cycles.
Primers were obtained from Shanghai Shenggong Biology Engineering Technology Service, Ltd. (Shanghai, China), and their sequences were: Beclin 1 (NM_003093), forward primer 5′-GGCTGAGAGACTGGATCAGG-3′ and reverse primer 5′-CTGCGTCTGGGCATAACG-3′; LC3-II (NM_003094), forward primer 5′-GAGAAGCAGCTTCCTGTTCTGG-3′ and reverse primer 5′-GTGTCCGTTCACCAACAGGAAG-3′; and GAPDH (NM_002046), forward primer 5′-TGGCACCCAGCACAATGAA-3′ and reverse primer 5′-CTAAGTCATAGTCCGCCTAGA-3′. GAPDH was used as an internal control, and the data were analyzed using the 2−ΔΔCq method (
Following treatment with COE, the cells were washed with PBS, collected by centrifugation at 1,500 ×
To detect the AVOs in COE-treated cells in the presence of 3-MA and rapamycin, cells were stained with acridine orange (Beyotime Institute of Biotechnology). In acridine orange-stained cells, the cytoplasm fluoresces bright green, whereas the acidic compartments fluoresce bright red (
The expression levels of Beclin 1, LC3, Bax, Bcl-2 and mTOR-p70S6K signaling proteins (Akt, Akt-308, mTOR, mTOR-Ser2448, p70S6K and p70S6K-Thr389) in HT-29 cells were determined by western blot analysis. Briefly, a cell lysis solution was prepared using Cytoplasmic Extraction Reagent II (Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). A 50-µg sample of supernatant was subjected to 10% SDS-PAGE and then transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The blot was blocked with 5% not-fat dry milk for 2 h at room temperature, and then incubated with primary antibodies (1:1,000) and agitated overnight at 4°C. Subsequently, the membranes were washed three times with washing buffer for 10 min and incubated with secondary antibodies (1:1,000; G130321; Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China) for 2 h at room temperature. β-actin (a housekeeping gene) was used as a loading control. ECL was used to detect the signals, using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) on a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometry was determined using Quantity One 1-D analysis software (Bio-Rad Laboratories, Inc.).
Results are expressed as the mean ± standard deviation. Each experiment was repeated ≥3 times. Statistical comparisons of ≥2 groups were conducted using analysis of variance followed by a Bonferroni post hoc test. All statistical analyses were performed using the SPSS 18.0 statistical software (SPSS, Inc., Chicago, IL, USA), and P<0.05 was considered to indicate a statistically significant difference.
The results of the MTT assay revealed that HT-29 cell viability decreased in a dose-dependent manner upon administration of COE. A strong dose-dependent inhibition of cell proliferation was also observed in the HT-29 cell line. Upon treatment with 149.65±0.49 mg/l of COE for 24 h, the cell viability was reduced to 50% (
The induction of autophagy is characterized by the formation of autophagosomes enclosed in a double membrane that fuse with lysosomes to form autolysosomes (
Under normal conditions, the protein LC3 is dispersed throughout the cytoplasm in a dissociated form (LC3-I) (
As shown in
The percentage of total apoptotic cells was calculated by adding the percentages of early apoptotic-gated cells [annexin V+, quadrant (Q) 3 area] and late apoptotic-gated cells (annexin V+/PI+, Q2 area) (
Members of the Bcl-2 family serve a vital role in the regulation of apoptosis (
Our data revealed that <20 mg/l COE had no significant impact on cell viability, while 80 mg/l COE significantly inhibited HT-29 cell viability (P<0.01;
To determine whether the PI3K-Akt-mTOR signaling pathway serves a role in mediating COE-induced apoptosis and autophagy, the levels of Akt, mTOR and p70S6K were determined by western blotting. Following COE treatment, there was a robust and sustained decrease in Akt-Thr308, mTOR-Ser2448 and p70S6K-Thr389 levels in HT-29 cells (
As surgery and adjuvant chemotherapy are associated with low survival rates in colorectal carcinoma, there is an urgent requirement to identify novel therapeutic agents for this type of cancer (
Autophagy is normally considered to be a cell survival mechanism induced by stress, starvation and other environmental cues, but recent reports have provided evidence of cell death mediated by autophagy (
It is well established that autophagy, mainly a self-digestion process, degrades intracellular structures in response to stress, leading to either cell survival or cell death (
Our experimental results also demonstrated that, in addition to activating autophagy, COE induced pro-apoptotic signaling pathways leading to cell death. Electron microscopy observation of the COE-treated HT-29 cells revealed chromatin condensation, nuclear collapse and apoptotic body formation in cells treated with 80 or 160 mg/l COE (
To further understand the mechanism of apoptosis and autophagy induced by COE, the PI3K/Akt/mTOR pathway was explored, which is frequently deregulated in cancer and is important for tumorigenesis, since this pathway can modulate apoptosis and autophagy (
In conclusion, COE-induced autophagy and apoptosis synergize to inhibit colorectal cancer growth. The present study provides novel evidence that COE may be a promising agent against human colorectal cancer, particularly in light of the fact that cancer cells can escape induction of apoptosis, resulting in drug resistance. The present study may aid in the development of new strategies for the formulation of effective therapeutic drugs that would selectively target cancer cells and induce autophagic cell death independently of the normal apoptotic pathway.
The present study was financially supported by grants from the National Natural Science Foundation of China (Beijing, China; grant nos. 81403232, 81274141 and 81450051) and the Natural Science Foundation of Jiangsu Province of China (Nanjing, China; grant nos. BK 2012686 and SBK 2014021480).
Reduction in HT-29 cell survival rates after the cells were treated with different concentrations of COE for varying lengths of time. Data are represented as the mean ± standard deviation (n=3). *P<0.05 and **P<0.01 compared with 0 mg/l COE treatment for 24 h; #P<0.05 and ##P<0.01 compared with 0 mg/l COE treatment for 48 and 72 h. COE,
COE triggered autophagy and apoptosis in HT-29 cells treated for 24 h. (A) Transmission electron microscope images of autophagosomes and cellular structures. The black arrows indicate autophagosome formation. (B) The expression levels of Beclin 1 and LC3-I/II in HT-29 cells were determined by western blot analysis. (C) The band intensities of Beclin 1 and LC3-II relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments. (D) Transmission electron microscope images showed chromatin condensation and nuclear collapse in the 80 and 160 mg/l COE-treated groups. (E) Flow cytometric analysis demonstrated that COE induced apoptosis in a dose-dependent manner. (F) Quantification of the apoptotic cell population. (G) The expression levels of Bcl-2 and Bax in HT-29 cells were determined by western blot analysis. (H) The band intensities of Bcl-2 and Bax relative to untreated control cells were quantified upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 compared with the untreated control. FITC, fluorescein isothiocyanate; PI, propidium iodide; LC, light chain; Bcl, B-cell lymphoma; Bax, Bcl-2-associated X; FU, fluorouracil; COE,
COE-induced cell death involves autophagy in HT-29 cells. (A) Detection of acidic vascular organelles in HT-29 cells treated with 80 mg/l COE for 24 h in the absence or presence of 3-MA or rapamycin by fluorescence microscopy with acridine orange staining. (B) Western blot analysis of the expression of LC3-II in HT-29 cells treated with 80 mg/l COE for 24 h in the absence or presence of 3-MA or rapamycin. (C) The band intensities of LC3-II were analyzed in HT-29 cells treated with 80 mg/l COE for 24 h in the absence or presence of 3-MA or rapamycin. (D) Effect of 80 mg/l COE treatment on the viability of HT-29 cells after 24 h in the absence or presence of 3-MA or rapamycin. *P<0.05 and **P<0.01 compared with the untreated control. LC, light chain; COE,
Protein expression of the PI3K/Akt/mTOR signaling pathway components in HT-29 cells treated with different concentrations of COE for 24 h. (A) The expression levels of PI3K, Akt, Akt-Ser308, mTOR, mTOR-Ser2448, p70S6K and p70S6K-Thr389 were analyzed by western blotting. (B) The band intensities of Akt-Ser308, mTOR-Ser2448 and p70S6K-Thr389 relative to the untreated control cells were determined upon normalizing to β-actin expression, and are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 compared with the untreated control. p, phosphorylated; FU, fluorouracil; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; mTOR, mechanistic target of rapamycin; p70S6K, p70 ribosomal protein S6 kinase; COE,