Contributed equally
Accumulating evidence indicates that aged black garlic extract (ABGE) may prove beneficial in preventing or inhibiting oncogenesis; however, the underlying mechanisms have not been fully elucidated. The present study aimed to investigate the effects of ABGE on the proliferation and apoptosis of HT29 colon cancer cells. Our results demonstrated that ABGE inhibited HT29 cell growth via the induction of apoptosis and cell cycle arrest. We further investigated the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signal transduction pathway and the molecular mechanisms underlying the ABGE-induced inhibition of HT29 cell proliferation. We observed that ABGE may regulate the function of the PI3K/Akt pathway through upregulating PTEN and downregulating Akt and p-Akt expression, as well as suppressing its downstream target, 70-kDa ribosomal protein S6 kinase 1, at the mRNA and protein levels. In conclusion, these findings suggest that the PI3K/Akt signal transduction pathway is crucial for the development of colon cancer. ABGE inhibited the growth and induced apoptosis in HT29 cells through the inhibition of the PI3K/Akt pathway, suggesting that ABGE may be effective in the prevention and treatment of colon cancer in humans.
Colon cancer is currently one of the most common types of gastrointestinal cancer, with an incidence rate of ~10–15% among cancer patients. Surgical resection has been the standard treatment for colon cancer; however, the local recurrence rate following surgical resection is 20–50% (
We previously reported the potential action of aged black garlic extract (ABGE) against gastric cancer by evaluating its effect on the inhibition of cell proliferation and induction of apoptosis in SGC-7901 human gastric cancer cells (
FBS, DMEM, penicillin, streptomycin, trypsin/EDTA, trypan blue dye, MTT, PI, bicinchoninic acid working solution and the ECL Plus light-emitting kit were purchased from Sigma (St. Louis, MO, USA). Antibodies against Akt, PTEN and β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Acrylamide and the protein assay kits were obtained from Bio-Rad (Hercules, CA, USA). Western blotting detection reagents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). The p-Akt, 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) and β-actin primers and the real-time kit were purchased from Bao Biological Engineering Co. (Dalian, China).
ABGE was provided by the Dalian Hua Valley Garlic Industry Co. (Dalian, China). The mashed aged black garlic was extracted with 95% ethanol for 24 h under mixing. Following precipitation, the cooled solution was filtered and evaporated under a reduced pressure at 47°C to provide a residue. The extract was then filtered through a 0.22-μm membrane, dissolved in 0.9% saline to prepare a solution at a concentration of 1 g/ml and stored at −80°C prior to use (
The HT29 human colon cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The HT29 cell line was cultured in DMEM containing 10% heat-inactivated FBS, glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a humidified incubator with 5% CO2. HT29 colon cancer cells in the logarithmic phase of growth were collected and inoculated in a 96-well culture plate, with 3×103 cells per well. After 24 h, the cells were exposed to varied doses of ABGE (20, 50 and 100 mg/ml). The cells in the control group were treated with 0.1% DMSO. There were 3 wells per drug concentration group.
The processed cells were cultured for 24, 48 and 72 h. MTT solution (8 μl) was added to each well, followed by the addition of 100 μl DMSO after 4 h. The absorbance value was measured by ELISA detection instrument at 570 nm and a reference wavelength of 630 nm (Universal Microplate Spectrophotometer; Thermo Fisher Scientific, Inc., Rockford, IL, USA). Each experiment was repeated three times.
The HT29 cells were treated with ABGE at different concentrations (20, 50 and 100 mg/ml, with 0/0.1% DMSO vehicle used as the control) for 24 h. The treated HT29 cells were detached in 2 ml of PBS with 2 mM EDTA, centrifuged at 15,000 × g for 5 min and resuspended in 250 μl of hypotonic fluorochrome solution (PBS, 50 μg PI, 0.1% sodium citrate and 0.1% Triton X-100) with RNase A (100 U/ml). The DNA content was analyzed by a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). All the events (n=20,000) were analyzed per sample and the cell cycle distribution and apoptosis were determined based on the DNA content and the sub-G1 cell population, respectively.
The treated HT29 cells were washed with ice-cold PBS and suspended in lysis buffer on ice for 30 min. The lysates were cleared by centrifugation at 15,000 × g for 15 min. Equal volumes of cell extracts (60 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with primary antibodies against human PTEN, Akt and β-actin, followed by horseradish-conjugated secondary antibodies. Anti-β-actin antibody was used as a loading control. Detection was performed using an ECL Plus light-emitting kit (Sigma).
Total RNA was extracted from HT29 cells treated with different concentrations of ABGE (20, 50 and 100 mg/ml, with 0/0.1%DMSO vehicle as the control) for 24 h, using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions, and was quantified by spectrophotometry. The reverse transcription reaction was performed using total RNA. The primers were designed by Bao Biological Engineering Co. as follows: p-Akt mRNA: upstream, 5′-CAATTCCGGTCTGAGGAA-3′ and downstream, 5′-CACATGGGAAGTGTTGTCTG-3′; and p70S6K1 mRNA: upstream, 5′-GGCAGTGATGGGCAACCT-3′ and downstream, 5′-GGTCCAACCCTTACTTCAGCA-3′. The qPCR conditions included an initial denaturation for 30 sec at 95°C, followed by 40 cycles of denaturation for 5 sec at 95°C and annealing for 20 sec at 60°C.
Data are expressed as the means ± standard deviation. The statistical analysis was conducted with SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA), using a two-sided Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.
ABGE inhibited the growth of HT29 cells in a time- and dose-dependent manner (
We investigated the effects of different concentrations of ABGE on the apoptosis of HT29 cells. The flow cytometric analysis confirmed that ABGE at concentrations of 20, 50 and 100 mg/ml achieved a dose-dependent induction of apoptosis. Additionally, ABGE at 20, 50 and 100 mg/ml induced the apoptosis of HT29 cells at 24 h with an apoptosis ratio of 11.21±0.86, 23.77±0.78 and 39.56±0.59%, respectively (P<0.05). Moreover, ABGE (20, 50 and 100 mg/ml) achieved a dose-dependent induction of G0/G1 cell cycle arrest and a G2/M+S decrease (
The effect of ABGE treatment on the expression of Akt and PTEN was assessed by western blot analysis. Compared to the control group, the Akt protein level in the HT29 cells treated with ABGE was lower, whereas the PTEN protein level was higher (
We investigated the effects of ABGE on p-Akt and its downstream target, p70S6K1, in HT29 cells by qPCR. The qPCR analysis demonstrated that treatment with ABGE at different concentrations for 24 h significantly reduced the mRNA levels of p-Akt and p70S6K1 in HT29 cells (
Garlic has been long recognized for its medicinal properties and has been used for sterilization and anti-inflammation throughout history. Allicin, the main active ingredient of garlic, is currently extensively investigated for antitumor therapy (
The PI3K/Akt signal transduction pathway plays an important biological role in the occurrence of apoptosis that is mediated through the regulation of apoptosis-related genes. PI3K/Akt signaling is deregulated through a variety of mechanisms, including overexpression or activation of growth factor receptors, mutations of the PI3K gene and mutation/amplification of the Akt gene (
Akt is a downstream target of PI3K and it is a 56-kDa serine/threonine kinase triggered by a lipid second messenger, phosphatidylinositol-3,4,5-trisphosphate, which is generated by PI3K. Akt may be phosphorylated to p-Akt by pyruvate dehydrogenase kinase-1, which is distributed across the cell membrane. p-Akt inactivates multiple effector molecules of apoptosis through a variety of mechanisms, promoting the proliferation and metastasis of tumor cells.
PTEN, the negative regulator of PI3K signaling, exhibits decreased expression in various types of cancer and may be downregulated through several mechanisms, including mutation, loss of heterozygosity, methylation, aberrant expression of regulatory microRNA and protein instability. The PTEN gene is an important effector molecule in the tumor signaling pathway, with the ability to inhibit growth and phosphatase activity in cancer cells. Low expression of PTEN may be associated with non-effective inhibition of the activation of Akt, resulting in the overactivation of the PI3K/Akt pathway. PTEN loss and Akt overexpression may significantly affect the progression of several advanced human cancers, including melanoma and breast, prostate and renal cancers (
p70S6K is the direct substrate of mTOR and forms downstream components of the PI3K/Akt signaling pathway. p70S6K was shown to be a signaling molecule, which is involved in the regulation of a series of physiological processes in protein synthesis. Once activated, p70S6K1 was shown to promote the reconstruction of actin filaments (
Apoptosis is the outcome of a gene expression cascade and is regulated by several gene products. In the present study, treatment with ABGE resulted in marked inhibition of cell growth, induction of apoptosis and cell cycle arrest in HT29 colon cancer cells. To further elucidate the mechanisms underlying the ABGE-induced growth suppression in HT29 cells, we investigated its effect on Akt, PTEN, p-Akt and p70S6K expression. The results revealed that ABGE modulated the PI3K/Akt signaling pathway in HT29 cells through the upregulation of PTEN and the downregulation of Akt expression and the reduction of p-Akt and p70S6K1 expression at the mRNA level.
By investigating the correlation between the PI3K/Akt signaling pathway and the growth inhibitory effect of ABGE on HT29 cells, as well as the underlying mechanisms, we concluded that the PI3K/Akt signaling pathway is critical for the development of colon cancer.
Our present data suggest that ABGE may be of therapeutic and/or adjuvant therapeutic value in the treatment of colon cancer, possibly via the modulation of the PI3K/Akt signaling pathway, the upregulation of PTEN and the downregulation of Akt and p-Akt expression. However, as ABGE may confer resistance to targeted drugs, further assessment of ABGE in clinical trials is required.
This study was supported by a grant from the Project of Army Medical Research in Science and Technology: Traditional Chinese Medicine and Optimized Modern Technology for Prevention and Treatment of Malignant Tumor (no. 06G034).
Effects of ABGE on the growth of HT29 cells in a time- and dose-dependent manner. HT29 cells were cultured for 24, 48 and 72 h in DMEM containing ABGE. The effects of ABGE on cell growth were determined by the MTT assay. The results are expressed as the means ± standard deviation from three independent experiments. Data were analyzed using the Student’s t-test. The values between the control group and the cells treated with ABGE differed significantly (P<0.05). ABGE, aged black garlic extract.
Effects of ABGE on the cell cycle and apoptosis of HT29 cells
Effects of ABGE on (A) Akt protein expression and (B) PTEN protein expression in HT29 cells treated with various concentrations of ABGE (20, 50 and 100 mg/ml, with 0/0.1% DMSO vehicle as the control) for 24 h. The cells were harvested and analyzed by western blotting. Anti-β-actin was used as the loading control. The results are expressed as the means ± standard deviation from three independent experiments. Data were analyzed using the Student’s t-test. *P<0.05, compared to the control group. ABGE, aged black garlic extract.
Effects of ABGE on p-Akt and p70S6K1 protein expression in HT29 cells. The values are expressed as means ± standard deviation. Data were compared with the control group and analyzed using the Student’s t-test. Significant decreases in the mRNA expression levels were observed for p-Akt (n=3) and p70S6K1 (experiments were repeated in triplicate). *P<0.05 compared to the control group. ABGE, aged black garlic extract.