Colorectal cancer (CRC) is the second leading cause of cancer mortality worldwide; however, there is still a lack of effective clinical anti-CRC agents. Naturally-occurring compounds have been considered a potentially valuable source of new antitumorigenic agents. Involucrasin A, a novel natural molecule, was isolated from
Colorectal cancer (CRC) has been ranked as the second most lethal and the third most prevalent type of cancer throughout the world (
Natural products are a key source of new antitumorigenic agents, as 80–83% of approved anticancer drugs are natural compounds or their derivatives (
For example, oroxylin A, one of the main bioactive flavonoids of Scutellariae Radix, can suppress the growth of CRC by reprogramming HIF1α-modulated fatty acid metabolism (
The Akt/murine double minute 2 homologue (MDM2)/p53 signaling pathway has significant roles in regulation of the cell cycle, proliferation and apoptosis (
In the present study, the antitumorigenic activities of involucrasin A in HCT-116 CRC cells were investigated and its regulatory effects on the Akt/MDM2/p53 pathway were examined.
The chemical structure of involucrasin A (C21H22O5) is shown in
In this study, all primary antibodies were used at a dilution of 1:1,000. Furthermore, the secondary antibodies were used at a dilution of 1:10,000. Primary antibodies against human proteins included anti-β-actin (cat. no. A5441; mouse monoclonal; MilliporeSigma); anti-cyclin A2 (cat. no. AF6624; rabbit polyclonal; Beyotime Institute of Biotechnology); anti-Bax (cat. no. 2772; rabbit polyclonal), anti-cleaved caspase-9 (cat. no. 9505; rabbit polyclonal), anti-cleaved caspase-6 (cat. no. 9761; rabbit polyclonal), anti-phosphorylated (p)-Akt (cat. no. 2965; rabbit monoclonal) and anti-Akt (cat. no. 9272; rabbit polyclonal) (all from Cell Signaling Technology, Inc.); anti-p53 (cat. no. sc-126; mouse monoclonal; Santa Cruz Biotechnology, Inc.); anti-p-MDM2 (cat. no. ab170880; rabbit monoclonal) and anti-MDM2 (cat. no. ab16895; mouse monoclonal) (both from Abcam). The anti-rabbit (cat. no. 211-035-109) and anti-mouse (cat. no. 715-035-150) secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc.
Colorectal cancer cell lines (HCT-116, Caco-2 and CT26), breast cancer cell lines (BT-549, SKBR3, HS578T, MDA-MB-436 and MDA-MB-453) and a liver cancer cell line (HepG2) were obtained from the American Type Culture Collection and grown in DMEM supplemented with 10% FBS and 1% antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin), and incubated at 5% CO2 and 37°C. CT26 is a murine CRC cell line from a BALB/c mouse. The HCT-116 p53 KO cell line was provided by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD, USA) and was cultured in DMEM supplemented with 10% FBS and 1% antibiotics, and incubated at 5% CO2 and 37°C (
To assess the effects of involucrasin A on the clonogenic capacity of HCT-116 cells, cells were plated into 12-well plates (1×103 cells/well) or 24-well plates (500 cells/well) and allowed to adhere overnight. Cells were then treated with three concentrations of involucrasin A (12.5, 25 and 50 µM) for 10 days, and incubated at 5% CO2 and 37°C. Colonies were washed with PBS and stained with 0.2% (w/v) crystal violet in buffered formalin for 20 min at room temperature. A colony was defined as consisting of at least 50 cells and was counted manually. Colony images were captured using an Epson Perfection V700 Photo (Epson Corporation).
Cell viability was determined using the SRB assay. Briefly, HCT-116 cells were seeded in 96-well plates at a density of 5×103 cells/well overnight. After incubation, fresh media containing different concentrations (0, 3.12, 6.25, 12.5, 25, 50, 100, and 200 µM) of involucrasin A was added, and further incubated at 5% CO2 and 37°C for 48 or 72 h. The cells were fixed with 50 µl ice-cold 50% (w/v) TCA at 4°C for 1 h. Then, wells were carefully rinsed with deionized water five times. After air drying, cells were subsequently stained with 0.4% (w/v) SRB for 30 min and rinsed with 1% acetic acid at room temperature. Finally, adhered cells were solubilized with 200 µl 10 mM Tris base solution (pH 10.5), and plates were agitated for 5 min before absorbance was measured using a SpectraMax paradigm microplate reader (Molecular Devices, LLC) at a wavelength of 515 nm.
Cell cycle analysis was performed by flow cytometry. Briefly, HCT-116 cells were plated in 6-well plates at a density of 2×105 cells/well and treated with involucrasin A (0, 25, 50 and 100 µM) in DMEM supplemented with 10% FBS and 1% antibiotics, and incubated at 5% CO2 and 37°C for 72 h. Subsequently, cells were washed with 1X PBS before trypsinization (3 min), centrifugation (300 × g for 5 min at room temperature), and fixation with 70% ethanol at −20°C for 2 h. Then, cells were stained with 500 µl propidium iodide (PI) (50 µg/ml)/RNase A (200 µg/ml; Nanjing KeyGen Biotech, Co., Ltd.) for 30 min at room temperature. Finally, cells were washed with 1X PBS, centrifuged at room temperature (300 × g for 5 min), and resuspended in 1X PBS, and their fluorescence was detected using a FACS Aria II flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software (V.10.4.1; FlowJo LLC).
The evaluation of apoptosis induction was performed using a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer's instructions. HCT-116 cells (2×105 cells/well) in a 6-well plate were treated with involucrasin A (0, 25, 50 and 100 µM) and incubated at 5% CO2 and 37°C for 48 or 72 h. Then, cells were washed with cold 1X PBS (pH 7.4) before trypsinization (3 min), centrifugation (300 × g for 5 min at room temperature), resuspension in 100 µl 1X binding buffer, staining with 2.5 µl FITC Annexin V and 2.5 µl PI, and incubation for 15 min in the dark at room temperature. The cellular analysis was performed using a flow cytometer (Beckman Coulter, Inc.).
Three different genes encode the isoforms of Akt, including Akt1, Akt2 and Akt3. Akt3 is expressed mainly in the testes and brain, Akt2 is highly expressed in insulin-responsive tissue, and Akt1 is ubiquitously expressed (
Western blotting was performed to detect the protein expression levels extracted from HCT-116 cells. Firstly, the protein samples were collected in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) with phosphatase inhibitor (Roche Diagnostics GmbH) and protease inhibitor cocktail (Roche Diagnostics GmbH). Next, a BCA Protein Assay Kit (Beyotime Institute of Biotechnology) was used to determine the concentration of the protein samples. Samples containing 20 µg of protein were separated by SDS-PAGE on 10–15% gels and transferred to PVDF membranes. Membranes were blocked with 3% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The blocked membranes were then incubated with primary antibodies diluted in TBST containing 5% bovine serum albumin (Beyotime Institute of Biotechnology) overnight at 4°C. Membranes were washed three times with TBST for 1 h at room temperature and then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies diluted in 5% skimmed milk in TBST for 1 h at room temperature. Membranes were washed three times with TBST and visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) via an Amersham Imager 800 (Cytiva). ImageJ software (version 1.48; National Institutes of Health) was used to semi-quantify protein levels after western blotting.
SPSS version 20.0 (IBM Corp.) was used for statistical analysis. All data are from at least three independent experiments. Data are presented as the mean ± standard deviation. Comparisons between two groups were performed using an unpaired Student's t-test; for comparisons of three or more groups, one-way ANOVA followed by Tukey's multiple comparison test was used. P<0.05 was considered to indicate a statistically significant difference.
To determine the effects of involucrasin A on CRC viability, HCT-116 cells were treated with involucrasin A at various doses for 72 h, and cell viability was analyzed by SRB assay. The results suggested that involucrasin A effectively inhibited the viability of the CRC cell line HCT-116 in a dose-dependent manner with an IC50 value of 37.92 mM (
In cancer, cancer cell proliferation is generally induced by a dysregulated cell cycle (
A close relationship exists between cell cycle progression, proliferation and apoptosis in cancer cells (
The Akt/MDM2/p53 signaling pathway is a well-known modulator of cell proliferation and apoptosis (
To confirm that involucrasin A exerts antitumorigenic functions by modulating the Akt/MDM2/p53 pathway, CA-Akt was exogenously expressed in HCT-116 cells. As shown in
To further investigate the dependency of p53 in involucrasin A-induced anti-proliferative and pro-apoptotic effects, the present study took advantage of the isogenic p53 KO HCT-116 cell line. In cells with p53 KO, the protein expression levels of p53 were significantly decreased compared with those of the control group (parental HCT-116 cells) (
To confirm the involvement of Bax in involucrasin A-induced cytotoxic effects on HCT-116 cells, the present study knocked down Bax using lentivirus-mediated RNA interference. In cells transduced with shBax, the protein expression levels of Bax were significantly decreased (
Although chemotherapy is the preferred treatment method for human CRC, adverse side effects and chemoresistance remain major hurdles to successful treatment. Natural flavonoids are a rich repository for colon cancer drug discovery (
Proliferation is a crucial part of cancer development and progression. Cancer therapy involving cytotoxic drugs kills cells that have a high basal level of proliferation. Therefore, excellent anticancer agents, such as vinblastine, homoharringtonine, etoposide, teniposide, docetaxel and camptothecin derivatives, can block proliferation, resulting in cell cycle arrest and apoptosis (
According to a previous report, flavonoids induce apoptosis by increasing levels of ROS in cancer cells (
In the present study, the results suggested that involucrasin A could significantly decrease the phosphorylation levels of Akt and MDM2, and upregulate p53 protein expression in HCT-116 cells. To confirm the involvement of the Akt/MDM2/p53 pathway in the antitumorigenic functions of involucrasin A, overexpressed CA-Akt and p53 KO in HCT-116 cells were assessed. The results showed that CA-Akt and p53 KO attenuated involucrasin A-induced anti-proliferative and pro-apoptotic effects. Bax has been verified to be involved in p53-mediated apoptosis (
Notably, the present study found that the expression levels of Bax were upregulated by involucrasin A. However, p53 KO attenuated involucrasin A-induced Bax expression. These results suggested that involucrasin A induced cancer cell apoptosis by p53-modulated Bax signaling. To confirm this hypothesis, Bax knockdown in HCT-116 cells was performed. The results showed that Bax knockdown attenuated involucrasin A-induced pro-apoptotic effects and proliferation inhibition. During apoptosis, Bax induces the release of cytochrome
In summary, the present study indicated that involucrasin A may exert a potent anticancer effect on colon cancer cells through inducing apoptosis and cell cycle arrest. Mechanistically, involucrasin A exhibited anticancer functions by modulating the Akt/MDM2/p53 pathway. For the present study, involucrasin A was isolated from
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
CW, ZY and WM conceived and designed the experiments. JD, YS, ZiW, QL, FZ, WL, ZhW and JC performed the experiments and analyzed the results. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. CW and WM wrote the manuscript. CW and WM confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Involucrasin A effectively inhibits HCT-116 cell proliferation. (A) Chemical structure of involucrasin A. (B) Dose-dependent effect of involucrasin A on the viability of HCT-116 cells at 72 h. (C) Colony formation of HCT-116 cells treated with involucrasin A (0, 12.5, 25 and 50 µM) for 10 days. (D) Dose-dependent effect of involucrasin A on the viability of different cancer cell lines at 72 h. Colorectal cancer cell lines include HCT116, Caco-2 and CT26. (E and F) HCT-116 cells were treated with involucrasin A (0, 25, 50 and 100 µM) for 72 h. The cell cycle assay was analyzed by flow cytometry. (G and H) Protein expression levels of cyclin A2 were analyzed using western blotting. β-actin served as a loading control. All data are from at least three independent experiments. *P<0.05, **P<0.01 vs. control (0 µM). PI, propidium iodide.
Involucrasin A effectively induces HCT-116 cell apoptosis. HCT-116 cells were treated with involucrasin A (0, 25, 50 and 100 µM) for 72 h. (A) HCT-116 cells were stained with Annexin V-FITC/PI, and apoptosis was quantified by flow cytometry. (B-E) Protein expression levels of cleaved-caspase 9, cleaved-caspase 6, p-Akt, Akt, p-MDM2, MDM2, p53 and Bax were examined via western blotting. β-actin served as a loading control. All data are from at least three independent experiments. **P<0.01 vs. control (0 µM). PI, propidium iodide; p, phosphorylated; MDM2, murine double minute 2 homologue.
CA-Akt attenuates involucrasin A-induced inhibition of proliferation and apoptosis. (A and B) In cells with CA-Akt, the protein expression levels of p-Akt and Akt were significantly increased. HCT116 cells transduced with the empty vector alone as the negative control. (C) HCT-116 cells were treated with involucrasin A at the indicated concentrations for 48 h. Cell viability was analyzed by sulforhodamine B assay. (D) Colony formation of control and CA-Akt HCT-116 cells treated with involucrasin A (0 or 25 µM) for 10 days. Cells were treated with involucrasin A (0 or 50 µM) for 48 h. (E) Apoptosis was quantified by flow cytometry assay. (F) Western blotting was used to analyze the protein expression levels of cleaved-caspase 9, cleaved-caspase 6 and Bax. β-actin served as a loading control. (G) Relative protein expression levels of Bax, cleaved-caspase 9, cleaved-caspase 6 were semi-quantified. (H and I) The protein levels of p53, p-Akt, Akt, p-MDM2 and MDM2 were analyzed by western blotting. All data are from at least three independent experiments. *P<0.05, **P<0.01 as indicated or vs. control. CA-Akt, constitutively active Akt; PI, propidium iodide; p, phosphorylated; MDM2, murine double minute 2 homologue.
p53 KO attenuates the anticancer functions of involucrasin A. Parental HCT-116 cells were used as the control group. (A and B) In the cells with p53 KO, the protein expression levels of p53 were significantly decreased. (C) Cells were treated with involucrasin A at the indicated concentrations for 48 h. Cell viability was analyzed by sulforhodamine B. (D) Control and p53 KO HCT-116 cells were treated with involucrasin A (0 or 25 µM) for 10 days, and colony formation was determined. (E and F) Protein expression levels of cyclin A2 were analyzed using western blotting. Cells were treated with 0 or 50 µM involucrasin A for 48 h. (G) Apoptosis was quantified by flow cytometry. (H and I) The protein expression levels of cleaved-caspase 9 and Bax were examined via western blotting. β-actin served as a loading control. All data are from at least three independent experiments. *P<0.05, **P<0.01 as indicated or vs. control. KO, knockout; PI, propidium iodide.
Bax knockdown attenuates the anticancer functions of involucrasin A. (A and B) In cells transduced with shBax, protein expression levels of Bax were significantly decreased. (C-E) Cells were treated with involucrasin A at the indicated concentrations (0 or 50 µM) for 48 h. The expression levels of cleaved-caspase 9 and Bax were examined using western blotting, and apoptosis was quantified by flow cytometry. (F) Cells were cultured with 0, 6.25, 12.5 25, 50 and 100 µM involucrasin A for 48 h. Cell viability was analyzed by sulforhodamine B. (G) For colony formation assay, cells were treated with 0 or 25 µM involucrasin A for 10 days. All data are from at least three independent experiments. **P<0.01 as indicated or vs. sh007. sh, short hairpin; PI, propidium iodide.