Skin cancer is one of the primary causes of mortality worldwide. With an increasing frequency of skin cancers, there is an urgent requirement for the development of numerous treatment options. The present study investigated the anticancer activity of caffeic acid n-butyl ester (CAE) against the A431 skin cancer cell line. Antiproliferative effects were investigated using an MMT assay. Apoptosis was examined by DAPI and Annexin V/fluorescein isothiocyanate and propidium iodide staining. Reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and cell cycle analyses were performed via flow cytometry. Protein expression was determined by western blotting. The findings of the present study demonstrated that among a variety of cancer cell lines, CAE exhibited significant anticancer activity against the A431 skin cancer cell line with a half-maximal inhibitory concentration of 20 µM. CAE was associated with apoptosis and cell cycle arrest of A431 cells, and induced ROS-mediated alterations in MMP. In addition, CAE considerably suppressed the expression of some of the important proteins of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) cascade. The results of the present study indicated that CAE exerted anticancer effects on the A431 skin carcinoma cell line via the induction of apoptosis and suppression of the PI3K/AKT/mTOR signaling pathway. Therefore, CAE may be beneficial for the development of chemotherapy for skin cancers.
Skin cancer is one among the primary causes of mortality worldwide (
The following chemicals were used in the present study. CAE, RNase A, Triton X-100 and dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Primary and secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The fluorescent probes DCFH-DA, DiOC6, DAPI, propidium iodide (PI), fetal bovine serum (FBS), RPMI-1640 medium, L-glutamine and antibiotics were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Human lung cancer cell line (A549), pancreas (MIA PaCa-2), prostate (PC-3), breast (MCF-7), gastric (SNU-5), colon (HTB-39), normal human fibroblast FR2 and skin cancer (A431) cell lines were obtained from the Cancer Research Institute of Beijing (Beijing, China) and were cultured continuously in RPMI-1640 supplemented with 10% FBS containing antibiotics, 100 µg/ml streptomycin and 100 U/ml penicillin G and maintained at 37°C and 5% CO2.
The anti-proliferative effects of CAE were investigated in the 8 aforementioned cell lines using an MTT assay. All cells were cultured at 37°C at a density of 1×106 cells/well in 96-well plates for 12 h. The cells were then subsequently treated with 0–200 µM CAE for 24 h. Subsequently, 20 µl MTT solution was added to each well. Prior to the addition of 500 µl DMSO, the medium was completely removed. The MTT formazan crystals were dissolved by adding 500 µl DMSO. The absorbance was detected using an ELISA plate reader (optical density at 570 nm). As CAE exhibited the lowest half-maximal inhibitory concentration (IC50) against A431 cells, subsequent experiments were conducted within this cell line at concentrations of 0, 10, 20 and 40 µM CAE.
The effect of CAE on the colony formation potential of A431 cells was investigated when cells were collected at the exponential phase of growth and the cells were then counted using a hemocytometer. Cells were seeded at a density of 200 cells/well and maintained at 37°C for 48 h to permit cell adherence. Subsequently, 0, 10, 20 and 40 µM CAE was administered. Following the treatment with CAE, cells were incubated at 37°C for 6 days. Following incubation, cells were washed with PBS and fixed with methanol at −20°C for 4 min and then stained with crystal violet for 30 min at room temperature, then counted under a light microscope (magnification, ×200).
A431 cells were seeded at the density of 1×106 cells/well in 6-well plates and subsequently treated with 0, 10, 20 and 40 µM CAE for 24 h, followed by DAPI staining at 25°C for 5 min. Then, the cell samples were examined via fluorescence microscopy (magnification, ×200).
A431 cells were plated at a density of 1×106 cells/well in 6-wellplates and treated with 0, 10, 20 and 40 µM CAE for 24 h. Subsequently, the cells were collected and washed with PBS. The cells were then incubated with Annexin V/FITC and PI for 15 min at 25°C, and apoptosis was estimated using flow cytometry and BD FACSuite software v1.0 (BD Biosciences, San Jose, CA, USA).
To investigate the dissemination of A431 cells in different phases of the cell cycle, ~1×105 cells/well in 6-well plates were maintained at 37°C overnight to allow cell adherence. Cells were then treated with 0, 10, 20 and 40 µM CAE and then the plates were incubated at 37°C for 24 h. Subsequently, the cells were trypsinized and resuspended in ice-cold PBS, followed by treatment with ethanol (70%) and allowed to fix overnight at −20°C. Following fixation with ethanol, cells were treated with ice-cold PBS twice and subjected to centrifugation (800 × g) for 10 min at 4°C. Cells were then resuspended in 1 ml PI/Triton-X 100 solution for 30 min in the dark. Finally, the dissemination of the cells at each phase were examined using 8,000 cells in a FACScan flow cytometer (BD Biosciences). The estimated percentage of cells in each phase of the cell cycle was quantified using WinMDI softwarev2.0 (Informer Technologies, Inc., Los Angeles, CA, USA).
A431 cells were seeded at a density of 2×105 cells/well in a 6-well plate and maintained for 24 h at 37°C and treated with 0, 10, 20 and 40 µM CAEfor 24 h at 37°C in 5% CO2 and 95% air. Subsequently, cells from all samples were collected, washed twice with PBS and re-suspended in 500 µl DCFH-DA (10 µM) for ROS quantification or 3,3-dihexyloxacarbocyanine iodide (1 µmol/l) for MMP analysis at 37°C in a dark room for 30 min. The samples were then examined instantly using a flow cytometer (WinMDI software version 2.0 (Informer Technologies, Inc.).
Cell migration analysis was performed using the Boyden chamber assay with some modifications. Cells at the density of 5×104 cells/well were suspended in RPMI-1640 medium supplemented with 2% FBS and placed in the upper chamber of 8-µm pore size Transwell inserts. Subsequently, RPMI-1640 medium medium supplemented with 10% FBS was added to lower chamber, followed by an incubation for 24 h at 37°C. On the upper surface of the membrane, non-migrated cells were removed and migrated cells on the lower surface of the membrane were fixed in 100% methanol and Giemsa stained at 20°C for 4 h. Cell migration was estimated by counting the number of the migrated cells under a microscope (Olympus CH20i, Binocular version; Olympus Corporation, Tokyo, Japan, magnification, ×200).
Protein expression was determined by western blot analysis. The cells were lysed in lysis buffer [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid, 350 mM NaCl, 20% glycerol, 1% Nonidet P 40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail and phosphatase inhibitor cocktail]. Briefly, proteins present in the cell extracts were resolved by 10% SDS-PAGE. This was followed by transfer on nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in PBS and then incubated with primary antibody primary antibody [AKT, sc-135829; phosphorylated (p)-AKT, sc-7985-R; PI3K, sc-136298; mTOR, sc-517464 and p-mTOR, sc-293133] for overnight at 4°C (all 1:1,000), followed by incubation with horse radish peroxidase-conjugated (cat. no. 9003-99-0) and anti-rabbit secondary antibody (sc-2372) for 1 h at room temperature. All antibodies were purchased from purchased from Santa Cruz Biotechnology, Inc. The western blots were visualized using an enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA). The western blots were quantified by my Image Analysis software v1.0 (Thermo Fisher Scientific, Inc.).
The experiments were performed in triplicate and data are presented as the mean ± standard deviation. Statistical analysis was performed sing a one-way analysis of variance followed by a Tukeys post-hoc test using GraphPad prism version 7 (GraphPad Software, Inc. La Jolla CA, USA). P<0.05 was considered to indicate statistically significant difference.
In order to investigate the anticancer effects of CAE on various cancer cell lines, cells were treated with CAE at various concentrations and the IC50 was determined for each cell line (
To examine whether the anticancer effects of CAE were due to the induction of apoptosis, A431 cells were treated with CAE and apoptosis was determined by DAPI staining. The results of the present study demonstrated that CAE induced A431 cell apoptosis in a dose-dependent manner, as demonstrated by an increase in the number of cells with white-colored nuclei (
The pro-apoptotic potential of CAE observed via Annexin V/FITC staining indicated that CAE induced accretion of intracellular ROS. Therefore, the ROS levels in cells treated with various doses of CAE for 24 h were investigated. The findings of the present study revealed that the intracellular ROS levels of CAE-treated cells increased by 210% in the 40 µM CAE-treated group (
ROS leads to an imbalance in the outer mitochondrial potential resulting in the release of apoptosis-inducing proteins (
To examine the impact of CAE on the cell cycle phase distribution of A431 cells, the cells were treated with 0, 10, 20 and 40 µM of CAE for 24 h. The number of cells in the G2 phase increased in a dose-dependent manner leading to a cell cycle arrest (
The effects of CAE on cell migration of A431 cell migration were investigated (
The mTOR/PI3K/AKT signalling cascade is considered to be one of the crucial pathways that maybe targeted for the treatment of cancer (
Skin cancers are among the principal causes of mortality in humans, particularly in the Caucasian population (
In conclusion, the findings of the present study indicated that CAE may be a potential treatment for skin cancer by targeting the mTOR/PI3K/AKT signaling cascade. As available treatment options are limited, naturally occurring CAE may be considered a potential treatment for skin cancer due to its associated low toxicity; however, further investigation
Effects of indicated concentrations of CAE on (A) cell viability and (B) colony formation. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.001, **P<0.001 and ***P<0.0001 vs. control. CAE, caffeic acid n-butyl ester.
Induction of apoptosis by caffeic acid n-butyl ester at indicated concentrations as depicted by DAPI staining. All experiments were carried out in triplicate.
Estimation of apoptotic cell populations at indicated concentrations of caffeic acid n-butyl ester by Annexin V/propidium iodide staining followed by flow cytometry. Experiments were carried out in triplicate.
Effect of CAE on the protein expression of Bax and Bcl-2 was determined using western blot analysis. The experiments were carried out in triplicates. *P<0.001, **P<0.001 and ***P<0.0001 vs. control. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X; CAE, caffeic acid n-butyl ester.
Effects of indicated concentration of CAE on (A) ROS generation and (B) MMP. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.001, **P<0.001 and ***P<0.0001 vs. control.MMP, mitochondrial membrane potential; ROS, reactive oxygen species. CAE, caffeic acid n-butyl ester.
CAE triggers G2/M cell cycle arrest at indicated doses of CAE. Experiments were performed in triplicate. CAE, caffeic acid n-butyl ester.
Effects of CAE on the migration of A431 cells at indicated concentrations. Experiments were performed in triplicate. *P<0.001, **P<0.001 and ***P<0.0001 vs. control.
Western blot analysis demonstrated the effect of indicated concentrations of CAE on expression of mTOR/PI3K/AKT signaling pathway proteins. The experiments were performed in triplicates. *P<0.001, **P<0.001 and ***P<0.0001 vs. control. p, phosphorylated; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase.
IC50 of CAE against different cancer cell lines as determined by MTT assay.
Cell line | IC50 (µM) |
---|---|
Gastric cancer SNU-5 | 30 |
Lung cancer A-549 | 30 |
Skin cancer carcinoma A431 | 20 |
Prostate PC-3 | 30 |
Breast MCF-7 | 40 |
Pancreas MIA PaCa-2 | 40 |
Colon HTB-39 | 30 |
Human normal fibroblasts (FR2) | >100 |
IC50, half-maximal inhibitory concentration.