Delphinidin is a member of the anthocyanidin family and is a natural pigment in red cabbage, berries, sweet potatoes and grapes. It possesses nutraceutical properties against various chronic diseases and types of cancer. However, little is known about its preventative effects on epithelial ovarian cancer, a disease that is associated with a low survival rate, a poor prognosis and a high rate of recurrence. The results of the present study demonstrated that the proliferation of SKOV3 cells decreased in a dose-dependent manner in response to treatment with delphinidin, and the phosphorylation of carcinogenic protein kinases associated with the progression of epithelial ovarian cancer was affected by delphinidin treatment. The levels of phosphorylated protein kinase B (AKT), ribosomal protein S6 kinase β-1 (P70S6K), ribosomal protein S (S6), extracellular signal-regulated kinase (ERK)1/2 and p38 were suppressed by increasing concentrations of delphinidin. Furthermore, the combination of certain pharmacological inhibitors, including phosphoinositide 3-kinase (PI3K; LY294002), ERK1/2 (U0126) and delphinidin significantly reduced the proliferation of SKOV3 cells and the phosphorylation of each of those target proteins. In addition, delphinidin treatment exerted anti-proliferative effects on paclitaxel-resistant SKOV3 cells, compared with treatment with paclitaxel alone. These results indicate that delphinidin inhibits the proliferation of SKOV3 cells through inactivation of PI3K/AKT and ERK1/2 mitogen-activated protein kinase signaling cascades, and that this cell signaling pathway may be a pivotal therapeutic target for the prevention of epithelial ovarian cancer, including paclitaxel-resistant ovarian cancer.
Flavonoids are the most abundant polyphenols in the human diet and may be subdivided into anthocyanidins, flavanols, flavanones, flavonols, flavones and isoflavones, according to structural and functional features (
Gynecological cancer subtypes are malignant carcinomas of the female reproductive tract, the most common of which is epithelial ovarian cancer (EOC); EOC is the fifth leading cause of cancer-associated mortalities in women, due to a lack of symptoms and effective biomarkers for diagnosis in the early stages (
Delphinidin was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ, USA). SB203580 and U0126 were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA) and LY294002 was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibodies against phosphorylated-AKT [serine (Ser)473] (catalog no. 4060), ERK1/2 [threonine (Thr)202/Tyr204, (catalog no. 9101; Cell Signaling Technology, Inc.)] c-Jun N-terminal kinase (JNK; Thr183/Thr185, catalog no. 4668), p38 (Thr180/Thr182; catalog no. 4511), ribosomal protein S6 kinase β-1 (P70S6K; Thr421/Ser424; catalog no. 9204) and S6 (Ser235/236, catalog no. 2211), and total AKT (catalog no. 9272), ERK1/2 (catalog no.4695), JNK (catalog no. 9252), p38 (catalog no. 9212), P70S6K (catalog no. 9202) and S6 (catalog no. 2217) were purchased from Cell Signaling Technology, Inc. All antibodies were diluted to 1:1,000 for western blot analysis. Cisplatin and paclitaxel were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).
SKOV3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in McCoy's 5A (modified) medium (cat. no. 16600-082; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in an atmosphere containing CO2.
Proliferation assays were conducted using a Cell Proliferation ELISA, 5-bromo-2′-deoxyuridine (BrdU) kit (catalog no. 11647229001; Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer's protocol. Briefly, SKOV3 cells (3×103 cells/100 µl) were seeded onto a 96-well plate and incubated at 37°C for 24 h in serum-free McCoy's 5A (modified) medium. Cells were then treated with 10 µM delphinidin alone or with various inhibitors of cell signaling proteins including 20 µM LY294002, 20 µM SB203580 and 10 µM U0126 or conventional chemotherapy including 20 µM paclitaxel and 50 µM cisplatin in a final volume of 100 µl/well. Following 48 h of incubation at 37°C, 10 µM BrdU was added to the cell culture and the cells were incubated for an additional 2 h at 37°C. After labeling cells with BrdU, the fixed cells were incubated with anti-BrdU-peroxidase (POD) working solution for 90 min at room temperature. The anti-BrdU-POD binds to BrdU incorporated in newly synthesized cellular DNA, and these immune complexes were detected by the reaction to 3,3′,5,5′-tetramethylbenzidine substrate. The absorbance values of the reaction product were quantified by measuring the absorbance at 370 and 492 nm using an ELISA reader. These experiments were performed in triplicate.
The SKOV3 cells (3×104 cells/300 µl) were seeded onto confocal dishes (catalog no. 100350; SPL Life Sciences Co., Ltd., Pocheon, Korea) and then incubated for 24 h at 37°C in serum-free McCoy's 5A. The cells were subsequently treated with delphinidin at a final concentration of 10 µM in 10 ml for 48 h at 37°C in a CO2 incubator. Following incubation, the cells were subjected to TUNEL staining using an
The concentration of proteins from whole-cell extracts was determined using a Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin (Sigma-Aldrich; Merck KGaA) as the standard. Proteins were denatured, separated using 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked using 5% bovine serum albumin in TBST buffer for 1 h at room temperature. Immunoreactive proteins were detected using goat anti-rabbit polyclonal antibodies against phosphorylated proteins and total proteins at a dilution of 1:1,000 (as mentioned in the previous section). The blots were developed using enhanced chemiluminescence detection (SuperSignal™ West Pico; Pierce; Thermo Fisher Scientific, Inc.) and quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light, using a ChemiDoc™ EQ system and Quantity One® software (version 4.5; Bio-Rad Laboratories, Inc.). Total protein normalization was used to quantify the abundance of the phosopho-proteins. As a loading control, total proteins were used to normalize the results from the detection of proteins by western blotting. Multiple exposures of each western blot were performed to ensure linearity of the chemiluminescent signals. These experiments were performed in triplicate.
Apoptosis of SKOV3 cells induced by delphinidin was analyzed using a fluorescein isothiocyanate (FITC)-Annexin V apoptosis detection kit I (BD Biosciences, Franklin Lakes, NJ, USA). The cells (4×105 cells) were seeded onto 6-well plates and incubated for 24 h at 37°C in a CO2 incubator in serum-free medium until the cells reached 70–80% confluence. The cells were then treated separately with 10 µM delphinidin, 20 µM LY294002, 10 µM U0126 or a combination of 10 µM delphinidin with each inhibitor for 48 h at 37°C in a CO2 incubator. Supernatants were removed from the culture dishes using a 10 ml serological pipette, and adherent cells were detached using 0.25% trypsin-EDTA. The cells were collected by centrifugation (300 × g) at room temperature for 5 min, washed with PBS and resuspended in 1X binding buffer (BD Biosciences). The cell suspension (100 µl) was then transferred into a 5 ml culture tube and incubated with 5 µl FITC-Annexin V and 5 µl PI for 15 min at room temperature in the dark. Subsequently, 400 µl 1X binding buffer was added to the 5 ml culture tube. Fluorescence intensity was analyzed using FACSCalibur™ at 488 nm (BD Biosciences).
SKOV3 cells (3×105 cells) were seeded onto 6-well plates and incubated for 24 h in serum-free medium at 37°C until the cells reached 70–80% confluence. The cells were then treated separately with 10 µM delphinidin, 20 µM LY294002, 10 µM U0126 or a combination of 10 µM delphinidin with each inhibitor for 48 h at 37°C in an incubator containing CO2. Supernatants were transferred from the culture dishes into collecting tubes and adherent cells were detached using 0.25% trypsin-EDTA. The cells were collected via centrifugation (300 × g) at room temperature for 5 min, washed twice with cold PBS and fixed in chilled-70% ethanol at 4°C overnight. Cells were subsequently washed twice with PBS, incubated with 10 µg/ml RNase A (Sigma-Aldrich; Merck KGaA) and 50 µg/ml PI (BD Biosciences) in PBS at room temperature for 30 min in the dark and subjected to flow cytometry (FACSCalibur™; BD Biosciences).
Data obtained from the proliferation and Annexin V and PI assays were subjected to two-way analysis of variance according to the general linear model (PROC-GLM) of the SAS program (SAS Institute, Cary, NC, USA) in order to detect the effects of treatments on SKOV3 cells proliferation. Data are presented as least square means with standard error of the mean, unless otherwise stated. P<0.05 was considered to indicate a statistically significant difference.
The current study aimed to determine the effects of delphinidin on SKOV3 cells derived from ovarian adenocarcinoma. The results demonstrated a dose-dependent effect of delphinidin to inhibit the proliferation of SKOV3 cells between concentrations of 0, 0.1, 1, 10, 50 and 100 µM (
It was examined whether the delphinidin-suppressed proliferation of SKOV3 cells is associated with the PI3K/AKT, ERK1/2 MAPK and P38 MAPK signal transduction pathways. Cell proliferation assays were conducted in the absence or presence of delphinidin (10 µM), LY294002 (20 µM; PI3K inhibitor), SB203580 (20 µM; P38 MAPK inhibitor) and U0126 (10 µM; ERK1/2 MAPK inhibitor;
In order to compare the apoptotic effects of various drug treatments (10 µM delphinidin, 20 µM LY294002, 10 µM U0126 and a combination of 10 µM delphinidin with each inhibitor) in SKOV3 cells, Annexin V/PI staining and cell cycle assays were performed. As illustrated in
To determine the regulatory effects of delphinidin and pharmacological inhibitors on the cell cycle distribution of SKOV3 cells, cell cycle assays were conducted via quantitation of DNA content using PI and flow cytometry (
To investigate chemotherapeutic effects of delphinidin on the proliferation of SKOV3 cells compared with conventional chemotherapy, the therapeutic efficacy of 10 µM delphinidin, 20 µM paclitaxel (a taxane-based drug) and 50 µM cisplatin (a platinum-based drug) alone and in combination were assessed (
The results of the present study indicate a functional role for delphinidin, a member of the anthocyanidin family, in suppressing the progression of EOC using SKOV3 cells as a model system. The anti-proliferative effects of delphinidin in SKOV3 cells were regulated by its inhibition of PI3K/AKT and ERK1/2 MAPK signal transduction protein phosphorylation. In addition, a combination of delphinidin and the pharmacological inhibitors of PI3K and ERK1/2 MAPK induced apoptosis and cell cycle arrest in SKOV3 cells. Furthermore, comparative analyses between delphinidin and conventional chemotherapy of EOC revealed that delphinidin exhibits a synergistic effect with paclitaxel in promoting anti-proliferative effects in SKOV3 cells. These findings support the hypothesis that delphinidin inhibits cell proliferation and induces apoptosis in EOC, and that appropriate therapeutic approaches using delphinidin may be useful for overcoming chemoresistance in ovarian cancer cells.
Delphinidin, a natural bioactive product belonging to the anthocyanidins, has a diphenylpropane-based polyphenolic ring structure, including a positive charge in its core ring (
EOC initiation and progression are complex events regulated by various intracellular signaling proteins (
Constitutive activation of the ERK1/2 MAPK pathway promotes the growth, proliferation and differentiation of ovarian cancer cells via the activation of downstream protein kinase or transcription factors that may enhance carcinogenesis (
For identifying interactions between delphinidin and the pharmacological inhibitors of PI3K and ERK1/2 MAPK signaling, apoptosis and cell cycle regulation in response to a combination of delphinidin with LY294002 or U0126 was investigated. As demonstrated in previous studies, delphinidin induces cell cycle arrest via the downregulation of certain cell cycle regulatory proteins, including cyclin-dependent kinase (Cdk)1, Cdk2, cyclin A and cyclin D1, and via the upregulation of p21WAF1 in a dose-dependent manner in prostate and colon cancer cells (
Despite initial positive responses to platinum- and taxane-based chemotherapies, persistent chemoresistance of EOC is consistently a problem in ≤80% of patients (
Collectively, the present study presents evidence, using the SKOV3 human ovarian cancer cell line, that delphinidin is a crucial physiological modulator in EOC, and is also an inhibitor of EOC cell proliferation. It was identified that delphinidin increased the rate of apoptosis via DNA fragmentation in SKOV3 cells. In addition, delphinidin suppressed the proliferation of SKOV3 cells through inactivating the phosphorylation of AKT, P70S6K, S6 and ERK1/2 proteins in the PI3K/AKT and ERK1/2 MAPK signaling pathways. Furthermore, blocking the ERK1/2 MAPK signaling pathways reduced the levels of S6 protein in SKOV3 cells, which regulates protein synthesis for cell death. Furthermore, interactions between delphinidin and various pharmacological inhibitors of PI3K/AKT and ERK1/2 MAPK, synergistically induced apoptosis by increasing the proportion of SKOV3 cells arrested in the sub-G1 phase of the cell cycle. Delphinidin-reduced proliferation of SKOV3 cells was also required for paclitaxel to decrease the proliferation of SKOV3 cells. In conclusion, these findings provide evidence for the synergistic anticancer effects of delphinidin combined with paclitaxel in human ovarian cancer SKOV3 cells, involving the inactivation of the PI3K/AKT and ERK1/2 MAPK signaling pathways. Thus, delphinidin may be developed as a novel therapeutic agent for the enhancement of survival in patients with EOC.
The authors thank Dr Fuller W. Bazer (Texas A&M University, College Station, USA) for their thoughtful editing and comments on this manuscript. The present study was supported by grants from the Basic Science Research Program (grant no. 2015R1D1A1A01059331) of the Republic of Korea and also supported by School of Life Sciences and Biotechnology for BK21 PLUS, Korea University.
Effects of delphinidin on the viability of SKOV3 ovarian adenocarcinoma cells. (A) Dose-response effects of delphinidin on the proliferation of SKOV3 cells were analyzed and are presented as a percentage relative to the non-treated controls (100%). (B)
Dose-response effects of delphinidin on the phosphorylation of target signaling proteins in SKOV3 cells. Serum-starved SKOV3 cells were treated with delphinidin (0, 0.1, 1 and 10 µM) for 30 min. Phosphorylation of (A) AKT, (B) P70S6K, (C) S6, (D) ERK1/2, (E) p38 and (F) JNK was estimated in SKOV3 cells incubated with delphinidin using western blot analysis. Blotting membranes were imaged to analyze the normalized values by calculating the abundance of phosphorylated protein relative to the total protein. **P<0.01; *P<0.05. ERK, extracellular-regulated kinase; JNK, c-Jun N-terminal kinase; p, phosphorylated; t, total protein; AKT, protein kinase B.
Delphinidin suppresses the proliferation of SKOV3 cells through the PI3K/AKT and ERK1/2 MAPK signaling pathways. (A) Effects of pharmacological inhibitors, including 20 µM LY294002 (a PI3K inhibitor), 20 µM SB203580 (a P38 MAPK inhibitor) or 10 µM U0126 (an ERK1/2 MAPK inhibitor), of cell signaling pathways on the proliferation of SKOV3 cells treated with delphinidin. The results exhibit relative differences in cell proliferation, as compared with non-treated SKOV3 cells set at 100%. Starved SKOV3 cells were pretreated with 20 µM LY294002 or 10 µM U0126, or with a combination of both inhibitors for 1 h and then incubated with 10 µM delphinidin for 30 min. Phosphorylation of (B) AKT, (C) ERK1/2, (D) P70S6K and (E) S6 was determined in SKOV3 cells by western blot analysis. Blotting membranes were imaged to analyze the normalized values by calculation of the abundance of phosphorylated protein relative to total protein. All quantitative data are presented as least square means with overall standard error of the mean. ***P<0.001; **P<0.01; *P<0.05, compared with delphinidin treatment.. PI3K, phosphoinositide-3 kinase; ERK, extracellular-regulated kinase; MAPK, mitogen-activated protein kinase; p, phosphorylated; t, total protein; AKT, protein kinase B.
Synergistic effects of delphinidin with pharmacological inhibitors of PI3K/AKT and ERK1/2 MAPK on the necrosis and apoptosis of SKOV3 cells. (A) Flow cytometric analysis of SKOV3 cells by Annexin V and propidium iodide staining. (B) The percentage of cells in the upper left quadrant indicates necrotic cells as compared with non-treated SKOV3 cells. (C) The percentage of cells in the upper right quadrant indicates late apoptotic cells as compared with non-treated SKOV3 cells. Different lowercase letters indicate the statistically significant effects of treatment (P<0.05): acompared with non-treated control; bcompared with delphinidin alone; ccompared with delphinidin plus LY294002; dcompared with delphinidin plus U0126; ecompared with LY294002 alone; fcompared with U0126 alone. PI3K, phosphoinositide-3 kinase; ERK, extracellular-regulated kinase; MAPK, mitogen-activated protein kinase; FITC, fluorescein isothiocyanate.
Delphinidin and pharmacological inhibitors regulate the cell cycle of SKOV3 cells. (A) Flow cytometric analysis was used to estimate DNA contents using PI in response to each treatment of SKOV3 cells. (B) The proportion of SKOV3 cells in each stage of the cell cycle was calculated. These results were performed in triplicate.
Effects of chemotherapeutic agents with delphinidin on proliferation of SKOV3 cells. Serum starved SKOV3 cells were treated with 10 µM delphinidin, 20 µM paclitaxel or 50 µM cisplatin or their combinations for 48 h. Results indicate relative differences in proliferation with values for non-treated SKOV3 cells set at 100%. ***P<0.001; **P<0.01; *P<0.05.