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. Author manuscript; available in PMC: 2016 Jan 28.
Published in final edited form as: Cancer Lett. 2014 Oct 7;356(2 0 0):418–433. doi: 10.1016/j.canlet.2014.09.023

Chaetoglobosin K induces apoptosis and G2 cell cycle arrest through p53-dependent pathway in cisplatin-resistant ovarian cancer cells

Bo Li a,e, Ying Gao a,e, Gary O Rankin b, Yon Rojanasakul c, Stephen J Cutler d, Youying Tu e,**, Yi Charlie Chen a,*
PMCID: PMC4351971  NIHMSID: NIHMS635101  PMID: 25304379

Abstract

Adverse side effects and acquired resistance to conventional platinum based chemotherapy have become major impediments in ovarian cancer treatment, and drive the development of more selective anticancer drugs. Chaetoglobosin K (ChK) was shown to have a more potent growth inhibitory effect than cisplatin on two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and was less cytotoxic to a normal ovarian cell line, IOSE-364, than to the cancer cell lines. Hoechst 33342 staining and Flow cytometry analysis indicated that ChK induced preferential apoptosis and G2 cell cycle arrest in both ovarian cancer cells with respect to the normal ovarian cells. ChK induced apoptosis through a p53-dependent caspase-8 activation extrinsic pathway, and caused G2 cell cycle arrest via cyclin B1 by increasing p53 expression and p38 phosphorylation in OVCAR-3 and A2780/CP70 cells. DR5 and p21 might play an important role in determining the sensitivity of normal and malignant ovarian cells to ChK. Based on these results, ChK would be a potential compound for treating platinum-resistant ovarian cancer.

Keywords: Chaetoglobosin K, Ovarian cancer, Apoptosis, Cell cycle arrest, p53, p38

Introduction

Ovarian cancer is one of the leading causes of cancer-related death among the gynecologic malignancies in the Western world. It affects approximately 204,000 women a year worldwide and is responsible for approximately 125,000 deaths [1]. The three most commonly used treatments for ovarian cancer are surgery, radiation, and chemotherapy, with chemotherapy being the predominant mode of treatment [2]. Although ovarian cancer initially responds to chemotherapy, patients with advanced or more aggressive tumors often experience chemoresistance and recurrence, leading to a poor long-term survival rate. The 5-year survival for patients with advanced stage ovarian cancer has remained less than 20% over the past 20 years [3].

Platinum drugs, such as cisplatin and its analogues, have been most frequently used for treatment of human cancer, including ovarian cancer. However, the obvious drawbacks of these agents, the normal tissue toxicity and acquired resistance to conventional platinum based chemotherapy are driving the development of more selective drugs that target cancer-specific defects [4,5]. The cell cycle is a set of organized and monitored events responsible of proper cell division into two daughter cells. This process involves four sequential phases that go from quiescence (G0 phase) to proliferation (G1, S, G2, and M phases) and back to quiescence. The mammalian cell cycle is a strictly regulated process controlled by the oscillating activities of cyclin-dependent kinases (CDKs). Cancer is a disease of inappropriate cell proliferation, and a number of potential molecular targets for novel anticancer drug discovery have been identified in cell cycle control mechanisms [6,7]. Apoptosis (programmed cell death) plays a crucial role for cell homeostasis. Recent studies suggested that a decreased susceptibility of ovarian cancer to apoptosis was strongly associated with drug resistance. Molecular mechanisms of failed apoptosis in chemoresistant ovarian cancer cells include expression of p-glycoproteins, p53 mutations, down-regulation of pro-apoptotic protein Bax, and up-regulation of anti-apoptotic protein Bcl-2 and other inhibitors of apoptosis that block caspases and stabilize the mitochondrial permeability pore. Thus, increasing the susceptibility of cancer to apoptosis is one of the potential strategies to overcome drug resistance in ovarian cancer cells [1,3].

Natural products have played a very important role as established cancer chemotherapeutic agents, either in their unmodified (naturally occurring) or synthetically modified forms [8]. Chaetoglobosin K (ChK) was first isolated from the fungus Diplodia macrospora in 1980 and was found to have inhibitory and toxic effects on plant growth [9]. Recently, this compound was shown to prevent organochlorine-induced inhibition of gap junctional communication in astrocytes and astroglial cells [10,11], inhibit both Akt and JNK phosphorylation at key activation sites in ras-transformed epithelial cells and human lung carcinoma cells [12], and effectively inhibit angiogenesis through downregulation of vascular epithelial growth factor (VEGF)-binding hypoxia-inducible factor 1α (HIF-1α) in ovarian cancer cells [13].

Although several studies have been carried out to understand the influence of ChK on cancer risk and growth, no efforts have been made to identify the beneficial effects of ChK on the apoptosis and cell cycle of ovarian carcinoma. Thus, the current study was undertaken to investigate the apoptotic and cell cycle arrest effects of ChK in two platinum-resistant ovarian cancer cell lines OVCAR-3 and A2780/CP70, and a normal ovarian surface epithelial cell line IOSE-364. The underlying signaling networks involved in the mechanism of action of ChK on the both ovarian cancer cells were also examined.

Materials and methods

Cell culture and reagents

Two platinum-resistant human ovarian cancer cell lines OVCAR-3 (p53 mutant) and A2780/CP70 (p53 wild-type) were kindly provided by Dr. Jiang at West Virginia University. IOSE-364, a normal ovarian surface epithelial cell line, was a gift from Dr. Auersperg at University of British Columbia, Canada. All cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 °C in a humidified incubator with 5% CO2. ChK, kindly provided by Dr. Cutler at the University of Mississippi, was prepared in dimethyl sulfoxide (DMSO) at 100 mM and stored at −20 °C. Cisplatin, pifithrin (PFT)-α and 2′,7′-dichlorofluorescein diacetate were purchased from Sigma-Aldrich. The primary antibodies against Bcl-xL, Bad, p21, phospho-p53 (ser15), p53, MDM2, phospho-ERK1/2, ERK1/2 (MK1) and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The primary antibodies against caspase-3, -8, and -9, Puma, Bax, Bcl-2, cyclin B1, phospho-cdc2 (Tyr 15), cdc2, Fas, Fas L, DR5, FADD, Phosphop38 MAPK (Thr180/Tyr182), p38 MAPK, Phospho-SAPK/JNK (Thr183/Tyr185) and SAPK/JNK were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

Cell growth assay

Cell growth inhibition or cell number was determined by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) dye absorbance or by trypan blue cell counting. 1 × 104 cells per well were seeded in 96-well microtiter plates for an MTT assay and 1 × 106 cells per well were seeded in 60-mm dishes for cell counting. Cells were allowed to attach to the bottom overnight, and then treated with different concentrations of ChK (0–10 μM) or cisplatin (0–80 μM) for 24 h. Control cells received an equal amount of DMSO only. For MTT assay, 20 μL of MTT (5 mg/ mL) was added to each well and incubated for 4 h at 37 °C in the dark. After removing the supernatant, formazan crystals formed were dissolved in 200 μL DMSO and the absorbance was measured at 570 nm. For trypan blue exclusion, cells from the culture supernatant and the bottom of dishes were collected and combined, incubated with isometrical 0.4% trypan blue solution for 3 min, and then counted under a phase contrast microscope with a hemocytometer.

Apoptosis assessment by Hoechst 33342 staining

OVCAR-3, A2780/CP70 and IOSE-364 cells were seeded in 24-well plates at 1 × 105 cells/well and incubated overnight. Cells were treated with various concentrations (0–4 μM) of ChK for 24 h. After treatment, cells were stained with 10 μg/mL Hoechst 33342 (Sigma, St. Louis, MO) in PBS for 10 min in the dark at 37 °C. Cell apoptosis was examined under a fluorescence microscope (ZEISS), and data were collected from three independent experiments.

Flow cytometry analysis of cell cycle

Cells treated with ChK for 24 h were digested by trypsin and collected by 3000 rpm centrifugation for 5 min and washed with ice-cold PBS. The cell pellet was suspended with 70% ethanol at −20 °C overnight, washed with PBS, then incubated with 180 μg/mL RNase A at 37 °C for 15 min. For flow cytometry, 50 μg/mL propidium iodide (final concentration) was added for 15 min staining in the dark at 37 °C. Flow cytometry (FACSCaliber system, BD Biosciences) was used for detection. Data were plotted and analyzed by using FCS Software (De Novo Software, Los Angeles, CA).

Caspase-3/7assay

OVCAR-3 and A2780/CP70 cells were seeded in 96-well plates at 1 × 104 cells/ well, incubated overnight, and treated with ChK (0–4 μM) for 24 h. After treatment, caspase-3/7 activities in both the cells were detected using a Caspase-Glo 3/7 Assay kit (Promega), and the total protein levels were measured with a BCA assay kit (Pierce). Caspase-3/7 activities were normalized by total protein levels, and were expressed as percentage of the untreated control.

Measurement of intracellular ROS

The intracellular reactive oxygen species (ROS) was detected by staining the cells with 2′,7′-dichlorofluorescein diacetate (DCF-DA). After treatment with ChK (0– 4 μM) for 24 h, cells were incubated with 10 μM DCF-DA for 30 min at 37 °C. Cells were washed twice with PBS (pH 7.4), and the fluorescence intensity was recorded by excitation at 485 nm and emission at 528 nm using a Synergy™ HT Multi-Mode Microplate Reader (BioTek). ROS generation was normalized by the total protein level, and was expressed as percentage of the untreated control.

Total RNA preparation and RT-PCR analysis

Total RNA was isolated from OVCAR-3 and A2780/CP70 cells using TRIzol reagent (Invitrogen). RNA samples were quantitated at OD 260/280 and 1 μg RNA was introduced to reverse transcription with AMV Reverse Transcriptase (Promega). cDNA, equivalent to 80 ng total RNA, was amplified by real-time PCR in triplicate with RT2 SYBR Green qPCR Master Mix (SuperArray) and a Chromo4 real-time detector coupled to a DNA Engine thermal cycler (Bia-Rad). GAPDH mRNA levels were quantified in each sample and were used as a normalization control. Relative mRNA expression was calculated by the mean value with the comparative Ct method (ΔΔCt). The following primer pairs for p53 and GAPDH were chosen from the Primer Bank website (http://medgen.ugent.be/rtprimerdb/index.php): 5′-TTGCAATAGGTGTGCGTCAGA-3′ and 5′-AGTGCAGGCCAACTTGTTCAG-3′ (p53); 5′-CATGAGAAGTATGACAACAGCCT-3′ and 5′-AGTCCTTCCACGATACCAAAGT-3′ (GAPDH).

Western blot

Cells were seeded in 60-mm dishes at 1 × 106 cells/dish, incubated overnight, and treated with 0–4 μM ChK for 24 h. After a double wash with cold PBS, cells were harvested with M-PER Mammalian Protein Extraction Reagent (Pierce) supplemented with Halt Protease and Phosphatase Inhibitor (Pierce), and total protein levels were assayed with BCA Protein Assay Kit (Pierce). Cell lysates were separated by SDS-PAGE and blotted onto nitrocellulose membrane with a Mini-Protean 3 System (Bio-Rad). The membrane was blocked with 5% skim milk in Tris-buffer saline containing 0.1% Tween 20 (TBST) and then incubated with specific primary polyclonal/ monoclonal antibodies. The membrane was washed with TBST, and then incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc.). After washing with TBST, the antigen-antibody complex was visualized with the ECL kit (Pierce). Protein bands were quantitated with NIH ImageJ software and normalized by GAPDH bands for analysis.

Transfection with small interfering RNA (siRNA)

OVCAR-3 and A2780/CP70 ovarian cancer cells were seeded in 60-mm dishes at 5 × 105 cells/dish, incubated overnight, and then transfected with p53 siRNA (Santa Cruz Biotechnology, Inc.) or p38 siRNA (Cell Signaling Technology) using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. After a 24 h transfection period, cells were treated with ChK for 24 h. Cell lysates were collected for Western blot analysis.

Statistical analysis

In this study, all samples were prepared and analyzed in triplicate. The data were presented as means ± standard deviations (SD) of three determinations. Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed with Student–Newman–Keuls (SNK) test. Significant differences among different treatments are indicated by different letters. Statistical differences between two groups were evaluated using the Student's t test. A p < 0.05 was considered statistically significant, and p < 0.01 was considered statistically highly significant. All computations were made by employing the SAS system for windows V8.

Results

ChK inhibits cell proliferation in OVCAR-3 and A2780/CP70 cells

To investigate the cytotoxic effect of ChK and to compare it with that of cisplatin in platinum-resistant ovarian cancer cells, MTT assays and trypan blue dye exclusion were performed after treatment of ovarian cancer and normal ovarian cells with ChK or cisplatin. As shown in Fig. 1A and B, ChK inhibited the growth of the three cell lines in a concentration-dependent manner. MTT assay showed that the cell viability with ChK treatment (1–10 μM) for 24 h ranged from 69.5% to 10.6% for OVCAR-3 cells, from 56.4% to 11.7% for A2780/ CP70 cells, and from 84.9% to 53.6% for IOSE-364 cells. The IC50 values of ChK for OVCAR-3, A2780/CP70 and IOSE-364 cells were estimated to be 2.2, 1.7 and 11.1 μM, respectively, which indicated that ChK had lower cytotoxic effect against normal ovarian cells than ovarian cancer cells (Fig. 1A). Trypan blue exclusion test showed that ChK treatment (1–10 μM) significantly decreased the cell number of OVCAR-3 and A2780/CP70 cells in a dose-depentdant manner ((p < 0.05), but had less inhibitory effect on the IOSE-364 cells (Fig. 1B). This result was consistent with that from MTT assay. The proliferation rate of IOSE 364 cells was observed to be obviously lower than that of OVCAR-3 and A2780/CP70 cells (Fig. 1B). The IC50 values of cisplatin from MTT assay were determined to be 39.6 and 47.9 μM in OVCAR-3 and A2780/CP70, respectively (Fig. 1C), suggesting that both platinum-resistant ovarian cancer cells were more sensitive to ChK than to cisplatin.

Fig. 1.

Fig. 1

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Effect of ChK and cisplatin on cell growth in human ovarian cancer cells OVCAR-3 and A2780/CP70 and normal ovarian cells IOSE-364. (A) ChK inhibits cell viability of OVCAR-3, A2780/CP70 and IOSE-364 cells at 24 h. Cell viability was determined by MTT assay. (B) ChK reduces cell number of OVCAR-3, A2780/CP70 and IOSE-364 cells at 24 h. Cell number was determined by trypan blue exclusion. (C) Cisplatin inhibits cell viability of OVCAR-3 and A2780/CP70 at 24 h. Cell viability was determined by MTT assay. Data represent means ± SD from three independent experiments. Significant differences among different treatments are indicated by different letters (p < 0.05).

ChK induces apoptosis in OVCAR-3 and A2780/CP70 cells

To determine whether ChK inhibited cell viability by inducing apoptosis in ovarian cancer and normal cells, the changes of nuclear morphology of OVCAR-3, A2780/CP70 and IOSE-364 cells treated with ChK (0–4 μM) for 24 h were analyzed under a fluorescence microscope by Hoechst 33342 DNA staining (Fig. 2). In the untreated group, the nuclei were stained less bright and intact. With treatment of ChK, both ovarian cancer cell lines exhibited numerous apoptotic cells with condensed or fragmented nuclei, which were much brighter. While less apoptotic cells were observed in the normal ovarian cells compared with both ovarian cancer cells after ChK treatment (Fig. 2A). The percentages of apoptotic cells were determined after counting 500–1000 cells/well in randomly selected fields. As shown in Fig. 2B, ChK significantly increased the number of apoptotic cells in a dose-dependent manner (p < 0.05), and the maximum apoptosis percentage reached to approximately 12% and 15% for OVCAR-3 and A2780/CP70 cells, respectively. However, the treatment of ChK almost could not induce the apoptosis of IOSE-364 cells within the test range of 0–4 μM. To confirm that ChK induced apoptosis, caspase-3/7 enzymatic activities were evaluated by Caspase-Glo 3/7 Assay kit and Western blot in OVCAR-3 and A2780/CP70 cells. As shown in Fig. 2C, treatment with ChK maximally increased the caspase-3/7 enzymatic activity to 1.27 and 1.16 folds of that in controls for OVCAR-3 and A2780/CP70 cells, respectively (p < 0.05). Fig. 2D showed that ChK, especially at 4 μM, significantly increased the protein level of cleaved caspase-3, and decreased the protein expression of procaspase-3 (p < 0.05). These results indicated that the ChK-induced growth inhibitory effect might be mediated at least partly by induction of apoptosis in platinum-resistant ovarian cancer cells.

Fig. 2.

Fig. 2

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ChK induces apoptosis in OVCAR-3 and A2780/CP70 cells. (A) Hoechst 33342 staining of OVCAR-3, A2780/CP70 and IOSE-364 cells detected by fluorescent microscopy after treatment with DMSO vehicle,1, 2 and 4 μM of ChK for 24 h (×400). Highly condensed or fragmented nuclei represent apoptotic cells. Intact nuclei represent viable cells. (B) Concentration-dependent rise in proportion of apoptotic OVCAR-3 and A2780/CP70 cells treated by ChK for 24 h. (C) Caspase 3/7 activity levels with ChK-treatment for 24 h. The caspase 3/7 activity of the control cells after treatment was arbitrarily expressed as 100%. (D) Protein expression levels of procaspase-3 and cleaved caspase-3 analyzed by Western blot. Protein lysates were prepared from OVCAR-3 and A2780/CP70 cells after treatment with ChK for 24 h. The quantification histograms are shown with error bars. All measurements were done in triplicates. Data represent means ± SD of three independent experiments. Significant differences among different treatments are indicated by different letters (p < 0.05).

ChK induces G2 phase cell cycle arrest

To identify whether the growth inhibitory effect of ChK is caused by specific perturbation of cell cycle-related events, cell cycle phase distribution of cells treated with ChK (0–4 μM) for 24 h was analyzed by flow cytometry after propidium iodide staining. As shown in Fig. 3 and Table 1, treatment of OVCAR-3, A2780/CP70 and IOSE-364 cells with ChK resulted in a significant increase in the proportion of cells at the G2 phase and a reduction in the proportion of cells at the G1 and S phases in a concentration-dependent manner (p < 0.05). The G2 phase percentage of the both ovarian cancer cells increased by approximate 45%, while that of IOSE-364 cells increased by around 12% after 4 μM ChK treatment for 24 h. These results indicated that ChK might have more potential to induce G2 cell cycle arrest for both ovarian cancer cells than the ovarian normal cells.

Fig. 3.

Fig. 3

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ChK induces cell cycle arrest in OVCAR-3, A2780/CP70 and IOSE 364 cells. Cells were treated with various concentrations (0, 1, 2, 4 μM) of ChK for 24 h, fixed in 70% ethanol, and stained with propidium iodide. DNA contents were determined by flow cytometry.

Table 1.

Cell cycle phase distribution of OVCAR-3, A2780/CP70 and IOSE-364 cells with ChK treatment.

Cell lines ChK (μM) Cell cycle phase distribution
%G1 %G2 %S
OVCAR-3 0 44.38 ± 1.93a 15.54 ± 0.91c 40.10 ± 2.84a
1 44.62 ± 2.17a 26.89 ± 0.69b 28.50 ± 1.48b
2 16.11 ± 3.30b 55.58 ± 2.23a 28.32 ± 1.06b
4 17.15 ± 0.91b 54.92 ± 0.30a 27.95 ± 0.62b
A2780/CP70 0 40.96 ± 1.78A 17.72 ± 0.64C 41.32 ± 1.15A
1 38.27 ± 0.B 42.98 ± 2.38B 18.86 ± 3.01B
2 13.87 ± 0.34C 65.15 ± 1.57A 20.98 ± 1.24B
4 15.45 ± 1.91C 62.09 ± 3.66A 22.46 ± 1.76B
IOSE-364 0 37.66 ± 0.04a 28.15 ± 0.87c 34.21 ± 0.83a
1 33.41 ± 2.63b 34.49 ± 3.25b 32.10 ± 0.62b
2 31.39 ± 0.69b 38.89 ± 2.36a 29.73 ± 1.68c
4 33.56 ± 2.31b 40.94 ± 1.97a 25.51 ± 0.33d
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Data represent means ± SD from three independent experiments. Values in a column followed by different letters are significantly different (p < 0.05).

Effect of ChK on the intrinsic apoptotic pathway

Initiation of apoptosis has been broadly separated into two main pathways: (I) the intrinsic or mitochondrial pathway and (II) the extrinsic or death receptor pathway [14]. To clarify whether the intrinsic pathway is involved in ChK-induced apoptosis, we firstly examined the protein expression of pro-apoptotic Bcl-2 family proteins (Puma, Bax and Bad), anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL), and caspase-9 by Western bolt. Fig. 4A shows ChK had no effect on these protein expressions (p > 0.05). ROS are believed to play an important role in the intrinsic apoptotic pathway [15]. We then determined the intracellular ROS level of OVCAR-3 and A2780/CP70 cells after 24h treatment with ChK. Fig. 4B shows ChK did not affect the intracellular ROS production in both cancer cells (p > 0.05). These results indicated that ChK induced apoptosis in OVCAR-3 and A2780/CP70 cells independent of the intrinsic apoptotic pathway.

Fig. 4.

Fig. 4

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Effect of ChK on the intrinsic apoptotic pathway in ovarian cancer cells. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. Significant differences among different treatments are indicated by different letters (p < 0.05). (A) Protein expression levels of Puma, Bcl-2, Bcl-xL, Bax, Bad and caspase-9 analyzed by Western blot. Protein lysates were prepared from OVCAR-3 and A2780/CP70 cells after treatment with various concentrations (0, 1, 2, 4 μM) of ChK for 24 h. (B) Determination of the intracellular ROS level. Cells were treated with different doses of ChK (0, 1, 2, 4 μM) for 24 h, and then stained with DCF-DA (10 μM) for 30 min.

Effect of ChK on the extrinsic apoptotic pathway

Next, we investigated whether ChK could induce apoptosis via the extrinsic apoptotic pathway. As shown in Fig. 5, ChK increased protein expression of DR5 and cleaved caspase-8 (p < 0.05), decreased the protein level of procaspase-8 (p < 0.05), and had no effect on Fas, Fas L and FADD expression (p > 0.05). These results suggested that ChK might induce apoptosis in OVCAR-3 and A2780/ CP70 cells through a DR5 and caspase-8 associated extrinsic pathway.

Fig. 5.

Fig. 5

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Effect of ChK on the extrinsic apoptotic pathway in ovarian cancer cells. Protein lysates were prepared from OVCAR-3 and A2780/CP70 cells after treatment with various concentrations (0, 1, 2, 4 μM) of ChK for 24 h. DR5, Fas, Fas L, FADD, procaspase-8 and cleaved caspase-8 protein levels were analyzed by Western blot. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. Significant differences among different treatments are indicated by different letters (p < 0.05).

Regulation of cell cycle G2-related proteins by ChK

Based on the observation that ChK induced a G2 arrest in OVCAR-3, A2780/CP70 and IOSE-364 cells, we evaluated the effect of ChK on cell cycle regulatory proteins that play important roles in G2 cell cycle progression by Western blot analysis. As shown in Fig. 6, ChK could effectively suppress cyclin B1 expression (p < 0.05), and had no effect on the protein levels of phospho-cdc2 (Tyr 15) and cdc2 in all three cell lines (p > 0.05). The p21 protein expression did not change in OVCAR-3 and A2780/CP70 cells (p > 0.05), but significantly increased in IOSE-364 cells (p < 0.05). These results implied that the down-regulation of cyclin B1 expression might be responsible for the G2 growth arrest induced by ChK in all three cell lines, and the protein p21 probably played an important role in ChK-induced G2 arrest in IOSE-364 cells.

Fig. 6.

Fig. 6

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Effect of ChK on cell cycle G2-related proteins in OVCAR-3,A2780/CP70 and IOSE-354 cells. Protein lysates were prepared from the both ovarian cancer cells after treatment with various concentrations (0, 1, 2, 4 μM) of ChK for 24 h. p21, cyclin B1, phospho-cdc2 and cdc2 protein expression levels were analyzed by Western blot. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. Significant differences among different treatments are indicated by different letters (p < 0.05).

Role of p53 in ChK-induced apoptosis and cell cycle arrest

The p53 tumor suppressor protein is a transcription factor that regulates the expression of numerous genes controlling different cellular outcomes such as cell cycle arrest and apoptosis [16]. To identify the role of p53 in ChK-induced apoptosis and cell cycle arrest, we first detected the phospho-p53 (Ser15), total p53 and murine double minute 2 (MDM2, a negative regulator of p53) protein expression by Western blot. As shown in Fig. 7A, ChK up-regulated the protein expression of p53 (p < 0.05), and had no effect on the protein levels of phospho-p53 (Ser15) and MDM2 in OVCAR-3 and A2780/ CP70 cells (p > 0.05). RT-PCR analysis showed ChK-treatment significantly increased mRNA expression of p53 in a concentration-dependent manner (p < 0.05) (Fig. 7B). These results indicated that ChK activated p53 at mRNA and protein levels independent of MDM2.

Fig. 7.

Fig. 7

Fig. 7

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Role of p53 in ChK-induced apoptosis and G2 cell cycle arrest in OVCAR-3 and A2780/CP70 cells. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. (A) The effects of ChK on the protein expression of phospho-p53 (Ser15), p53 and MDM2 determined by Western blot. Significant differences among different treatments are indicated by different letters (p < 0.05). (B) The effect of ChK on the mRNA expression of p53 determined by RT-PCR. Significant differences among different treatments are indicated by different letters (p < 0.05). (C) The effect of PFT-α on the apoptotic rates of OVCAR-3 and A2780/ CP70 cells treated by ChK. Cells were pretreated with 20 μM PFT-α for 2 h followed by ChK treatment for 24 h. *p < 0.05, ** p < 0.01, compared with respective controls without PFT-α treatment. (D) The effects of p53 siRNA (50 nM) on the protein expression of DR5, procaspase-8 and cyclin B1 determined by Western blot. *p < 0.05, ** p < 0.01, compared with respective controls.

Next, pifithrin (PFT)-α, a pharmacological inhibitor of p53 and p53 siRNA were used to further clarify the effect of p53 on apoptosis and G2 cell cycle arrest. Fig. 7C shows that pre-incubation of 20 μM PFT-α significantly decreased the apoptotic rates of OVCAR-3 and A2780/CP70 cells induced by ChK (p < 0.05 or 0.01) (The dose of PFT-α used in this study was determined according to our pre-experiment results that 20 μM PFT-α had no effect on cell viability and apoptosis, and obviously attenuated ChK-induced overexpression of p53 in OVCAR-3 and A2780/CP70 cells). Fig. 7D shows that knockdown of p53 by specific siRNA (50 nM) resulted in significant inhibition of p53 expression after ChK treatment (p < 0.05 or 0.01). This p53 depletion had no effect on DR5 protein level (p > 0.05), but led to abrogation of ChK-induced decrease in cyclin B1 and procaspase-8 protein expression (p < 0.05). These results suggested that p53 was a pivotal mediator of ChK-induced apoptosis and G2 cell cycle arrest in OVCAR-3 and A2780/CP70 cells.

Role of MAPK in ChK-induced apoptosis and cell cycle arrest

It was suggested that the p53 protein can functionally interact with the mitogen-activated protein kinase (MAPK) pathways, including the stress-activated protein kinase [SAPK/c-Jun N-terminal protein kinase (JNK)], the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal related kinase (ERK) [17]. Therefore, we tested whether MAPKs mediated ChK-induced apoptosis and cell cycle arrest in OVCAR-3 and A2780/CP70 cells. As shown in Fig 8A, treatment with ChK resulted in a concentration-dependent increase in the phosphorylation of p38 MAPK in the both cells (p < 0.05). However, the protein levels of total p38, phospho- and total JNK, phospho- and total ERK1/2 remained relatively constant after the ChK treatment (p > 0.05).

Fig. 8.

Fig. 8

Fig. 8

Fig. 8

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Role of MAPKs in ChK-induced apoptosis and G2 cell cycle arrest in OVCAR-3 and A2780/CP70 cells. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. (A) The effects of ChK on the protein expression of phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2 and ERK1/2 determined by Western blot. Significant differences among different treatments are indicated by different letters (p < 0.05). (B) The effects of p38 siRNA (50 nM) on the protein expression of phospho-p53 (Ser 15), p53, procaspase-8 and cyclinB1 determined by Western blot. *p < 0.05, ** p < 0.01, compared with respective controls. (C) The effects of p53 siRNA on the protein expression of phospho-p38 and p38 determined by Western blot. *p < 0.05, ** p < 0.01, compared with respective controls.

To further analyze the role of p38 MAPK in ChK-induced cell death, p38 MAPK was selectively knocked down by siRNA approach. Fig. 8B shows that the protein levels of phospho-p53 (Ser15), p53, and procaspase-8 were not obviously affected after treatment with 50 nM p38 siRNA (p > 0.05), indicating p38 MAPK was not involved in the p53-mediated apoptosis induced by ChK in OVCAR-3 and A2780/ CP70 cells. Compared with oligonucleotide-transfected control cells, the inhibition of cyclin B1 expression by ChK was attenuated in the both ovarian cancer cell lines transfected with p38 MAPK siRNA (p < 0.05 or 0.01), which indicated that p38 MAPK might influence the cell cycle arrest induced by ChK. Furthermore, the effect of p53 on the phospho-p38 and total p38 expression were examined. As shown in Fig. 8C, knockdown of p53 did not affect the protein expression of total p38 (p > 0.05), but significantly increased the phospho-p38 level (p < 0.05 or 0.01). This result indicated that p53 inhibited the phosphorylation of p38 in OVCAR-3 and A2780/CP70 cells.

Discussion

Chemotherapy adverse side effects and resistance to current anticancer agents have been the pressing problems in the success of ovarian cancer therapy [1]. New chemotherapy strategies are urgently needed for ovarian cancer treatment. Although ChK has been known for about 30 years and received considerable attention for its anti-cancer activity, the study of its pharmacological activity is still limited [12,13]. In the present work, we demonstrated that ChK had a more potent growth inhibitory effect than cisplatin in two cisplatin-resistant ovarian cancer cell lines OVCAR-3 and A2780/CP70, and was less cytotoxic to a normal ovarian cell line IOSE-364 than to both the ovarian cancer cell lines. The preferential cell growth inhibitory effect might be at least partly attributed to the apoptosis and G2 cell cycle arrest induced by ChK.

One mechanism by which tumor cells develop resistance to cytotoxic agents and radiation is related to resistance to apoptosis. In pre-clinical disease models, agents that target the apoptotic pathway have been shown to sensitize tumor cells to chemotherapy and radiotherapy [18]. Two major pathways leading to apoptosis have been delineated: the intrinsic or mitochondrial pathway, and the extrinsic or receptor-mediated pathway. The intrinsic apoptotic pathway is characterized by permeabilization of the mitochondria and release of cytochrome c into the cytoplasm. ROS are known triggers of the intrinsic apoptotic cascade via interactions with proteins of the mitochondrial permeability transition complex. Cytochrome c forms a multi-protein complex known as the ‘apoptosome’ and initiates activation of the caspase cascade through caspase-9. Proteins of the Bcl-2 family have either pro- or anti-apoptotic activities and regulate the mitochondrial pathway of apoptosis by controlling the permeabilization of the outer mitochondrial membrane and the release of the pro-apoptotic factors. The balance between pro- and anti-apoptotic family members determines whether or not a cell will undergo apoptosis [19–21]. In the extrinsic pathway, tumor ne crosis factor-related apoptosis-inducing ligand (TRAIL), such as Apo2L/TRAIL and Fas ligand (Fas L), engage their respective death receptors, DR4/DR5 or Fas, which homotypically bind to the adaptor protein FAS-associated death domain protein (FADD). FADD then recruits the initiating caspases-8 and -10 through homophilic death effector domain (DED) interactions to form the death inducing signaling complex (DISC). Activation of either the intrinsic or extrinsic pathway of apoptosis subsequently results in activation and cleavage of procaspase-3, -6 and -7 culminating in cell death [14,22,23]. In this study, our data indicated that ChK within the test concentrations had no effect on the apoptosis of IOSE-364 cells, but induced apoptosis through a DR5 and caspase-8 associated extrinsic pathway in OVCAR-3 and A2780/CP70 cells. It was reported that the differential induction of apoptosis in cancer versus normal cells involved direct targeting of mitochondria associated with alterations in the balance of Bcl-2 proteins [24]. However, our data showed that ChK did not influence the mitochondrial pathway. Another explanation is that normal cells are resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) due to the high TRID levels, whereas tumor lines carrying TRAIL-receptors in most cases can not be protected [25]. Numerous evidences from clinical trials has indicated that targeting DR5 selectively eliminates tumor cells while sparing normal cells [26]. The present work showed DR5 was upregulated in both ovarian cancer cells, which might at least partly explain for the preferential induction of apoptosis by ChK. Previous study showed that the cellular caspase-8 protein level was an important determinant of sensitivity to rhTRAIL-induced apoptosis in A2780/CP70 cells which had lower caspase-8 protein levels compared with A2780 cells [27]. Agents that can up-regulate the cell surface expression of DR4 or DR5 have the potential for clinical application in combination with TRAIL [28]. These results indicated that the combination of ChK with TRAIL and/or platinum compounds might be a promising therapy for platinum-resistant ovarian cancer, and is worth investigating in the future.

Although our data suggested that ChK induced apoptosis in both ovarian cancer cells, the higher reduction of cell viability (60–70% at 4 μM ChK) with respect to the percentage of apoptotic cells (12– 15% at 4 μM ChK) indicated other mechanisms existed for the cell proliferation-inhibitory activity of ChK. Cancer cells evolve in part by overriding normal cell cycle regulation. The defective cell-cycle checkpoint is an intrinsic property of the cancer, and offers a relatively untapped area of potentially selective cytotoxic therapies for a range of cancers [29]. It was revealed that many cancer cells have defective G1 checkpoint mechanisms, and depend on G2 checkpoint during cell replication far more than normal cells. These insights have given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer cell specific therapy [30]. Several compounds were reported to inhibited cancer cell proliferation through G2/M cell cycle arrest [31–33]. The DNA mismatch repair system (MMR) may link G2 arrest with cell death [34]. Our cell cycle analysis revealed ChK induced more prominent G2 arrest in OVCAR-3 and A2780/CP70 cells than in IOSE-364 cells, suggesting that in addition to apoptosis, G2 cell cycle arrest was another factor that contributed to the preferential cell growth inhibition. The sub G1 peak is indicative of cells that have undergone some DNA degradation due to apoptosis [35]. However, no obvious sub-G1 peak was observed in our cell cycle analysis, which might be due to the low percentage of apoptotic cells. It is known that progression through G1 and entry into S-phase is regulated by the Cyclin A-Cdk2 and Cyclin E-Cdk2 complexes, respectively, and the G2/M phase transition is driven by Cyclin B-Cdc2 [36]. The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and -independent mechanisms, and causes G1, G2 and S-phase arrest in response to many stimuli. In addition to being an inhibitor of cell proliferation, p21 acts as an inhibitor of apoptosis in a number of systems [37]. Interestingly, previous studies showed p21 mediated differential sensitivity of normal and cancer cells to G2-M arrest and apoptosis induced by some compounds. p21 induction may inhibit apoptosis and decrease G2-M arrest due to suppression of the S-G2 checkpoint [38,39]. Our data showed that ChK downregulated cyclin B1 in all three cell lines, but just increased p21 protein level in IOSE-364 cells. These results provided a possible explanation for the observed G2 arrest induced by ChK treatment in all three cell lines, and suggested that p21 may play an important role in determining the sensitivity of normal and malignant ovarian cells to ChK.

p53 is a multifunctional tumor suppressor that regulates transcription, DNA repair, cell cycle arrest, differentiation, senescence, genomic instability, apoptosis and survival as well as glucose metabolism, oxidative stress and angiogenesis [40]. It stimulates apoptosis by a wide network of signals including death receptor or pathway such as Killer/DR5 and Fas/APO-1, and mitochondrial pathway including proteins such as Bax, Puma and Noxa [41]. Activated p53 causes a G1 arrest by inducing expression of p21 and the consequent inhibition of cyclin D/Cdks. p53 also functions at the G2/M checkpoint by decreasing cyclin B1 transcription and synthesis, or inactivates Cdc25C and, consequently, Cdc2 activity thorough 14-3-3σ [42]. A number of previous studies in vitro and in vivo have shown that in a variety of cancers p53 alterations have been linked to the failure of radiotherapy and chemotherapy by loss of function, dominant-negative activity, or gain of oncogenic function [43]. Post-translational modification of p53 by phosphorylation has been proposed to be an important mechanism by which p53 stabilization and function are regulated. Serine 15 is a major phosphorylation site of p53 in vivo. It has been found that mutation of only this residue in the full-length protein greatly reduces phosphorylation to almost the same extent as mutation of all of the other potential N-terminal phosphorylation sites together [44,45]. Mouse double minute 2 homolog (MDM2) acts as the negative regulator of p53 in a feedback auto-regulatory loop, inactivating the apoptotic and cell cycle arrest functions of p53 [46]. It is known that A2780/ CP70 cell line has a wild-type p53 gene sequence [47]. Although OVCAR-3 has a point mutation in the p53 gene which results in single amino acid changes, p53 still play an important role in the apoptosis and cell cycle arrest of OVCAR-3 cells induced by some cytokines and compounds [48–50]. The present work suggested that ChK regulated p53 at either the transcriptional and translational levels, but not at a post-translational level in OVCAR-3 and A2780/CP70 cells. p53 was involved in the apoptosis and G2 cell cycle arrest induced by ChK through regulating caspase 8 and cyclin B1. These results were in agreement with the previous reports that caspase-8 was an essential mediator of the p53/p73-dependent apoptosis induced by etoposide in HNSCC cells [51], and p53 prevented G2/M transition by decreasing intracellular levels of cyclin B1 protein in human ovarian cells [52]. In some cancer cell types, DR5 is a downstream target of p53. Some conventional chemotherapeutic drugs, such as etoposide and doxorubicin, can induce apoptosis via DR5 upregulation in a p53-dependent manner. However, DR5 can also be regulated in a cell type-specific, trigger-dependent, and p53-independent manner, but the underlying mechanisms remain largely unclear [53,54]. Our data showed p53 was not associated with DR5 upregulation in ChK-treated OVCAR-3 and A2780/CP70 cells, which was in agreement with the previous study conducted in human ovarian cancer SKOV3 cells induced by CHM-1 [55]. This result indicated that p53-dependent pathway might not be the unique mechanism of apoptosis induced by ChK in OVCAR-3 and A2780/ CP70 cells. In addition to p53, other proteins including NF-kB and CCAAT enhancer-binding protein homologous protein (CHOP) have been shown to upregulate DR5 [56,57]. It is interesting to further investigate the mechanism of DR5 overexpression in platinum-resistant ovarian cancer cells induced by ChK in the future work.

MAPK pathways are evolutionarily conserved kinase modules that link extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, migration and apoptosis, and play an important role in cancer [58]. In the present study, ChK treatment increased p38 MAPK phosphorylation in OVCAR-3 and A2780/CP70 cells, and had no effect on the protein levels of phospho/total JNK and phospho/ total ERK1/2. Previous studies showed that some drugs such as salinomycin and cisplatin could induce apoptosis in human ovarian cancer cell line OV2008 by activating p38 MAPK [59,60]. However, our results showed that p38 activation was not important for ChK-induced apoptosis in both ovarian cancer cell lines. Furthermore, we found that p38 activation might play an important role in G2 cell cycle arrest of ChK-treated OVCAR-3 and A2780/CP70 cells through downregulating cyclin B1, which was consistent with the previous report that the p38 signaling pathway triggered cyclin B1 proteolysis [61].

It has become clear that the p53 protein can functionally interact with the MAPK pathways. Upon exposure to stressful stimuli, MAP kinases phosphorylate and activate p53, leading to p53-mediated cellular responses. A number of studies have showed that p38 plays an important role in activation of p53 via a mechanism involving phosphorylation. On the other hand, p53 acts as an upstream activator to regulate MAPK signaling via the transcriptional activation of the dual specificity of phosphatase family [17]. In this study, no change of phospho-p53 (Ser15) and total p53 protein levels were observed after p38 knockdown by siRNA, while p53 siRNA remarkably increased the phosphorylation of p38 without affecting total p38 expression. This result was consistent with the previous reports that p53 selectively inactivated p38 by specific dephosphorylation of its conserved threonine residue via wip1 phosphatase [62], and raised the possibility that p53 played dual roles in the cell cycle arrest induced by ChK in OVCAR-3 and A2780/ CP70 cells. p53 might directly decrease the cyclin B1 expression, but attenuate the downregulation of cyclin B1 inhibition of p38 phosphorylation. However, ChK finally upregulated the p53 expression and p38 phosphorylation at the same time, and decreased the expression of cyclin B1.

In conclusion, our study demonstrated that ChK had a potent and preferential cell growth inhibitory effect on two cisplatin-resistant ovarian cancer cell lines with respect to normal cells via apoptosis and G2 cell cycle arrest. ChK induced apoptosis through a p53-dependent caspase-8 activation extrinsic pathway, and caused G2 arrest via cyclin B1 by increasing p53 expression and p38 phosphorylation in both ovarian cancer cells. DR5 and p21 might play an important role in determining the sensitivity of normal and malignant ovarian cells to ChK. Based on these results, ChK would be a potential compound for treating platinum-resistant ovarian cancer.

Acknowledgements

We thank Mr. Zhaoliang Li for expert technical assistance. We thank Dr. Kathy Brundage from the Flow Cytometry Core at West Virginia University for providing technical help on cell cycle analysis.

Funding

We acknowledge financial support from the West Virginia Higher Education Policy Commission/Division of Science Research. This research was also supported by NIH grants P20RR016477 from the National Center for Research Resources and P20GM103434 from the National Institute for General Medical Sciences (NIGMS) awarded to the West Virginia IDeA Network of Biomedical Research Excellence. This study was also supported by Grant Number P20GM104931 from NIGMS, a component of the National Institutes of Health (NIH) and its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIGMS or NIH. This study was also supported by CoBRE grant GM102488/RR032138, ARIA S10 grant RR020866, FORTESSA S10 grant OD016165 and INBRE grant GM103434.

Footnotes

Conflicts of interest

Authors declare that there is no conflict of interest involved in this research.

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