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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 1  |  Page : 107-112

A3 adenosine receptor agonist induce G1 cell cycle arrest via Cyclin D and cyclin-dependent kinase 4 pathways in OVCAR-3 and Caov-4 cell lines


1 Medical Laboratory Research Center, Golestan University of Medical Sciences, Gorgan, Iran
2 Cellular and Molecular Research Center, Zahedan University of Medical Sciences, Zahedan; Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences; Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
4 Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences; Bioinformatics Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
5 Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Web Publication03-Feb-2017

Correspondence Address:
Mahmoud Aghaei
Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, P.O. Box: 81746-73461, Isfahan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.199381

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 > Abstract 

Aim of the Study: The cell cycle, a vital process that involves in cells' growth and division, lies at the heart of cancer. It has been shown that IB-MECA, an A3 adenosine receptor agonist inhibits the proliferation of cancer cells by inducing cell cycle arrest in several tumors. In this study, we evaluated the role of IB-MECA inhibition in cell cycle progression in ovarian cancer cells.
Materials and Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in Caov-4 and OVCAR-3. Analysis of cell cycle distribution was carried out by flow cytometry. To determine the mechanisms of IB-MECA-mediated induction of cell cycle arrest, the expression of cell cycle regulatory proteins Cyclin D1 and cyclin-dependent kinase 4 (CDK4) was evaluated.
Results: Our results showed that IB-MECA significantly reduced cell viability in a dose-dependent manner. Moreover, our results indicated that a low concentration of IB-MECA induced G1 cell cycle arrest. Reduction of Cyclin D1 and CDK4 protein levels was also observed after treating cancer cells with IB-MECA.
Conclusion: This study demonstrated that IB-MECA induces G1 phase cell cycle arrest through Cyclin D1/CDK4-mediated pathway in ovarian cancer cells.

Keywords: A3 adenosine receptor, cell cycle, G1 arrest


How to cite this article:
Joshaghani HR, Jafari SM, Aghaei M, Panjehpour M, Abedi H. A3 adenosine receptor agonist induce G1 cell cycle arrest via Cyclin D and cyclin-dependent kinase 4 pathways in OVCAR-3 and Caov-4 cell lines. J Can Res Ther 2017;13:107-12

How to cite this URL:
Joshaghani HR, Jafari SM, Aghaei M, Panjehpour M, Abedi H. A3 adenosine receptor agonist induce G1 cell cycle arrest via Cyclin D and cyclin-dependent kinase 4 pathways in OVCAR-3 and Caov-4 cell lines. J Can Res Ther [serial online] 2017 [cited 2019 Nov 12];13:107-12. Available from: http://www.cancerjournal.net/text.asp?2017/13/1/107/199381


 > Introduction Top


The cell cycle is an ordered set of events that directs the growth and proliferation of cells.[1] The cell cycle consists of four distinct phases: G1, S, G2, and M.[2] The key parts of the cell cycle machinery are controlled by expression and activation of the cyclin-dependent kinases (CDKs) and the regulatory proteins which are called cyclins.[3] The cell cycle lies at the heart of cancer, thus identifying therapeutic targets, and the cell cycle can be used for cancer therapy.[4],[5] Adenosine is a purine nucleoside that is released from cells under injury or stress.[6] Adenosine, as a regulatory metabolite, plays a major role in growth, proliferation, and death of many different cell types.[7] Numerous studies show that many effects of adenosine are mediated via the stimulation of adenosine receptors.[8] These receptors are members of the superfamily of G protein-coupled receptors and classified into four subtypes: A1, A2A, A2B, and A3.[9] Pharmacological studies demonstrated that among these receptors, A3 adenosine receptor (A3 AR) has a key role in the antiproliferative effects of adenosine.[10] Our previous study indicated the messenger RNA and protein expression and functionality of four adenosine receptors, namely, A1, A2A, A2B, and A3 in three ovarian cancer cell lines OVCAR-3, Caov-4, and SKOV-3. This study also showed a strong relationship between A3 AR and ovarian cancer.[11] Several studies revealed that selective A3 AR agonists (IB-MECA and Cl-IB-MECA) suppress the proliferation of different tumor cell types including melanoma, thyroid colon, breast, leukemia, thyroid, and prostate cancer cell lines.[12],[13],[14],[15],[16],[17] Cell death induced by A3 AR agonists could be due to apoptosis,[18] necrosis,[19] or cell cycle arrest in cancer cells.[20] A mediatory role for the A3 AR in the control of cell cycle has been reported.[21] An in vitro study shows that A3 AR agonist is effective in the suppression of leukemia HL-60 cells with cytostatic effect, this effect was mediated through the induction of cell cycle arrest in the G0/G1 phase.[22] It was demonstrated that Thio-Cl-IB-MECA, a novel A3 AR agonist, inhibits the growth of human lung cancer cells via a mechanism that involves arresting cell cycle progression in G0/G1 phase and induction of apoptosis.[23] The effect of IB-MECA on DU-145, PC3, and LNCaP prostate cancer cells proliferation was investigated and reported that IB-MECA induces G1 cell cycle arrest in androgen-dependent and androgen-independent prostate cancer cell lines through P53-dependent, CDK4/Cyclin D1-mediated pathway.[17] In our previous papers,[24] we showed that high concentrations of IB-MECA lead to apoptotic death in ovarian cancer cell lines. In the present study, we have focused on the inhibitory role of IB-MECA in cell cycle progression in the OVCAR-3 and Caov-4 human ovarian cancer cells.


 > Materials And Methods Top


Chemicals and reagents

RPMI 1640 medium, fetal bovine serum, and penicillin-streptomycin were purchased from GIBCO (Life Technologies Corporation, New York, USA). Cell culture plastic ware was obtained from SPL (Lifesciences Co. Ltd., Seoul, Korea). Cell cycle analysis kit from BioVision Incorporated (Milpitas, CA, USA), A3 receptor agonist, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-CDK4, anti-Cyclin D1 antibodies, and horseradish peroxidase-conjugated anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Hyper film ECL was obtained from Amersham (Little Chalfont, United Kingdom) and Clarity ECL Western Blot Substrate was obtained from Bio-Rad (Bio-Rad Laboratories, Inc., USA).

Cell culture

OVCAR-3 and Caov-4 ovarian cancer cells lines were purchased from the Pasture Institute of Iran. The cell lines were grown adherently in RPMI-1640 media supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in 5% CO2/95% air.

Analysis of cell viability

Cell viability was evaluated using the MTT assay as previously reported.[25] In brief, OVCAR-3 and Caov-4 cells were seeded at 5000 cells per well in a 96-well plate and incubated for 24 h at 37°C. After cells were grown to 70% confluency, they were treated with different concentrations of IB-MECA ranging from 0.001 to 10 μM. Following 48 h of treatment, MTT tetrazolium salt was added to the culture media, and the cells were allowed to incubate for 4 h at 37°C. Mitochondrial dehydrogenases of viable cells cleave MTT into purple formazan crystals. Absorbance of the samples was measured at 570 nm in a microplate reader (Tekan Sunrise Instruments, Austria) to evaluate the number of viable cells. Cell viability was calculated as percentage using the following formula: (Mean optical density [OD] of treated cells/mean OD of control cells) ×100. The results expressed as percentage of control cells which no treated.

Analysis of cell cycle

The effect of IB-MECA on cell cycle progression was evaluated by flow cytometry according to the method described by Nicoletti et al.[26] as described previously.[17] Cancer cells were synchronized by serum deprivation for 48 h and then the cells were seeded at 3 × 105 cells per well in a 6-well plate. After treating cells with various concentrations of IB-MECA ranging from 0.001-10 μM for 48 h, the floating cells were collected and then transferred to the attached cells harvested by trypsinization. Cells were resuspended in phosphate-buffered saline (PBS), fixed with 2 ml of ice-cold 70% ethanol, and incubated for 30 min at 4°C. The pellets were collected by centrifugation and resuspended in PBS solution, containing 20 mg/ml of propidium iodide (PI), 0.1% Triton X-100, and 100 mg/ml of RNAse. After incubation for 30 min in dark at 37°C, the cells were analyzed for DNA content using a FACSCalibur flow cytometer. Cell distribution among cell cycle phases and the percentage of apoptotic cells were determined. The cell cycle distribution is shown as the percentage of cells containing 2n (G1 phase), 4n (G2 and M phases), and 4n>3>2n DNA amount (S phase) judged by PI staining.[24]

Western blot analysis

Western blot analysis was done as described previously.[27] Cells were serum deprived for 24 h prior to treatments. At the end of IB-MECA treatment, the cells were harvested at 4°C in a lysis buffer (20 mM Tris-HCl [pH 7.5], 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 100 mM b-glycerol 3-phosphate, and 0.5% protease inhibitor cocktail) and disrupted by sonication and centrifuged (14,000 rpm, 10 min, 4°C). The protein concentration of each lysates was determined by Quick Start™ Bradford Protein Assay (Bio-Rad Laboratories, Inc., USA). Each protein (30–50 μg) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto poly (vinylidene fluoride) membranes. The membranes were incubated with blocking buffer (5% nonfat dry milk in PBS containing 0.1% Tween-20 [PBST]) for 1 h at room temperature. Membranes were then incubated with mouse monoclonal antibody against Cyclin D1, CDK4 (Santa Cruz Biotechnology, USA) overnight at 4°C, and washed three times (each for 5 min) with PBST. Membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature. After washing with PBST, the proteins were detected by Clarity™ Western ECL Substrate from Bio-Rad. The expression of GAPDH was used as an internal standard.

Statistical analysis

The results are presented as mean ± standard deviation, and statistical analysis was performed by nonparametric test of variance between groups (ANOVA) followed by Dunnett's post hoc test. All experiments were repeated at least three times independently. Statistical analyses were carried out using the software SPSS 18 (Statistical Package ver. 18.0; SPSS Inc., Chicago, IL, USA). A difference was regarded statistically significant at P< 0.05.


 > Results Top


Inhibitory effect of IB-MECA on the growth of human ovarian cancer cell lines Caov-4 and OVCAR-3

To evaluate the effect of IB-MECA on the ovarian cancer cells, Caov-4 and OVCAR-3 cells were plated in a 96-well plate after treated with various concentrations of IB-MECA (0.001–10 μM) for 48 h, and the inhibition of cell proliferation was assessed using the MTT colorimetric assay. The proliferation of Caov-4 cells was inhibited significantly by the IB-MECA in a dose-dependent manner starting at 0.001 μM and increased up to 10 μM (from 96.3% ±1.7% for 0.001 μM to 64.3% ± 6.7% for 10 μM vs. control 100%) [Figure 1]a. In addition, a significant reduction in the OVCAR-3 cells viability was observed (93.2% ± 2.3% for 0.001 μM to 66.2% ± 4.2% for 10 μM vs. control 100%) [Figure 1]b. This result demonstrated that IB-MECA has an inhibitory effect on the growth of human ovarian cancer cell lines such as Caov-4 and OVCAR-3 (P < 0.05). No significant difference in the antiproliferative effect of IB-MECA was observed between Caov-4 cells compared to OVCAR-3 cell lines.
Figure 1: The effect of IB-MECA on cell viability in ovarian cancer cell lines, (a) Caov-4 and (b) OVCAR-3. Cells were treated with different concentrations of IB-MECA for 48 h, and cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. IB-MECA induced ovarian cancer cell death in a dose-dependent manner. Data represent mean ± standard deviation of three independent experiments. *P < 0.05 represents significant differences compared to control values

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Induction of cell cycle arrest in Caov-4 and OVCAR-3 cells by IB-MECA

To determine how IB-MECA inhibits Caov-4 and OVCAR-3 cells' growth, the cells were plated in a 6-well plate and treated with various concentrations of IB-MECA (0.01–10 μM) for 48 h and then cell cycle analysis was performed by flow cytometry to observe the cells' DNA content. Alterations in sub-G1, G1, S, and G2/M phases of cell cycle under various concentrations of IB-MECA are shown in [Figure 2]a and [Figure 3]a. In Caov-4 cells, 6.9% of the control cells were in sub-G1 phase, 55.4% in G1 phase, 14.1% in S phase, and 23.6% in G2/M phase [Figure 2]b. In OVCAR-3 cells, 5.2% of the control cells were in sub-G1 phase, 53.6% in G1 phase, 17.8% in S phase, and 23.4% in G2/M phase [Figure 3]b. Treatment of cells with 0.01–10 μM doses of IB-MECA for 48 h induces an accumulation of Caov-4 and OVCAR-3 cells in the G1 phase of the cell cycle in comparison to the controls (P < 0.05). Moreover, results showed that Treatment of Caov-4 cells with 10 μM doses of IB-MECA an accumulation in G2/M phase and also treatment of OVCAR-3 cells with 10 μM doses of IB-MECA a decrease in G2/M phase in comparison to the controls (P > 0.05).
Figure 2: Effect of IB-MECA in cell cycle distribution of Caov-4 cells. (a) Cells were treated with 0.5, 1, and 10 μM IB-MECA for 48 h, and then stained with propidium iodide. The DNA content was evaluated by flow cytometry. (b) The cell cycle distributions were analyzed and data represent mean ± standard deviation of three independent experiments. *P < 0.05 represents significant differences compared to control values

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Figure 3: Effect of IB-MECA in cell cycle distribution of OVCAR-3 cells. (a) Cells were treated with 0.5, 1, and 10 μM IB-MECA for 48 h, and then stained with propidium iodide. The DNA content was evaluated by flow cytometry. (b) The cell cycle distributions were analyzed and data represent mean ± standard deviation of three independent experiments. *P < 0.05 represents significant differences compared to control values

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The results showed IB-MECA Inhibit growth of ovarian cancer cells via G1 cell-cycle arrest.

Downregulation of Cyclin D1 expression and cyclin-dependent kinase 4 in Caov-4 and OVCAR-3 cells by IB-MECA

To investigate the potential mechanisms of IB-MECA-mediated induction of cell cycle arrest, the effects of IB-MECA on the expression of CDK4 and Cyclin D1, which are necessary for cell cycle progression, were evaluated. Caov-4 and OVCAR-3 cells were treated with various concentrations of IB-MECA (0.01–10 μM) for 48 h, and the expression levels of Cyclin D1 and CDK4 proteins were analyzed by Western blot analysis. IB-MECA significantly decreases the protein levels of CDK4 (from 71% ±0.7% for 0.01 μM, 49% ± 0.61% for 0.1, and 34% ± 0.052% for 10 μM vs. control 100%) and Cyclin D1 (from 60% ± 0.54% for 0.01 μM, 25% ± 0.58% for 0.1, and 14% ± 0.037% for 10 μM vs. control 100%) in Caov-4 cells [Figure 4]a and [Figure 4]b. In addition, there was a significant decrease in the levels of Cyclin D1 and CDK4 proteins in OVCAR-3 cells observed after treatment with IB-MECA (CDK4 from 83% ±0.8% for 0.01 μM, 55% ± 0.1% for 0.1, and 40% ± 0.08% for 10 μM and Cyclin D1 from 81% ± 0.074% for 0.01 μM, 50% ± 0.12% for 0.1, and 35 ± 0.07% for 10 μM vs. control 100%) [Figure 5]a and [Figure 5]b. These results showed that relative to untreated control, IB-MECA in a dose-dependent manner suppressed the levels of Cyclin D1 and CDK4 in ovarian cancer cells (P < 0.05).
Figure 4: IB-MECA affects cell cycle regulatory proteins in Caov-4 cell lines. (a) The effects of IB-MECA on the expression of cell cycle regulatory proteins in Caov-4 cell lines. Cells were treated with different concentrations of IB-MECA for 48 h, and then the expression of proteins was analyzed by Western blotting. (b) The band intensity of cyclin-dependent kinase 4 and Cyclin D1 levels relative to glyceraldehyde-3-phosphate dehydrogenase. Data represent mean ± standard deviation of three independent experiments. *P < 0.05 represents significant differences compared to control values

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Figure 5: IB-MECA affects cell cycle regulatory proteins in OVCAR-3 cell lines. (a) The effects of IB-MECA on the expression of cell cycle regulatory proteins in OVCAR-3 cell lines. Cells were treated with different concentrations of IB-MECA for 48 h, and then the expression of proteins was analyzed by Western blotting. (b) The band intensity of cyclin-dependent kinase 4 and Cyclin D1 levels relative to glyceraldehyde-3-phosphate dehydrogenase. Data represent mean ± standard deviation of three independent experiments. *P < 0.05 represents significant differences compared to control values

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 > discussion Top


A3 AR plays an important role in the regulation of cell cycle and apoptosis in various human cancer cell lines.[26],[28] Moreover, in animals and humans, A3 ARshowed that it is a therapeutic target in inflammatory diseases, and A3 AR agonists such as IB-MECA and Cl-IB-MECA have a very good safety profile in clinical trial studies.[29] Lee et al.[22] reported that IB-MECA has a biphasic effect on cancer cells. At low concentrations, it inhibits cancer cell growth, and at high concentrations, it induces cell death by apoptosis. In our previous papers,[29] we showed that high concentrations of IB-MECA lead to apoptotic death in ovarian cancer cell lines. At present, there are no reports available in literature on the effect of low concentrations of IB-MECA in ovarian cancer cell lines. In this study, we evaluated the effect of low concentrations of IB-MECA on cell proliferation in ovarian cancer cell lines such as OVCAR-3 and Caov-4. In the first phase of this study, we indicated that low concentrations of IB-MECA (0.001–10 μM) decrease the viability of ovarian cancer cell lines. We also found that IB-MECA inhibited the proliferation of ovarian cancer cells in vitro by the suppression of cell cycle progression. A3 receptor activity by IB- MECA halts cells into G1-late cell cycle phase that appeared to be the mechanism of inhibition of IB-MECA in ovarian cancer cells. In mammalian cells, cell cycle progression is tightly regulated through the activation of CDKs whose association with the corresponding regulatory cyclins is required for their activation.[30] It is well known that G1 to S phase transition is regulated by complexes formed by Cyclin D and CDK4.[31] Having determined the mechanism of IB-MECA-induced cell cycle arrest in ovarian cancer cells, we evaluate the effect of IB-MECA treatment on protein levels of G1-S-specific cyclins. Our data showed that IB-MECA treatment significantly reduces the protein level of Cyclin D1 and CDK4 in OVCAR-3 cells. Furthermore, the protein levels of Cyclin D1 and CDK4 were also downregulated in Caov-4 cells after treatment with IB-MECA.


 > Conclusion Top


Finally, our study showed that the IB-MECA -induced cell cycle arrest was associated by downregulating expression cyclin D1 protein levels and cyclin-dependent kinase 4 (Cdk4).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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