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Year : 2019  |  Volume : 15  |  Issue : 5  |  Page : 1105-1108

Anticancer activity of britannin through the downregulation of cyclin D1 and CDK4 in human breast cancer cells

1 Traditional Medicine and Materia Medica Research Center; Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
3 Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran

Date of Web Publication4-Oct-2019

Correspondence Address:
Faranak Fallahian
Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_517_17

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 > Abstract 

Aim of the Study: Both apoptotic induction and cell cycle blockade in cancer cells are effective strategies to eliminate cancer cells. Many conventional cancer drugs that induce apoptosis and inhibit cell cycle progression have been reported as potential therapeutics for various types of cancer. Britannin is a natural sesquiterpene lactone that its profound anticancer properties were revealed in our previous study. In this study, we evaluated the effects of britannin on the cell cycle distribution and also cell cycle-related proteins.
Materials and Methods: Analysis of cell cycle distribution was carried out using flow cytometer. The effects of britannin on cyclin D1 and CDK4 expression were evaluated using the Western blot.
Results: The obtained results show that britannin at the low concentrations induces cell growth inhibition mainly through G1-phase arrest while it seems that apoptosis contributes to cell growth inhibitory effect of high doses of britannin. Reduction of cyclin D1 and CDK4 protein levels were also observed after treating cancer cells with britannin.
Conclusion: The obtained results reveal that britannin can inhibit MCF-7 and MDA-MB-468 breast cancer cells proliferation through arresting cell cycle progression through cyclin D1/CDK4-mediated pathway.

Keywords: Britannin, CDK4, cell cycle, cyclin D1, sesquiterpene lactone

How to cite this article:
Hamzeloo-Moghadam M, Aghaei M, Abdolmoham Madi MH, Fallahian F. Anticancer activity of britannin through the downregulation of cyclin D1 and CDK4 in human breast cancer cells. J Can Res Ther 2019;15:1105-8

How to cite this URL:
Hamzeloo-Moghadam M, Aghaei M, Abdolmoham Madi MH, Fallahian F. Anticancer activity of britannin through the downregulation of cyclin D1 and CDK4 in human breast cancer cells. J Can Res Ther [serial online] 2019 [cited 2020 Jul 14];15:1105-8. Available from: http://www.cancerjournal.net/text.asp?2019/15/5/1105/237387

 > Introduction Top

Breast cancer is the most common cause of cancer-related death in women, and the incidence rate is predicted to increase over the coming years.[1] Despite advances in therapies using chemotherapy, surgery, and radiation, breast carcinoma remains a great challenge for clinical therapy. In the recent years, there has been growing interest in the use of natural products with more potent anticancer properties and reduced adverse effects.[2],[3] Increasing evidence reveals that plant-derived compounds could alter the natural history of cancer.[4] Botanical medicines are generally plentiful and relatively nontoxic in clinical practice. Vincristine, vinblastine, etoposide, and paclitaxel are classic examples of plant-derived anticancer compounds that are currently in clinical use.[5],[6] Sesquiterpene lactones (SLs) are one of the most relevant secondary metabolites in the Asteraceae family has been used in traditional medicine for the treatment of human diseases such as inflammation, headache, and infections.[7],[8] During the last decades, extensive research work has been carried out to characterize the anticancer properties and the potential chemotherapeutic application of SLs.[9] Many STLs have proved to have significant cytotoxic activity against different tumor cell lines that make them attractive for cancer therapy. Artemisinin, thapsigargin, and parthenolide are examples of SLs that have already reached clinical cancer trials.[7],[8]

Britannin is a SL which in our previous works was isolated from the Inula aucheriana aerial parts, and its anticancer activity and possible mechanisms of action were investigated in two human breast cancer cells, MCF-7 and MDA-MB-468.[10] The results revealed that britannin can inhibit the proliferation of breast cancer cells through inducing mitochondrial apoptotic pathway. Since there is not any report about the effects of britannin on the cell cycle distribution and also cell cycle-related proteins, the current study was therefore conducted to address the cell cycle inhibitory effects of britannin.

 > Materials and Methods Top

Chemicals and reagents

Culture media, penicillin/streptomycin, growth supplements, and 0.25% trypsin-EDTA solution were obtained from Gibco (BRL, Eggenstein, Germany). Mouse monoclonal anti-CDK4, anti-cyclin D1antibodies, and horseradish peroxidase-conjugated anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Hyper film enhanced chemiluminescence (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).

Isolation and purification of Britannin

Briefly, the chloroform fraction of this species was successively subjected to vacuum liquid chromatography (VLC) and solid-phase extraction (SPE) chromatographic methods. For VLC, silica gel 40–63 μm, was eluted with EtOAc, EtOAc-MeOH (2:1), EtOAc-MeOH (1:1) and MeOH, successively. The fractions eluted with EtOAc-MeOH (2:1) were then gone through further separation using SPE chromatography [2.5 cm × 7.5 cm silicagel (40–63 μm) in each cartridge]. The mobile phase consisted of CH2 Cl2, CH2 Cl2-EtOAc (10:1)-(1:1), EtOAc, EtOAc-MeOH (3:1), EtOAc-MeOH (1:1) and MeOH in turn. Britannin crystals [Figure 1] were isolated from the middle fractions. They were washed with a mixture of n-hexane: EtOAc (1:1) to give pure crystals.
Figure 1: The structure of britannin from Inula aucheriana

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Cell culture

The human breast cancer cell lines, MCF-7 and MB-MDA-468, were obtained from the National Cell Bank of Iran. The cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin and were maintained at 37°C in a humidified incubator with 5% CO2.

Cell cycle analysis by flow cytometry

Propidium iodide (PI) staining was used to analyze cell cycle distribution, according to the method described by Nicoletti et al.[12] MCF-7 and MB-MDA-468 cells were exposed to test compound for 48 h and then harvested and fixed with 70% ice-cold ethanol. After fixation, cells were washed twice with ice-cold phosphate-buffered saline ( PBS) and stained with 0.1% Triton-X 100 (Sigma), 0.1% sodium citrate (Sigma), 0.1 mg/ml of RNase A (Sigma), and 0.05 mg/ml of PI (Sigma) at 37°C for 30 min in the dark. The DNA content of cells and the cell phase distribution was then measured using a FACSCalibur™ flow cytometer.

Western blot analysis

The protein content of cyclin D1 and CDK4 was determined using the Western blot analysis. Cells were harvested at 4°C in a lysis buffer (20 mM Tris–HCl, 0.5% Nonidet P-40, 0.5 mM PMSF, 100 mM b-glycerol 3-phosphate, and 0.5% protease inhibitor cocktail) and disrupted using sonication and centrifugation (14,000 rpm, 10 min, and 4°C). Equal amounts of total protein were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat dry milk in PBS containing 0.1% Tween-20 (PBST) for 1 h at room temperature, the membranes were incubated overnight at 4°C with the monoclonal antibodies against cyclin D1 and CDK4. Membranes were washed with PBST and incubated with corresponding secondary antibodies for 1 h at room temperature. Membranes were again washed with PBST and visualized by ECL detection reagent (Amersham Corp., Arlington Heights, IL). B-actin was used as a control for normalization. The software ImageJ (National Institutes of Health, USA) was employed for optical density measurement.

Statistical analysis

The results of quantitative studies are reported as the mean ± standard deviation (SD) IC50 value was determined using GraphPad Prism5 software (GraphPad, San Diego, California, USA). All experiments were repeated at least three times. To compare data, one-way analysis of variance with Dunnett's post hoc test was used. In all cases, a value of P < 0.05 was taken as the level of statistical significance.

 > Results Top

Britannin-induced G1-phase arrest in MCF-7 and MDA-MB-468

A common mechanism for chemotherapeutic drugs is blocking the cell cycle. To further clarify the suppressive mechanisms of britannin on MCF-7 and MDA-MB-468 cells, we monitored changes in cell cycle distribution using flow cytometer. Alterations in sub-G1, G1, S, and G2/M phases of cell cycle under various concentrations of britannin are shown in [Figure 2]a and [Figure 2]b. Treatment of MCF-7 and MDA-MB-468 cells with 1 and 10 μM of britannin resulted in the accumulation of cells in the G1 phase and a dose-dependent decrease in G2/M and S phases, compared to controls (P < 0.05). More importantly, an apparent sub-G1 peak was observed in MCF-7 and MDA-MB-468 cells as the concentration of britannin was increased.
Figure 2: Britannin-induced cell cycle arrest at the G1 phase in MCF-7 (a) and MDA-MB-468 (b) cells. Cells were treated with britannin for 48 h, and the distribution of cells in each phase of cell cycle was analyzed using flow cytometer. Data are expressed as the mean ± standard deviation (n = 3), with results representative of three independent experiments shown. A value of *P < 0.05 and **P < 0.01 compared to the untreated control group

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Britannin regulated the expression of cell cycle-related proteins

Since cell growth inhibition is related to cell cycle arrest, we investigated the expression of cyclin D1 and CDK4, as cell cycle regulators that control the G1 to S phase of the cell cycle. The cells were treated with 1, 1, and 10 μM for 48 h and the Western blot was performed. As expected, the proteins of cyclin D1 and CDK4 were markedly downregulated using britannin treatment [Figure 3].
Figure 3: The effects of britannin on the expression of cell-cycle regulatory proteins in MCF-7 (a) and MDA-MB-468 (b) cancer cells. Cells were treated with the indicated concentrations of britannin for 48 h and then analyzed the expression of proteins using Western blotting. Protein quantification of the Western blot analysis in MCF-7 (c) and MDA-MB-468 cells (d). Protein levels were normalized to the β-actin level and are shown relative to the untreated control cells. Significant differences between control group versus each treated cell line are indicated by the value of * P < 0.05, **P < 0.01, and ***P < 0.001

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 > Discussion Top

Despite numerous advances in cancer treatments, the lack of effective treatments has prompted a great deal of research into novel anticancer drugs derived from natural compounds. Medicinal herbals have gained increasing attention for their effectiveness and relatively minor side effects.[13] As reported earlier, about 74% of the known anticancer medicines are derived from various plant species.[14]

SLs comprise a large group of secondary plant metabolites with various biological activities.[7],[8] As evidenced from the previous study, britannin, a SL isolated from Inula aucheriana, was found to exert profound activity against MCF-7 and MDA-MB-468 human breast cancer cells.[10] Analysis of apoptosis-related proteins revealed that britannin trigger the mitochondria-mediated apoptosis pathway through increased ratio of Bax/Bcl-2, which led to the loss of mitochondrial membrane potential (ΔΨm), releasing of cytochrome c from mitochondria, and sequential activation of caspase-3 and-9.[10]

Deregulation of the cell cycle machinery plays a significant role in cancer initiation and progression.[15] Both apoptotic induction and cell cycle blockade in cancer cells are effective strategies to eliminate cancer cells. Many conventional cancer drugs that induce apoptosis and inhibits cell cycle progression have been reported as potential therapeutics for various types of cancer.[16] Therefore, to elucidate the mechanism of cell growth inhibition by the britannin, we determined its effect on cell cycle distribution. The obtained results indicated that exposure of MCF-7 and MDA-MB-468 cells to britannin at concentrations ≤10 μM resulted in a significant G1-phase arrest.

Eukaryotic cell cycle progression involves sequential activation of CDKs with corresponding regulatory cyclins. CyclinD1-CDK4 plays a vital role in the G1 phase of the cell cycle.[15],[17] Consistent with this notion, we revealed that britannin downregulated the expression of Cdk-4 and cyclin D1. In addition to the noted effect of G1-cell cycle arrest, britannin at concentrations >10 μM caused a significant increase in the sub-G1 phase population, which is a hallmark of apoptosis. This finding is consistent with the previous data showing apoptosis-inducing properties of britannin in MCF-7 and MDA-MB-468 cells.[10] These data suggest that britannin at the low concentration (0.1–10 μM) induces cell growth inhibition mainly through G1-phase arrest in human breast cancer cells while it seems that apoptosis contributes to cell growth inhibitory effect of high doses of britannin (100 μM).

Taken together, the results presented here, and those of the previous study suggest that britannin play its inhibitory roles on MCF-7 and MDA-MB-468 cells through the induction of both cell-cycle arrest and apoptosis. The findings will provide the potential britannin usage in the cancer drug development. Further efforts to explore the more precise molecular mechanisms and to evaluate its anticancer activity in vivo are necessary.

Financial support and sponsorship

This work has financial supports of Cellular and Molecular Research Center of Qom University of Medical Sciences and also Shahid Beheshti University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.

 > References Top

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Koehn FE, Carter GT. The evolving role of natural products in drug discovery. Nat Rev Drug Discov 2005;4:206-20.  Back to cited text no. 3
Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012;75:311-35.  Back to cited text no. 4
Kaczirek K, Schindl M, Weinhäusel A, Scheuba C, Passler C, Prager G, et al. Cytotoxic activity of camptothecin and paclitaxel in newly established continuous human medullary thyroid carcinoma cell lines. J Clin Endocrinol Metab 2004;89:2397-401.  Back to cited text no. 5
Nobili S, Lippi D, Witort E, Donnini M, Bausi L, Mini E, et al. Natural compounds for cancer treatment and prevention. Pharmacol Res 2009;59:365-78.  Back to cited text no. 6
Ghantous A, Gali-Muhtasib H, Vuorela H, Saliba NA, Darwiche N. What made sesquiterpene lactones reach cancer clinical trials? Drug Discov Today 2010;15:668-78.  Back to cited text no. 7
Merfort I. Perspectives on sesquiterpene lactones in inflammation and cancer. Curr Drug Targets 2011;12:1560-73.  Back to cited text no. 8
Gach K, Janecka A. A-methylene-γ-lactones as a novel class of anti-leukemic agents. Anticancer Agents Med Chem 2014;14:688-94.  Back to cited text no. 9
Hamzeloo-Moghadam M, Aghaei M, Fallahian F, Jafari SM, Dolati M, Abdolmohammadi MH, et al. Britannin, a sesquiterpene lactone, inhibits proliferation and induces apoptosis through the mitochondrial signaling pathway in human breast cancer cells. Tumour Biol 2015;36:1191-8.  Back to cited text no. 10
Moghadam MH, Hajimehdipoor H, Saeidnia S, Atoofi A, Shahrestani R, Read RW, et al. Anti-proliferative activity and apoptotic potential of britannin, a sesquiterpene lactone from inula aucheriana. Nat Prod Commun 2012;7:979-80.  Back to cited text no. 11
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139:271-9.  Back to cited text no. 12
Graham JG, Quinn ML, Fabricant DS, Farnsworth NR. Plants used against cancer-an extension of the work of Jonathan hartwell. J Ethnopharmacol 2000;73:347-77.  Back to cited text no. 13
Al-Asmari AK, Albalawi SM, Athar MT, Khan AQ, Al-Shahrani H, Islam M, et al. Moringa oleifera as an anti-cancer agent against breast and colorectal cancer cell lines. PLoS One 2015;10:e0135814.  Back to cited text no. 14
Casimiro MC, Crosariol M, Loro E, Li Z, Pestell RG. Cyclins and cell cycle control in cancer and disease. Genes Cancer 2012;3:649-57.  Back to cited text no. 15
Preya UH, Lee KT, Kim NJ, Lee JY, Jang DS, Choi JH, et al. The natural terthiophene α-terthienylmethanol induces S phase cell cycle arrest of human ovarian cancer cells via the generation of ROS stress. Chem Biol Interact 2017;272:72-9.  Back to cited text no. 16
Molenaar JJ, Ebus ME, Koster J, van Sluis P, van Noesel CJ, Versteeg R, et al. Cyclin D1 and CDK4 activity contribute to the undifferentiated phenotype in neuroblastoma. Cancer Res 2008;68:2599-609.  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3]


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