Synergistic effect of coumarine derivatives imperatorin and osthol on 5-fluorourasil cytotoxicity on nonsmall cell lung cancer cells

Abstract

BACKGROUND:

Imperatorin and osthol, the two coumarin derivatives from Apiacea family, have several anticancer activities. A large body of evidence demonstrates that these two coumarin derivatives regulates apoptosis, proliferation and invasion in different cancer types including ovarian, cervical, colon and prostate cancers as well as chronic myeloid leukemia etc., which are mediated by multiple signal transduction cascades.

OBJECTIVE:

In present study, the effects of imperatorin and osthol were compared with a known anticancer natural agent curcumin on 5-fluorourasil resistance in A549 cells.

METHODS:

Imperatorin (100–200 μM), osthol (100–200 μM), curcumin (100 μM) and/or 5-FU (64 μM) were applied on NSCLC cell line A549 and human bronchial epithelial cell line (Beas-2b) after a general concentration screening between 10 nM–1 mM and xCELLigence Real Time Cell Analysis (RTCA) was conducted to evaluate cytotoxic effects.

RESULTS:

100 μM curcumin indicated cytotoxic effect in A549 cells and when 5-FU alone did not show cytotoxic activity, it was observed that the cytotoxic effect of curcumin appeared to be similar when combined with 5-FU. 200 μM imperatorin/osthol, when used alone or in combination with 5-FU, showed a stronger cytotoxic effect on both cell lines.

CONCLUSIONS:

Imperatorin/osthol synergistically increased the efficacy of 5-FU in combination, but the same effect on healthy cells was observed as a limiting factor for future studies.

Keywords

Osthol imperatorin curcumin A549 Beas-2b

1 Introduction

Lung cancer is the most common reason of cancer death in the world and its survival is quite poor after diagnosis. Although new chemotherapy agents and radiotherapy have improved survival and quality of life of patients, the overall effect in the last decade has been mainly on palliation rather than reduction in mortality [1].

Curcumin, most active curcuminoid in turmeric, has chemotherapeutic activities and affects multiple signaling pathways; including survival, cytoprotective, metastatic and angiogenic. Curcumin inhibits cancer development and progression, targeting multiple steps in the pathway to malignancy. Curcumin has activity as both a blocking agent, inhibiting the initiation step of cancer by preventing carcinogen activation, and as a suppressing agent, inhibiting malignant cell proliferation during promotion and progression of carcinogenesis [2].

Imperatorin and osthol, coumarin derivatives, have several anticancer activities. Imperatorin, 9-(3-Methylbut-2-enyloxy)-7H-furo[3,2-g]chromen-7-one, is a furanocoumarin isolated from the root of Angelica dahurica. Imperatorin induces apoptosis mediated by both death receptor and mitochondrial pathways [3]. Osthol, 7-methoxy-8-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one, first isolated from Cnidium plant, is highly enriched in mature fruit of Cnidium monnieri (Fructus Cnidii). Osthol inhibits cell proliferation, induces cell cycle arrest, and affects multiple signaling pathways [4].

5-Fluorouracil (5-FU) which is a pyrimidine antimetabolite take part in a relatively large group of anticancer agents that structurally resemble natural substrates, but differ enough to interfere with their metabolism [5]. 5-FU acts in several ways, but principally acts as a thymidylate synthase inhibitor. Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which is a nucleoside required for DNA replication. Thymidylate synthase methylates deoxyuridine monophosphate to form thymidine monophosphate (dTMP). Administration of 5-FU causes a scarcity in dTMP, so rapidly dividing cancerous cells undergo cell death via thymineless death [6].

Measurement of cell viability is a fundamental step in cell biology. There are many traditional cell viability methods, such as MTT reduction and trypan blue assay. By the development of technology, new alternative viability methods with increased sensitivity and specificity have been developed. One of these new methods is the xCELLigence System Real-Time Cell Analyzer. Cellular biological events can be analyzed in real time by the xCELLigence system without labeling. The xCELLigence System Real-Time Cell Analyzer is used for label-free and real-time monitoring of cell proliferation, cytotoxicity, and migration. This system is based on recording the electronic impedance of specially designed plates containing interdigitated gold microelectrodes [7].

In our present study, we conducted experiments with the xCELLigence system and investigated the cytotoxicity of imperatorin, osthol, curcumin alone and in combination with 5-FU on A549 and Beas-2b cell lines by real-time and continuous monitored the cell growth, proliferation and viability.

2 Materials methods

2.1 Materials

Dimethyl sulfoxide (DMSO, cat. A36720100), Roswell Park Memorial Institute 1640 Medium (RPMI, cat. 11875093) were purchased from Applichem, Gibco™ respectively. Ham’s F-12 Medium (cat. N4888), fetal bovine serum (FBS, cat. F2442), L-glutamine solution (cat. G7513), imperatorin (cat. I6659), osthole (cat. 78889), curcumin (cat. C1386), 5-Fluorouracil (5-FU, cat. F6627), penicillin-streptomycin (cat. P4333), trypsin-EDTA (cat. T3924) were purchased from Sigma-Aldrich.

2.2 Cell culture

A549 (ATCC, CCL-222) cell line, Beas-2b (ATCC, CRL-9609) cell line purchased from American Type Culture Collection (ATCC). A549 (12,500 cells/well) and Beas-2b cells (10,000 cells/well) cultured with Ham’s F-12 and RPMI respectively which are contain with FBS 10%, L-glutamine 1%, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were grown to 80% confluence at 37°C and humidified atmosphere with CO₂ 5%. When cells reached approximately 80% confluence, we detached cells with 0.25% trypsin-EDTA. The cells were centrifuged with the Universal 320R (Hettich, Zentrifugen, 1406 Germany) at 1000 rpm for 5 min at 25°C and seeded on 96 wells e-plate xCELLigence analysis. When cells reached log growth phase approximately 24 h later from seeding to e-plate, cells were treated with 100 μM, 200 μM imperatorin and osthol; 100 μM curcumin and 64 μM 5-FU concentrations.

2.3 xCELLigence Real-Time Cell Analysis (RTCA)

Cytotoxic effect of these molecules was monitored with xCELLigence Real Time Cell Analysis (RTCA) system as described by manufacturer’s instructions (Roche Applied Science and ACEA Biosciences) with slight modifications. Firstly, optimal seeding concentration of A549 and Beas-2b cells were determined and then the cells were seeded in E-plate 96 wells. Cell proliferation, viability and spreading were monitored every 15 minutes via the impedance of E-plate wells. Approximately 24 h post-seeding when the cells were in the log growth phase, we treated cells with these molecules and controls received only medium and replicated 4-times.

2.4 Cell growth and proliferation assay using xCELLigence system

At the end of experiment all calculations were done with the RTCA-integrated software of the xCELLigence system. An unit-less parameter termed cell index (CI) is derived to represent cell status based on the measured relative change in electrical impedance that occurs in the presence and absence of cells in the wells, which is calculated based on the following formula: CI = (Z_i–Z₀)/15, where Z_i is the impedance at an individual point of time during the experiment and Z0 is the impedance at the start of the experiment. RTCA software performs the curve-fitting of selected “sigmoidal dose-response equation” and calculated logarithmic half maximum effect of concentration [log (IC₅₀)] values at a given time point based on log concentration producing 50% reduction of CI value relative to the control CI value (100%) [8].

3 Results

Taking into account the concentrations used for nonsmall cell lung cancer (NSCLC) studies with curcumin, imperatorin and osthol a general logarithmic concentration screening has been performed between 10 nM–1 mM and it was decided to use 100–200 μM of imperatorin and osthol and 100 μM curcumin for further studies in combination with 5-FU. The concentration of 5-FU has been decided as 64 μM which was determined after concentration screening.

According to the results, 100 μM imperatorin did not show cytotoxic effect in Beas-2b cells, nor in A549 cells. However, when combined with 5-FU, cytotoxic effect was observed in healthy cells, but the combination at this concentration did not show cytotoxic effect in cancer cells (Fig. 1a, 1g). 200 μM imperatorin, when used alone or in combination, showed a stronger cytotoxic effect on both cells. When assessed in terms of the primary aim of this study, imperatorin synergistically increased the efficacy of 5-FU in combination, but the same effect on healthy cells was observed as a limiting factor for future studies (Fig. 1b, 1h).

Fig.1

Real-time monitoring effects of the compounds on A549 and Beas-2b cells using the xCELLigence system. (a): Effects of imperatorin (100 μM) alone and in combination with 5-FU (64 μM) on A549 cell line (b): Effects of imperatorin (200 μM) alone and in combination with 5-FU (64 μM) on A549 cell line (c): Effects of osthol (100 μM) alone and in combination with 5-FU (64 μM) on A549 cell line (d): Effects of osthol (200 μM) alone and in combination with 5-FU (64 μM) on A549 cell line (e): Effects of curcumin (100 μM) alone and in combination with 5-FU (64 μM) on A549 cell line (f): Effects of curcumin (100 μM) alone and in combination with 5-FU (64 μM) on Beas-2b cell line (g): Effects of imperatorin (100 μM) alone and in combination with 5-FU (64 μM) on Beas-2b cell line (h): Effects of imperatorin (200 μM) alone and in combination with 5-FU (64 μM) on Beas-2b cell line (i): Effects of osthol (100 μM) alone and in combination with 5-FU (64 μM) on Beas-2b cell line (j): Effects of osthol (200 μM) alone and in combination with 5-FU (64 μM) on Beas-2b cell line.

100 μM osthol did not show cytotoxic effect in Beas-2b cells, nor in A549 cells. However, when combined with 5-FU, cytotoxic effect was observed in healthy cells, but the combination at this concentration did not show cytotoxic effect in cancer cells (Fig. 1c, 1i). 200 μM osthol, when used alone or in combination, showed a stronger cytotoxic effect on healthy cells. When assessed in terms of the primary aim of this study, osthol synergistically increased the efficacy of 5-FU in combination, but the same effect on healthy cells was stronger than the effects on cancer cells which limits the further studies at this concentration (Fig. 1d, 1j).

100 μM curcumin indicated cytotoxic effect in A549 cells while 5-FU alone did not show cytotoxic activity, it was observed that the cytotoxic effect of curcumin appeared to be the same when combined with curcumin. However, the 100 μM curcumin was also toxic to healthy cells at this concentration (Fig. 1e, 1f).

4 Discussion

Non–small cell lung cancer (NSCLC) accounts for 75–85% of all lung cancer cases. The side effects of standard chemotherapeutics limit their use. Therefore, it has become a common approach to investigate the combined use of natural compounds, which might reduce the side effects on healthy cells, while increasing the efficacy of chemotherapeutic drugs in treatment. In the present study, we examined the cytotoxic effects of curcumin, osthol and imperatorin in the presence of 5-FU.

xCELLigence RTCA equipment which is a non-invasive, impedance-based biosensor system that can measure cell viability, growth, spreading and proliferation was used. The xCELLigence system is well-suited to analyze drug effects on cell proliferation, cytostasis and cytotoxicity in real-time [9]. Sometimes the drug candidate might cause an acute effect and this effect can be noticed via real time monitoring.

Curcumin exhibit antitumor actions in several cancer cell lines [10 –13]. Curcumin is a hydrophobic molecule and passes easily through the plasma membrane into the cytosol. Pillai et al. has revealed that this phenolic substance has the ability to reduce the viability of lung cancer cells through induction of apoptosis by activating the preexisting apoptosis machinery. Induction of apoptosis by curcumin involves multiple pathways, it upregulates some of pro-apoptotic genes like caspase-3 and at the same time down-regulate some of the anti-apoptotic genes like that Bcl X_L [14]. Curcumin has anticancer effects both alone and in combination with other anticancer drugs (e.g., gemcitabine, 5-fluorouracil, and oxaliplatin) and the related sturdies about the anticancer effects of curcumin including the molecular mechanisms in pancreatic cancer has been well reviewed [15].

Coumarins are derivatives of 2H-1-benzopyran-2-one which can be obtained chiefly from cyclization of a C-2 oxygenated cis-cinnamic acid. These compounds are widely found in plants in the form of free coumarins or their glycosides. Coumarins have important anticancer properties with low adverse effects based on the functional groups in the original structure. Coumarine derivatives may affect different cellular pathways, including suppression of angiogenesis, cell proliferation as well as inhibition of the enzymes chiefly involved in the pathophysiology of cancer [16].

Anticancer effects of imperatorin has been shown in many studies [17 –19]. The underlying anticancer mechanisms are not well understood. Imperatorin activated caspase-9 which is an apical caspase in the initiation of the caspase cascade during chemical-induced apoptosis in HL-60 cells. It occurs release of cytochrome c from mitochondria into cytosol, and thus the activation of caspase-9 [19]. The cytotoxic effects of osthol has been shown before [20, 21] via some mechanisms such as inducing G2/M arrest and apoptosis. Xu et al. has showed that treating A549 cells with osthol resulted in decreased expression of Cdc2 and Cyclin B1, suggesting that decreasing of Cdc2 and Cyclin B1 expression could be the molecular mechanism through which osthol induced G2/M arrest. Osthol increased proapoptotic Bax expression and decreased anti-apoptotic Bcl-2 expression, leading to up-regulation of the ratio of Bax/Bcl-2 and this could be one of the molecular mechanisms through which osthol induces apoptosis [22]. Xu et al. [23] has further revealed that the effects of various concentrations of osthol on the migratory and invasive potential of A549 cells. Osthol dose-dependently exerted inhibitory effects on lung cancer cells and effectively suppressed proliferation, migration and invasion of cancerous cells. Cellular mechanisms which are essential for these effects were associated with the inhibition of MMP-2 and MMP-9 expression in the human lung cancer cells which have a significant role in cell invasion and migration. Taking into account those studies related to the efficacy of imperatorin and osthol on lung cancer cells we have investigated the synergistic effect of these coumarines on 5-FU treatment in NSCLC. According to our results osthol and imperatorin increased the efficacy of 5-FU on these cells however they have also showed cytotoxic effect on the healthy cells in the concentrations that were effective which limits their use in combination for further studies.

5 Conclusion

Results obtained from studies evaluating anticancer potential of coumarins have confirmed its protective and therapeutic effect on various types of malignancies including ovarian cancer, cervical cancer, chronic myeloid leukemia, lung adenocarcinoma cells, glioma as well as glioblastoma multiform cells, invasive mammary carcinoma cells, colon cancer, and prostate cancer. These compounds might synergistically increase the efficacy of conventional therapies and reduce the side effects. However the concentrations those do not affect the healthy cells should be identified and the molecular mechanisms of their synergistic effect should be further investigated.

Footnotes

Acknowledgments

We thank to Emine Bektaş, Gamze Nur Keser and Oğuz Kalkanoğlu for their help on experiments.

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