Protective effect of quercetin and luteolin in human melanoma HMB-2 cells

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Abstract

Multifunctional effects of flavonoids are reported to be markedly connected with their structure and the functional groups in the molecule. The important role in the activity play C2–C3 double bond, hydroxyl group at C3 and the number of hydroxyl groups at phenyl ring (B). In this paper, the DNA protective free radical scavenging potential of quercetin (QU) and luteolin (LU) against H2O2 and their clastogenic effect alone and in combination with melphalan (MH) were investigated in human melanoma HMB-2 cells. Elevated frequency of chromosomal aberrations induced by MH, that at high doses have shown a variety of toxic side effects, was statistically decreased by studied flavonoids regarding to control (QU at the concentration of 50 μM and LU already at the concentration of 20 μM). The results concerning DNA protective potential against free radicals in HMB-2 cells demonstrated that QU and LU have significant effect in dose dependent manner. The percentage of QU protective effect is 40% at the concentration 20 μM, resp. 80% at the concentration 100 μM. Comparable values were obtained with LU. Results are correlated to their structural arrangement and organization of the hydroxyl groups.

Introduction

Polyphenols are secondary plant metabolites that have been reported to possess antihepatotoxic, anti-inflammatory, antiatherogenic, antiallergic, antiosteoporotic and anti-carcinogenic activities [1]. Described effects are related to their interaction with several enzymes [1], [2], antioxidant activity, which includes chain-breaking and metal-chelating abilities in the same molecule [3], [4], [5] and to synergistic effects with other antioxidants [6].

The multifunctional effects of flavonoids are very intimately connected with the structure and its functional groups that consist of a benzene ring (A) fused with a pyrone ring (C) that in position 2 or 3 carries a phenyl ring (B) as a substituent. Besides, the important role in the activity play C2–C3 double bond, hydroxyl group at C3 and the number of hydroxyl groups at B-ring which act as electron donors and are responsible for radical scavenging activity of flavonoids [7].

At the present study, attention has been made on ubiquitous quercetin (QU) and luteolin (LU). They diverse in the number of hydroxyl groups in the skeleton (Fig. 1). Numerous experiments on QU have shown their protective effect against DNA damage induced by hydrogen peroxide [8], [9], [10]. QU and LU can inhibit protein kinase C, lipoxygenase, histamine-release activities and angiogenesis, they can modulate cell cycle [11]. LU has reduced the frequency of micronucleated reticulocytes in mouse peripheral blood cells [12], inhibited the doxorubicin-induced elevation of the peroxide level in mouse heart and bone marrow [13].

Melphalan (MH) is a phenylalanine derivative of nitrogen mustard currently used in cancer therapy—against multiple myeloma, ovarian, breast and colorectal cancers and malignant melanoma. MH at high doses shows a diversity of toxic side effects. Leukopenia and thrombocytopenia occur during therapy, secondary malignancies such as acute nonlymphocytic leukemia, myeloproliferative syndrom, carcinoma have been reported after long-lasted therapy [14]. Futhermore, MH has been observed to produce chromosomal aberrations in human cells in vitro and in vivo. For the study of chromosomal aberrations is useful to study sister chromatid exchange, chromosomal translocations, marker chromosomes [15].

We attempt to establish the protective antioxidant effect of QU and LU against H2O2-induced DNA damage in human melanoma HMB-2 cells using the comet assay. The futher part of our study was focused on the protective effect of mentioned flavonoids on chromosomal aberrations induced by MH in HMB-2 cells.

We attempt to study the protective effects of QU and LU against free radicals induced by H2O2 and against chromosomal aberration production induced by MH in human melanoma HMB-2 cells. The investigation has been performed by two different assays—the comet and cytogenetic assays. The results were further evaluated considering the structural dissimilarity of studied flavonoids.

Section snippets

Cell culture

The human melanoma cell line (HMB-2) was obtained from metastasis of malignant melanoma to be removed from right axial lymph node region of 51-year-old woman [16]. The cell line was cultured in Eagle's minimum essential medium (MEM-E) supplemented with 20% fetal bovine serum (FBS), streptomycin (0.1 mg/ml) and gentamycin (0.05 mg/ml). The cells were cultivated on plastic Petri dishes (Ø60 mm) and were incubated at 37 °C in a humidified atmosphere of 5% CO2:95% air. All in vitro experiments were

Cytotoxicity of flavonoids

The cytotoxicity of QU and LU were performed by the cell counting after 24 h pre-treatment with flavonoid. The results are summarized in the Table 1.

Comet assay

The level of DNA single strand breaks induced in HMB-2 cells by H2O2 was determined by the SCGE. The optimal concentration of H2O2 in our experiments was 100 μmol/l. The percentage of tail DNA corresponding to the level of DNA strand breaks at this H2O2 concentration, was 60. This damage induced by H2O2 was regarded as positive controls. The

Discussion

A plethora of methods applied to the investigation of the antioxidant activity of pure compounds, food constituents, and body fluids have been reported, and in the case of the former, many attempts have been made to establish structure–activity relationships. An assessment of the relative antioxidant activities of flavonoids against free radicals generated in the aqueous phase as well as the relationship with their structural arrangement and organization of the hydroxyl functions responsible

Acknowledgements

This work was supported by the National Program of Research and Development Use of Cancer Genomics to Improve the Human Population Health No. 2003SP00/0280800/0280801 and Slovak Grant Agency VEGA (Grant No. 2/4005/04).

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