In vitro antileukaemic activity of extracts from berry plant leaves against sensitive and multidrug resistant HL60 cells
Introduction
The food of plant origin, especially fruits and vegetables draw increased attention because of its potential benefits to human health. Epidemiological studies showed that there is a significant positive correlation between intake of fruits and vegetables and reduced rate of heart diseases, cancer mortality and degenerative diseases [1], [2], [3], [4]. Phytochemicals, especially phenolics, in fruits and vegetables are suggested to be the major bioactive compounds for the health benefits because they act as antioxidants and scavenge free radicals [5].
Recently, the attempts of many researchers are focused on the identification of phytochemicals able to inhibit cancer cell growth. There is an increasing number of evidence indicating in vitro antiproliferative activity of fruit extracts against various cancer cell lines. Hibasami et al. [6] observed that the growth of human stomach cancer KATO III cells was inhibited by genistein of soybean koji. It was also found that green tea catechins: (−)-epicatechin, (−)-epigallocatechin and (−)-epigallocatechin gallate inhibited the growth of HCT 116 colorectal and HepG2 hepatocellular carcinoma cells [7]. There is also a recent study showing that theaflavin digallate (black tea), epigallocatechin gallate (green tea) and oolong tea polyphenol trimer caused the growth inhibition and induction of apoptosis in human colon cancer COLO 205 cells [8]. Saleem et al. [9] observed antiproliferative effect of fruit extract of Terminalia chebula on human prostate cancer PC-3, human MCF-7 and mouse S115 breast cancer and human osteosarcoma HOS-1 cell lines. It is also proposed that some phenolics namely flavonoids: catechins, quercetin, kaempferol, anthocyanins and phenolic acids: derivatives of hydroxybenzoic and hydroxycinnamic acids, especially ellagic acid could exibit a high antiproliferative activity against cancer cells [10], [11], [12], [13]. These phytochemicals are abundantly occurring in fruits and vegetables. Published data demonstrated that the ability of phenolic compounds to inhibit the growth of leukaemic and carcinoma cells could be related to the G1 or G2/M phase arrest as well as the induction of apoptosis through cytochrome c release and activation of caspase 9 and caspase 3 [14], [15], [16], [17].
However, one of the most important problems in cancer chemotherapy is the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1), belonging to the ATP-binding cassette protein family [18], [19], [20], [21]. Some reports presented the properties of plant compounds (e.g. genistein, green tea polyphenols, compounds present in bitter melon Momocardia charantia extracts) as reversal agents able to restore the activity of antitumour agents, especially doxorubicin and daunorubicin against multidrug resistant tumour cells overexpressing P-glycoprotein [22], [23], [24], [25]. In contrast, it should be noted that although there are numerous data demonstrating the ability of phytochemicals to inhibit themselves the growth of various cancer cells, up to now very little is known about their antiproliferative activity towards multidrug resistant tumour cells. Thus, the search for substances better protecting against cancer is ongoing all over the world. Recent study showed that the pentacyclic triterpenes isolated from Chrysobalanaceae species inhibited the growth and induced apoptosis of erythroleukaemia K562 cell line and its multidrug resistant subline K562/DOX overexpressing P-glycoprotein [26]. Apart from edible fruits and vegetables, other plant materials as the sources of biologically active substances, especially rich in ellagic acid are studied: leaves and other parts of pomegranate [27], fruits of Terminalia sp. or leaves of Alangium javanicum, branches of Anisophyllea apetala, flowers, leaves and twigs of Crypteronia paniculata, roots of Mouririi sp., stem bark and stem wood of Schlotzia parviflora [28]. Also non-edible parts of strawberry: green fruits, achenes and leaves were found richer in ellagic acid than ripe fruits [29], [30]. Likewise, it was evidenced that young leaves of strawberry, raspberry and blackberry had higher total phenolic content than the respective fruit tissues and old leaves [31].
The aim of the present study was to determine in vitro antileukaemic activity of extracts obtained from selected berry plant leaves against promyelocytic HL60 cell line and its multidrug resistant sublines exhibiting two different MDR phenotypes: HL60/VINC (overexpressing P-glycoprotein) and HL60/DOX (overexpressing MRP1 protein).
Section snippets
Chemicals
HCl, NaOH and methanol were of analytical grade and purchased from POCh Gliwice, Poland. HPLC chemicals were of HPLC purity and purchased from Sigma–Aldrich (USA).
Cell culture
HL60 human promyelocytic leukemia (Division of Biology, Kansas State University, Manhattan, KS 66506, USA) and their resistant sublines: HL-60/VINC (overexpressing P-glycoprotein) [32] and HL-60/DOX (overexpressing MRP1) [33], [34] were cultured. The cells were grown in RPMI 1640 (Gibco Limited) medium supplemented with 2 mM glutamine
Composition of strawberry, raspberry and blueberry leaf extracts
The dry weight of strawberry, raspberry and blueberry leaves used for extract preparation is shown in Table 1. The highest dry weight content in fresh material was observed in blueberry leaves (39.51±1.97 g/100 g) and the lowest in strawberry leaves (32.85±1.64 g/100 g). In contrast, the values of dry weight content of the leaves after room temperature air-drying were very similar for the three berry plant species studied (about 92–93 g/100 g).
The representative chromatograms of phenolic compounds
Discussion
Multidrug resistance (MDR) constitutes the major problem in cancer therapy. Tumour cells become resistant to a wide array of chemotherapeutic agents (e.g. anthracyclines, vinca alkaloids, podophylotoxins, colchicine), structurally diverse and having different mechanisms of action [35]. Thus, many efforts are focused on the search of agents conserving the antitumour activity against multidrug resistant cells [36], [37], [38], [39]. Alternatively, the identification of effective chemosensitizers
Acknowledgements
This work was supported by the grant BW/HK/11/2004 of Agricultural Academy of Szczecin, Poland. The authors wish to thank Katarzyna Cholewińska, MSc for supplying strawberry and raspberry leaves as well as Dorota Zaborowska, MSc and Adam Zaborowski, MSc for supplying blueberry leaves for this study.
References (52)
- et al.
Dietary factors and cancer mortality among atomic-bomb survivors
Mutat. Res.
(2004) - et al.
Screening of selected flavonoids and phenolic acids in 19 berries
Food Res. Int.
(1999) - et al.
Inhibition of green tea catechins against the growth of cancerous human colon and hepatic epithelial cells
Cancer Lett.
(2001) - et al.
Inhibition of cancer cell growth by crude extract and phenolics of Terminalia chebula retz
Fruit, J. Ethnopharm.
(2002) - et al.
Dietary flavonols: chemistry, food content, and metabolism
Nutrition
(2002) - et al.
Antioxidant interactions of catechin, cyanidin, caffeic acid, quercetin, and ellagic acid on human LDL oxidation
Food Chem.
(1998) - et al.
Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells
Eur. J. Cancer
(1999) - et al.
Studies on the inhibitory effects of quercetin on the growth of HL-60 leukemia cells
Biochem. Pharmacol.
(1997) P-Glycoprotein–a mediator of multidrug resistance in tumour cells
Eur. J. Cancer
(1996)- et al.
MDR1/P-glycoprotein in haematological neoplasms
Eur. J. Cancer
(1996)