Jolanta Tarasiuk

In Vitro Antileukaemic Activity of Extracts from Chokeberry (Aronia melanocarpa [Michx] Elliott) and Mulberry (Morus alba L.) Leaves against Sensitive and Multidrug Resistant HL60 Cells

The aim of the present study was to determine in vitro antileukaemic activities of extracts obtained from chokeberry (Aronia melanocarpa [Michx] Elliot) and mulberry (Morus alba L.) 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). It was found that the extracts from chokeberry and mulberry leaves were active against the sensitive leukaemic cell line HL60 and retained the in vitro activity against multidrug resistant sublines (HL60/VINC and HL60/DOX). The values of resistance factor (RF) found for these extracts were very low lying in the range 1.2–1.6. Copyright

Transport of new non-cross-resistant antitumor compounds of the benzoperimidine family in multidrug resistant cells.

Multidrug resistance (MDR) phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or multidrug resistance-associated protein (MRP1). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the pump-mediated efflux. Here, we studied three members of a new family of benzoperimidine antitumor compounds which exhibit comparable cytotoxicity towards resistant cells expressing P-glycoprotein, or MRP1, and sensitive cells. We used spectrofluorometric methods to determine the kinetics of the uptake and release of these three drugs in different cell lines: the erythroleukemia cell line K562 and the resistant K562/Adr expressing P-glycoprotein, the small-cell lung cancer cell line GLC4 and resistant GLC4/Adr expressing MRP1. We also studied, using confocal microscopy, the intracellular distribution of these drugs in NIH/3T3 cells. Our data show that (i) the kinetics for the uptake of these drugs is very rapid, higher than 2 x 10(-17) mole cell(-1) s(-1), (ii) the drugs are strongly accumulated in the nucleus and lysosomes, (iii) the three drugs are recognized and pumped out by both transporters, as shown by the inhibition of P-glycoprotein- and MRP1-mediated efflux of pirarubicin by benzoperimidine, with inhibitory constants of 1.5 and 2.1 microM for P-glycoprotein and MRP1, respectively, suggesting that benzoperimidine is transported by the two transporters with K(m) approximately 2 microM. In conclusion, the fast uptake kinetics of the benzoperimidines counterbalance their efflux by P-glycoprotein and MRP1.

Comparative effects of selected plant polyphenols, gallic acid and epigallocatechin gallate, on matrix metalloproteinases activity in multidrug resistant MCF7/DOX breast cancer cells.

The aim of the study was to investigate the effect of selected polyphenols: gallic acid (GA) and epigallocatechin gallate (EGCG) on matrix metalloproteinase (MMP-2 and MMP-9) activity in multidrug resistant (MDR) human breast adenocarcinoma cells: MCF7/DOX cells and obtained recently in our laboratory MCF7/DOX500 cells by the permanent selection of MCF7/DOX cells with 500 nM doxorubicin (DOX). The activity of MMP-2 and MMP-9 and the effect of studied polyphenols on these matrix proteases were examined by gelatin zymography assays. We have found that the activity of MMP-2 and MMP-9 significantly increased in resistant MCF7/DOX and MCF7/DOX500 cells whereas they were not detected in sensitive MCF7 cells. It was also observed that GA (30, 60, 100 and 120 µM) and EGCG (5, 10 and 20 µM) caused a comparable concentration-dependent inhibition of MMP-2 and MMP-9 activity in MCF7/DOX and MCF7/DOX500 cells. Control experiments confirmed that examined compounds in these ranges of concentration did not affect the cell growth of MCF7/DOX and MCF7/DOX500 sublines (80-100% of control cell growth was observed in the presence of studied polyphenols).

Retaining cytotoxic activity of anthrapyridone CO1 against multidrug resistant cells is related to the ability to induce concomitantly apoptosis and lysosomal death of leukaemia HL60/VINC and HL60/DOX cells

The effect of anthrapyridone compound CO1 retaining cytotoxic activity against multidrug resistant (MDR) tumour cells on inducing cell death of the sensitive leukaemia HL60 cell line and its MDR sublines (HL60/VINC and HL60/DOX) was examined.

Anthraquinone antitumour agents, doxorubicin, pirarubicin and benzoperimidine BP1, trigger caspase-3/caspase-8-dependent apoptosis of leukaemia sensitive HL60 and resistant HL60/VINC and HL60/DOX cells

We examined the effect of selected anthraquinone antitumour agents – doxorubicin (DOX), pirarubicin (PIRA) and benzoperimidine BP1 – on inducing apoptosis of the sensitive leukaemia HL60 cell line and its multidrug resistance sublines overexpressing P-glycoprotein (HL60/VINC) and multidrug resistance-associated protein 1 (HL60/DOX). All agents used at IC50 and IC90 were able to influence the cell cycle of sensitive HL60 and resistant cells and induce apoptosis. Interestingly, it was seen that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by the studied anthraquinone compounds compared with HL60 and HL60/DOX cells. However, the examined agents did not change the expression of Fas receptors on the surface of HL60-sensitive and-resistant cells.

Role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase. Implications for increasing the activity against sensitive and multidrug-resistant leukaemia HL60 cells

The aim of this study was to examine the role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially with regard to multidrug resistant (MDR) tumour cells. It was found that at a high NADPH concentration (500 &mgr;mol/l), the anthracenedione agent ametantrone, with an unmodified quinone structure, was susceptible to CPR-dependent reductive activation. In contrast, it was shown that compounds with modified quinone grouping (benzoperimidine BP1, anthrapyridone CO1 and pyrazolopyrimidoacridine PPAC2) did not undergo reductive activation by CPR. This suggests that the presence of a modified quinone function is the structural factor excluding reductive activation of antitumour anthraquinone derivatives and analogues by CPR. In the second part of the work, the ability of antitumour anthraquinone derivatives and analogues to inhibit the growth of the human promyelocytic, sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. A significant increase in the activity of ametantrone with an unmodified quinone structure after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells, whereas in the case of quinone-modified compounds (BP1, CO1 and PPAC2), the presence of the activation system had no effect on their activity against the sensitive and MDR tumour cells examined.

Bioreductive activation of mitoxantrone by NADPH cytochrome P450 reductase does not change its apoptotic stimuli properties in regard to sensitive and multidrug resistant leukaemia HL60 cells

The objective of this study was to examine the effect of bioreductive activation of antitumour drug, mitoxantrone (MX), by liver NADPH cytochrome P450 reductase (CPR) on inducing apoptosis of human promyelocytic sensitive leukaemia HL60 cell line and its multidrug resistance (MDR) sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX). It was found that non-activated as well as CPR-activated form of MX used at IC90 were able to influence cell cycle of sensitive HL60 as well as resistant cells and induce apoptosis. Interestingly, it was evidenced that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by both studied forms of MX compared to HL60 and HL60/DOX cells. However, the examined agent did not change the expression of Fas receptors on the surface of HL60 sensitive as well as resistant cells regardless of its form used in the study. Obtained results suggest that CPR-dependent reductive activation of MX does not change its apoptotic stimuli properties in regard to sensitive HL60 and multidrug resistant (HL60/VINC and HL60/DOX) leukaemia cells. Nevertheless, taking into account that side toxic effects observed in course of patient treatment with antitumour drugs are dose-dependent, it seems that the reported increase in antiproliferative activity and ability to induce apoptosis of MX after its reductive activation by exogenous CPR against the MDR cells overexpressing both P-glycoprotein and MRP1 at much more lower concentrations of this drug could be of clinical importance for the treatment of tumours resistant to classical chemotherapy.

Effect of selected NAD + analogues on mitochondria activity and proliferation of endothelial EA.hy926 cells

The aim of the study was to examine the effect of 1-methylnicotinamide (MNA) and 1-methyl-3-nitropyridine (MNP) on mitochondria activity and proliferation of endothelial EA.hy926 cells. The activity of MNA was also referred to nicotinamide (NAM) being MNA metabolic precursor. NAM and MNA used at high concentrations (up to 1 mM) had no effect on mitochondria metabolism and proliferation of EA.hy926 cells. It could be related to the fact that these compounds hardly cross the cell membrane. It supports the results of our previous study suggesting that anti-inflammatory and anti-thrombotic effects of MNA could be associated with its ability to bind to glycosaminoglycans, especially heparins, located on the endothelium membrane without entering into target cells. In contrast, MNP caused substantial changes in mitochondria activity and proliferation of EA.hy926 cells. This compound used at low concentrations (below 100 microM) blocked the cell cycle of EA.hy926 cells in G1 phase and was very effective in inhibiting cell growth (IC50=13.8+/-2.4 microM). At higher concentrations (0.1-1 mM) MNP caused a significant reduction of cell survival. The observed effects of MNP could be related, at least in part, to its ability to influence the ATP and NAD+ intracellular levels. MNP caused also important changes in Ca2+ intracellular concentration, significant decrease in inner mitochondrial membrane potential and high increase in mitochondrial respiration of EA.hy926 cells. The observed effects of MNP may be related in part to its cellular metabolites detected after 45 min incubation with 250 microM MNP.

The ability of selected pyridinium salts to increase the cytotoxic activity of vincristine but not doxorubicin towards sensitive and multidrug resistant promyelocytic leukaemia HL60 cells

The aim of this study was to examine the effect of selected pyridinium salts, 1‐methyl‐3‐nitropyridine chloride (MNP+Cl−) and 3,3,6,6,10‐pentamethyl‐3,4,6,7‐tetrahydro‐[1,8(2H,5H)‐dion]acridine chloride (MDION+Cl−), on the activity of doxorubicin (DOX) and vincristine (VINC) towards human promyelocytic leukaemia HL60 cells as well as its multidrug resistant (MDR) sublines exhibiting two different phenotypes of MDR related to the overexpression of P‐glycoprotein (HL60/VINC) or MRP1 (HL60/DOX). MNP and MDION salts were much less cytotoxic themselves (about 100‐fold and 2000‐fold compared with DOX and VINC, respectively) against HL60 cells but, in contrast to DOX and VINC, they conserved an important cytotoxic activity towards resistant HL60/VINC and HL60/DOX cells (resistance factor, RF = 2–4.5). It was shown that MNP+Cl− and MDION+Cl− increased the cytotoxicity of non‐bioreductive antitumour agent VINC towards human promyelocytic leukaemia HL60 cells and its resistant sublines HL60/VINC and HL60/DOX. However, in the case of DOX the decrease in its cytotoxic activity towards all studied cell lines was observed in the presence of MNP+Cl− and MDION+Cl−. Presented data suggest that the bioreductive drug DOX, in contrast to VINC, could compete with pyridinium salts (MNP+Cl− and MDION+Cl−) for NADPH‐dependent oxidoreductases and for undergoing cellular reductive activation. This could explain the inefficiency of these salts to increase the cytotoxic activity of DOX against examined leukaemic HL60 cell line and its MDR sublines, HL60/VINC and HL60/DOX.

Invasion and metastasis of tumour cells resistant to chemotherapy

Metastatic tumours resistant to chemotherapy are the major cause of the clinical failure in the treatment of malignant diseases. It is observed often that drugs active against primary tumours do not exhibit the same efficacy towards metastatic tumour cells having modified signaling pathways. Among cellular factors involved in the development of the metastatic potential of multidrug resistant tumour cells are some oncoproteins, antiapoptotic proteins, mutated suppressor proteins, integrins and CD44 receptor. It was also demonstrated that numerous chemotherapeutics have the effect on the emergence of the metastatic potential and multidrug resistance (MDR) phenomenon of tumour cells. The results of numerous studies suggest that genes involved in the development of MDR and metastatic phenotype of tumour cells are regulated by the same signaling pathways. They lead to the activation of transcription factors e.g. HIF-1α, NF-κB, Ets1 and AP-1 controlling the expression of genes involved in the development of the metastatic potential of multidrug resistant tumour cells. The identification of key cellular factors responsible for the emergence of the metastatic potential of MDR tumour cells could lead to the development of new efficient strategies for the treatment of metastatic tumours resistant to the conventional chemotherapy.