Zofia Jóźwiak

Aclarubicin-induced ROS generation and collapse of mitochondrial membrane potential in human cancer cell lines

The cytotoxicity of aclarubicin (ACL) in A549 (human non-small lung), HepG2 (human hepatoma) and MCF-7 (human breast adenocarcinoma) cancer cell lines was evaluated and compared with that of doxorubicin (DOX). Changes in mitochondrial transmembrane potential (DeltaPsim), and production of reactive oxygen species (ROS) of drug-treated cells were monitored. Moreover, morphological changes associated with apoptosis were examined using double staining with Hoechst 33258-propidium iodide (PI). The results showed that ACL was much more cytotoxic than DOX in all investigated cell lines. Furthermore, ACL induced a concentration- and time-dependent increase in ROS production and decrease in mitochondrial membrane potential. The drugs, especially ACL, also induced ROS mediated apoptosis and necrosis pathways in all cell lines depending on the length of the post-treatment time. All these processes were partially inhibited by the antioxidants: N-acetylcysteine (NAC) and alpha-tocopherol. Of both drugs, DOX caused considerably weaker depolarization of the mitochondrial membrane. Its 10-fold higher concentration, as compared to ACL, was required to induce a similar effect, in accordance with the highly distinct cytotoxicity of these drugs towards investigated cells. In conclusion, ROS production preceded a decrease in mitochondrial membrane potential, but only changes in DeltaPsim were correlated with drug cytotoxicity in particular cell line. These results suggest that the impairment of DeltaPsim and an increase in ROS level might be important mechanisms of ACL cytotoxicity in cancer cells in solid tumors.

Doxorubicin-transferrin conjugate selectively overcomes multidrug resistance in leukaemia cells

Neoplastic cells frequently have an increased number of transferrin receptors. Coupling transferrin to an anti-neoplastic drug has the potential to overcome multidrug resistance (MDR). The purpose of this study was to examine the distribution and action of doxorubicin-transferrin conjugate (DOXTRF) in a leukaemia cell line (HL60), a multidrug-resistant leukaemia cell line (HL60ADR) and a normal tissue cell line (human fibroblasts). The intracellular accumulation of DOX and DOX-TRF was monitored by direct fluorescence. More DOX-TRF than free DOX was delivered to the tumour cells, and consecutively the levels of DNA double-strand breaks and apoptosis increased even in the multidrug-resistant cell line. In the normal tissue cell line, DOX-TRF did not accumulate, and therefore, the levels of DNA double-strand breaks and apoptosis did not increase. Cell viability was determined using the MTT assay. The IC50 for DOX-TRF was lower than the IC50 value for the free drug in both leukaemia cell lines. The IC50 values for the HL60 cells were 0.08 μM for DOX and 0.02 μM for DOX-TRF. The IC50 values for HL60ADR cells were 7 μM for DOX and 0.035 μM for DOX-TRF. In conclusion, DOX-TRF was able to overcome MDR in the leukaemia cell lines while having only a very limited effect on normal tissue cells.

The role of reactive oxygen species in WP 631-induced death of human ovarian cancer cells: a comparison with the effect of doxorubicin.

In the present study, we investigated the anticancer activity of WP 631, a new anthracycline analog, in weakly doxorubicin-resistant SKOV-3 ovarian cancer cells. We studied the time-course of apoptotic and necrotic events: the production of reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in human ovarian cancer cells exposed to WP 631 in the presence and absence of an antioxidant, N-acetylcysteine (NAC). The effect of WP 631 was compared with the activity of doxorubicin (DOX), the best known first-generation anthracycline. Cytotoxic activity was determined by the MTT assay. The morphological changes characteristic of apoptosis and necrosis in drug-treated cells were analyzed by double staining with Hoechst 33258 and propidium iodide (PI) using fluorescence microscopy. The production of reactive oxygen species and changes in mitochondrial membrane potential were studied using specific fluorescence probes: DCFH2-DA and JC-1, respectively. The experiments showed that WP 631 was three times more cytotoxic than DOX in the tested cell line. It was found that the new anthracycline analog induced mainly apoptosis and, marginally, necrosis. Apoptotic cell death was associated with morphological changes and a decrease in mitochondrial membrane potential. In comparison to DOX, the novel bisanthracycline induced a significantly higher level of ROS and a greater drop in the membrane potential. The results provide direct evidence that the novel anthracycline WP 631 is considerably more cytotoxic to human SKOV-3 ovarian cancer cells than doxorubicin. The drug can produce ROS, which are immediately involved in the induction of apoptotic cell death.

Interaction of doxorubicin and idarubicin with red blood cells from acute myeloid leukaemia patients

Doxorubicin (DOX) and idarubicin (IDA) are anthracycline antibiotics, widely used in human cancer treatment. The present study addressed the effects of these two drugs on lipid bilayer fluidity, protein conformation and microviscosity in erythrocytes from acute myeloid leukaemia patients, using electron spin resonance (ESR) spectroscopy and fluorescence measurements. Only DOX caused statistically significant changes in the parameters examined. Within 30 min of drug injection, changes were observed in the fluidity of the hydrophobic parts of the lipid bilayer and erythrocyte membrane protein conformation. These changes persisted for up to 24 h. Analysis of the EPR Tempamine spectrum also showed that the microviscosity of the erythrocyte interior increased during the early stages of the drug effect. Idarubicin, in contrast, caused no identifiable change in any of the parameters studied and therefore seems to be safe for erythrocytes. We conclude that IDA is markedly less toxic than DOX to erythrocytes from acute myeloid leukaemia patients.

Damage to the cell antioxidative system in human erythrocytes incubated with idarubicin and glutaraldehyde.

Encapsulation of antineoplastic drugs within erythrocytes is one of the studied strategies to diminish the toxic side effects of anthracycline antibiotics. Glutaraldehyde is often used as crosslinking agent to link the drugs, including idarubicin (IDA) to the cells. The previous studies indicated that in glutaraldehyde-treated human erythrocytes the elevated level of drug was observed but also the various changes in the organization of the red cells were noted. In this study, we continue our investigations and now we concentrate on the effect of these compounds on antioxidative system in erythrocytes. We determined reactive oxygen species (ROS) production, glutathione content and alterations in the activity of enzymes responsible for maintaining glutathione in reduced form in human erythrocytes. Measurements of both reduced and total glutathione levels and the activity of glutathione reductase and glucose-6-phosphate dehydrogenase were performed spectrophotometrically. The results show that ROS were produced in erythrocytes treated with IDA and with IDA and glutaraldehyde. IDA at a concentration of 10 microg/ml did not cause any changes in total or reduced glutathione levels. When IDA-preincubated erythrocytes were treated with glutaraldehyde, significant changes in the determined parameters were observed in a glutaraldehyde concentration dependent manner. It was correlated with decreased activity of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD). Together with the significant changes in reduced form of glutathione (GSH)/total glutathione ratio, the exposure of phosphatidylserine at the cell surface was also observed.

Activation of apoptotic pathway in normal, cancer ovarian cells by epothilone B

The epothilones, a new class of microtubule-targeting agents, seem to be a very promising alternative to the current strategy of cancer treatment. We have analyzed the aspects of epothilone B (Epo B) on cellular metabolism of tumor (OV-90) and normal (MM 14) ovarian cells. The observed effects were compared with those of paclitaxel (PTX), which is now a standard for the treatment of ovarian cancer. The results provide direct evidence that Epo B is considerably more cytotoxic to human OV-90 ovarian cancer cells than PTX. We have found, that antitumor efficacy of this new drug is related to its apoptosis-inducing ability, which was confirmed during measurements typical markers of the process. Epo B induced changes in morphology of cells, mitochondrial membrane potential and cytochrome c release. Also a slight increase of the intracellular calcium level was observed. Moreover, we have found that ROS production, stimulated by Epo B, is directly involved in the induction of apoptosis via mitochondrial pathway.

Aclarubicin-induced apoptosis and necrosis in cells derived from human solid tumours

In the present study, we investigated the response of A549 (non-small cell lung-cancer), HepG2 (human hepatoma) and MCF-7 (human breast adenocarcinoma) cell lines to treatment with aclarubicin (ACL). The aim of this research was to compare the ability of ACL to induce apoptosis or necrosis in solid tumours. The mode of cell death induced by ACL was evaluated by flow-cytometry and fluorescence microscopy. We show that the drug induced both apoptosis and necrosis in the cells. Apoptotic cell death was associated with morphological changes, DNA fragmentation, changes in activity of poly(ADP-ribose)polymerase (PARP) and drug-mediated activation of caspase-3 and caspase-8. The occurrence of all these events was time-dependent. The extent of apoptosis was also dependent on the kind of cell line, the sensitivity to ACL and the intracellular drug content. This study demonstrates that the cells most sensitive to ACL, A549, accumulated a significantly higher level of the drug and were also more susceptible to apoptosis than the other cells. In contrast, the relatively less sensitive HepG2 and MCF-7 cell lines appeared more resistant to apoptosis induction. On the basis of these results, it seems that aclarubicin is able to induce apoptosis in human solid tumours.

Combined effect of low-power laser irradiation and anthraquinone anticancer drug aclarubicin on survival of immortalized cells : Comparison with mitoxantrone

The photodynamic response of the anthraquinone anticancer drug aclarubicin (ACL) was investigated in vitro and compared with that of mitoxantrone (MTX). Cultured immortalized rodent B14 and NIH 3T3 cells were used in the experiments as a model for cells with neoplastic phenotype. Long‐term cytotoxicity and inhibition of cell proliferation assayed by the clonal growth and MTT‐tetrazolium methods were estimated to compare the efficacy of aclarubicin and mitoxantrone in photosensitizing cells and their death after non‐thermal exposure to monochromatic laser light. Green He—Ne (543.5 nm) or red semiconductor (670 nm) low‐power laser (LPL) irradiations were applied. Different dose‐responses of both cell lines to aclarubicin and mitoxantrone were found so that the cytotoxicity of MTX was considerably greater than the cytotoxicity of ACL. Phototherapy response (P < 0.0001) was observed only for B14 cells after sensitisation with aclarubicin. Under the same conditions no significant effect of red light irradiation (semiconductor 670 nm laser) on survival of both cell lines treated with mitoxantrone was found.

The induction of apoptosis by daunorubicin and idarubicin in human trisomic and diabetic fibroblasts

In this study, we investigated apoptosis induced in human trisomic and diabetic fibroblasts by daunorubicin (DNR) and its derivative, idarubicin (IDA). The cells were incubated with DNR or IDA for 2 h and then cultured in a drug-free medium for a further 2–48 h. The apoptosis in the cultured cell lines was assessed by biochemical analysis. We found that both drugs induced a timedependent loss of mitochondrial membrane potential, and a significant increase in intracellular calcium and caspase-3 activity. Mitochondrial polarization and changes in the level of intracellular calcium were observed during the first 2–6 h after drug treatment. Caspase-3 activation occurred in the late stages of the apoptotic pathway. Our findings also demonstrated that idarubicin was more cytotoxic and more effective than daunorubicin in inducing apoptosis in trisomic and diabetic fibroblasts.

Relevance of drug uptake, cellular distribution and cell membrane fluidity to the enhanced sensitivity of Down's syndrome fibroblasts to anticancer antibiotic-mitoxantrone.

Sensitivity of human fibroblasts derived from Downs syndrome (DS) individuals (S-240, T-158, T-74, T-164) and normal donors (S-126, WA-1) to anticancer antibiotic-mitoxantrone (1,4-dihydroxy-5,8-bis((2-((2-hydroxy-ethyl)amino)ethyl)amino)-9,10-anthracenedione dihydrochloride; MIT) and its relationship to the transport rate, cellular distribution and interaction with cell membrane were studied. The survival assay showed that MIT was more toxic to trisomic fibroblast lines than to normal cells. Studies of transport kinetics indicated that the amount of drug taken up and extruded by DS cells was diminished, compared to control cells. In contrast, the cellular level of MIT associated with DNA was greater in trisomic than in normal cells. The fluorescence anisotropy measurements of TMA-DPH and 12-AS demonstrated that the fluidity of the polar region of the outer lipid monolayer of DS cell membrane was decreased in comparison with normal cells. MIT treatment decreased fluidity of the inner hydrophobic region of plasma membrane, but only slightly influenced the fluidity of the outer surface of the cell membrane. Finally, we concluded that lowered membrane fluidity, diminished amount of MIT extruded by cells and the enhanced level of the drug associated with DNA could be responsible for the enhanced sensitivity of DS fibroblasts to the MIT treatment.