Marzena Szwed

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.

Transferrin as a drug carrier: Cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells.

Leukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin-transferrin conjugate (DOX-TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX-TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX-TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX-TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX-TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX-TRF conjugate and free drug.

Gliclazide may have an antiapoptotic effect related to its antioxidant properties in human normal and cancer cells

Experimental and clinical studies suggest that gliclazide may protect pancreatic β-cells from apoptosis induced by an oxidative stress. However, the precise mechanism(s) of this action are not fully understood and requires further clarification. Therefore, using human normal and cancer cells we examined whether the anti-apoptotic effects of this sulfonylurea is due to its free radical scavenger properties. Hydrogen peroxide (H2O2) as a model trigger of oxidative stress was used to induce cell death. Our experiments were performed on human normal cell line (human umbilical vein endothelial cell line, HUVEC-c) and human cancer cell lines (human mammary gland cell line, Hs578T; human pancreatic duct epithelioid carcinoma cell line, PANC-1). To assess the effect of gliclazide the cells were pre-treated with the drug. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was employed to measure the impact of gliclazide on cell viability. Generation of reactive oxygen species, mitochondrial membrane potential (∆Ψm), and intracellular Ca2+ concentration [Ca2+] were monitored. Furthermore, the morphological changes associated with apoptosis were determined using double staining with Hoechst 33258-propidium iodide (PI). Gliclazide protects the tested cells from H2O2-induced cell death most likely throughout the inhibition of ROS production. Moreover, the drug restored loss of ΔΨm and diminished intracellular [Ca2+] evoked by H2O2. Double staining with Hoechst 33258-PI revealed that pre-treatment with gliclazide diminished the number of apoptotic cells. Our findings indicate that gliclazide may protect both normal and cancer human cells against apoptosis induced by H2O2. It appears that the anti-apoptotic effect of the drug is most likely associated with reduction of oxidative stress.

Induction of apoptosis by doxorubicin–transferrin conjugate compared to free doxorubicin in the human leukemia cell lines

In our research we compared the effect of doxorubicin (DOX) and doxorubicin-transferrin (DOX-TRF) conjugate on the induction of programmed cell death. All experiments were carried out on human leukemia cells: CCRF-CEM, K562 sensitive and resistant to DOX, (K562/DOX), which are the molecular model for the chronic and acute form of hematological malignancies, respectively. At the same time, studies were also performed on normal, peripheral blood mononuclear cells (PBMCs). The first stages of apoptosis, connected with externalization of phosphatidylserine (PS), were evaluated after comparing the viability of tested cell lines treated with DOX-TRF conjugate or free DOX. Morphological changes of nuclei connected with apoptosis were analyzed by double staining Hoechst 33258/propidium iodide. Subsequently, we conducted a more accurate evaluation of DOX-TRF-trigged cell death by using DNA ladder assay, measuring the activation of caspase-3, -8 and -9 and changes in poly-ADP ribose polymerase (PARP) activity. The percentage of apoptotic cells reached its maximum at 24 and 48 h incubation. Prolonged treatment time with DOX-TRF conjugate progressively increased the level of necrotic cells. At 24-48 h time points, we observed a significant increase in the activity of apoptosis-characterized enzymes (caspases -8, -9, -3). This study provided the evidence that DOX-TRF conjugate triggers apoptotic pathway connected with DNA damage mediated by the activation of pro-caspases and PARP cleavage.

Relationship between therapeutic efficacy of doxorubicin-transferrin conjugate and expression of P-glycoprotein in chronic erythromyeloblastoid leukemia cells sensitive and resistant to doxorubicin

BackgroundConjugation of anti-neoplastic agents with human proteins is a strategy to diminish the toxic side effects of anthracycline antibiotics. We have developed a novel doxorubicin-transferrin (DOX-TRF) conjugate aimed to direct anticancer drugs against therapeutic targets that display altered levels of expression in malignant versus normal cells. Our previous work has shown that the cellular bio-distribution of the conjugate is dependent on a dynamic balance between influx and efflux processes. Here, we set out to investigate whether P-glycoprotein (P-gp) expression may affect DOX-TRF conjugate-induced cellular drug accumulation and cytotoxicity.ResultsAll experiments were carried out on human erythromyeloblastoid cells exhibiting P-gp over-expression (K562/DOX) and its drug sensitive parental line (K562). MTT cytotoxicity, flow cytometry, fluorescence microscopy and RT-PCR assessments revealed that the investigated conjugate (DOX-TRF) possesses a greater cytotoxic potential than free DOX.ConclusionOur data suggest that the newly developed DOX-TRF conjugate is a less P-gp dependent substrate than free DOX and, consequently, may be used in a clinical setting to increase treatment efficacy in resistant human tumors.

Efficacy of doxorubicin-transferrin conjugate in apoptosis induction in human leukemia cells through reactive oxygen species generation

BackgroundDoxorubicin (DOX) is a small molecular cytotoxic agent that can be transferred efficiently to cancer cells by nanocarriers. This anthracycline antibiotic serves as an effective anti-neoplastic drug against both hematological and solid malignancies. Here, we set out to assess the capacity of a novel doxorubicin - transferrin conjugate (DOX-TRF) to provoke apoptosis in human normal and leukemia cells through free radicals produced via a redox cycle of doxorubicin (DOX) when released from its conjugate.MethodsAfter DOX-TRF exposure, we determined the time-course of apoptotic and necrotic events, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, as well as alterations in cytochrome c levels and intracellular calcium concentrations in human leukemia-derived cell lines (CCRF-CEM, K562 and its doxorubicin-resistant derivative K562/DOX) and normal peripheral blood-derived mononuclear cells (PBMC).ResultsWe found that DOX-TRF can induce apoptosis in all leukemia-derived cell lines tested, which was associated with morphological changes and decreases in mitochondrial membrane potential. In comparison to free DOX treated cells, we observed a time-dependency between a higher level of ROS generation and a higher drop in mitochondrial membrane potential, particularly in the doxorubicin-resistant cell line. In addition, we found that the apoptotic cell death induced by DOX-TRF was directly associated with a release of cytochrome c from the mitochondria and an increase in intracellular calcium level in all human leukemia-derived cell lines tested.ConclusionsOur data indicate that DOX-TRF is considerably more cytotoxic to human leukemia cells than free DOX. In addition, we show that DOX-TRF can effectively produce free radicals, which are directly involved in apoptosis induction.

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.

Doxorubicin-transferrin conjugate triggers pro-oxidative disorders in solid tumor cells.

The formation of reactive oxygen species (ROS) is a widely accepted mechanism of doxorubicin (DOX) toxicity toward cancer cells. However, little is known about the potential of new systems, designed for more efficient and targeted doxorubicin delivery (i.e. protein conjugates, polymeric micelles, liposomes, monoclonal antibodies), to induce oxidative stress (OS) in tumors and hematological malignancies. Therefore, the objective of our study was to determine the relation between the toxicity of doxorubicin-transferring (DOX-TRF) conjugate and its capability to generate oxidative/nitrosative stress in solid tumor cells. Our research proves that DOX-TRF conjugate displays higher cytotoxicity towards lung adenocarcinoma epithelial (A549) and hepatocellular carcinoma (HepG2) cell lines than the reference free drug (DOX) and induces more extensive OS, characterized by a significant decrease in the total cellular antioxidant capacity, glutathione level and amount of -SH groups and an increase in hydroperoxide content. The intracellular redox imbalance was accompanied by changes in the transcription of genes encoding key antioxidant enzymes engaged in the sustaining of cellular redox homeostasis: superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST) and glutathione peroxidase (GP).

The Connection between the Toxicity of Anthracyclines and Their Ability to Modulate the P-Glycoprotein-Mediated Transport in A549, HepG2, and MCF-7 Cells

Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of solid tumors. We compared the resistance of the most popular solid tumors, breast adenocarcinoma (MCF-7 cell line) and nonsmall cell lung (A549 cell line) hepatocellular liver carcinoma (HepG2 cells), to aclarubicin (ACL) and doxorubicin (DOX). This research aimed at determining the relation between the toxicity of ACL and DOX, their cell accumulation, and then effect on P-glycoprotein functionality. ACL is more cytotoxic for tumor cells compared to DOX. The intracellular concentration of drugs in cancer cells was dependent on the dose of the drugs and the time of incubation. The P-gp inhibitor Verapamil (V) increased DOX accumulation in all tested cell lines. By contrast, the intracellular level of ACL was not affected by this modifying agent. The assessment of the uptake of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) or Rhodamine 123 (R123) allows the evaluation of the different influence of drugs on P-gp activity which is in agreement with the estimation of expression measured by MDR-1 shift assay. These data suggest that ACL is less P-gp dependent than DOX and consequently may be used in a clinical setting to increase treatment efficacy in resistant human tumors.

Nanoradiopharmaceuticals for breast cancer imaging: development, characterization, and imaging in inducted animals

Monoclonal antibodies as polymeric nanoparticles are quite interesting and endow this new drug category with many advantages, especially by reducing the number of adverse reactions and, in the case of radiopharmaceuticals, also reducing the amount of radiation (dose) administered to the patient. In this study, a nanoradiopharmaceutical was developed using polylactic acid (PLA)/polyvinyl alcohol (PVA)/montmorillonite (MMT)/trastuzumab nanoparticles labeled with technetium-99m (99mTc) for breast cancer imaging. In order to confirm the nanoparticle formation, atomic force microscopy and dynamic light scattering were performed. Cytotoxicity of the nanoparticle and biodistribution with 99mTc in healthy and inducted animals were also measured. The results from atomic force microscopy showed that the nanoparticles were spherical, with a size range of ~200–500 nm. The dynamic light scattering analysis demonstrated that over 90% of the nanoparticles produced had a size of 287 nm with a zeta potential of −14,6 mV. The cytotoxicity results demonstrated that the nanoparticles were capable of reaching breast cancer cells. The biodistribution data demonstrated that the PLA/PVA/MMT/trastuzumab nanoparticles labeled with 99mTc have great renal clearance and also a high uptake by the lesion, as ~45% of the PLA/PVA/MMT/trastuzumab nanoparticles injected were taken up by the lesion. The data support PLA/PVA/MMT/trastuzumab labeled with 99mTc nanoparticles as nanoradiopharmaceuticals for breast cancer imaging.