Katarzyna D. Kania

Differential regulation of the human MRP2 and MRP3 gene expression by glucocorticoids

Multidrug resistance proteins, which catalyse the detoxification of xenobiotics and excretion of metabolites, are very often controlled at the transcriptional level by interaction of exogenous compounds or hormones with nuclear receptors. Since synthetic glucocorticoids have found extensive use as anti-inflammatory drugs, also in the inhaled form in the treatment of asthma, lung cancer is potentially highly prone to transcriptional induction of multidrug resistance proteins by these steroids. MRP3 and MRP2 are major active anionic conjugate transporters in human cells and play a significant role in clinical multidrug resistance in cancer. A549 cells (non-small-cell lung cancer cell line) were challenged with glucocorticoids (dexamethasone, hydrocortisone and prednisone) at physiologically and therapeutically relevant concentrations for 24h and changes in MRP2 and MRP3 expression were followed on four levels: promoter regulation (luciferase reporter constructs), mRNA level (semi-quantitative real-time PCR), protein level (Western blotting) and activity (drug resistance and cellular transport of the model substrate calcein). DEX and HCT in the submicromolar concentration range caused a 2-fold induction of transcriptional activity at the MRP3 promoter construct, while MRP2 expression was not activated. All investigated glucocorticoids caused a modest stimulation of organic anion transport activity. We conclude that glucocorticoids used in clinical practice have the ability to transcriptionally upregulate human MRP3 gene expression in lung-derived cells where this protein is a major component of the organic anion extrusion system. This phenomenon has to be taken into account when designing treatments for lung cancer, especially for patients treated simultaneously with glucocorticoids against inflammatory symptoms.

L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation

BackgroundThe consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells.MethodsThree cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA.ResultsL- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA).ConclusionsOur data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.

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.

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).

Molecular damage caused by generation of reactive oxygen species in the redox cycle of doxorubicin–transferrin conjugate in human leukemia cell lines

Abstract In this study we focused on evaluation of the pro-oxidant properties of doxorubicin–transferrin (DOX-TRF) conjugate and its potency to damage macromolecules which are components of cellular compartments. Our experiments were performed on two human leukemia cell lines: K562 (chronic erythromyeloblastoid leukemia) and CCRF-CEM (acute lymphoblastic leukemia). We determined the reactive oxygen species (ROS) production and programmed cell death (PCD) induction by free DOX and its conjugate. Besides this, the lipid peroxidation and protein damage which can be provoked by DOX alone and DOX-TRF conjugate were assessed. ROS were produced in leukemia cells incubated with free DOX and DOX-TRF conjugate and the extent of apoptosis and necrosis was strongly dependent on the cell line, sensitivity to drug and time of incubation with the investigated compounds. The role of ROS in DOX-TRF conjugate-induced cell death was confirmed by the diminution effects of the antioxidant vitamin C.

Interleukin-4 Enhances PARP-Dependent DNA Repair Activity In Vitro

Eukaryotic cells possess several DNA repair mechanisms, including homologous recombination and the non-homologous end-joining (NHEJ) system. There are two known NHEJ systems. The major mechanism depends on the catalytic unit of DNA-dependent protein kinase (DNA-PKcs) and DNA ligase IV, and an alternative mechanism (B-NHEJ) depends on poly(ADP-ribose) polymerase (PARP). These systems are upregulated by genotoxic agents. Interleukin 4 (IL-4) is an immunoregulatory cytokine that is secreted by immune cells upon contact with certain genotoxic compounds and is known to regulate several genes encoding components of DNA repair systems in human monocytes. We have investigated the possible effects of IL-4 on the DNA repair process within murine and human cells exposed to selected genotoxic compounds. In a series of experiments, including the comet assay, cell surface annexin V staining, analysis of histone H2AX phosphorylation, and a DNA end-joining assay, we observed that IL-4 decreased DNA damage in murine fibroblasts and human glioblastoma cells exposed to genotoxic agents and increased DNA ligation activity in the nuclei of these cells in a process that depended on PARP. These observations suggest that IL-4 is capable of upregulating the alternative NHEJ DNA repair mechanism in murine and human cells.

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms.

Toxicity of doxorubicin-transferrin conjugate is connected to the modulation of Wnt/β-catenin pathway in human leukemia cells.

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by constitutive activation of the BCR/ABL tyrosine kinase. However, the tyrosine kinase inhibitors like imatinib mesylate are not effective in the patients with advanced-stage of CML. Hence, there is an urgent need for new approaches to overcome a cancer cells resistance in CML long term therapy. Development of new drug carriers, is presently one of the most challenging tasks in experimental oncology. In this report we investigated whether the toxicity of newly synthetized doxorubicin transferrin conjugate (DOX-TRF) may be connected to the limitation of multidrug resistance in CML cells by the alternations of Wnt/β-catenin signaling pathway. The studies were performed on human chronic myeloid leukemia cell lines sensitive (K562) and resistant (K562/DOX) to doxorubicin. Our research proves that DOX-TRF conjugate displays higher cytotoxicity toward both examined cell lines than the reference free drug (DOX) and induces more extensive pro-apoptotic changes. Moreover, by the of engagement of Wnt pathway agonist (LiCl) and antagonist (ICG 001) we demonstrate that DOX-TRF conjugate effectively reduces transcription of key genes involved in β-catenin signaling transduction trial (Wnt3a, DVL-1, FZD-3, LRP5, β-catenin, DKK2) and triggers morphology alternations of CML cells.

Mycobacterium tuberculosis RecA is indispensable for inhibition of the mitogen‐activated protein kinase‐dependent bactericidal activity of THP‐1‐derived macrophages in vitro

Our knowledge about the mechanisms utilized by Mycobacterium tuberculosis to survive inside macrophages is still incomplete. One of the mechanism that protects M. tuberculosis from the hosts microbicidal products and allows bacteria to survive involves DNA repair systems such as the homologous recombination (HR) and nonhomologous end‐joining (NHEJ) pathways. It is accepted that any pathway that contributes to genome maintenance should be considered as potentially important virulence factor. In these studies, we investigated reactive oxygen species, nitric oxide and tumor necrosis factor‐α production by macrophages infected with wild‐type M. tuberculosis, with an HR‐defective mutant (∆recA), with an NHEJ‐defective mutant [∆(ku,ligD)], with a mutant defective for both HR and NHEJ [∆(ku,ligD,recA)], or with appropriate complemented strains. We also assessed the involvement of extracellular signal‐regulated kinases (ERKs) 1 and 2 in the response of macrophages to infection with the above‐mentioned strains, and ERK1/2 phosphorylation in M. tuberculosis‐infected macrophages. We found that mutants lacking RecA induced a greater bactericidal response by macrophages than did the wild‐type strain or an NHEJ‐defective mutant, and activated ERK1/2 was involved only in the response of macrophages to recA deletion mutants [∆(ku,ligD,recA) and ∆recA]. We also demonstrated that only the triple mutant induced ERK1/2 phosphorylation in phorbol‐12‐myristate‐13‐acetate‐stimulated macrophages. Moreover, HR‐defective mutants induced lower amounts of tumor necrosis factor‐α secretion than did the wild‐type or ∆(ku,ligD). Our results indicate that RecA contributes to M. tuberculosis virulence, and also suggest that diminished ERK1/2 activation in macrophages infected with M. tuberculosis possessing recA may be an important mechanism by which wild‐type mycobacteria escape intracellular killing.

Assessment of pro-apoptotic activity of doxorubicin-transferrin conjugate in cells derived from human solid tumors.

Conjugates of anthracyclines are a new possibility for anticancer agent delivery, which seems to be a very promising alternative to the currently used cancer treatment strategies. In our study, we investigated the ability of a doxorubicin-transferrin (DOX-TRF) conjugate to induce cell death in two solid tumor cell lines: non-small cell lung cancer (A549) and hepatocellular liver carcinoma (HepG2). The observed effects of the DOX-TRF conjugate on these cell cultures were compared with those of free doxorubicin (DOX), a widely used antineoplastic therapeutic agent. Our results provided direct evidence that the investigated conjugate is considerably more cytotoxic to the examined human cancer cell lines than is DOX alone. Moreover, we confirmed that the antitumor efficacy of DOX-TRF conjugate is related to its apoptosis-inducing ability, which was shown during measurements of typical features of programmed cell death. In solid tumor cell lines, the DOX-TRF conjugate induced changes in cellular morphology, mitochondrial membrane potential and caspases-3 and -9 activities. Furthermore, all of the analyzed hallmarks of apoptosis were confirmed by the oligonucleosomal DNA fragmentation assay and by a real-time PCR quantitative study, which displayed the superiority of the conjugate-induced programmed cell death over free drug-triggered cell death.