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

Doxorubicin–transferrin conjugate alters mitochondrial homeostasis and energy metabolism in human breast cancer cells

Doxorubicin (DOX) is considered one of the most powerful chemotherapeutic agents but its clinical use has several limitations, including cardiomyopathy and cellular resistance to the drug. By using transferrin (Tf) as a drug carrier, however, the adverse effects of doxorubicin as well as drug resistance can be reduced. The main objective of this study was to determine the exact nature and extent to which mitochondrial function is influenced by DOX–Tf conjugate treatment, specifically in human breast adenocarcinoma cells. We assessed the potential of DOX–Tf conjugate as a drug delivery system, monitoring its cytotoxicity using the MTT assay and ATP measurements. Moreover, we measured the alterations of mitochondrial function and oxidative stress markers. The effect of DOX–Tf was the most pronounced in MDA-MB-231, triple-negative breast cancer cells, whereas non-cancer endothelial HUVEC-ST cells were more resistant to DOX–Tf conjugate than to free DOX treatment. A different sensitivity of two investigate breast cancer cell lines corresponded to the functionality of their cellular antioxidant systems and expression of estrogen receptors. Our data also revealed that conjugate treatment mediated free radical generation and altered the mitochondrial bioenergetics in breast cancer cells.This work was supported in part by Grant No. B1511000001026.02 of the University of Lodz, Poland

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

Cytotoxicity of paclitaxel loaded in polyelectrolytes multicore nanocapsules in breast cancer cells

Opis plików: (1) dokumenty pdf z fizykochemiczną charakterystyką badanych nanokapsuł polielektrolitowych (pomiary potencjału zeta, średnicy hydrodynamicznej nanocząstek, ich indeksu polidyspersyjności oraz stężenia), obrazy przedstawiające optymalizację warunków syntezy, wykresy obrazujące widmo UV-Vis dla pustych nanokapsuł i tych z enkapsulowanym paklitakselem, zdjęcia z mikroskopu elektronowego obrazujące morfologię badanych nanokapsuł polielektrolitowych typu SDS/PLL i SDS/PLL/PGA. (2) Pliki MS Excel z wynikami dla przeżywalności komórek HMEC-1, MCF-7 i MDA-MB-231 poddanych działaniu nanokapsuł SDS/PLL i SDS/PLLPGA. Oznaczenia były wykonywane z zastosowaniem testu redukcji MTT. Dodatkowo w jednym z podfolderów znajdują się zdjęcia obrazujące morfologię badanych komórek prawidłowych i nowotworowych poddanych działaniu badanych nanomateriałów po 96 godzinnej inkubacji w warunkach in vitro. (3) Pliki MS Excel z wynikami dla przeżywalności komórek HMEC-1, MCF-7 i MDA-MB-231 poddanych działaniu wolnego paklitakselu oraz dla leku umieszczonego w badanych nanokapsułach to jest dla preparatów: SDS/PLL/PTX i SDS/PLL/PGA/PTX. Pomiary były dokonywane w oparciu o dwa testy cytotoksyczne (CellTiterGlo i MTT). Dodatkowo w jednym z podfolderów znajdują się wyselekcjonowane zdjęcia obrazujące morfologię badanych komórek prawidłowych i nowotworowych poddanych działaniu paklitakselu wolnego i leku zamkniętego w nośnikach po 72 godzinach inkubacji w warunkach in vitro. (4) Pliki MS Excel z wynikami dla oceny stopnia proliferacji komórek HMEC-1, MCF-7 i MDA-MB-231 poddanych działaniu wolnego paklitakselu oraz dla leku umieszczonego w badanych nanokapsułach to jest dla preparatów: SDS/PLL/PTX i SDS/PLL/PGA/PTX. Pomiary były dokonywane w oparciu o inkorporację sondy CyQuant do genomowego DNA badanych komórek. Dodatkowo w jednym z podfolderów znajdują się wyselekcjonowane zdjęcia obrazujące zmianę tempa podziałów komórek prawidłowych i nowotworowych poddanych działaniu wolnego paklitakselu i leku zamkniętego w nośnikach po 72 godzinach inkubacji w warunkach in vitro. (5) Pliki MS Excel z wynikami dla analizy rozkładu faz cyklu komórkowego dla komórek HMEC-1, MCF-7 i MDA-MB-231 poddanych 48 godzinnemu działaniu wolnego paklitakselu oraz dla leku umieszczonego w badanych nanokapsułach to jest dla preparatów: SDS/PLL/PTX i SDS/PLL/PGA/PTX oraz wolnego PTX. Pomiary były dokonywane w oparciu o wybarwianie DNA badanych komórek jodkiem propidyny, po ich uprzednim utrwaleniu w etanolu. (6) Pliki MS Excel z wynikami dla eksternalizacji fosfatydyloseryny, jako jednego z markerów wczesnych etapów apoptozy, dla komórek HMEC-1, MCF-7 i MDA-MB-231 poddanych działaniu wolnego paklitakselu oraz dla leku umieszczonego w badanych nanokapsułach to jest dla preparatów: SDS/PLL/PTX i SDS/PLL/PGA/PTX oraz wolnego PTX. Pomiary były dokonywane po uprzednim wybarwieniu komórek specjalistycznymi sondami molekularnymi, będącymi składowymi zestawu odczynników Annexin V detection kit. (7) Pliki MS Excel z wynikami dla zmian aktywności kaspazy 3/7 dla komórek HMEC-1 i MDA-MB-231 poddanych działaniu wolnego paklitakselu oraz dla leku umieszczonego w badanych nanokapsułach to jest dla preparatów: SDS/PLL/PTX i SDS/PLL/PGA/PTX oraz wolnego. Komórki MCF-7 nie posiadają tego enzymu. Pomiary były dokonywane z wykorzystaniem komercyjnego zestawu odczynników Caspase 3/7 assay kit.The 21st century has brought the rapid expansion in nanotechnology, a new science branch focused on the particles’ production that submicron sizes ranging from 1 to 200 nm. Sequential adsorption of oppositely charged polyelectrolytes, also known as layer by layer method (LbL) is a convenient tool for a production of various nano- drug delivery systems. The main objective of the project was to verify a hypothesis that newly synthesized polyelectrolytes multicore nanocapsules based on water-soluble surfactant - sodium dodecyl sulphate (SDS) could be used as drug delivery systems for water-insoluble anticancer drugs (Paclitaxel, PTX). Two different breast cancer cellular models (MCF-7 and MDA-MB-231 cells) were proposed in this project. In parallel, experiments were performed on non-cancer HMEC-1 cell line. The project was carried out in three stages. Initially, the cytotoxicity of empty, multicore nanocapsules against normal and breast cancer cells was evaluated. Then, we selected those nanocapsules with the lowest cytotoxic parameters and the properties of encapsulated paclitaxel in the investigated nanocapsules were compared with the free PTX. Subsequently, we compared the proliferation rate and measured the cell cycle distribution in normal and cancer cells treated with both forms of PTX. Finally, the caspase -7 activity and the externalization of phosphatidylserine were estimated. Our scientific task is a pioneering research, that proposes the application of SDS- based, multicore polyelectrolyte nanocapsules as a tool for highly hydrophobic drug delivery systems. The obtain data allow to build the better competence for the use of new nanocarriers in chemotherapy.Wiek XXI przyniósł gwałtowny rozwój wielu dziedzin nauki i technologii, w tym technologii w skali nano. Technologia ta umożliwiła wytworzenie niespotykanych dotąd cząstek o submikronowych rozmiarach od 1 do 200 nm - nanocząstek. Dogodnym narzędziem wytwarzania nanocząstek ukierunkowanych na dostarczania leków przeciwnowotworowych (nanonokapsułek) jest sekwencyjna adsorpcja przeciwnie naładowanych polielektrolitów (LbL). Głównym celem projektu była weryfikacja hipotezy badawczej, że nowo zsyntetyzowane multirdzeniowe nanokapsułki oparte na wodorozpuszczalnym surfaktancie- dodecylosiarczanie sodu (SDS) mogą być w przyszłości wykorzystane jako nanosystemy do dostarczania leków przeciwnowotworowych nierozpuszczalnych w wodzie (np. paklitakselu, PTX). Materiałem badawczym w zakończonym zadaniu badawczym były dwa różne modele komórkowe raka piersi - komórki MCF-7 i MDA-MB-231. Równolegle doświadczenia wykonywano na nienowotworowych komórkach śródbłonka człowieka HMEC-1. W projekcie początkowo oceniona została cytotoksyczność multirdzeniowych, pozbawionych leku nanokapsułek, wobec ludzkich prawidłowych oraz nowotworowych komórek ludzkiego raka piersi. Następnie wybrano takie nanokapsułki, które cechowały się najniższymi parametrami cytotoksycznymi, a jednocześnie optymalnymi właściwości fizykochemicznymi dla stosowanego w leczeniu raka piersi hydrofobowego paklitakselu. Ostatecznie, właściwości enkapsulowanego paklitakselu w badanych, multirdzeniowych nanonośnikach porównano z wolną formą leku oceniając tempo proliferacji badanych komórek poddanych działaniu obu form paklitakselu oraz przeanalizowano zmiany w rozkładzie faz cyklu komórkowego. Następnie wykonano oznaczenie aktywności kaspazy 3 oraz zmierzono eksternalizację fosfatydyloseryny. Zakończony projekt jest pionierską propozycją zastosowania multirdzeniowych nanokapsułek polielektrolitowych jako nośników leków przeciwnowotworowych. Efekty doświadczeń prowadzonych w projekcie, umożliwią stworzenie zaplecza wyników wstępnych, koniecznego do późniejszego pogłębienia wiedzy na temat zastosowania potencjalnych nanonośników w chemioterapii.Narodowe Centrum Nauki, MINIATURA 4, nr 2020/04/X/NZ3/0077

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

Background Conjugation 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 DOXTRF conjugate-induced cellular drug accumulation and cytotoxicity. Results All 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. Conclusion Our data suggest that the newly developed DOXTRF 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.Grant no. 545/ 756 of the University of Lodz, Poland

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

Biological response and cytotoxicity induced by lipid nanocapsules

Lipid nanocapsules (LNCs) are promising vehicles for drug delivery. However, since not much was known about cellular toxicity of these nanoparticles in themselves, we have here investigated the mechanisms involved in LNC-induced intoxication of the three breast cancer cell lines MCF-7, MDA-MD-231 and MDA-MB-468. The LNCs used were made of Labrafac™ Lipophile WL1349, Lipoid® S75 and Solutol® HS1

New SDS-Based Polyelectrolyte Multicore Nanocarriers for Paclitaxel Delivery—Synthesis, Characterization, and Activity against Breast Cancer Cells

The low distribution of hydrophobic anticancer drugs in patients is one of the biggest limitations during conventional chemotherapy. SDS-based polyelectrolyte multicore nanocarriers (NCs) prepared according to the layer by layer (LbL) procedure can release paclitaxel (PTX), and selectively kill cancer cells. Our main objective was to verify the antitumor properties of PTX-loaded NCs and to examine whether the drug encapsulated in these NCs retained its cytotoxic properties. The cytotoxicity of the prepared nanosystems was tested on MCF-7 and MDA-MB-231 tumour cells and the non-cancerous HMEC-1 cell line in vitro. Confocal microscopy, spectrophotometry, spectrofluorimetry, flow cytometry, and RT PCR techniques were used to define the typical hallmarks of apoptosis. It was demonstrated that PTX encapsulated in the tested NCs exhibited similar cytotoxicity to the free drug, especially in the triple negative breast cancer model. Moreover, SDS/PLL/PTX and SDS/PLL/PGA/PTX significantly reduced DNA synthesis. In addition, PTX-loaded NCs triggered apoptosis and upregulated the transcription of Bax, AIF, cytochrome-c, and caspase-3 mRNA. Our data demonstrate that these novel polyelectrolyte multicore NCs coated with PLL or PLL/PGA are good candidates for delivering PTX. Our discoveries have prominent implications for the possible choice of newly synthesized, SDS-based polyelectrolyte multicore NCs in different anticancer therapeutic applications

Relationship between Oxidative Stress and Imatinib Resistance in Model Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) develops due to the presence of the BCR-ABL1 protein, a target of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), used in a CML therapy. CML eradication is a challenge due to developing resistance to TKIs. BCR-ABL1 induces endogenous oxidative stress leading to genomic instability and development of TKI resistance. Model CML cells susceptible or resistant to IM, as well as wild-type, non-cancer cells without the BCR-ABL1 protein were treated with IM, hydrogen peroxide (H2O2) as a model trigger of external oxidative stress, or with IM+H2O2. Accumulation of reactive oxygen species (ROS), DNA damage, activity of selected antioxidant enzymes and glutathione (GSH), and mitochondrial potential (MMP) were assessed. We observed increase in ROS accumulation in BCR-ABL1 positive cells and distinct levels of ROS accumulation in IM-susceptible cells when compared to IM-resistant ones, as well as increased DNA damage caused by IM action in sensitive cells. Depletion of GSH levels and a decreased activity of glutathione peroxidase (GPx) in the presence of IM was higher in the cells susceptible to IM. IM-resistant cells showed an increase of catalase activity and a depletion of MMP. BCR-ABL1 kinase alters ROS metabolism, and IM resistance is accompanied by the changes in activity of GPx, catalase, and alterations in MMP