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Overcoming the limitations for efficient and selective drug delivery is one of the most challenging obstacles for newly designed anticancer agents. In this study, we present two types of platinum-based nanoparticles (NP), ultrasmall 2 nm PtNPs and core-shell 30 nm Au@Pt, which can be highly cytotoxic in an oxidative environment and remain biologically inactive in cells with lower oxidative status. Our research highlighted the differences in platinum nanoparticle-induced chemotoxicity and is the first study examining its mechanism as a substantial aspect of Au@Pt/PtNPs biological activity. Selectively induced oxidative stress was found to be a primary trigger of NPs’ toxicity. Significant differences between Au@Pt and PtNPs were observed especially during 24 h treatment, due to successful intranuclear PtNPs location (~13% of internalized fraction). Reactive oxygen species (ROS)-level induced from both NPs types were similar, while reduction of reduced glutathione (GSH) intracellular content was stronger after treatment with PtNPs. Any biological activity was found in HER2+ breast cancer cells, which have only slightly increased oxidative status. Platinum-containing nanoparticles are an interesting tool for the improvement of selectivity in anticancer therapies against hepatocellular carcinoma (HCC). Due to intranuclear uptake, 2 nm PtNPs seems to be more promising for further research for HCC therapy.

BackgroundLipid 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®HS15.ResultsHigh resolution SIM microscopy showed that the DiD-labeled LNCs ended up in lysosomes close to the membrane. Empty LNCs, i.e. without encapsulated drug, induced not only increased lysosomal pH, but also acidification of the cytosol and a rapid inhibition of protein synthesis. The cytotoxicity of the LNCs were measured for up to 72 h of incubation using the MTT assay and ATP measurements in all three cell lines, and revealed that MDA-MB-468 was the most sensitive cell line and MCF-7 the least sensitive cell line to these LNCs. The LNCs induced generation of reactive free oxygen species and lipid peroxidation. Experiments with knock-down of kinases in the near-haploid cell line HAP1 indicated that the kinase HRI is essential for the observed phosphorylation of eIF2α. Nrf2 and ATF4 seem to play a protective role against the LNCs in MDA-MB-231 cells, as knock-down of these factors sensitizes the cells to the LNCs. This is in contrast to MCF-7 cells where the knock-down of these factors had a minor effect on the toxicity of the LNCs. Inhibitors of ferroptosis provided a large protection against LNC toxicity in MDA-MB-231 cells, but not in MCF-7 cells.ConclusionsHigh doses of LNCs showed a different degree of toxicity on the three cell lines studied, i.e. MCF-7, MDA-MD-231 and MDA-MB-468 and affected signaling factors and the cell fate differently in these cell lines.

Simple Summary Ovarian cancer has the highest mortality rate among gynecological malignancies and is the second most commonly diagnosed gynecological cancer. Acquired resistance to platinum therapy remains a major problem in gynecological oncology. Considering the unique physicochemical properties of the metallacarboranes and antimetabolite activity of nucleoside derivatives, we designed and synthesized the adenosine conjugates with metallacarboranes containing iron, cobalt, or chromium as semi-abiotic compounds that influence cisplatin-resistance of cancer cells. The iron-containing conjugate of metallacarborane and adenosine sensitized resistant cancer cells and highly resistant multicellular cancer spheroids to cisplatin, increasing cell cycle arrest, apoptosis or necrosis, and reactive oxygen species production. The presence of nucleosides in the structure of the conjugates was revealed to be indispensable for protecting cells against the development of cross-resistance to cisplatin, carboplatin, doxorubicin, paclitaxel, or gemcitabine in long-term-cultures. The findings indicate that adenine nucleoside modified with metallacarboranes may help sensitize ovarian cancer cells to chemotherapeutic agents in combination therapy. Abstract Platinum compounds remain the first-line drugs for the treatment of most lethal gynecological malignancies and ovarian cancers. Acquired platinum resistance remains a major challenge in gynecological oncology. Considering the unique physicochemical properties of the metallacarboranes modifier and the significant role of nucleoside derivatives as anticancer antimetabolites, we designed and synthesized a set of adenosine conjugates with metallacarboranes containing iron, cobalt, or chromium as semi-abiotic compounds that influence the cisplatin sensitivity of ovarian cancer cells. Adherent cultures of ovarian carcinoma cell lines and multicellular spheroids, ranging from sensitive to highly resistant including experimental cell lines “not responding” to platinum drugs were used. Iron-containing metallacarborane conjugates showed the best anticancer activity, especially against resistant cells. Compound modified at the C2′ nucleoside position showed the best activity in resistant cancer cells and highly resistant cancer spheroids exposed to cisplatin, increasing cell cycle arrest, apoptosis or necrosis, and reactive oxygen species production. Moreover, it showed high cellular accumulation and did not induce cross-resistance to cisplatin, carboplatin, doxorubicin, paclitaxel, or gemcitabine in long-term cultures. The reference nido-carborane derivative (no metal ions) and unmodified nucleosides were not as effective. These findings indicate that metallacarborane modification of adenosine may sensitize ovarian cancer cells to cisplatin in combination treatment.

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.

Lipid nanocapsules (LNCs) have proven their efficacy in delivering different drugs to various cancers, but no studies have yet described their uptake mechanisms, paclitaxel (PTX) delivery or resulting cytotoxicity towards breast cancer cells. Herein, we report results concerning cellular uptake of LNCs and cytotoxicity studies of PTX-loaded LNCs (LNCs-PTX) on the three breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468. LNCs-PTX of sizes 50 ± 2 nm, 90 ± 3 nm and 120 ± 4 nm were developed by the phase inversion method. Fluorescence microscopy and flow cytometry were used to observe the uptake of fluorescently labeled LNCs and cellular uptake of LNCs-PTX was measured using HPLC analyses of cell samples. These studies revealed a higher uptake of LNCs-PTX in MDA-MB-468 cells than in the other two cell lines. Moreover, free PTX and LNCs-PTX exhibited a similar pattern of toxicity towards each cell line, but MDA-MB-468 cells appeared to be more sensitive than the other two cell lines, as evaluated by the MTT cytotoxicity assay and a cell proliferation assay based upon [3H]thymidine incorporation. Studies with inhibitors of endocytosis indicate that the cellular uptake is mainly via the Cdc42/GRAF-dependent endocytosis as well as by macropinocytosis, whereas dynamin-dependent processes are not required. Furthermore, our results indicate that endocytosis of LNCs-PTX is important for the toxic effect on cells. Western blot analysis revealed that LNCs-PTX induce cytotoxicity by means of apoptosis in all the three cell lines. Altogether, the results demonstrate that LNCs-PTX exploit different mechanisms of endocytosis in a cell-type dependent manner, and subsequently induce apoptotic cell death in the breast cancer cells here studied. The article also describes biodistribution studies following intravenous injection of fluorescently labeled LNCs in mice.

Even though application of nanoparticles in medicine seems to provide unique solutions for drug delivery and diagnosis diseases, understanding interactions between nanoscale materials and biological systems is imperative. Therefore, this study determined the effect of different types of nanoparticles (NPs) on human endothelial cells and examined the types of toxicity responses they can induce. Four different types of NPs were tested (PLA/MMT/TRASTUZUMAB, PLA/EDTMP, PLGA/MDP, and Pluronic F127 MICELLES), representing three putative areas of application: anticancer therapy, scintigraphy, and cosmetology. The experiments were performed on immortalized human umbilical vein endothelial cells (HUVEC-STs). Light contrast phase microscopy as well as cell viability assays showed that only Pluronic F127 MICELLES decreased the number of HUVEC-STs in contrast to PLA/MMT/TRASTUZUMAB, PLA/EDTMP, and PLGA/MDP NPs, which altered cell morphology, but not their confluency. The tested NPs induced not only DNA strand-breaks and alkali-labile sites, but also internucleosomal DNA fragmentation, visualized as a DNA ladder pattern typical of apoptosis. Moreover, generation of free radicals and subsequent mitochondrial membrane potential collapse showed the significance of free radical production during interactions between NPs and endothelial cells. High concentrations of NPs had different degrees of toxicity in human endothelial cells and affected cell proliferation, redox homeostasis, and triggered mitochondrial dysfunction.

Background: In this study, the effect of low concentrations of poly(amido)amine dendrimers (G2-G4) on human lymphocytes was studied. Some works revealed that PAMAMs can adversely affect the morphology of blood components and mitochondria functions. In this context, the present report aimed to investigate the in vitro cationic dendrimers’ effect on mitochondrial respiration and cell morphology in lymphocytes isolated from human blood. Methods: To monitor the mitochondrial changes, the high-resolution respirometer was used, whereas the cell morphology was analyzed using a flow cytometer and fluorescence microscopy. Results: The concentration-dependent dendrimers’ influence on lymphocytes morphology was shown. Changes in mitochondrial respiration revealed the concentration- and generation-dependent differences between dendrimer activity. There were no alterations in the routine respiration and in the state of the inner mitochondrial membrane (L/E), but decreased ADP- and FCCP-stimulated respirations were detected after treatment with G3 and G4 dendrimers. The markers of mitochondrial membrane integrity (RCR) and OXPHOS efficiency (P/E) significantly decreased regardless of the dendrimer generation used. Conclusion: Based on these in vitro evaluations, we state that cationic PAMAM dendrimers can impair both the morphology and the bioenergetics of human lymphocytes, even when used at low concentrations and in a short time (up to 1 h). However, these results do not imply that similar findings could be possible for in vivo observations.

Disturbances in adipose tissue significantly contribute to the development of metabolic disorders, which are connected with hyperglycemia (HG) and underlain by epigenetics-based mechanisms. Therefore, we aimed to evaluate the effect of hyperglycemia on proliferating, differentiating and maturating human visceral pre/adipocytes (HPA-v). Three stages of cell culture were conducted under constant or variable glycemic conditions. Adipogenesis progress was assessed using BODIPY 505/515 staining. Lipid content typical for normal and hyperglycemic conditions of adipocytes was analyzed using Raman spectroscopy and imaging. Expression of adipogenic markers, PPAR&gamma, and C/EBP&alpha, was determined at the mRNA and protein levels. We also examined expression of miRNAs proven to target PPAR&gamma, (miR-34a-5p) and C/EBP&alpha, (miR-137-3p), employing TaqMan Low-Density Arrays (TLDA) cards. Hyperglycemia altered morphology of differentiating HPA-v in relation to normoglycemia by accelerating the formation of lipid droplets and making their numbers and volume increase. Raman results confirmed that the qualitative and quantitative lipid composition under normal and hyperglycemic conditions were different, and that the number of lipid droplets increased in (HG)-treated cells. Expression profiles of both examined genes markedly changed either during adipogenesis under physiological and hyperglycemic conditions, orat particular stages of adipogenesis upon chronic and/or variable glycemia. Expression levels of PPAR&gamma, seemed to correspond to some expression changes of miR-34a-5p. miR-137-3p, whose expression was rather stable throughout the culture, did not seem to affect C/EBP&alpha, Our observations revealed that chronic and intermittent hyperglycemia change the morphology of visceral pre/adipocytes during adipogenesis. Moreover, hyperglycemia may utilize miR-34a-5p to induce some expression changes in PPAR&gamma

Disturbances in adipose tissue significantly contribute to the development of metabolic disorders, which are connected with hyperglycemia (HG) and underlain by epigenetics-based mechanisms. Therefore, we aimed to evaluate the effect of hyperglycemia on proliferating, differentiating and maturating human visceral pre/adipocytes (HPA-v). Three stages of cell culture were conducted under constant or variable glycemic conditions. Adipogenesis progress was assessed using BODIPY 505/515 staining. Lipid content typical for normal and hyperglycemic conditions of adipocytes was analyzed using Raman spectroscopy and imaging. Expression of adipogenic markers, PPARγ and C/EBPα, was determined at the mRNA and protein levels. We also examined expression of miRNAs proven to target PPARγ (miR-34a-5p) and C/EBPα (miR-137-3p), employing TaqMan Low-Density Arrays (TLDA) cards. Hyperglycemia altered morphology of differentiating HPA-v in relation to normoglycemia by accelerating the formation of lipid droplets and making their numbers and volume increase. Raman results confirmed that the qualitative and quantitative lipid composition under normal and hyperglycemic conditions were different, and that the number of lipid droplets increased in (HG)-treated cells. Expression profiles of both examined genes markedly changed either during adipogenesis under physiological and hyperglycemic conditions, orat particular stages of adipogenesis upon chronic and/or variable glycemia. Expression levels of PPARγ seemed to correspond to some expression changes of miR-34a-5p. miR-137-3p, whose expression was rather stable throughout the culture, did not seem to affect C/EBPα. Our observations revealed that chronic and intermittent hyperglycemia change the morphology of visceral pre/adipocytes during adipogenesis. Moreover, hyperglycemia may utilize miR-34a-5p to induce some expression changes in PPARγ.

For optimal exploitation of nanoparticles (NPs) in biomedicine, and to predict nanotoxicity, detailed knowledge of the cellular responses to cell-bound or internalized NPs is imperative. The final outcome of NP-cell interaction is dictated by the type and magnitude of the NP insult and the cellular response. Here, this has been systematically studied by using poly(alkylcyanoacrylate) (PACA) particles differing only in their alkyl side chains; butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), respectively. Surprisingly, these highly similar NPs induced different stress responses and modes of cell death in human cell lines. The POCA particles generally induced endoplasmic reticulum stress and apoptosis. In contrast, PBCA and PEBCA particles induced oxidative stress and lipid peroxidation depending on the level of the glutathione precursor cystine and transcription of the cystine transporter SLC7A11. The latter was induced as a protective response by the transcription factors ATF4 and Nrf2. PBCA particles strongly activated ATF4 downstream of the eIF2α kinase HRI, whereas PEBCA particles more potently induced Nrf2 antioxidant responses. Intriguingly, PBCA particles activated the cell death mechanism ferroptosis; a promising option for targeting multidrug-resistant cancers. Our findings highlight that even minor differences in NP composition can severely impact the cellular response to NPs. This may have important implications in therapeutic settings.

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.

Doxorubicin (DOX) is an effective antineoplastic drug against many solid tumors and hematological malignancies. However, the clinical use of DOX is limited, because of its unspecific mode of action. Since leukemia cells overexpress transferrin (Tf) receptors on their surface, we proposed doxorubicin&ndash, transferrin (DOX&ndash, Tf) conjugate as a new vehicle to increase drug concentration directly in cancer cells. The data obtained after experiments performed on K562 and CCRF-CEM human leukemia cell lines clearly indicate severe cytotoxic and genotoxic properties of the conjugate drug. On the other hand, normal peripheral blood mononuclear cells (PBMCs) were more resistant to DOX&ndash, Tf than to DOX. In comparison to free drug, we observed that Tf-bound DOX induced apoptosis in a TRAIL-dependent manner and caused DNA damage typical of programmed cell death. These fatal hallmarks of cell death were confirmed upon morphological observation of cells incubated with DOX or DOX&ndash, Tf. Studies of expression of TNF-&alpha, IL-4, and IL-6 at the mRNA and protein levels revealed that the pro-inflammatory response plays an important role in the toxicity of the conjugate. Altogether, the results demonstrated here describe a mechanism of the antitumor activity of the DOX&ndash, Tf conjugate.

In the present study we report the interactions of four types of different nanoparticles with normal peripheral blood mononuclear cells. To our research we chose four types of nanoparticles which possess therapeutic properties (Trastuzumab, ethylene-diamine-tetra-methylene-phosphonic for breast and bone cancers treatment, respectively) or can be used as the ingredients of sun-protected films (nanoemulsions with or without chitosan). By carrying out XTT survival assay we observed that both types of tested nanoemulsions suppressed the proliferation of normal lymphocytes. However, the survival of peripheral blood mononuclear cells after incubation neither with Trastuzumab nor with ethylene-diamine-tetra-methylene-phosphonic nanoparticles decreased below 80%. If the investigated nanoparticles were analyzed for their effectiveness to the induction of programmed cell death, we proved that only nanoemulsions with or without chitosan provoked an increase of the fraction of apoptotic cells. Moreover we noticed the characteristic, typical for apoptosis changes of cells morphology, which appeared in lymphocytes after all tested nanoparticles treatment. Interestingly, representative for necrosis swollen, enlarged cells were observed after nanoemulsions treatment.

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).

For optimal exploitation of nanoparticles (NPs) in biomedicine, and to predict nanotoxicity, detailed knowledge on the cellular responses to cell-bound or internalized NPs is imperative. The outcome of NP-cell interaction is dictated by the type and magnitude of the NP insult and the cellular response. Here, we have systematically studied the impact of minor differences in NP composition on cellular stress responses and viability by using highly similar poly(alkylcyanoacrylate) (PACA) particles. Surprisingly, PACA particles differing only in their alkyl side chains; butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), respectively, induced different stress responses and modes of cell death in human cell lines. POCA particles induced endoplasmic reticulum stress and apoptosis. In contrast, PBCA and PEBCA particles induced lipid peroxidation by depletion of the main cellular antioxidant glutathione (GSH), in a manner depending on the levels of the GSH precursor cystine, and transcription of the cystine transporter SLC7A11 regulated by ATF4 and Nrf2. Intriguingly, these particles activated the recently discovered cell death mechanism ferroptosis, which constitutes a promising alternative for targeting multidrug-resistant cancer stem-like cells. Of the two, PBCA was the strongest inducer. In summary, our findings highlight the cellular sensitivity to nanoparticle composition and have important implications for the choice of PACA monomer in therapeutical settings.

Background Doxorubicin (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. Methods After 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). Results We 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. Conclusions Our 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.

Doxorubicin (DOX), one of the oldest member of the anthracycline antibiotics, has been administered for over 50 years to patients with leukemias and solid tumors. However, the high unspecified DOX toxicity, related to reactive oxygen species (ROS), affects its limitation in clinical application. Therefore we proposed the usage of human transferrin as a doxorubicin carrier in order to improve the quality of doxorubicin application in conventional chemotherapy. In this study we continue our investigations related to the mechanism of the toxicity of doxorubicin-transferrin (DOX-TRF) conjugate in human leukemia cells. Consequently, we are now concentrating on the influence of this compound on the antioxidative system in K562 and CCRF-CEM cell lines (chronic erythromyeloblastoid leukemia and acute lymphoblastic leukemia cells, respectively). We carried out a neutral red cytotoxicity assay, reduced (GSH) and total (GSH + GSSG) glutathione content, alterations in the activity of catalase and enzymes responsible for maintaining glutathione in reduced form. Exposure of leukemia cells to the investigated anticancer agents caused a time-dependent depletion of intracellular GSH, accompanied by an increase of catalase activity. Moreover, analysis of GSH-related enzymes showed a significant increase in the activities of thioredoxin reductase and glutathione peroxidase after DOX-TRF application. In contrast, glutathione reductase activity was reduced by conjugate treatment to 50%. Significant differences between the pro-oxidative actions of the investigated anticancer compounds were observed in RT-PCR experiments, which confirmed that changes in the activity of catalase and GSH-related enzymes are strictly correlated with their gene transcription changes.

Although methacrylic acid derivatives in their polymeric form are considered to be safe, insufficient polymerization and the release of monomers due to either mechanical or enzymatical factors can lead to their reaching millimolar concentrations in local tissue. The present study evaluates the effect of two methacrylate monomers - ethylene glycol dimethacrylate (EGDMA) and diethylene glycol dimethacrylate (DEGDMA) - on human gingival fibroblasts (HGFs). Both monomers were found to reduce cells viability in MTT assay, increase apoptosis and cause cell cycle arrest in G1/G0 phase. They also increased intracellular reactive oxygen species (ROS) production as measured by DCFH-DA and DHE probes and increased expression of GPx4 and SOD2. Both monomers increased DNA damage in comet assay. Moreover, HGFs were not able to repair those lesions within 120min of repair incubation. However, the monomers were not found to have any effect on the integrity of isolated plasmids. We postulate that EGDMA and DEGDMA exhibit their cytotoxic and genotoxic properties via increased production of ROS, which cause DNA damage, affect apoptosis, viability and cell cycle. Further studies are needed to better understand the properties of methacrylic acid monomers and to evaluate the risk that they cause for patients, dentists and dental technicians.

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.

Mitoxantrone is an antineoplastic agent approved for clinical use in the secondary progressive phase of multiple sclerosis (MS). Several scientific reports indicate that mitoxantrone acts through the induction of short-term cell lysis at high concentrations and long-term induction of programmed cell death at lower concentrations of antigen-presenting cells. In this paper, we present the potential cytotoxic effects of mitoxantrone on the cells of the immune system, whose activity is associated with their degenerative effects on axonal myelin sheaths. The article also evaluates the results from the hospital treatment of patients diagnosed with MS. The presented data indicate that, apart from the cytostatic properties, mitoxantrone also exhibits side effects of its clinical application. This drug has high cardiotoxicity, and is associated with decreased left ventricular ejection fraction and increased risk of congestive heart failure. Therefore researchers are currently looking for new substances that can reduce the toxic effects of mitoxantrone in healthy tissues, resulting in the generation of reactive oxygen species during its metabolism.

Michelle Alvares Sarcinelli,1,2 Marta de Souza Albernaz,3 Marzena Szwed,4 Alexandre Iscaife,2 Kátia Ramos Moreira Leite,2 Mara de Souza Junqueira,5 Emerson Soares Bernardes,6 Emerson Oliveira da Silva,1 Maria Ines Bruno Tavares,1 Ralph Santos-Oliveira7 1Instituto de Macromoléculas Professora Eloisa Mano Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; 2Laboratory of Medical Investigation, Faculty of Medicine, São Paulo University, São Paulo, Brazil; 3Radiopharmacy Sector, University Hospital Clementino Fraga Filho, Rio de Janeiro, Brazil; 4Department of Thermobiology, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 5Laboratory of Experimental Oncology, Faculty of Medicine, São Paulo University, São Paulo, Brazil; 6Radiopharmacy Center, Instituto de Pesquisas Energéticas e Nucleares (IPEN), São Paulo, Brazil; 7Laboratory of Nanoradiopharmaceuticals, Zona Oeste State University, Rio de Janeiro, Brazil Abstract: 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. Keywords: radiopharmaceuticals, nanotechnology, oncology, breast cancer, molecular imaging, nanomedicine, nuclear pharmacy

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 (H(2)O(2)) 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 (∆Ψ(m)), and intracellular Ca(2+) concentration [Ca(2+)] 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 H(2)O(2)-induced cell death most likely throughout the inhibition of ROS production. Moreover, the drug restored loss of ΔΨ(m) and diminished intracellular [Ca(2+)] evoked by H(2)O(2). 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 H(2)O(2). It appears that the anti-apoptotic effect of the drug is most likely associated with reduction of oxidative stress.

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.

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.

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 cell's 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.

Treatment of chronic diseases in children is a special medical problem. Maintaining constant access to the central vascular system is necessary for long-term hemato-oncological and nephrological therapies as well as parenteral nutrition. Providing such access enables chemotherapic treatment, complete parenteral nutrition, long-term antibiotic therapy, hemodialysis, treatment of intensive care unit patients, monitoring blood pressure in the pulmonary artery and stimulation of heart rate in emergency situations as well as treatment of patients suffering from complications, especially when chances of access into peripheral veins are exhausted. Continuous access to the central vascular system is desirable in the treatment of chronically ill children. Insertion of a central venous catheter line eliminates the unnecessary pain and stress to a child patient accompanying injection into peripheral vessels. In order to gain long-term and secure access to the central venous system, respecting the guidelines of the Center for Disease Control and Prevention contained in the updated ‘Guidelines for the Prevention of Intravascular CatheterRelated Infections’ is necessary (Adv Clin Exp Med 2014, 23, 6, 1001–1009).

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.

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.

Doxorubicin (DOX) is an effective anthracycline antibiotic against a wide spectrum of tumors and hematological malignancies. It mainly interacts with DNA, but can also generate reactive oxygen species (ROS), which damage cell components. Unfortunately, numerous side effects, such as severe cardiotoxicity and bone-marrow suppression, limit its use. To reduce this obstacle and improve its pharmacokinetics, we conjugated DOX to transferrin (TRF), a human plasma protein. In our study, we compared the effect of DOX and the doxorubicin-transferrin conjugate (DOX-TRF) on human leukemic lymphoblasts (CCRF-CEM), and on normal peripheral blood mononuclear cells (PBMC). In parallel, experiments were carried out on two human chronic myeloid leukemia (CML) cell lines derived from K562 cells, of which one was sensitive and the other resistant to doxorubicin (K562/DOX). By use of the alkaline comet assay, the effect of the agents on the induction of DNA damage in normal human cells and human leukemia cells was determined. Oxidative and alkylating DNA damage were assayed by a slightly modified comet assay that included the use of the DNA-repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). To investigate whether DNA breaks are the result of apoptosis, we examined the induction of DNA fragmentation visualized as oligosomal ladders after simple agarose electrophoresis under neutral conditions. Modifications of the genome induced by the different drugs were analyzed following assessment of the cell-cycle phase. The DOX-TRF conjugate caused more DNA damage than the free drug, the degree of DNA fragmentation being dependent on the duration of treatment and the cell type analyzed. With neutral agarose electrophoresis we showed that the test compounds caused the formation of a characteristic DNA-ladder pattern. Furthermore, the DOX-TRF conjugate generated a higher percentage of apoptotic cells in the subG1 fraction and blocked more cells in the G2/M phase of the cell cycle than did free DOX. In summary, both agents induced DNA damage in cancer cells, but the DOX-TRF conjugate generated more genotoxic effects and apoptosis than the unconjugated drug.

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.

The use of nanoparticles is increasing each day. Although only a few nanoparticle drugs are on the market, their research and development are immense. In the field of cancer, the number of new drugs is very large, however, the efficacies of those drugs are very limited. In this study we developed a new radioactive nanoparticle for gastrointestinal stromal tumour imaging and early detection. The results suggest that this nanoparticle may be used for imaging as well as for treatment due the efficacy and inherent quality of nanoparticle systems, especially regarding the higher enhanced permeability and retention effect.

Alendronate sodium is a medicine, which is commonly used in osteoporosis treatment. Nowadays, this substance is given to patients in tablets, but in future it is planned that it will be administrated into human organisms as intravenous infusions; therefore, significant interactions of this medicine with erythrocytes will be inevitable. It is the reason why we decided to investigate the interaction of alendronate sodium with human erythrocytes. The effect of this medicine on acetylcholinesterase activity, lipid and protein peroxidation, as well as cellular thiol content was examined. Moreover, the effect of alendronate sodium on alterations in erythrocytes morphology was assessed. Human erythrocytes were incubated with alendronate sodium in the concentrations ranging from 0.33 to 100 μM for 1 h and 24 h. No changes have been observed in the parameters examined after 1 h of incubation of the erythrocytes with this medicine excluding the carbonyl groups level. Moreover, no alterations in the activity of acetylcholinesterase, the level of thiols as well as in morphology of the erythrocytes incubated with alendronate sodium for 24 h have been observed. It was also proven that alendronate sodium increased the level of reactive oxygen species (ROS) but only after 24 h of incubation. We have not observed any severe changes in cells studied even at the highest dose of bisphosphonates examined, thus their usage should not be dangerous for the erythrocytes of people treated with these medicines.

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.

Epothilones are a new group of compounds with action mechanisms similar to taxanes. The aim of this study was to compare the effects of epothilone A (Epo A) and epothilone B (Epo B) on ovarian cancer cell line SKOV-3 with those of paclitaxel (PTX), a classic taxane. We evaluate glycoprotein P (P-gp) activity in the ovarian cancer cell line. Apoptotic and necrotic cell levels were measured by double staining with Hoechst 33258 and propidium iodide (PI) as well as Annexin V staining. The production of reactive oxygen species (ROS) and changes in mitochondrial membrane potential (ΔΨm) in cells exposed to Epo A, Epo B and PTX were studied using specific fluorescence probes, DCFH(2)-DA (2',7'-dichlorodihydrofluorescein diacetate) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine). The cytotoxic activity of the drugs was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) test. All probes were analyzed in both the presence and absence of the antioxidant N-acetylcysteine (NAC). The results obtained demonstrated that the antiproliferative capacity of Epo A and Epo B in SKOV-3 cell line (measured as IC(50) after 72 h continuous treatment) was six and five times greater than that of PTX's respectively. Epothilones induced timecourse-dependent apoptosis and necrosis. Apoptotic and necrotic events were partially blocked by NAC, indicating ROS played a substantial role in epothilone-induced apoptosis. Cell death was also associated with a decrease in mitochondrial membrane potential, which was more pronounced after treatment with epothilones as compared to paclitaxel.

Cancer cells often accumulate spontaneous and treatment-induced DNA damage i.e. potentially lethal DNA double strand breaks (DSBs). Targeting DSB repair mechanisms with specific inhibitors could potentially sensitize cancer cells to the toxic effect of DSBs. Current treatment for glioblastoma includes tumor resection followed by radiotherapy and/or temozolomide (TMZ) - an alkylating agent inducing DNA damage. We hypothesize that combination of PARP inhibitor (PARPi) with TMZ in glioblastoma cells displaying downregulation of DSB repair genes could trigger synthetic lethality. In our study, we observed that PARP inhibitor (BMN673) was able to specifically sensitize DNA ligase 4 (LIG4)-deprived glioblastoma cells to TMZ while normal astrocytes were not affected. LIG4 downregulation resulting in low effectiveness of DNA-PK-mediated non-homologous end-joining (D-NHEJ), which in combination with BMN673 and TMZ resulted in accumulation of lethal DSBs and specific eradication of glioblastoma cells. Restoration of the LIG4 expression caused loss of sensitivity to BMN673+TMZ. In conclusion, PARP inhibitor combined with DNA damage inducing agents can be utilized in patients with glioblastoma displaying defects in D-NHEJ.