Effects of chemotherapeutic drugs on the antioxidant capacity of human erythroleukemia cells with MDR phenotype.

In this work, we identified that different chemotherapeutic drugs may select cells with different antioxidant capacities. For this, we evaluated the sensitivity of two multidrug-resistant (MDR) erythroleukemia cell lines: Lucena (resistant to vincristine, VCR) and FEPS (resistant to daunorubicin, DNR) derived from the same sensitive cell K562 (non-MDR) to hydrogen peroxide. In addition, we evaluated how the cell lines respond to the oxidizing agent in the absence of VCR/DNR. In absence of VCR, Lucena drastically decreases cell viability when exposed to hydrogen peroxide, while FEPS is not affected even without DNR. To analyze whether selection by different chemotherapeutic agents may generate altered energetic demands, we analyzed the production of reactive oxygen species (ROS) and the relative expression of the glucose transporter 1 gene (glut1). We observed that the selection through DNR apparently generates a higher energy demand than VCR. High levels of transcription factors genes expression (nrf2, hif-1α, and oct4) were kept even when the DNR is withdrawn from the FEPS culture for one month. Together, these results indicate that DNR selects cells with greater ability to express the major transcription factors related to the antioxidant defense system and the main extrusion pump (ABCB1) related to the MDR phenotype. Taking into account that the antioxidant capacity of tumor cells is closely related to resistance to multiple drugs, it is evident that endogenous antioxidant molecules may be targets for the development of new anticancer drugs.

Anti-MDR Effects of Quercetin and its Nanoemulsion in Multidrug-Resistant Human Leukemia Cells.

BACKGROUND: Quercetin has potential against the Multidrug Resistance (MDR) phenotype, but with low bioavailability. The increase in the bioavailability can be obtained with nanostructures. OBJECTIVE: To analyze the effects of quercetin and its nanoemulsion on MDR and non-MDR cells. METHODS: We used high-pressure homogenization for nanoemulsion production; Trypan Blue for cytostatic/cytotoxicity assays; Epifluorescence microscope (with specific probes) for apoptosis and DNA damage; Real-Time PCR for gene expression; AutoDock Vina for docking and Flow Cytometry for efflux analysis. Quercetin exerted antiproliferative impact, induced apoptosis, necrosis and DNA damage on cells. RESULTS: Quercetin combined with vincristine showed an effect similar to verapamil (an ABCB1 inhibitor), and docking showed that it binds to ABCB1 in a similar region. Quercetin was also capable of altering ABCB1 gene expression. Quercetin in nanoemulsion maintained the cytotoxic and cytostatic effects of quercetin, which may increase bioavailability. Besides, the unloaded nanoemulsion was able to inhibit per se the efflux activity of ABCB1, demonstrating pharmacological action of this structure. CONCLUSION: Quercetin may be considered as a prospective drug to overcome resistance in cancer cells and its nanoemulsion can be an alternative for in vivo application.

Interaction Between Near-Infrared Radiation and Temozolomide in a Glioblastoma Multiform Cell Line: A Treatment Strategy?

Photodynamic therapy (PDT) is a potential therapeutic modality against cancer, resulting from the interaction of a photosensitizer (PS) and radiation that generates damage to tumor cells. The use of near-infrared radiation (IR-A) is relevant because presents recognized biological effects, such as antioxidant, neuroprotective and antitumor effects. Glioblastoma is the most aggressive central nervous system (CNS) neoplasm with high proliferation and tissue invasion capacity and is resistant to radio and chemotherapy. Here, we evaluated in vitro the possible interaction of temozolomide (TMZ) with IR-A in a glioblastoma cell line (C6) and in a human keratinocyte cell line (HaCat) how non-tumor cell model, in an attempt to search for a new treatment strategy. The effects of TMZ, IR-A and the interaction between TMZ and IR-A was evaluated by viability exclusion with trypan blue. To perform the interaction experiments, we have chosen 10 µM TMZ and 4.5 J/cm2 of IR-A. From this, we evaluated cytotoxicity, cell proliferation, intracellular reactive oxygen species levels (ROS), as well as the process of cell migration and the P-gp and MRP-1 activity. Cell death mainly due to apoptosis, followed by necrosis, decreased cell proliferation, increased ROS levels, decreased cell migration and decreased P-gp and MRP1 activity were observed only when there was interaction between TMZ and IR-A in the C6 cell line. The interaction between TMZ and IR-A was not able to affect cell proliferation in the HaCat non-tumor cell line. Our results suggest that this interaction could be a promising approach and that in the future may serve as an antitumor strategy for PDT application.

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations.

BACKGROUND: PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype. OBJECTIVE: In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR). MATERIALS AND METHODS: In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR). RESULTS: Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments. CONCLUSION: Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

Silencing the OCT4-PG1 pseudogene reduces OCT-4 protein levels and changes characteristics of the multidrug resistance phenotype in chronic myeloid leukemia.

Cancer stem cells show epigenetic plasticity and intrinsic resistance to anti-cancer therapy, rendering capable of initiating cancer relapse and progression. Transcription factor OCT-4 regulates various pathways in stem cells, but its expression can be regulated by pseudogenes. This work evaluated how OCT4-PG1 pseudogene can affect OCT-4 expression and mechanisms related to the multidrug resistance (MDR) phenotype in FEPS cells. Considering that OCT-4 protein is a transcription factor that regulates expression of ABC transporters, level of gene expression, activity of ABC proteins and cell sensitivity to chemotherapy were evaluated after OCT4-PG1 silencing. Besides we set up a STRING network. Results showed that after OCT4-PG1 silencing, cells expressed OCT-4 gene and protein to a lesser extent than mock cells. The gene and protein expression of ABCB1, as well as its activity were reduced. On the other hand, ALOX5 and ABCC1 genes was increased even as the activity of this transporter. Moreover, the silencing cells become sensitive to two chemotherapics tested. The network structure demonstrated that OCT4-PG1 protein interacts directly with OCT-4, SOX2, and NANOG and indirectly with ABC transporters. We conclude that OCT4-PG1 pseudogene plays a key role in the regulation OCT-4 transcription factor, which alters MDR phenotype in the FEPS cell line.

Multidrug resistance phenotype: Relation between phenotype induction and its characteristics in erythroleukemia cells.

Chemotherapy may be followed by multiple drug resistance (MDR). This is an obstacle in the treatment of cancer. It is therefore essential to understand the mechanisms underlying tumor resistance, especially those involved in the cell target/MDR relationship. To investigate this, the effects of exposing cells to UVB (to target DNA), UVA, and H2 O2 (to target the cell membrane) were observed in K562 (non MDR) and FEPS (MDR) cell lines. The K562 cells were more sensitive to UVA than the FEPS cells. The FEPS cell line was more resistant to H2 O2 than K562, only presenting cytotoxicity 72 h after being exposed to 40 mM, with no ROS increase until 48 h. Both cell lines were sensitive to UVB, presenting cytotoxicity after 24 h, mainly by apoptosis, and showed an increase in ROS levels. Our results indicate that agents acting on DNA may be able to overcome the MDR phenotype.

Combination of ω-3 fatty acids and cisplatin as a potential alternative strategy for personalized therapy of metastatic melanoma: an in-vitro study.

The recently developed therapeutic strategies have led to unprecedented improvements in the control of metastatic melanoma and in the survival of specific subgroups of patients. However, drug resistance, low response rates, and undesired side effects make these treatments not suitable or tolerable for all the patients, and chemotherapeutic treatments appear still indispensable, at least for subgroups of patients. New combinatory strategies are also under investigation as tailored treatments or salvage therapies, including combined treatments of immunotherapy with conventional chemotherapy. On this basis, and in consideration of the antineoplastic properties of ω-3 polyunsaturated fatty acids, we have here investigated the potential of these bioactive dietary factors to revert the resistance frequently exhibited by this form of cancer to cisplatin (CDDP, cis-diamminedichloroplatinum). We demonstrated that docosahexenoic acid (DHA, 22:6ω-3) sensitizes the cells to the CDDP-induced inhibition of cell growth and migration by reverting CDDP effects on DNA damage and ERCC1 expression, as well as on the DUSP6 and p-ERK expressions, which regulate ERCC1 activation upwardly. In line, DUSP6 gene silencing prevented the effect of DHA, confirming that DHA acted on the DUSP6/p-ERK/ERCC1 repair pathways to sensitize melanoma cells to the anticancer effect of CDDP. Similar effects were obtained also with eicosapentaenoic acid (20:5ω-3). Overall, our findings suggest that the combination of CDDP treatment with a dietary supplementation with ω-3 polyunsaturated fatty acids could potentially represent a new therapeutic strategy for overcoming CDDP resistance in metastatic melanoma.

Cytoprotection of lipoic acid against toxicity induced by saxitoxin in hippocampal cell line HT-22 through in silico modeling and in vitro assays.

Saxitoxins (STXs) are potent neurotoxins that block voltage-gated channels in neurons and induce cytotoxicity. These toxins not only can generate reactive oxygen species but also can alter antioxidant levels, promoting oxidative stress. Under this pro-oxidant situation, the use of the antioxidant lipoic acid (LA) can represent a chemoprotective alternative to minimize the deleterious effects induced by neurotoxins as STXs. P-glycoprotein (P-gp) is a well-known ATP-binding cassette (ABC) transporter that plays a crucial role in the extrusion of toxic substances, decreasing their accumulation and potential intracellular effects in virtue of its broad substrate specificity, its expression in many excretory tissues and its large efflux capacity. The interaction of STXs with LA was evaluated by ab initio simulation, molecular docking and bioassays using the cell line HT-22. The interaction of STXs with LA occurs by physisorption. Molecular docking indicated that STXs can be a substrate of P-gp and, estimating the Free Energy of Binding (FEB), LA has lower amino acids residues binding sites, similar to verapamil, while STX and STX+LA_1 have similar amino acids residues and binding sites with similar FEB between this ligands.Cells were exposed to STXs and LA for 30min and 24h. LA treatment minimizes STX cytotoxicity, evaluated by trypan blue and MTT assay and both STX and STX-LA treatments were efficient to induce P-gp activity measured by rhodamine 123 dye extrusion. LA and STX+LA treatments induced low reactive oxygen species levels and low oxygen consumption. Based on our results, it can be concluded that LA was able to induce cytoprotection, including induction of cellular glutathione levels, and that STX+LA interaction reduced toxicity effects induced by STX. Overall, the in vitro results corroborated the semi-empirical evidences found using density functional theory ab initio simulation and molecular docking.

Protective effect of infrared-A radiation against damage induced by UVB radiation in the melan-a cell line.

The present work evaluated the infrared-A (IR-A) protective effect using a light-emitting diode (LED) lamp against the cytotoxic effects of ultraviolet B radiation (UVB). Effects on cell viability (Trypan blue assay), DNA damage (comet assay), lipid peroxidation (FOX method), reactive oxygen species production and antioxidant capacity were analyzed in melan-a, a non-tumoral murine melanocytic cell line. To define the doses used in the interaction experiments between IR-A+UVB, dose/response curves were made after exposure to IR-A or UVB. The IR-A dose chosen was 0.8J/cm(2) because this dose caused no significant inhibition of proliferation effects and viability decreased. For UVB exposure, a dose of 0.015J/cm(2), which showed a decrease in viable cell number by approximately 50% in relation to control until 72h, was selected. For IR-A+UVB, cell proliferation recovery was showed, decreasing DNA damage and lipid peroxide content when compared to UVB alone. Besides, the results obtained for ROS and antioxidant capacity showed that the protection observed was probably not related to decreased oxidative stress. In conclusion, non-thermal IR-A was capable of protecting the melan-a cells from UVB induced damage.

Infrared radiation influence on molt and regeneration of Neohelice granulata Dana, 1851 (Grapsidae, Sesarminae).

This paper analyzes the influence of infrared radiation (IR) on regeneration, after autotomy of limb buds of Neohelice granulata and consequently the time molt. Eyestalks were ablated to synchronize the start of molt. Afterward, animals were autotomized of five pereopods and divided into control and irradiated groups. The irradiated group was treated for 30 min daily until molt. Limb buds from five animals of days 4, 16 and 20 were collected and histological sections were made from them. These sections were photographed and chitin and epithelium content measured. Another group was made, and after 15 days limb buds were extracted to analyze mitochondrial enzymatic activity from complex I and II. The irradiated group showed a significant reduction in molt time (19.38+/-1.22 days) compared with the control group (32.69+/-1.57 days) and also a significant increase in mitochondrial complex I (388.9+/-27.94%) and II (175.63+/-7.66%) in the irradiated group when compared with the control group (100+/-17.90; 100+/-7.82, respectively). However, these effects were not accompanied by histological alterations in relation to chitin and epithelium. This way, it was possible to demonstrate that IR increases complex I and II activity, reduces the time molt and consequently increases the appendage regeneration rate.

Photodynamic action of benzo[a]pyrene in K562 cells.

Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo-oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA-protein cross-link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA.

Sensitivity to microcystins: a comparative study in human cell lines with and without multidrug resistance phenotype.

Multidrug resistance (MDR) is an obstacle in cancer treatment. An understanding of how tumoral cells react to oxidants can help us elucidate the cellular mechanism involved in resistance. Microcystins are cyanobacteria hepatotoxins known to generate oxidative stress. The aim of this study was to compare the sensitivity to microcystins of human tumoral cell lines with (Lucena) and without (K562) MDR phenotype. Endpoints analyzed were effective microcystins concentration to 50% of exposed cells (EC50), antioxidant enzyme activity, lipid peroxidation, DNA damage, reactive oxygen species (ROS) concentration, and tubulin content. Lucena were more resistant and showed lower DNA damage than K562 cells (P<0.05). Although microcystins did not alter catalase activity, a higher mean value was observed in Lucena than in K562 cells. Lucena cells also showed lower ROS concentration and higher tubulin content. The higher metabolism associated with the MDR phenotype should increase ROS concentration and make for an improved antioxidant defense against the toxic effects of microcystins.