Multimodal doxorubicin loaded magnetic nanoparticles for VEGF targeted theranostics of breast cancer

In presented paper we have developed new system for cancer theranostics based on vascular endothelial growth factor (VEGF) targeted magnetic nanoparticles. Conjugation of anti-VEGF antibodies with bovine serum albumin coated PEGylated magnetic nanoparticles allows for improved binding with murine breast adenocarcinoma 4T1 cell line and facilitates doxorubicin delivery to tumor cells. It was shown that intravenous injection of doxorubicin loaded VEGF targeted nanoparticles increases median survival rate of mice bearing 4T1 tumors up to 50%. On the other hand magnetic resonance imaging (MRI) of 4T1 tumors 24 h after intravenous injection showed accumulation of nanoparticles in tumors, thus allowing simultaneous cancer therapy and diagnostics.     

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H2O2) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H2O2 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H2O2-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H2O2 in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H2O2 and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H2O2 against PC12 cells. Ethanol may enhance the H2O2-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.     

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H₂O₂ across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H₂O₂ release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H₂O₂ release, assessed by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H₂O₂ release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.     

The induction of reactive oxygen species and loss of mitochondrial Omi/HtrA2 is associated with S-nitrosoglutathione-induced apoptosis in human endothelial cells

The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.     

High effectiveness of platinum(IV) complex with adamantylamine in overcoming resistance to cisplatin and suppressing proliferation of ovarian cancer cells in vitro

[(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand - adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC(50) and IC(90) concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC(50) or IC(90)) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G(2)/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC(50) or IC(90) of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied.     

Exposure of human skin to benzo[a]pyrene: Role of CYP1A1 and aryl hydrocarbon receptor in oxidative stress generation

Exposure to benzo[a]pyrene (BaP) can induce inflammatory skin diseases and skin cancer, which are both associated to oxidative stress. BaP is known to bind with high specificity to the aryl hydrocarbon receptor (AhR), modifying the expression of CYP1A1, involved both in cancer and inflammation.     

Multimodal imaging for a theranostic approach in a murine model of B-cell lymphoma with engineered nanoparticles

Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.     

Identification and functional analysis of genes which confer resistance to cisplatin in tumor cells

The efficacy of cisplatin during cancer chemotherapy is often impaired by the emergence of cancer cells which become resistant to chemotherapeutic agents. While various mechanisms have been proposed to explain resistance to cisplatin, the genes involved in this process still remain unclear. By using DNA microarrays, we performed a genome-wide analysis of cisplatin-resistant HeLa cells in order to identify genes involved in resistance. We identified nine genes (NAPA, CITED2, CABIN1, ADM, HIST1H1A, EHD1, MARK2, PTPN21, and MVD), which were consistently upregulated in two cisplatin-resistant HeLa cell lines. The upregulated genes, here referred to as cisplatin resistance genes (CPR), were further analyzed for their ability to modify the response of HEK293 cells to cisplatin. Short-hairpin RNA (shRNA) knockdown of CPR genes, individually or in combination, was shown to sensitize HEK293 cells to cisplatin, but not to vincristine or taxol, suggesting that CPR genes may be involved specifically in cisplatin resistance. Among the treatments performed, shRNA knockdown of NAPA was the most efficient treatment able to sensitize cells to cisplatin. Furthermore, shRNA knockdown of a single CPR gene was sufficient to partially reverse acquired cisplatin resistance in HeLa cells. Sensitization to cisplatin following knockdown of CPR genes was also observed in the tumorigenic cell lines Sk-ov-3, H1155, and CG-1. Based on these results, we propose that the CPR genes identified here may represent potential candidates for novel target therapies aimed at preventing resistance to cisplatin during chemotherapy.     

Protective effect of pine (Pinus morrisonicola Hay.) needle on LDL oxidation and its anti-inflammatory action by modulation of iNOS and COX-2 expression in LPS-stimulated RAW 264.7 macrophages.

The protective effects of pine (Pinus morrisonicola Hay.) needle on low-density lipoprotein (LDL) oxidation and nitric oxide production in macrophages as well as its bioactive compounds were investigated. Of the four solvent extracts, the ethyl acetate extract of pine needle (EAE-PN) exhibited the strongest scavenging action on free radicals. EAE-PN significantly inhibited copper-induced LDL oxidation through prolonging the lag phase of conjugated dienes formation and decreasing the relative electrophoretic mobility of LDL. Lipid accumulation and foam cell formation were significantly reduced when EAE-PN (75 microg/mL) was added to the medium co-incubated with macrophages cells and copper-induced LDL. EAE-PN also markedly inhibited reactive oxygen species production in RAW 264.7 cells stimulated with lipopolysaccharide (LPS). As regards NO production in cells, EAE-PN showed dose-dependent inhibitory effect on nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells. The inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions in LPS-stimulated RAW 264.7 cells were inhibited by EAE-PN. RT-PCR analysis indicated that the iNOS and COX-2 mRNA expression were suppressed by EAE-PN. The major phenolic compounds in EAE-PN were epicatechin and p-coumaric acid by HPLC analysis. The presence of epicatechin and p-coumaric acid in EAE-PN may be partially responsible for the biological action of EAE-PN. Taken together, these results suggest that EAE-PN may provide potential protective effects against LDL oxidation and attenuating excessive NO generation at inflammatory sites; consequently, this may contribute to anti-atherosclerotic and anti-inflammatory effects of EAE-PN.     

Development and applications of photo-triggered theranostic agents

Theranostics, the fusion of therapy and diagnostics for optimizing efficacy and safety of therapeutic regimes, is a growing field that is paving the way towards the goal of personalized medicine for the benefit of patients. The use of light as a remote-activation mechanism for drug delivery has received increased attention due to its advantages in highly specific spatial and temporal control of compound release. Photo-triggered theranostic constructs could facilitate an entirely new category of clinical solutions which permit early recognition of the disease by enhancing contrast in various imaging modalities followed by the tailored guidance of therapy. Finally, such theranostic agents could aid imaging modalities in monitoring response to therapy. This article reviews recent developments in the use of light-triggered theranostic agents for simultaneous imaging and photoactivation of therapeutic agents. Specifically, we discuss recent developments in the use of theranostic agents for photodynamic-, photothermal- or photo-triggered chemotherapy for several diseases.     

Investigating oxidative stress and inflammatory responses elicited by silver nanoparticles using high-throughput reporter genes in HepG2 cells: Effect of size,surface coating,and intracellular uptake

Silver nanoparticles (Ag NP) have been shown to generate reactive oxygen species; however, the association between physicochemical characteristics of nanoparticles and cellular stress responses elicited by exposure has not been elucidated. Here, we examined three key stress-responsive pathways activated by Nrf-2/ARE, NFκB, and AP1 during exposure to Ag NP of two distinct sizes (10 and 75 nm) and coatings (citrate and polyvinylpyrrolidone), as well as silver nitrate (AgNO₃), and CeO₂ nanoparticles. The in vitro assays assessed the cellular response in a battery of stable luciferase-reporter HepG2 cell lines. We further assessed the impact of Ag NP and AgNO₃ exposure on cellular redox status by measuring glutathione depletion. Lastly, we determined intracellular Ag concentration by inductively coupled plasma mass spectroscopy (ICP-MS) and re-analyzed reporter-gene data using these values to estimate the relative potencies of the Ag NPs and AgNO₃. Our results show activation of all three stress response pathways, with Nrf-2/ARE displaying the strongest response elicited by each Ag NP and AgNO₃ evaluated here. The smaller (10-nm) Ag NPs were more potent than the larger (75-nm) Ag NPs in each stress-response pathway, and citrate-coated Ag NPs had higher intracellular silver concentrations compared with both PVP-coated Ag NP and AgNO₃. The cellular stress response profiles after Ag NP exposure were similar to that of AgNO₃, suggesting that the oxidative stress and inflammatory effects of Ag NP are likely due to the cytotoxicity of silver ions.