Tolerance to drug-induced cell death favours the acquisition of multidrug resistance in Leishmania

The control of the protozoan parasite Leishmania relies on few drugs with unknown cellular targets and unclear mode of action. Several antileishmanials, however, were shown to induce apoptosis in Leishmania and this death mechanism was further studied in drug-sensitive and drug-resistant Leishmania infantum. In sensitive parasites, antimonials (SbIII), miltefosine (MF) and amphotericin B (AMB), but not paromomycin (PARO), triggered apoptotic cell death associated with reactive oxygen species (ROS). In contrast, Leishmania mutants resistant to SbIII, MF or AMB not only failed to undergo apoptosis following exposure to their respective drugs, but also were more tolerant towards apoptosis induced by other antileishmanials, provided that these killed Leishmania via ROS production. Such tolerance favored the rapid acquisition of multidrug resistance. PARO killed Leishmania in a non-apoptotic manner and failed to produce ROS. PARO resistance neither protected against drug-induced apoptosis nor provided an increased rate of acquisition of resistance to other antileishmanials. However, the PARO-resistant mutant, but not SbIII-, MF- or AMB-resistant mutants, became rapidly cross-resistant to methotrexate, a model drug also not producing ROS. Our results therefore link the mode of killing of drugs to tolerance to cell death and to a facilitated emergence of multidrug resistance. These findings may have fundamental implications in the field of chemotherapeutic interventions.     

Antimicrobial activity of metal based nanoparticles against microbes associated with diseases in aquaculture

The emergence of diseases and mortalities in aquaculture and development of antibiotics resistance in aquatic microbes, has renewed a great interest towards alternative methods of prevention and control of diseases. Nanoparticles have enormous potential in controlling human and animal pathogens and have scope of application in aquaculture. The present investigation was carried out to find out suitable nanoparticles having antimicrobial effect against aquatic microbes. Different commercial as well as laboratory synthesized metal and metal oxide nanoparticles were screened for their antimicrobial activities against a wide range of bacterial and fungal agents including certain freshwater cyanobacteria. Among different nanoparticles, synthesized copper oxide (CuO), zinc oxide (ZnO), silver (Ag) and silver doped titanium dioxide (Ag–TiO₂) showed broad spectrum antibacterial activity. On the contrary, nanoparticles like Zn and ZnO showed antifungal activity against fungi like Penicillium and Mucor species. Since CuO, ZnO and Ag nanoparticles showed higher antimicrobial activity, they may be explored for aquaculture use.     

Development of a multifunctional luciferase reporters system for assessing endoplasmic reticulum-targeting photosensitive compounds

Photodynamic therapy (PDT) is a recently developed antitumor modality utilizing the generation of reactive oxygen species (ROS), through light irradiation of photosensitizers (PSs) localized in tumor. Interference with proper functioning of endoplasmic reticulum (ER) by ER-targeting PDT is a newly proposed strategy to achieve tumor cell death. The aim of this study is to establish a multifunctional model to screen and assess ER-targeting PSs based on luciferase reporters system. Upregulation of GRP78 is a biomarker for the onset of ER stress. CHOP is a key initiating player in ER stress-induced cell death. Here, the most sensitive fragments of GRP78 and CHOP promoters responding to ER-targeting PDT were mapped and cloned into pGL3-basic vector, forming −702/GRP78-Luc and −443/CHOP-Luc construct, respectively. We demonstrated that −702/GRP78-Luc expression can be used to indicate the ER-targeting of PSs, meanwhile estimate the ROS level induced by low-dose ER-targeting PDT. Moreover, the luciferase signaling of −443/CHOP-Luc showed highly consistence with apoptosis rate caused by ER-targeting PDT, suggesting that −443/CHOP-Luc can evaluate the antitumor properties of PSs. Hypericin, Foscan® and methylene blue were applied to verify the sensitivity and reliability of our model. These results proved that GRP78-CHOP model may be suitable to screen ER-targeting photosensitive compounds with lower cost and higher sensitivity than traditional ways.     

Toxicity of commercially available engineered nanoparticles to Caco-2 and SW480 human intestinal epithelial cells

The effects of ingestion of engineered nanoparticles (NPs), especially via drinking water, are unknown. Using NPs spiked into synthetic water and cell culture media, we investigated cell death, oxidative stress, and inflammatory effects of silver (Ag), titanium dioxide (TiO₂), and zinc oxide (ZnO) NPs on human intestinal Caco-2 and SW480 cells. ZnO NPs were cytotoxic to both cell lines, while Ag and TiO₂ NPs were toxic only at 100 mg/L to Caco-2 and SW480, respectively. ZnO NPs led to significant cell death in synthetic freshwaters with 1 % phosphate-buffered saline in both cell lines, while Ag and TiO₂ NPs in buffered water led to cell death in SW480 cells. NP exposures did not yield significant increased reactive oxygen species generation but all NP exposures led to increased IL-8 cytokine generation in both cell lines. These results indicate cell stress and cell death from NP exposures, with a varied response based on NP composition.     

Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases

Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT.     

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis.     

Synthesis and biological evaluation of glucose conjugated phthalocyanine as a second-generation photosensitizer

Photodynamic therapy (PDT) is a treatment method using light and photosensitizers (PSs), which is categorized as a non-invasive surgery treatment for cancers. When the tumor is exposed to a specific light, the PSs become active and generate reactive oxygen species (ROS), mainly singlet oxygen which kills nearby cancer cells. PDT is becoming more widely recognized as a valuable treatment option for localized cancers and pre-cancers of skin as it has no long-term effects on the patient. But, due to the limited penetration rate of light into the skin and other organs, PDT can’t be used to treat large cancer cells or cancer cells that have grown deeply into the skin or other organs. Hence, in this study, our focus centers on synthesizing glucose-conjugated phthalocyanine (Pc) compatible with near-infrared (NIR) irradiation as second-generation photosensitizer, so that PDT can be used in a wider range to treat cancers without obstacles.     

Mitochondrial dysfunction mediates neuronal cell response to DMMB photodynamic therapy

Photodynamic therapy (PDT) is a process in which a photosensitizer (PS) is exposed to specific wavelengths and generates reactive oxygen species (ROS) which act within nanometers. The low invasive nature and directed cytotoxicity of this approach render it attractive to the treatment of different conditions, including the ones that affect the central nervous system (CNS). The effect of PDT on healthy neurons is one main concern over its use in the CNS, since neuronal-like cells were shown to be particularly sensitive to certain PSs. Among available PSs, 1,9-dimethyl-methylene blue (DMMB) stands out as being resistant to reduction to its inactive leuco form and by being able to produce high levels of singlet?oxygen. In this study, we aimed to investigate DMMB photodamage mechanisms in the hippocampal cell line HT22. Our results demonstrate that DMMB-PDT decrease in cell viability was linked with an increase in cell death and overall ROS production. Besides, it resulted in a significant increase in mitochondrial ROS production and decreased mitochondria membrane potential. Furthermore, DMMB-PDT significantly increased the presence of acidic autolysosomes, which was accompanied by an increase in ATG1 and ATG8 homologue GaBarap1 expression, and decreased DRAM1 expression. Taken together our results indicated that mitochondrial and autophagic dysfunction underlie DMMB-PDT cytotoxicity in neuronal cells.     

Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung

Zinc oxide (ZnO) nanoparticles are one of the important industrial nanoparticles. The production of ZnO nanoparticles is increasing every year. On the other hand, it is known that ZnO nanoparticles have strong cytotoxicity. In vitro studies using culture cells revealed that ZnO nanoparticles induce severe oxidative stress. However, the in vivo influence of ZnO nanoparticles is still unclear. In the present study, rat lung was exposed to ZnO nanoparticles by intratracheal instillation, and the influences of ZnO nanoparticles to the lung in the acute phase, particularly oxidative stress, were examined. Additionally, in vitro cellular influences of ZnO nanoparticles were examined using lung carcinoma A549 cells and compared to in vivo examinations. The ZnO nanoparticles used in this study released zinc ion in both dispersions. In the in vivo examinations, ZnO dispersion induced strong oxidative stress in the lung in the acute phase. The oxidative stress induced by the ZnO nanoparticles was stronger than that of a ZnCl(2) solution. Intratracheal instillation of ZnO nanoparticles induced an increase of lipid peroxide, HO-1 and alpha-tocopherol in the lung. The ZnO nanoparticles also induced strong oxidative stress and cell death in culture cells. Intracellular zinc level and reactive oxygen species were increased. These results suggest that ZnO nanoparticles induce oxidative stress in the lung in the acute phase. Intracellular ROS level had a high correlation with intracellular Zn(2+) level. ZnO nanoparticles will stay in the lung and continually release zinc ion, and thus stronger oxidative stress is induced.     

Mitochondrial reactive oxygen species and nitric oxide-mediated cancer cell apoptosis in 2-butylamino-2-demethoxyhypocrellin B photodynamic treatment

Photodynamic therapy (PDT) is a novel and promising cancer treatment which employs a combination of a photosensitizing chemical and visible light to induce apoptosis in cancer cells. Singlet oxygen has been recognized as the main origin of oxidative stress in PDT. However, the precise mechanism of PDT-induced apoptosis is not well characterized, especially the dualistic role of nitric oxide (NO). To dissect the apoptosis pathways triggered by PDT, the intracellular free radicals in MCF-7 cells were investigated by examining a novel photosensitizer 2-butylamino-2-demethoxyhypocrellin B (2-BA-2-DMHB)-mediated PDT. It was found that exposure of the cells to 2-BA-2-DMHB and irradiation resulted in a significant increase of intracellular ROS in minutes, and then followed by cytoplasmic free calcium enhancement, mitochondrial nitric oxide synthase (mtNOS) activation, cytochrome c release, and apoptotic death. Scavengers of singlet oxygen or NO could attenuate PDT-induced cell viability loss, nucleus morphology changes, cytochrome c release, mitochondria swelling, and apo-apoptosis gene p53 and p21 mRNA levels. The results suggested that both ROS and NO played important roles in the apoptosis-induced by PDT.     

Selective Sensitization of Zinc Finger Protein Oxidation by Reactive Oxygen Species through Arsenic Binding

Cysteine oxidation induced by reactive oxygen species (ROS) on redox-sensitive targets such as zinc finger proteins plays a critical role in redox signaling and subsequent biological outcomes. We found that arsenic exposure led to oxidation of certain zinc finger proteins based on arsenic interaction with zinc finger motifs. Analysis of zinc finger proteins isolated from arsenic-exposed cells and zinc finger peptides by mass spectrometry demonstrated preferential oxidation of C3H1 and C4 zinc finger configurations. C2H2 zinc finger proteins that do not bind arsenic were not oxidized by arsenic-generated ROS in the cellular environment. The findings suggest that selectivity in arsenic binding to zinc fingers with three or more cysteines defines the target proteins for oxidation by ROS. This represents a novel mechanism of selective protein oxidation and demonstrates how an environmental factor may sensitize certain target proteins for oxidation, thus altering the oxidation profile and redox regulation.     

Subcellular localization of Photofrin determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: when plasma membranes are the main targets

Photodynamic therapy (PDT) is a kind of photochemo-therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane-localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 micro g/ml, and at higher dose (28 micro g/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin-PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c-Jun N-terminal kinase, (iii) delayed activation of caspase-3 and cleavage of polyADP-ribose polymerase and p21-activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin-PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.     

Expression of oxidored nitro domain-containing protein 1(NOR1) impairs nasopharyngeal carcinoma cells adaptation to hypoxia and inhibits PDK1 expression

Drug repositioning has been considered a promising approach to discover novel treatments against neglected diseases. Among the major protozoan diseases, leishmaniasis remains a public health threat with few therapeutic alternatives, affecting 12 million people in 98 countries. In this study, we report the in vitro antileishmanial activity of the imidazole drugs clotrimazole, and for the first time in literature, econazole and bifonazole and their potential action to affect the regulation of reactive oxygen species (ROS) of the parasites. The lethal action of the imidazoles was investigated using spectrofluorimetric techniques to detect ROS content, plasma membrane permeability, and mitochondrial membrane potential. The imidazoles showed activity against L. (L.) infantum chagasi promastigotes with IC₅₀ values in a range of 2–8 μM; econazole was also effective against Leishmania intracellular amastigotes, with an IC₅₀ value of 11 μM, a similar in vitro effectiveness to miltefosine. Leishmania promastigotes rapidly up-regulated the ROS release after incubation with the imidazoles, but econazole showed a marked increase in ROS content of approximately 1,900 % higher than untreated parasites. When using SYTOX^® Green as a fluorescent probe, the imidazoles demonstrated considerable interference in plasma membrane permeability at the early time of incubation; econazole resulted in the higher influx of SYTOX^® Green at 60 min. Despite cellular alterations, no depolarization could be observed to the mitochondrial membrane potential of Leishmania until 60 min. The lethal action of econazole involved strong permeabilization of plasma membrane of promastigotes, with an overloaded ROS content that contributed to the death of parasites. Affecting the ROS regulation of Leishmania via small molecules would be an interesting strategy for new drugs.     

Hydrophilic Acylated Surface Protein A (HASPA) of <Emphasis Type="Italic">Leishmania donovani</Emphasis>: Expression,Purification and Biophysico-Chemical Characterization

Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a “non-classical” pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils. Studies in mouse macrophage RAW264.7 cell lines indicate that r-LdHASPA enhances reactive oxygen species (ROS) production. Co-immunoprecipitation (IP) studies indicate that r-LdHASPA interacts with certain macrophage proteins which however could not be identified unambiguously. The present study provides key insights into the structural and functional aspects of an important Leishmania protein, HASPA, which we believe could be useful for further research on vaccine/drug development.     

ω-Hydroxyundec-9-enoic acid induces apoptosis through ROS-mediated endoplasmic reticulum stress in non-small cell lung cancer cells

ω-Hydroxyundec-9-enoic acid (ω-HUA), a hydroxyl unsaturated fatty acid derivative, is involved in the antifungal activity of wild rice (Oryza officinalis). Here, we investigated the anti-cancer activity of ω-HUA on a non-small cell lung cancer (NSCLC) cell line. ω-HUA increased apoptosis and induced cleavages of caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP). ω-HUA treatment significantly induced endoplasmic reticulum (ER) stress response. Suppression of CHOP expression and inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly attenuated the ω-HUA treatment-induced activation of caspase-6, caspase-9, and PARP, and subsequent apoptotic cell death, indicating a role for ER stress in ω-HUA-induced apoptosis. In addition, cells subjected to ω-HUA exhibited significantly increased quantity of reactive oxygen species (ROS), and the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited ω-HUA-induced apoptotic cell death and ER stress signals, indicating a role for ROS in ER stress-mediated apoptosis in ω-HUA-treated cells. Taken together, these results suggest that sequential ROS generation and ER stress activation are critical in ω-HUA treatment-induced apoptosis and that ω-HUA represents a promising candidate for NSCLC treatment.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Hypericin‐PDT‐induced rapid necrotic death in human squamous cell carcinoma cultures after multiple treatment

PDT (photodynamic therapy) has been used for the treatment of NMCC (non‐melanoma cutaneous cancer) particularly, human SCC (squamous cell carcinoma). However, the nature of the photosensitizer, the activation light source and the mode of cell death induced post‐PDT remains elusive. We tried to optimize PDT using the light‐activated (320–400 nm) St John's Wort‐derived compound, Hyp (hypericin). The work highlights the potential mode of cell death and the increased efficacy of the technique associated with multiple Hyp‐PDT treatment. SCC cells were exposed to different concentrations of Hyp and activated with light at 1 J/cm² for 1 or 2 days. Thereafter with the optimum dose of Hyp proliferation, ROS (reactive oxygen species), and apoptosis were analysed by XTT [2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide] assay, FACS analysis and Fluorescent/Phase contrast microscopy was carried out for morphological studies. Hyp‐PDT produces more ROS after 1 day compared with 2 days and the mode of cell death is a necrotic caspase‐independent mechanism. We propose a novel ‘double‐hit/2‐day’ strategy to reduce the viability in SCC using Hyp‐based PDT as an adjunctive treatment modality.