In vitro and in vivo antimelanoma effect of ethyl ester cyclohexyl analog of ethylenediamine dipropanoic acid.

Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortality and increasing morbidity. Multiple available chemotherapeutic and immunotherapeutic modalities failed to improve survival in advanced disease, and the search for new agents is ongoing. The aim of this study was to investigate antimelanoma effects of O,O-diethyl-(S,S)-ethylenediamine-N,N'di-2-(3-cyclohexyl) propanoate dihydrochloride (EE), a previously synthesized and characterized organic compound. Mouse melanoma B16 cell viability was assessed using acid phosphatase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sulforhodamine B, and lactate dehydrogenase assays. Apoptosis and autophagy were investigated using flow cytometry, fluorescence and electron microscopy, and western blotting. In vivo antitumor potential was assessed in subcutaneous mouse melanoma model after 14 days of treatment with EE. Tumor mass and volume were measured, and RT-PCR was used for investigating the expression of autophagy-related, proapoptotic, and antiapoptotic molecules in tumor tissue. Investigated organic compound exerts significant cytotoxic effect against B16 cells. EE induced apoptosis, as confirmed by phosphatidyl serine externalisation, caspase activation, and ultrastructural features typical for apoptosis seen on fluorescence and electron microscopes. The apoptotic mechanism included prompt disruption of mitochondrial membrane potential and oxidative stress. No autophagy was observed. Antimelanoma action and apoptosis induction were confirmed in vivo, as EE decreased mass and volume of tumors, and increased expression of several proapoptotic genes. EE possesses significant antimelanoma action and causes caspase-dependent apoptosis mediated by mitochondrial damage and reactive oxygen species production. Decrease in tumor growth and increase in expression of proapoptotic genes in tumor tissue suggest that EE warrants further investigation as a candidate agent in treating melanoma.

Photodynamic antibacterial effect of graphene quantum dots.

Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. In the present study we report that electrochemically produced graphene quantum dots (GQD), a new class of carbon nanoparticles, generate reactive oxygen species when photoexcited (470 nm, 1 W), and kill two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. Bacterial killing was demonstrated by the reduction in number of bacterial colonies in a standard plate count method, the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining with a redox-sensitive fluorochrome dihydrorhodamine 123. Neither GQD nor light exposure alone were able to cause oxidative stress and reduce the viability of bacteria. Importantly, mouse spleen cells were markedly less sensitive in the same experimental conditions, thus indicating a fairly selective antibacterial photodynamic action of GQD.

Graphene quantum dots as autophagy-inducing photodynamic agents.

The excellent photoluminescent properties of graphene quantum dots (GQD) makes them suitable candidates for biomedical applications, but their cytotoxicity has not been extensively studied. Here we show that electrochemically produced GQD irradiated with blue light (470 nm, 1W) generate reactive oxygen species, including singlet oxygen, and kill U251 human glioma cells by causing oxidative stress. The cell death induced by photoexcited GQD displayed morphological and/or biochemical characteristics of both apoptosis (phosphatidylserine externalization, caspase activation, DNA fragmentation) and autophagy (formation of autophagic vesicles, LC3-I/LC3-II conversion, degradation of autophagic target p62). Moreover, a genetic inactivation of autophagy-essential LC3B protein partly abrogated the photodynamic cytotoxicity of GQD. These data indicate potential usefulness of GQD in photodynamic therapy, but also raise concerns about their possible toxicity.

A novel C,D-spirolactone analogue of paclitaxel: autophagy instead of apoptosis as a previously unknown mechanism of cytotoxic action for taxoids.

The design, synthesis and biological evaluation of a novel C,D-spirolactone analogue of paclitaxel is described. This is the first paclitaxel analogue without an oxetane D-ring that shows a significant cytotoxic effect (activity one order of magnitude lower than paclitaxel). More importantly, its cytotoxicity is a result of a different mechanism of action, involving mTOR inhibition-dependent autophagy instead of G(2)/M cell cycle arrest-dependent apoptosis.

In vitro comparison of the photothermal anticancer activity of graphene nanoparticles and carbon nanotubes.

The present study compared the photothermal anticancer activity of near-infrared (NIR)-excited graphene nanoparticles and carbon nanotubes (CNT). Despite lower NIR-absorbing capacity, suspension of polyvinylpyrrolidone-coated graphene sheets exposed to NIR radiation (808 nm, 2 W/cm(2)) generated more heat than DNA or sodium dodecylbenzenesulfonate-solubilized single-wall CNT under the same conditions. Accordingly, graphene nanoparticles performed significantly better than CNT in inducing photothermal death of U251 human glioma cells in vitro. The superior photothermal sensitivity of graphene sheets could be largely explained by their better dispersivity, which has been supported by a simple calculation taking into account thermodynamic, optical and geometrical properties of the two type of carbon nanoparticles. The mechanisms of graphene-mediated photothermal killing of cancer cells apparently involved oxidative stress and mitochondrial membrane depolarization resulting in mixed apoptotic and necrotic cell death characterized by caspase activation/DNA fragmentation and cell membrane damage, respectively.

Protective effect of autophagy in laser-induced glioma cell death in vitro.

BACKGROUND AND OBJECTIVE: Laser phototherapy could be potentially used for cancer treatment, but the mechanisms of laser-induced cell death are not completely understood. Autophagy is the process in which the damaged cellular proteins and organelles are engulfed by and destroyed in acidified multiple-membrane vesicles. The aim of the present study was to investigate the role of autophagy in laser-induced tumor cell death in vitro. STUDY DESIGN/MATERIALS AND METHODS: The monolayers of U251 human glioma tumor cells were exposed to 532 nm laser light from a single mode frequency-doubled Nd-YVO4 laser. A flattened Gaussian radial profile of laser beam (0.5-4 W) was used to uniformly illuminate entire colony of cells for various amounts of time (15-120 seconds) in the absence of cell culture medium. The cells were grown for 24 hours and the cell viability was determined by crystal violet or MTT assay. The presence of autophagy was assessed after 16 hours by fluorescence microscopy/flow cytometric analysis of acridine orange-stained autophagolysosomes and Western blot analysis of the autophagosome-associated LC3-II protein. The concentration of the principal pro-autophagic protein beclin-1 was determined after 6 hours by cell-based ELISA. RESULTS: The intracytoplasmic accumulation of autophagic vesicles, increase in LC3-II and up-regulation of beclin-1 expression were clearly observed under irradiation conditions that caused approximately 50% cytotoxicity. Post-irradiation addition of three different autophagy inhibitors (bafilomycin A1, chloroquine, or wortmannin) further increased the laser-induced cytotoxicity, without affecting non-irradiated cells. CONCLUSIONS: These data indicate that beclin-1-dependent induction of autophagy can protect glioma cells from laser-mediated cytotoxicity.

Opposite effects of nanocrystalline fullerene (C(60)) on tumour cell growth in vitro and in vivo and a possible role of immunosupression in the cancer-promoting activity of C(60).

In the present study, we compared the effects of nanocrystalline fullerene suspension (nanoC(60)) on tumour cell growth in vitro and in vivo. NanoC(60) suspension was prepared by solvent exchange using tetrahydrofuran to dissolve C(60). In vitro, nanoC(60) caused oxidative stress, mitochondrial depolarization and caspase activation, leading to apoptotic and necrotic death in mouse B16 melanoma cells. Biodistribution studies demonstrated that intraperitoneally injected radiolabeled (125I) nanoC(60) readily accumulated in the tumour tissue of mice subcutaneously inoculated with B16 cells. However, intraperitoneal administration of nanoC(60) over the course of two weeks starting from melanoma cell implantation not only failed to reduce, but significantly augmented tumour growth. The tumour-promoting effect of nanoC(60) was accompanied by a significant increase in splenocyte production of the immunoregulatory free radical nitric oxide (NO), as well as by a reduction in splenocyte proliferative responses to T- and B-cell mitogens ConcanavalinA and bacterial lipopolysaccharide, respectively. A negative correlation between NO production and splenocyte proliferation indicated a possible role of NO in reducing the proliferation of splenocytes from nanoC(60)-injected mice. These data demonstrate that nanoC(60), in contrast to its potent anticancer activity in vitro, can potentiate tumour growth in vivo, possibly by causing NO-dependent suppression of anticancer immune response.

The protection of cells from nitric oxide-mediated apoptotic death by mechanochemically synthesized fullerene (C(60)) nanoparticles.

The influence of fullerene (C(60)) nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide gamma-cyclodextrin or the copolymer ethylene vinyl acetate-ethylene vinyl versatate. C(60) nanoparticles were characterized by UV-vis and atomic force microscopy. While readily internalized by mouse L929 fibroblasts, C(60) nanoparticles were not cytotoxic. Moreover, they partially protected L929 cells from the cytotoxic effect of NO-releasing compounds sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholino-sydnonimine (SIN-1). C(60) nanoparticles reduced SNP-induced apoptotic cell death by preventing mitochondrial depolarization, caspase activation, cell membrane phosphatidylserine exposure and DNA fragmentation. The protective action of C(60) nanoparticles was not exerted via direct interaction with NO, but through neutralization of mitochondria-produced superoxide radical in NO-treated cells, as demonstrated by using different redox-sensitive reporter fluorochromes. These data suggest that C(60) complexes with appropriate host molecules might be plausible candidates for preventing NO-mediated cell injury in inflammatory/autoimmune disorders.