Human cancer genomes harbor the mutational signature of tobacco-specific nitrosamines NNN and NNK.

Tobacco usage is linked to multiple cancer types and accounts for a quarter of all cancer-related deaths. Tobacco smoke contains various carcinogenic compounds, including polycyclic aromatic hydrocarbons (PAH), though the mutagenic potential of many tobacco-related chemicals remains largely unexplored. In particular, the highly carcinogenic tobacco-specific nitrosamines NNN and NNK form pre-mutagenic pyridyloxobutyl (POB) DNA adducts. In the study presented here, we identified genome-scale POB-induced mutational signatures in cell lines and rat tumors, while also investigating their role in human cancer. These signatures are characterized by T>N and C>T mutations forming from specific POB adducts damaging dT and dC residues. Analysis of 2,780 cancer genomes uncovered POB signatures in ∼180 tumors; from cancer types distinct from the ones linked to smoking-related signatures SBS4 and SBS92. This suggests that, unlike PAH compounds, the POB pathway may contribute uniquely to the mutational landscapes of certain hematological malignancies and cancers of the kidney, breast, prostate and pancreas.

Bone regeneration response in an experimental long bone defect orthotopically implanted with alginate-pullulan-glass-ceramic composite scaffolds.

In the present study, scaffolds based on alginate-pullulan-bioactive glass-ceramic with 0.5 and 1.5 mol % copper oxide were orthotopically implanted in experimental rat models to assess their ability to heal an induced bone defect. By implying magnetic resonance and imaging scans together with histological evaluation of the processed samples, a progressive healing of bone was observed within 5 weeks. Furthermore, as the regenerative process continued, new bone tissue was formed, enhancing the growth of irregular bone spicules around the scaffolds. A significantly higher amount of new bone was formed (37%) in the defect that received the composite with 1.5 mol % CuO (in glass-ceramic matrix) content implant. Nevertheless, the bone regeneration obtained by scaffold with 0.5 mol % CuO implanted is comparable with the alginate-pullulan-ß-tricalcium phosphate/hydroxiapatite composite implant. The assessed amount of new bone formed was found to be between 29.75 and 37.15% for all the composition involved in the present study. During this process a regeneration process was shown when the alginate-pullulan composite materials were involved, fact that indicate the great potential of these materials to be used in tissue engineering.

Comparison of two models of inflammatory bowel disease in rats.

BACKGROUND: There is a need for experimental animal models for inflammatory bowel diseases (IBD), but no proposed model has been unanimously accepted. OBJECTIVES: The aim of this study was to develop 2 affordable models of IBD in rats and to compare them. MATERIAL AND METHODS: We produced IBD in rats using either dextran sodium sulfate (DSS) or 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). The requirements for experimental models were: a predictable clinical course, histopathology and inflammation similar to human ulcerative colitis (UC) and Crohn's disease (CD). The effect of acute administration of DSS and TNBS on oxidative stress (as measured by the assessment of glutathione peroxidase - GPx) was verified. The activity of whole blood GPx was measured using a commercially available Randox kit (Crumlin, UK). RESULTS: The administration of DSS increased GPx activity compared to the control and TNBS-treated groups, but not to a statistically significant degree. Histological examination of the colonic mucosa following the administration of DSS showed multifocal erosions with minimal to mild inflammatory infiltrate, mainly by polymorphonuclear cells (PMN), lymphocytes and plasma cells. For TNBS-induced colitis, the histological changes manifested as multifocal areas of ulcerative colitis with mild to severe inflammatory infiltrate. Whole blood GPx values displayed a direct dependence on the chemical agent used. Our results show a correlation between histopathology, proinflammatory state and oxidative stress. CONCLUSIONS: The experimental DSSor TNBS-induced bowel inflammation used in this study corresponds to human IBD and is reproducible with characteristics indicative of acute inflammation in the case of the protocols mentioned.

Laser thermal ablation of multidrug-resistant bacteria using functionalized gold nanoparticles.

The issue of multidrug resistance (MDR) has become an increasing threat to public health. One alternative strategy against MDR bacteria would be to construct therapeutic vectors capable of physically damaging these microorganisms. Gold nanoparticles hold great promise for the development of such therapeutic agents, since the nanoparticles exhibit impressive properties, of which the most important is the ability to convert light into heat. This property has scientific significance since is exploited to develop nano-photothermal vectors to destroy bacteria at a molecular level. The present paper summarizes the latest advancements in the field of nanotargeted laser hyperthermia of MDR bacteria mediated by gold nanoparticles.

Selective ex vivo photothermal nano-therapy of solid liver tumors mediated by albumin conjugated gold nanoparticles.

We have used albumin (BSA) bound to gold nanoparticles (GNPs) as active vectors to target liver cells. Our incentive to develop an original model of living liver cancer sprang from the ethical drawbacks that hindered the assessment of the selective character and the therapeutic capacity of these nano-biosystems in cancer patients. Ex vivo-perfused liver specimens were obtained from hepatocellular carcinoma patients similarly to the surgical technique of transplantation. Albumin bound to GNPs was inoculated intra-arterially onto the resulting specimen and determined the specific delivery of the nano-bioconjugate into the malignant tissue by means of the capillary bed. The extent of necrosis was considerable following laser therapy and at the same time surrounding parenchyma was not seriously affected. The selective photothermal ablation of the malignant liver tissue was obtained after the selective accumulation of BSA bound to GNPs into tumor cells following ex-vivo intra-vascular perfusion.

Selective in vitro photothermal nano-therapy of MRSA infections mediated by IgG conjugated gold nanoparticles.

There are serious systemic infections associated with methicillin-resistant Staphylococcus aureus (MRSA) and several other types of bacteria leading to the deaths of millions of people globally. This type of mortality is generally caused by the increasing number of antibiotic-resistant organisms, a consequence of evolution via natural selection. After the synthesis of gold nanoparticles (GNPs) by wet chemistry, bio-functionalization with IgG molecules was performed. Following administration of IgG-GNPs to MRSA cultures at various concentrations and various incubation time laser irradiation was performed. To assess the selectivity and specificity of the proposed treatment the following methods were used: flow cytometry, contrast phase microscopy, and by fluorescence microscopy. The results in our study indicate that following administration of IgG-GNPs biomolecule an extended and selective bacterial death occurs following laser irradiation in a dose dependent manner. Therefore, the new findings might impel studies on these antibacterial nanomaterials and their biological and medical applications.

Selective Laser Ablation of Methicillin-Resistant Staphylococcus Aureus with IgG Functionalized Multi-Walled Carbon Nanotubes.

Severe infections caused by Methicillin-resistant Staphylococcus aureus (MRSA) and other bacteria are responsible for millions of deaths each year. One of the main objectives of future antibiotic strategies is to develop new anti-infective agents, which would be highly effective and drug-resistant (antimicrobial resistance being currently exhibited by MRSA), using specific antibodies conjugated to thermally active nanomaterials such as NIR-responsive photothermal contrast agents. Multi-walled carbon nanotubes (MWCNTs) covalently functionalized with immunoglobulin G (IgG, an antagonist of Staphylococcal protein A-SpA, which is a MRSA membrane associated protein) were selectively delivered (at various concentrations and incubation times) into MRSA bacteria. Following treatment, cultures were irradiated using an 808 nm 2 w laser diode. The post irradiation death rate ranged from 39.6% (for 1 mg/L) to 79.2% (for 50 mg/L) at 60 seconds (p < 0.001), while at 30 minutes, the death rate increased from 45.2% (1 mg/L) to 85.72% (50 mg/L), p < 0.001. Irradiated MRSAs treated with MWCNTs alone (control) for 60 seconds and 30 minutes, at concentrations ranging from 1 mg/L to 50 mg/L, resulted in significantly lower death rates (7.1-34.1% for 60 seconds, 11.7-48.8% for 30 minutes). Using IgG molecules bound to MWCNTs, followed by laser irradiation, we obtained a very efficacious nanoshell-mediated laser therapy of individual MRSA agents providing highly localized killing effects for IgG-MWCNTs targeted bacteria.

Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus-ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor.

We present a method of enhanced laser thermal ablation of HepG2 cells based on a simple gold nanoparticle (GNP) carrier system such as serum albumin (Alb), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. HepG2 or hepatocytes were treated with Alb-GNPs at various concentrations and various incubation times, and further irradiated using a 2 W, 808 nm laser. Darkfield microscopy and immunochemical staining was used to demonstrate the selective internalization of Alb-GNPs inside the HepG2 cells via Gp60 receptors targeting. The postirradiation apoptotic rate of HepG2 cells treated with Alb-GNPs ranged from 25.8% (for 5 µg/mL) to 48.2% (for 50 µg/mL) at 60 seconds, while at 30 minutes the necrotic rate increased from 35.7% (5 µg/mL) to 52.3% (50 µg/mL), P-value <0.001. Significantly lower necrotic rates were obtained when human hepatocytes were treated with Alb-GNPs in a similar manner. We also showed by means of immunocytochemistry that photothermal treatment of Alb-conjugated GNPs in liver cancer initiates Golgi apparatus-endoplasmic reticulum dysfunction with consequent caspase-3 apoptotic pathway activation and cellular apoptosis. The presented results may become a new method of treating cancer cells by selective therapeutic vectors using nanolocalized thermal ablation by laser heating.

Photothermal Treatment of Human Pancreatic Cancer Using PEGylated Multi-Walled Carbon Nanotubes Induces Apoptosis by Triggering Mitochondrial Membrane Depolarization Mechanism.

Pancreatic cancer (PC) is one of the most lethal solid tumor in humans, with an overall 5-year survival rate of less than 5%. Thermally active carbon nanotubes have already brought to light promising results in PC research and treatment. We report here the construct of a nano-biosystem based on multi-walled carbon nanotubes and polyethylene glycol (PEG) molecules validated through AFM, UV-Vis and DLS. We next studied the photothermal effect of these PEG-ylated multi-walled carbon nanotubes (5, 10 and 50 µg/mL, respectively) on pancreatic cancer cells (PANC-1) and further analyzed the molecular and cellular events involved in cell death occurrence. Using cell proliferation, apoptosis, membrane polarization and oxidative stress assays for ELISA, fluorescence microscopy and flow cytometry we show here that hyperthermia following MWCNTs-PEG laser mediated treatment (808 nm, 2W) leads to mitochondrial membrane depolarization that activates the flux of free radicals within the cell and the oxidative state mediate cellular damage in PC cells via apoptotic pathway. Our results are of decisive importance especially in regard with the development of novel nano-biosystems capable to target mitochondria and to synergically act both as cytotoxic drug as well as thermally active agents in order to overcome one of the most common problem met in oncology, that of intrinsic resistance to chemotherapeutics.

Surface plasmon resonance-induced photoactivation of gold nanoparticles as mitochondria-targeted therapeutic agents for pancreatic cancer.

BACKGROUND: Noble metal nanoparticles such as gold nanoparticles can strongly absorb light in the visible region by inducing coherent collective oscillation of conduction band electrons in strong resonance with visible frequencies of light. This phenomenon is frequently termed as surface plasmon resonance (SPR). OBJECTIVES: The main objective was to study the effects of laser photoactivated gold nanoparticles (by means of SPR) on human pancreatic cancer cells. RESULTS: Gold nanoparticles obtained using standard wet chemical methods (with sodium borohydride as a reducing agent) underwent photoexcitation using 2w 808 nm laser and further administered to 1.4E7 pancreatic cancer cell lines. Flow cytometry, transmission electron microscopy, phase contrast microscopy, quantitative proteomics and confocal microscopy combined with immunochemical staining were used to examine the interaction between photo excited gold nanoparticles and pancreatic cancer cells. CONCLUSION: The study shows that phonon-phonon interactions following laser photoexcitation of gold nanoparticles exhibit increased intracellular uptake, as well as mitochondrial swelling, closely followed by mitochondrial inner membrane permeabilization and depolarization. This unique data may represent a major step in mitochondria-targeted anticancer therapies using laser-activated gold nanoparticles.

Selective ex-vivo photothermal ablation of human pancreatic cancer with albumin functionalized multiwalled carbon nanotubes.

The process of laser-mediated ablation of cancer cells marked with biofunctionalized carbon nanotubes is frequently called "nanophotothermolysis". We herein present a method of selective nanophotothermolisys of pancreatic cancer (PC) using multiwalled carbon nanotubes (MWCNTs) functionalized with human serum albumin (HSA). With the purpose of testing the therapeutic value of these nanobioconjugates, we have developed an ex-vivo experimental platform. Surgically resected specimens from patients with PC were preserved in a cold medium and kept alive via intra-arterial perfusion. Additionally, the HSA-MWCNTs have been intra-arterially administered in the greater pancreatic artery under ultrasound guidance. Confocal and transmission electron microscopy combined with immunohistochemical staining have confirmed the selective accumulation of HSA-MWCNTs inside the human PC tissue. The external laser irradiation of the specimen has significantly produced extensive necrosis of the malign tissue after the intra-arterial administration of HSA-MWCNTs, without any harmful effects on the surrounding healthy parenchyma. We have obtained a selective photothermal ablation of the malign tissue based on the selective internalization of MWCNTs with HSA cargo inside the pancreatic adenocarcinoma after the ex-vivo intra-arterial perfusion.

Enhanced laser thermal ablation for the in vitro treatment of liver cancer by specific delivery of multiwalled carbon nanotubes functionalized with human serum albumin.

The main goal of this investigation was to develop and test a new method of treatment for human hepatocellular carcinoma (HCC). We present a method of carbon nanotube-enhanced laser thermal ablation of HepG2 cells (human hepatocellular liver carcinoma cell line) based on a simple multiwalled carbon nanotube (MWCNT) carrier system, such as human serum albumin (HSA), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. Both HepG2 cells and hepatocytes were treated with HSA-MWCNTs at various concentrations and at various incubation times and further irradiated using a 2 W, 808 nm laser beam. Transmission electron, phase contrast, and confocal microscopy combined with immunochemical staining were used to demonstrate the selective internalization of HSA-MWCNTs via Gp60 receptors and the caveolin-mediated endocytosis inside HepG2 cells. The postirradiation apoptotic rate of HepG2 cells treated with HSA-MWCNTs ranged from 88.24% (for 50 mg/L) at 60 sec to 92.34% (for 50 mg/L) at 30 min. Significantly lower necrotic rates were obtained when human hepatocytes were treated with HSA-MWCNTs in a similar manner. Our results clearly show that HSA-MWCNTs selectively attach on the albondin (aka Gp60) receptor located on the HepG2 membrane, followed by an uptake through a caveolin-dependent endocytosis process. These unique results may represent a major step in liver cancer treatment using nanolocalized thermal ablation by laser heating.