Impact of Methadone on Cisplatin Treatment of Bladder Cancer Cells.

BACKGROUND: Cisplatin-based chemotherapy is the treatment of choice for advanced bladder cancer. Since many tumor cells show inherent or acquired cisplatin resistance, research is needed to improve the therapeutic efficacy. Since the analgesic methadone is discussed as being a sensitizer for chemotherapy, we tested its effects on the cisplatin treatment of bladder cancer cells. MATERIALS AND METHODS: T24 and HT-1376 bladder cancer cells were incubated with cisplatin in combination with methadone. Cytotoxicity was examined using the WST-1 viability assay and induction of apoptosis was analyzed via phase-contrast microscopy, flow cytometry, and western blot analysis. RESULTS: Methadone was shown to enhance the cytotoxic effects of cisplatin on T24 cells based on the induction of apoptosis. In contrast, HT-1376 cells were identified as non-responders to methadone. CONCLUSION: Methadone could act as a chemosensitizer in the future treatment of advanced bladder cancer. Further research is needed to identify the underlying molecular mechanisms.

In Vitro Evaluation of Humanized/De-immunized Anti-PSMA Immunotoxins for the Treatment of Prostate Cancer.

BACKGROUND: We generated humanized/de-immunized immunotoxins targeting the prostate-specific membrane antigen (PSMA) and tested their cytotoxic activity against prostate cancer cells in vitro. MATERIALS AND METHODS: The humanized/de-immunized version of our murine anti-PSMA single-chain antibody fragment (scFv) D7, termed hD7-1(VL-VH), was ligated to the 40-kDa toxin domain of Pseudomonas aeruginosa exotoxin A (PE40), and to the deimmunized 24-kDa toxin domains PE24 or PE24mut. The immunotoxins designated as hD7-1(VL-VH)-PE40, hD7-1(VL-VH)-PE24 and hD7-1(VL-VH)-PE24mut were bacterially expressed and purified by affinity chromatography. Binding and cytotoxicity were examined by flow cytometry and viability assay, respectively. RESULTS: All immunotoxins revealed strong binding to prostate cancer cells expressing PSMA and specific cytotoxicity, with half-maximal inhibitory concentration values in the picomolar range. CONCLUSION: We successfully created powerful anti-PSMA immunotoxins with reduced immunogenicity for further clinical development and application against advanced prostate cancer.

Synergistic cytotoxicity of a prostate cancer-specific immunotoxin in combination with the BH3 mimetic ABT-737.

In many tumors, including prostate cancer, anti-apoptotic members of the Bcl-2 family are overexpressed and cause cell death resistance, which is a typical hallmark of cancer. Different therapeutic approaches, therefore, aim to restore the death mechanisms for enhanced apoptosis. Our recombinant immunotoxin D7(VL-VH)-PE40 is composed of the scFv D7(VL-VH) against the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer cells and of the cytotoxic domain of the bacterial toxin Pseudomonas Exotoxin A (PE40). Since Pseudomonas Exotoxin A-based immunotoxins are known to preferentially inhibit the expression of the anti-apoptotic protein Mcl-1, the rationale was to test our immunotoxin in combination with the BH3 mimetic ABT-737, which specifically inhibits Bcl-2, Bcl-xl, and Bcl-w for enhanced induction of apoptosis in prostate cancer cells. The immunotoxin showed high and specific binding and cytotoxicity against PSMA expressing prostate cancer cells marked by a direct inhibition of Mcl-1. The combination of the immunotoxin with a subtoxic concentration of ABT-737 caused additive or even synergistic effects, which were based on an enhanced apoptosis induction as detected by poly(ADP-ribose) polymerase (PARP) and Caspase-3 cleavage in Western blot. Our study shows that the combination therapy of immunotoxin plus ABT-737 is a promising approach for the future treatment of advanced prostate cancer to improve therapeutic efficacy and to reduce adverse side effects.

Contemporary follow-up imaging after endovascular repair of lower extremity atherosclerotic lesions.

Atherosclerotic disease is currently one of the most important problems of modern medicine because it is a leading cause of increased morbidity, morbidity and mortality, and disability in the Western World. Atherosclerosis of the lower limbs (peripheral arterial disease - PAD) significantly affects the quality of life and in a considerable proportion of patients is a cause of disability. Radical treatment of PAD, both surgical and endovascular, aims at revascularisation of ischaemic tissues distal to obstructed arteries. Surveillance imaging is an important part of patient management after endovascular repair of PAD. Apart from availability and contraindications, challenges of imaging include calcifications, flow dynamics, and stent-related artefacts. The aim of this paper was to review the current literature on imaging methods for follow-up after endovascular repair of atherosclerotic lesions, with special attention paid to novel techniques. As a non-invasive modality, ultrasound is still the first-line examination, but computed tomography angiography remains a current state-of-the art technique for follow-up. However, since current imaging recommendations seem not to adhere to contemporary imaging possibilities, more attention should be paid to recent improvements in magnetic resonance angiography technology.

Methadone as a "Tumor Theralgesic" against Cancer.

Methadone has beneficial characteristics as an analgesic against cancer pain, including high bioavailability, multiple receptor affinities, and lack of active metabolites that might induce adverse side effects. However, methadone has an own pharmacological profile that should be considered in the treatment of cancer patients. There is evidence from preclinical studies that methadone could also elicit antitumor activity by downregulating the threshold of apoptosis and to enhance the effects of different chemotherapeutic agents. This confirms the concept of using methadone as a chemosensitizer in the future treatment of cancer. Our article discusses major issues about the role of methadone as a possible "tumor theralgesic," combining tumor therapeutic and analgesic activities.

The effect of hydrazine derivatives of 3-formylchromones on angiogenic basic fibroblast growth factor and fibroblast growth factor receptor-1 in human melanoma cell line WM-115.

The hydrazine derivatives of benzopyrones remain an unexplored group of chemical compounds. This preliminary study investigates the influence of A-5, CH-3 and K-2 derivatives at concentrations of 1, 10, 100 nM and 1 µM on selected biochemical factors of a melanoma cell line WM-115, with regard to their potential angiogenic properties. The studied compounds were found to influence cell proliferation, as well as total protein, bFGF and FGFR1 concentration.

In vitro and in vivo effects of a recombinant anti-PSMA immunotoxin in combination with docetaxel against prostate cancer.

Docetaxel (DOC) is used for the first-line treatment of castration resistant prostate cancer (CPRC). However, the therapeutic effects are limited, only about one half of patients respond to the therapy and severe side effects possibly lead to discontinuation of treatment. Therefore, actual research is focused on the development of new DOC-based combination treatments. In this study we investigated the antitumor effects of a recombinant immunotoxin targeting the prostate specific membrane antigen (PSMA) in combination with DOC in vitro and in vivo. The immunotoxin consists of an anti-PSMA single chain antibody fragment (scFv) as binding and a truncated form of Pseudomonas aeruginosa Exotoxin A (PE40) as toxin domain. The immunotoxin induced apoptosis and specifically reduced the viability of androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cells. A synergistic cytotoxic activity was observed in combination with DOC with IC50 values in the low picomolar or even femtomolar range. Moreover, combination treatment resulted in an enhanced antitumor activity in a C4-2 SCID mouse xenograft model. This highlights the immunotoxin as a promising therapeutic agent for a future DOC-based combination therapy of CPRC.

Pseudomonas Exotoxin A: optimized by evolution for effective killing.

Pseudomonas Exotoxin A (PE) is the most toxic virulence factor of the pathogenic bacterium Pseudomonas aeruginosa. This review describes current knowledge about the intoxication pathways of PE. Moreover, PE represents a remarkable example for pathoadaptive evolution, how bacterial molecules have been structurally and functionally optimized under evolutionary pressure to effectively impair and kill their host cells.

Biofilm formation on the surface of polylactide during its biodegradation in different environments.

The research was aimed at determining the abundance and viability of biofilm formed on the surface of polylactide (PLA) during its biodegradation in different environments. It was also aimed at isolating biofilm forming bacteria, determining their hydrolytic activity and taxonomic status. The first step was to evaluate PLA biodegradability in lake water, compost and soil, using OxiTop Control. The next step was to assess the ability of isolated bacteria to form biofilm in the investigated environments and to evaluate the biofilm structure. The results indicate that PLA is sensitive to biodegradation in any environment, particularly in compost. During this process biofilm of high viability was observed on the surface of PLA. Based on the 16S rRNA gene sequence, the biofilm-forming bacteria were classified as the following species: Acidovorax sp. LW9, Chryseobacterium sp. LW2, Aeromonas veronii LW8, Arthrobacter aurescens LG2, Arthrobacter sp. LG12, A. aurescens LG9, Elizabethkingia meningoseptica LK3, A. aurescens LK9, A. aurescens and LK7. The results show that different bacterial species formed biofilm of different abundance and hydrolytic activitiy levels.

Isolation and characterization of collagen from the skin of Brama australis.

Collagen was extracted from the skin of Brama australis, the fish from warm-water sea. The yield of collagen from skin of B. australis was about 1.5% on a wet weight basis of raw material. The isolated protein was confirmed as collagen by different physico-chemical techniques such as: FTIR, SDS-PAGE, and amino acid analysis. The denaturation temperature (T(d)) of obtained collagen was found to be 24°C, what is promising as an advantage for cosmetic application. According to the electrophoretic pattern, collagen consisted of two different α-chains (α1 and α2) and was classified as type I collagen. Although T(d) of obtained collagen is higher than 20 °C it is still far from T(d) of mammalian collagen.

Estimation of the use of fibrin and collagen membranes as carriers for platelet-derived growth factor-BB (PDGF-BB) in the presence of amoxicillin.

The effect of homogeneous fibrin (Fb), collagen (Coll) and composite fibrin-heparin (Fb-Hp), fibrin-collagen (Fb-Coll) membranes on in vitro release of platelet-derived growth factor (PDGF-BB) was evaluated in the presence or absence of amoxicillin using of the ELISA immunoassay test. Amoxicillin concentration was determined spectrophotometrically at 272 nm. The process of the PDGF-BB growth factor and amoxicillin release from the studied membranes was of a two-phase nature in the majority of the systems analysed. The PDGF-BB was released in the highest amount from the Coll membrane (M7) without the presence of amoxicillin--546.2 ± 7.47 pg, t0.5 = 0.88 h and 202.5 ± 6.83 pg, t0.5 = 26.65 h during the first phase and second phase, respectively. The lowest PDGF-BB release was observed from composite M4 (Fb-Hp) membrane--5.88 ± 0.81 pg, t0.5 = 1.69 h; and 110.2 ± 6.48 pg, t0.5 = 855.6 h during first and second phase respectively. An optimal release of amoxicillin was observed in the case of the composite M6 (Fb-Coll) membrane--only in the second phase: 64.2 ± 7.8 µg, t0.5 = 83.5 h. The lowest and delayed amoxicillin release was achieved for M4 membrane (approx. 17.1 ± 1.12 µg, t0.5 = 46.5 h). The results of the PDGF-BB release and amoxicillin from membranes indicated a correlation between the level of release and composition of the film. Our results suggested that fibrin and collagen membranes may be beneficial to enhance periodontal bone regeneration.

The influence of UV-irradiation on thermal and mechanical properties of chitosan and silk fibroin mixtures.

In the present paper the results regarding the influence of UV-irradiation with 254 nm wavelength on the thermal and mechanical properties and the intrinsic viscosity of chitosan/silk fibroin mixtures are presented. The mixture of chitosan and silk fibroin in solution and thin films made of chitosan/silk fibroin mixture obtained by solvent evaporation were submitted to the treatment with UV irradiation (wavelength 254 nm) for different time intervals. Mechanical properties of thin films made of chitosan/silk fibroin blends before and after UV-irradiation have been investigated by mechanical testing machine and compared with mechanical properties of chitosan films. The changes in such mechanical properties as ultimate breaking strength, percentage of elongation at break and Young Modulus have been investigated. The results have shown, that the mechanical properties of the blends were greatly affected by time of exposure to UV irradiation. Ultimate tensile strength and ultimate percentage of elongation decreased after UV irradiation of the blend. Increasing UV irradiation led to the decrease in Young's Modulus of the chitosan/silk fibroin blend. Viscosity of chitosan/silk fibroin mixtures decreased after UV-irradiation. Thermal properties of the mixtures have been only slightly altered by UV-irradiation.

Fibrinogen, bFGF and VEGF levels during antibiotic therapy in gynecologic cancer: a preliminary report.

The role of angiogenesis in the development of neoplasia has been identified and characterized. However, antiangiogenic therapeutic intervention still requires more evidence to become recognized and successful. The aim of this study was to evaluate levels of selected proangiogenic factors, such as fibrinogen, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in serum of patients with the gynecologic cancer on the first, third and sixth day of antibiotic therapy, routinely administered as a perioperative treatment. In addition, serum concentrations of gamma-gamma dimers and alpha-polymers of cross-linked fibrin structure and the degree of bFGF binding with the fibrin network were investigated. Immunohistochemistry staining of the excised tumor tissue was also performed. We observed higher levels of bFGF, VEGF, as well as fibrinogen in patients with gynecologic malignancy, as compared to healthy women. In cancer patients, the concentration of alpha-polymers and gamma-gamma dimers of fibrin network increased. Further only gamma-gamma dimers fraction of fibrin was found to bind to bFGF. Immunohistochemical analysis indicated the presence of bFGF in an excised tumor tissue. In conclusion, the decrease of proangiogenic bFGF and fibrinogen levels in a clinical trial of gynecologic patients may confirm anti-angiogenic properties of selected antibiotic therapy.

The knowledge and awareness of hypertension among patients with hypertension in central Poland: a pilot registry.

We assessed the differences in the knowledge and level of awareness of hypertension among patients with hypertension from Central Poland; 248 (57.6% females) patients diagnosed with hypertension completed a questionnaire. Most (79%) of the patients were unaware of the optimal blood pressure (BP) range. The elderly patients did not know the symptoms of hypertension (23.7%), were not willing to make lifestyle changes (57%-65%), and had a poor awareness of hypertension therapy in the absence of symptoms (28.7%). Poor BP control occurred mainly in rural residents (10.7%) and in people with higher education (39.3%). Untreated patients with hypertension did not know the symptoms of hypertension (29.2%), rarely measured BP (37.5%), but were more likely to engage in regular physical activity (70.8%). Efforts should be made to improve knowledge of hypertension, especially among the rural population, the elderly patients, those with a low-education level, and in young males who had the highest BP.

Influence of NADPH oxidase inhibition on oxidative stress parameters in rat hearts.

BACKGROUND: The aim of this study was to assess whether apocynin, an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocker, influences lipid peroxidation TBARS, hydrogen peroxide (H2O2) content, protein level, heart edema, tumor necrosis factor α (TNF-α) concentration or the glutathione redox system in heart homogenates obtained from endothelin 1 (ET-1)-induced oxidative stress rats. METHODS: Experiments were carried out on adult male Wistar-Kyoto rats. The animals were divided into 4 groups: Group I: saline-treated control; Group II: saline followed by ET-1 (3 µg/kg b.w., iv); Group III: apocynin (5 mg/kg b.w., iv) administered half an hour before saline; Group IV: apocynin (5 mg/kg b.w., iv) administered half an hour before ET-1 (3 µg/kg b.w., iv). RESULTS: Injection of ET-1 alone showed a significant (p < 0.001) increase in thiobarbituric acid reactive substances (TBARS) and the hydrogen peroxide level (p < 0.01) vs. control, as well as a decrease (p < 0.001) in the GSH level. Apocynin significantly decreased TBARS (p < 0.001) and H2O2 (p < 0.05) level (vs. control) as well as improved protein level (p < 0.001) in the heart. Apocynin also prevented ET-1-induced heart edema (p < 0.05). The presence of ET-1 increased the concentration of TNF-α (p < 0.05) while apocynin decreased it (p < 0.05). Our results indicate that ET-1 may induce oxidative stress in heart tissue by reducing the GSH/GSSG ratio, stimulating lipid peroxidation and increasing TNF-α concentration. Apocynin diminished these measures of oxidative stress and TNF-α. CONCLUSION: ET-1-induced formation of ROS in the heart is at least partially regulated via NADPH oxidase.

Effect of phosphorohydrazone derivatives of chromone on fibrin polymerization in the presence of bFGF.

The effect of ten phosphorohydrazone derivatives of chromone on the fibrin formation process was evaluated in the presence of basic fibroblast growth factor (bFGF), which plays an important role in tumor angiogenesis. The relationship between the chemical properties of the derivatives and the resulting fibrin structure was also examined. The structure of fibrin was analyzed by UV spectrophotometry and the degree of bFGF binding to fibrin was estimated by Western blotting. The measurements were taken at 3 different pH values: 6.64, 7.60 and 8.40. Three of the analyzed phosphorohydrazone derivatives (compounds 2, 7 and 10) demonstrated the most significant influence on reduction of polymerization at the studied pH values.

The influence of chromone based hydrazones on lipid peroxidation and bFGF concentration in the HL-60 cell line.

Natural and synthetic derivatives of benzo-γ-pyrones (i.e. flavones, chromones, and coumarins) and their synthetic analogues possess a wide range of biological properties in vitro and in vivo. In this paper we investigated the influence of two hydrazone compounds of chromones, 3-{[(2-dimethoxytiophosphoryl)-2-methylhydrazono]-methyl}-chromen-4-one (CH-3) and 2-amino-6-chloro-3-[(2-hydroxyethyl)-hydrazonomethyl]-chromen-4-one (A-12), on lipid peroxidation and bFGF concentration in the HL-60 cells. Both of the studied compounds had a significant influence on bFGF and TBARS in ranges -137.20 ~ 380.26% and -81.66 ~ -28.68%, respectively, in comparison with the control (counted as 0%).

The effect of cadmium on the coagulation and fibrinolytic system in women with uterine endometrial cancer and myoma.

OBJECTIVES: Cadmium (Cd) is a persistent and widespread environmental pollutant, which may constitute a potential risk factor for hormone-dependent tumors such as endometrial cancer. The vascular endothelium is an important target of cadmium toxicity, which may interfere with the coagulation cascade and fibrinolytic system. The aim of this research was to investigate whether in female patients with uterine endometrial cancer or myoma in comparison to healthy women, the concentration of cadmium in blood affects the process of coagulation and fibrinolysis. MATERIALS AND METHODS: The study group comprised 91 women: 35 healthy (A-control), 39 with uterine myoma (B) and 17 with endometrial cancer (C), in which blood cadmium concentrations (BCd), coagulation and selected fibrinolysis parameters in plasma were assayed. RESULTS: In the women with myoma and especially in those with endometrial cancer disturbances in coagulation and fibrinolysis were detected when compared to the healthy women. In the group of women with endometrial cancer significant changes in prothrombin index, levels of fibrinogen, fibrin D-dimer and t-PA were observed. Whereas, in the patients with myoma significant changes in prothrombin time, index of vWillebrand Factor and fibrin D-dimer level were noted. Mean BCd concentrations in subsequent groups were as follows: B - 0.91±0.81; C - 0.78±0.45 µg Cd/l and did not differ significantly in comparison with the control group (0.86±0.35 µg Cd/l). However, in each study group smokers had approximately twice as high BCd as non-smokers. Studies also showed significant associations between BCd and fibrinogen level and thrombin time among the women with myoma and endometrial cancer, as well as in healthy women. Moreover, thrombin time significantly correlated with fibrinogen level in the women studied. CONCLUSIONS: In the patients with myoma and especially in these with endometrial cancer disturbances in coagulation and fibrinolysis parameters leading to hypercoagulability were detected. Exposure to cadmium can be one of the factors inducing these changes.

Influence of block of NF-kappa B signaling pathway on oxidative stress in the liver homogenates.

The aim of the present study was to assess whether BAY 11-7082, a nuclear factor-kappaB (NF- κ B) inhibitor, influences the level of reactive oxygen species (ROS), tumor necrosis factor alpha (TNF- α), and NF- κ B related signaling pathways in the liver. The animals were divided into 4 groups: I: saline; II: saline + endothelin-1 (ET-1) (1.25 µg/kg b.w., i.v.); III: saline + ET-1 (12.5 µg/kg b.w., i.v.); and IV: BAY 11-7082 (10 mg/kg b.w., i.v.) + ET-1 (12.5 µg/kg b.w., i.v.). Injection of ET-1 alone at a dose of 12.5 µg/kg b.w. showed a significant (P < 0.001) increase in thiobarbituric acid reactive substances (TBARS) and hydrogen peroxide (H2O2) level and decrease (P < 0.01) in GSH level (vs. control). ET-1 administration slightly downregulated gene expression of p65 of NF- κ B but potently and in a dose-dependent way downregulated p21-cip gene expression in the liver. BAY 11-7082 significantly decreased TBARS (P < 0.001), H2O2 (P < 0.01) and improved the redox status (P < 0.05), compared to ET-1 group. The concentration of TNF- α was increased in the presence of ET-1 (P < 0.05), while BAY 11-7082 decreased TNF- α concentration (P < 0.01). Inhibition of IkB α before ET-1 administration downregulated gene expression of p21-cip but had no effect on p65.

Visualization of vascular inflammation in the atherosclerotic mouse by ultrasmall superparamagnetic iron oxide vascular cell adhesion molecule-1-specific nanoparticles.

OBJECTIVE: Noninvasive imaging of atherosclerosis remains challenging in clinical applications. Here, we applied noninvasive molecular imaging to detect vascular cell adhesion molecule-1 in early and advanced atherosclerotic lesions of apolipoprotein E-deficient mice. METHODS AND RESULTS: Ultrasmall superparamagnetic iron oxide particles functionalized with (P03011) or without (P3007) vascular cell adhesion molecule-1-binding peptide were visualized by ultra high-field (17.6 T) magnetic resonance. Injection of P03011 resulted in a marked signal loss in the aortic root of apolipoprotein E-deficient mice fed a Western diet for 8 and 26 weeks in vivo and ex vivo, compared with preinjection measurements, P3007-injected mice, and P03011- or P3007-injected age-matched C57BL/6 controls. Histological analyses revealed iron accumulations in the intima, in colocalization with vascular cell adhesion molecule-1-expressing macrophages and endothelial cells. Coherent anti-Stokes Raman scattering microscopy demonstrated iron signals in the intima and media of the aortic root in the P03011-injected but not untreated apolipoprotein E-deficient mice, localized to macrophages, luminal endothelial-like cells, and medial regions containing smooth muscle cells. Electron microscopy confirmed iron particles enclosed in endothelial cells and in the vicinity of smooth muscle cells. CONCLUSIONS: Using a combination of innovative imaging modalities, in this study, we demonstrate the feasibility of applying P03011 as a contrast agent for imaging of atherosclerosis.