Low Doses of Celecoxib Might Promote Phenotype Switching in Cutaneous Melanoma Treated with Dabrafenib-Preliminary Study.

Background: Cutaneous melanoma is a heterogeneous tumor with a rapidly switching molecular and cellular phenotype. The invasive phenotype switching characterized by MITFlow/AXLhigh predicts early resistance to multiple targeted drugs in melanoma. Celecoxib proved to be a valuable adjuvant in cutaneous melanoma in preclinical studies. Our in vitro study evaluated for the first time whether celecoxib could prevent phenotype switching in two human melanoma cell lines treated with dabrafenib. Methods: All in vitro experiments were carried out on BRAF-V600E-positive A375 and SK-MEL-28 human melanoma cell lines, and subjected to a celecoxib and dabrafenib drug combination for 72 h. Melanoma cells were already in the MITFlow/AXLhigh end of the spectrum. Of main interest was the evaluation of the key proteins expressed in phenotype switching (TGF-ß, MITF, AXL, YAP, TAZ), as well as cell death mechanisms correlated with oxidative stress production. Results: Celecoxib significantly enhanced the apoptotic effect of dabrafenib in each melanoma cell line compared to the dabrafenib group (p < 0.0001). Even though celecoxib promoted low MITF expression, this was correlated with high receptor tyrosine kinase AXL levels in A375 and SK-MEL-28 cell lines (p < 0.0001), a positive marker for the phenotype switch to an invasive state. Conclusion: This preliminary study highlighted that celecoxib might promote MITFlow/AXLhigh expression in cutaneous melanoma treated with dabrafenib, facilitating phenotype switching in vitro. Our results need further confirmation, as this finding could represent an important limitation of celecoxib as an antineoplastic drug.

Celecoxib as a Valuable Adjuvant in Cutaneous Melanoma Treated with Trametinib.

BACKGROUND: Melanoma patients stop responding to targeted therapies mainly due to mitogen activated protein kinase (MAPK) pathway re-activation, phosphoinositide 3 kinase/the mechanistic target of rapamycin (PI3K/mTOR) pathway activation or stromal cell influence. The future of melanoma treatment lies in combinational approaches. To address this, our in vitro study evaluated if lower concentrations of Celecoxib (IC50 in nM range) could still preserve the chemopreventive effect on melanoma cells treated with trametinib. MATERIALS AND METHODS: All experiments were conducted on SK-MEL-28 human melanoma cells and BJ human fibroblasts, used as co-culture. Co-culture cells were subjected to a celecoxib and trametinib drug combination for 72 h. We focused on the evaluation of cell death mechanisms, melanogenesis, angiogenesis, inflammation and resistance pathways. RESULTS: Low-dose celecoxib significantly enhanced the melanoma response to trametinib. The therapeutic combination reduced nuclear transcription factor (NF)-kB (p < 0.0001) and caspase-8/caspase-3 activation (p < 0.0001), inhibited microphthalmia transcription factor (MITF) and tyrosinase (p < 0.05) expression and strongly down-regulated the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway more significantly than the control or trametinib group (p < 0.0001). CONCLUSION: Low concentrations of celecoxib (IC50 in nM range) sufficed to exert antineoplastic capabilities and enhanced the therapeutic response of metastatic melanoma treated with trametinib.

Genetic predisposition in pediatric oncology.

Identifying patients with a genetic predisposition for developing malignant tumors has a significant impact on both the patient and family. Recognition of genetic predisposition, before diagnosing a malignant pathology, may lead to early diagnosis of a neoplasia. Recognition of a genetic predisposition syndrome after the diagnosis of neoplasia can result in a change of treatment plan, a specific follow-up of adverse treatment effects and, of course, a long-term follow-up focusing on the early detection of a second neoplasia. Responsible for genetic syndromes that predispose individuals to malignant pathology are germline mutations. These mutations are present in all cells of conception, they can be inherited or can occur de novo. Several mechanisms of inheritance are described: Mendelian autosomal dominant, Mendelian autosomal recessive, X-linked patterns, constitutional chromosomal abnormality and non-Mendelian inheritance. In the following review we will present the most important genetic syndromes in pediatric oncology.

Flexural strength, biocompatibility, and antimicrobial activity of a polymethyl methacrylate denture resin enhanced with graphene and silver nanoparticles.

OBJECTIVE: The study evaluates the effect of adding graphene-Ag nanoparticles (G-AgNp) to a PMMA auto-polymerizing resin, with focus on antibacterial activity, cytotoxicity, monomer release, and mechanical properties. MATERIALS AND METHODS: Auto-polymerizing acrylic resin (M) was loaded with 1 wt% G-AgNp (P1) and 2 wt% G-AgNp (P2). Methyl methacrylate monomer release (MMA) was measured after immersion of the samples in chloroform and cell medium respectively. Cell viability was assessed on dysplastic oral keratinocytes (DOK) and dental pulp stem cells. Oxidative stress and inflammatory response following exposure of dysplastic oral keratinocytes to the experimental resins was evaluated. Antibacterial activity against Staphylococcus aureus, Streptococcus mutans and Escherichia coli and also flexural strength of the resins were assessed. RESULTS: Residual monomer: For samples immersed in chloroform, MMA concentration reached high levels, 10.27 µg/g for sample P1; MMA increased at higher G-AgNp loading; 0.63 µg/g MMA was found in medium for P1, and less for sample P2. Cell viability: Both cell lines displayed a viability decrease, but remained above 75%, compared to controls, when exposed to undiluted samples. Inflammation: proinflammatory molecule TNF-α decreased when DOK cultures were exposed to G-AgNp samples. MDA levels indicated increased oxidative stress damage in cells treated with PMMA, confirmed by the antioxidant mechanism activation, while samples containing G-AgNp induced an antioxidant effect. All tested samples showed antibacterial properties against Gram-positive bacteria. Samples containing G-AgNp also exhibited bactericide action on E. coli. Mechanical properties: both samples containing G-AgNp improved flexural strength compared to the sample resin, measured through elastic strength parameters. CONCLUSIONS: PMMA resin loaded with G-AgNp presents promising antibacterial activity associated with minimal toxicity to human cells, in vitro, as well as improved flexural properties. CLINICAL RELEVANCE: These encouraging results obtained in vitro support further in vivo investigation, to thoroughly check whether the PMMA loaded with graphene-silver nanoparticles constitute an improvement over current denture materials.

Aberrant crypt foci are regionally affected by zinc treatment in a 1,2-dimethylhydrazine induced colon carcinogenesis model.

Zinc is a trace element widely known for its marked antioxidant properties. To gain more insight into the site- and time- specific mechanisms by which it induces chemoprevention, this study was elaborated over a pre-cancerous model of colon carcinogenesis. Colon cancer was induced by 1,2-dimethylhydrazine (DMH) in mice (20 mg/kg for 2 weeks) and groups of animals were supplemented with or without zinc sulfate (ZnSO4, 200 mg/L) in drinking water for 4, 10 or 14 weeks. Colon tissues were collected for pathological observation, analyzing aberrant crypt (AC) and aberrant crypt foci (ACF) formations, multiplicity and distribution. Similarly, histological assessment and mucin production, as well as oxidative stress markers estimation was performed for the different groups. Results showed a significant increase in ACF and AC numbers, ACF multiplicity and demonstrated stronger distal occurrence than in the proximal after DHM administration. Histopathological analysis presented marked structural alterations and mucin loss in the distal than the proximal colons. A significant increase in myeloperoxidase (MPO), nitric oxide (NO), L-ornithine and malondialdehyde (MDA) levels was observed followed by a significant decrease in antioxidant markers (superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH)). Oral ZnSO4 supplementation (continuous or partial) induced significant decrease in ACF, AC numbers and multiplicity, restored histological architecture and mucin production, and a significant decrease in proinflammatory markers while it reduced antioxidants to normal levels. From this study, insight was obtained on the use of ZnSO4 as a chemopreventive agent and shed light on its potential, as a supplement in nutraceutical approaches.

Photodynamic Therapy in Melanoma - Where do we Stand?

BACKGROUND: Malignant melanoma is one of the most aggressive malignant tumors, with unpredictable evolution. Despite numerous therapeutic options, like chemotherapy, BRAF inhibitors and immunotherapy, advanced melanoma prognosis remains severe. Photodynamic therapy (PDT) has been successfully used as the first line or palliative therapy for the treatment of lung, esophageal, bladder, non melanoma skin and head and neck cancers. However, classical PDT has shown some drawbacks that limit its clinical application in melanoma. OBJECTIVE: The most important challenge is to overcome melanoma resistance, due to melanosomal trapping, presence of melanin, enhanced oxidative stress defense, defects in the apoptotic pathways, immune evasion, neoangiogenesis stimulation. METHOD: In this review we considered: (1) main signaling molecular pathways deregulated in melanoma as potential targets for personalized therapy, including PDT, (2) results of the clinical studies regarding PDT of melanoma, especially advanced metastatic stage, (3) progresses made in the design of anti-melanoma photosensitizers (4) inhibition of tumor neoangiogenesis, as well as (5) advantages of the derived therapies like photothermal therapy, sonodynamic therapy. RESULTS: PDT represents a promising alternative palliative treatment for advanced melanoma patients, mainly due to its minimal invasive character and low side effects. Efficient melanoma PDT requires: (1) improved, tumor targeted, NIR absorbing photosensitizers, capable of inducing high amounts of different ROS inside tumor and vasculature cells, possibly allowing a theranostic approach; (2) an efficient adjuvant immune therapy. CONCLUSION: Combination of PDT with immune stimulation might be the key to overcome the melanoma resistance and to obtain better, sustainable clinical results.

Effects of different hypoxia degrees on endothelial cell cultures-Time course study.

INTRODUCTION: Exposure of the endothelial cells to hypoxia, the decrease in oxygen supply can trigger an endothelial response. This response is involved in inflammatory diseases, tumorigenesis, and also with the micro vascular damage associated with aging. The aim of our study was to determine the hypoxia/re-oxygenation induced response in vitro, using human umbilical vein endothelial cells (HUVEC) cultures, at different time points with focus on cell viability, apoptosis oxidative stress and angiogenesis stimulation. MATERIALS AND METHODS: Cells were exposed to 10%, 5% or 0% O2 for 6h, 12h, and 24h. Viability was measured through colorimetry, apoptosis - annexin V-FITC staining, DNA lesions (γH2AX), endothelial activation (sICAM1), angiogenesis (HIF1α), oxidative stress (malondialdehyde, superoxidismutase and NFκB activation) were determined by ELISA, Western Blot and spectrophotometry. RESULTS AND DISCUSSION: Hypoxia decreased viability, increased apoptosis, oxidative stress, endothelial activation and angiogenesis, depending on O2 concentration and time exposure. Short exposures to 5% and 10% O2, efficiently activated anti-apoptotic mechanisms through NFκB activation, HIF1α and γH2AX related DNA damage repair pathways. However, severe hypoxia and longer exposures to mild hypoxia induced high oxidative stress related damage and eventually led to apoptosis, through strong increases of HIF1α and accumulating DNA lesions.

Toxicity and efficiency study of plant extracts-based bleaching agents.

OBJECTIVES: Tooth bleaching is one of the most required dental esthetic treatments. However, it can generate side effects like oral irritation, enamel alteration, tooth sensitivity, especially caused by hydrogen peroxide, the main bleaching component of the commercial products. Therefore, development of new tooth bleaching agents, based on natural products, with comparable esthetic results and lower side effects is needed. The aim of this study was to evaluate the biological effects and bleaching efficacy of four experimental bleaching agents, derived from fruit juices, against the commercially available Opalescence (Ultradent, USA). MATERIALS AND METHODS: Organic acid composition of the gels was characterized by HPLC. Bleaching efficiency was tested by spectrophotometry on composite restorative materials. Biological testing was done in vitro, on human fibroblasts. Cells were exposed to dilutions of the bleaching gel-conditioned medium. Viability was measured by MTS, apoptosis by FACS-AnnexinV FITC/Propidium iodide, NF-kB activation by western blot, malondyaldehide, and superoxide dismutase activity by spectrophotometry. RESULTS: All gels exhibited physical stability and dental bleaching capabilities. Experimental gels induced significantly better viability and apoptosis rates, lower lipid peroxidation, and increased antioxidant defense, compared to Opalescence. CONCLUSIONS: The studied experimental gel formulations exhibited a good safety profile in vitro, as well as bleaching efficiency on restorative composite materials. CLINICAL RELEVANCE: These data open new possibilities for the use of new natural products in dental bleaching treatments that can insure significant esthetic results and lower side effects.

Melanogenesis and DNA damage following photodynamic therapy in melanoma with two meso-substituted porphyrins.

Melanoma, a cancer derived from melanocytes is very difficult to treat, especially in advanced cases. There are several encouraging studies of the efficacy of photodynamic therapy (PDT) in melanoma. However, PDT has to overcome the main defense mechanisms like: defects in the apoptotic pathways, pigmentation, sequestration of the photosensitisers (PS) inside melanosomes and increased oxidative stress defense. Two meso-substituted porphyrins, meso-5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin (THOPP) and meso-5-(4-hydroxyphenyl)-10, 15, 20-tris (4-methoxyphenyl) porphyrin (THOMPP) were used as PS to investigate several mechanisms underlining the PDT anti-melanoma effects, on a lightly pigmented melanoma cell line (WM35), in vitro. γH2AX foci formation (a measure of DNA double strand brakes) was used for the assessment of DNA damage by means of immune-fluorescence and western blot. Cytoskeleton alterations were detected by phalloidin staining. Tyrosinase activity and melanin pigment were quantified by spectrophotometry, tyrosinase protein by western blot, total peroxidase activity by resorurfin reaction (Amplex Red). PDT induced high levels of DNA damage, cytoskeleton alterations and enhanced pigmentogenesis. THOPP mediated PDT was the most efficient. The melanogenesis stimulated by PDT was directly correlated to the PDT induced cellular damage and provided no protection against therapy. Thus, PDT induced melanogenesis combined with severe DNA damage was able to overcome the mechanisms of resistance and increased the efficiency of PDT in WM35 melanoma cells. These results are encouraging for a possible use of PDT, as an adjuvant therapy in lightly pigmented melanomas.

Photodynamic therapy of melanoma using new, synthetic porphyrins and phthalocyanines as photosensitisers - a comparative study.

UNLABELLED: Melanoma, a cancer that arises from melanocytes, is one of the most unresponsive cancers to known therapies and has a tendency to produce early metastases. Several studies showed encouraging results of the efficacy of photodynamic therapy (PDT) in melanoma, in different experimental settings in vitro and in vivo, as well as several clinical reports. AIMS: Our study focuses on testing the antimelanoma efficacy of several new, synthetic photosensitisers (PS), from two different chemical classes, respectively four porphyrins and six phthalocyanines. METHODS: These PS were tested in terms of cell toxicity and phototoxicity against a radial growth phase melanoma cell line (WM35), in vitro. Cells were exposed to different concentrations of the PS for 24h, washed, then irradiatied with red light (630 nm) 75 mJ/cm(2) for the porphyrins and 1 J/cm(2) for the phthalocyanines. Viability was measured using the MTS method. RESULTS: Two of the synthetic porphyrins, TTP and THNP, were active photosensitizers against WM35 melanoma in vitro. Phthalocyanines were effective in producing a dose dependent PDT-induced decrease in viability in a dose-dependent manner. The most efficient was Indium (III) Phthalocyanine chloride, a metal substituted phthalocyanine. CONCLUSIONS: The most efficient photosensitizers for PDT in melanoma cells were the phthalocyanines in terms of tumor cell photokilling and decreased dark toxicity.

Efficiency of photodynamic therapy on WM35 melanoma with synthetic porphyrins: Role of chemical structure, intracellular targeting and antioxidant defense.

Photodynamic therapy (PDT) could be an adjuvant therapy in melanoma, an aggressive cancer that arises from melanocytes. Several reports showed encouraging results of the efficacy of PDT in melanoma on experimental models and in clinical trials. Therefore, we studied the efficacy of two derivatives of tetraphenylporphyrin (TPP): meso-5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin (THOPP) and meso-5-(4-hydroxyphenyl)-10,15,20-tris (4-methoxyphenyl) porphyrin (THOMPP) as photosensitizers for PDT, compared to FDA approved delta aminolevulinic acid (ALA) against a lightly pigmented, melanoma cell line, WM35, in vitro. Both porphyrins were more efficient as photosensitizers, compared to ALA, without dark toxicity. The efficiency depended on the intracellular localization and the molecule structure. THOPP, the most efficient porphyrin localized mainly in mitochondria, while THOMPP accumulated in lysosomes; both showed melanosomal localization. The symmetric THOPP molecule was able to generate increased oxidative stress damage and apoptosis. THOPP also induced a low effect on the defense mechanisms like antioxidant enzyme SOD (superoxide dismutase), NF-kB (nuclear transcription factor kB) activation and MITF (microphthalmia transcription factor). The lower efficiency of the asymmetric molecule, THOMPP was probably due to a diminished photoactivation, which led to a lower ROS induced damage, combined with higher activation of the defense mechanisms.

Usefulness of real-time elastography strain ratio in the assessment of bile duct ligation-induced liver injury and the hepatoprotective effect of chitosan: an experimental animal study.

The purpose of the study described here was to evaluate the usefulness of the elastographic strain ratio in the assessment of liver changes in an experimental animal setting and the hepatoprotective effects of chitosan. Ultrasonography and Strain Ratio calculation were performed before and after bile duct ligation (BDL) in three groups of Wistar albino rats (n = 10 animals per group): (i) rats subjected to bile duct ligation only; (ii) rats subjected to bile duct ligation and administered chitosan for 14 d; (iii) rats subjected to bile duct ligation and administered chitosan for 7 d. The results were compared with the laboratory data and pathologic findings. Strain ratios revealed an increase in liver stiffness after bile duct ligation (p < 0.05), except in the group with chitosan administered for 7 d, and agreed with laboratory and pathology data. In conclusion, strain ratio can be used as an experimental research instrument in the assessment of liver response to injury. To the best of our knowledge, this is the first study reporting on the usefulness of the sonoelastographic liver-to-kidney strain ratio in assessing the effects of experimentally induced liver lesions.