The Bioactive Properties of Carotenoids from Lipophilic Sea buckthorn Extract (Hippophae rhamnoides L.) in Breast Cancer Cell Lines.

In women, breast cancer is the most commonly diagnosed cancer (11.7% of total cases) and the leading cause of cancer death (6.9%) worldwide. Bioactive dietary components such as Sea buckthorn berries are known for their high carotenoid content, which has been shown to possess anti-cancer properties. Considering the limited number of studies investigating the bioactive properties of carotenoids in breast cancer, the aim of this study was to investigate the antiproliferative, antioxidant, and proapoptotic properties of saponified lipophilic Sea buckthorn berries extract (LSBE) in two breast cancer cell lines with different phenotypes: T47D (ER+, PR+, HER2-) and BT-549 (ER-, PR-, HER2-). The antiproliferative effects of LSBE were evaluated by an Alamar Blue assay, the extracellular antioxidant capacity was evaluated through DPPH, ABTS, and FRAP assays, the intracellular antioxidant capacity was evaluated through a DCFDA assay, and the apoptosis rate was assessed by flow cytometry. LSBE inhibited the proliferation of breast cancer cells in a concentration-dependent manner, with a mean IC50 of 16 µM. LSBE has proven to be a good antioxidant both at the intracellular level, due to its ability to significantly decrease the ROS levels in both cell lines (p = 0.0279 for T47D, and p = 0.0188 for BT-549), and at the extracellular level, where the ABTS and DPPH inhibition vried between 3.38-56.8%, respectively 5.68-68.65%, and 35.6 mg/L equivalent ascorbic acid/g LSBE were recorded. Based on the results from the antioxidant assays, LSBE was found to have good antioxidant activity due to its rich carotenoid content. The flow cytometry results revealed that LSBE treatment induced significant alterations in late-stage apoptotic cells represented by 80.29% of T47D cells (p = 0.0119), and 40.6% of BT-549 cells (p = 0.0137). Considering the antiproliferative, antioxidant, and proapoptotic properties of the carotenoids from LSBE on breast cancer cells, further studies should investigate whether these bioactive dietary compounds could be used as nutraceuticals in breast cancer therapy.

Modulatory effect of curcumin analogs on the activation of metalloproteinases in human periodontal stem cells.

Periodontitis progresses due to increased levels of active metalloproteinases (MMPs) and the imbalance between MMPs and their tissue inhibitors (TIMPs). Natural curcumin limits the lytic activity of MMPs but has low cellular uptake. Use of synthetic curcumin analogs could be a means of overcoming this limitation of treatment efficiency. Human periodontal stem cells were isolated from gingival tissue, gingival ligament fibers, periodontal ligament, and alveolar bone. The effect of five synthetic curcumin analogs was compared with that of natural curcumin by assessing cytotoxicity [by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay], the cellular uptake (by fluorometry), the proteolytic activities of MMP-2 and -9 (by zymography), and the levels of TIMP-1 (by ELISA). Our results indicated increased cytotoxicity of synthetic curcumins for doses between 100 and 250 µM. At a concentration of 10 µM, cellular uptake of synthetic curcumins varied depending on their chemical structure. The curcumin compounds modulated pro-MMP-2 levels and increased TIMP-1 production. There was no detectable synthesis of pro-MMP-9 and no activation of MMPs 2 and 9. Gingival tissue and gingival ligament fiber stem cells were most responsive to treatment, showing inverse correlations between pro-MMP-2 and TIMP-1 levels. In conclusion, synthetic curcumins influenced the balance between pro-MMP-2 and TIMP-1 in human periodontal stem cells in vitro, and this could open perspectives for their application as adjuvants in periodontal therapy.

Synthesis, structural characterization and biological activity of novel Knoevenagel condensates on DLD-1 human colon carcinoma.

Biologically active Knoevenagel condensates (1-14) of diarylheptanoids: 1,7-bis(3-methoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione and 1,7-bis(3-ethoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione, were synthesized and structurally characterized. Compounds 1-14 exhibited cytotoxicity against colon carcinoma cells, and their antiproliferative effect was associated with a significant decrease of multidrug resistance proteins. One of the underlying mechanisms of these effects is the reduction of intracellular and extracellular SOD enzymes by compounds 1, 12 and 14, which render the tumor cells more vulnerable to oxidative stress.

Metformin plus sorafenib highly impacts temozolomide resistant glioblastoma stem-like cells.

PURPOSE: Glioblastoma stem cells (GSCs), responsible for the dismal disease prognosis after conventional treatments, are driven by overactive signaling pathways, such as PI3K/ AKT/mTOR and RAS/RAF/MAPK. The objective of our study was to target in vitro-GSCs by combining metformin (Met) as a mTOR inhibitor, with sorafenib (Soraf) as a RAF inhibitor. METHODS: GSCs cultured under basal conditions were treated with Met, temozolomide (TMZ), Soraf, Met+TMZ and Met+Soraf; as untreated arm served as control. At 4 hrs of drug exposure, we measured the level of reactive oxygen species (ROS) by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, apoptosis by prodium iodide (PI)-V Annexin staining and efflux pump activity by using the fluorescent dye rhodamine 123. At 24 hrs, we measured cell proliferation by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis and malondialdehyde (MDA) levels. MTT results were compared with corresponding measurements on cultures of non-stem glioblastoma cells and osteoblasts. RESULTS: Met+Soraf exerted the highest antiproliferative effects in GSCs and non-stem glioblastoma cells (p<0.001). Both Met and Soraf monotherapy exhibited a selective cytotoxic effect on GSCs (p<0.001), while no effect was detected on non-stem glioblastoma cells (p>0.05). Soraf, but not Met, impacted the proliferation of normal cells. Soraf displayed synergism with Met in producing high levels of ROS, decreasing efflux pump activity and generating the highest apoptotic rates when compared to either drug alone (p<0.001). CONCLUSION: GSCs were highly sensitive to the combination of Met and Soraf which reduced cell proliferation, increased oxidative stress, inhibited efflux pump activity and ultimately killed GSCs. We strongly believe that these results warrant further in vivo exploration.

Oxaliplatin induces different cellular and molecular chemoresistance patterns in colorectal cancer cell lines of identical origins.

BACKGROUND: Cancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs. Chemotherapy with oxaliplatin is among the leading treatments for colorectal cancer with a response rate of 50%, inducing intrastrand cross-links on the DNA. Despite of this drug's efficiency, resistance develops in nearly all metastatic patients. Chemoresistance being of crucial importance for the drug's clinical efficiency this study aimed to contribute to the identification and description of some cellular and molecular alterations induced by prolonged oxaliplatin therapy. Resistance to oxaliplatin was induced in Colo320 (Colo320R) and HT-29 (HT-29R) colorectal adenocarcinoma cell lines by exposing the cells to increasing concentrations of the drug. Alterations in morphology, cytotoxicity, DNA cross-links formation and gene expression profiles were assessed in the parental and resistant variants with microscopy, MTT, alkaline comet and pangenomic microarray assays, respectively. RESULTS: Morphology analysis revealed epithelial-to-mesenchymal transition in the resistant vs parental cells suggesting alterations of the cells' adhesion complexes, through which they acquire increased invasiveness and adherence. Cytotoxicity measurements demonstrated resistance to oxaliplatin in both cell lines; Colo320 being more sensitive than HT-29 to this drug (P < 0.001). The treatment with oxaliplatin caused major DNA cross-links in both parental cell lines; in Colo320R small amounts of DNA cross-links were still detectable, while in HT-29R not. We identified 441 differentially expressed genes in Colo320R and 613 in HT-29R as compared to their parental counterparts (at least 1.5 -fold up- or down- regulation, p < 0.05). More disrupted functions and pathways were detected in HT-29R cell line than in Colo320R, involving genes responsible for apoptosis inhibition, cellular proliferation and epithelial-to-mesenchymal transition. Several upstream regulators were detected as activated in HT-29R cell line, but not in Colo320R. CONCLUSIONS: Our findings revealed a more resistant phenotype in HT-29R as compared to Colo320R and different cellular and molecular chemoresistance patterns induced by prolonged treatment with oxaliplatin in cell lines with identical origins (colorectal adenocarcinomas).

Superior cytotoxicity and DNA cross-link induction by oxaliplatin versus cisplatin at lower cellular uptake in colorectal cancer cell lines.

Platinum-based chemotherapeutic agents are considered among the most potent anticancer drugs used in the treatment of human tumors. Cisplatin is efficient in the treatment of testicular, ovarian, bladder, and head and neck carcinomas, although its use is limited by severe nephrotoxicity and ototoxicity and resistance. Oxaliplatin has consistently exerted antitumor activity in colon, ovarian, and lung cancers and shown less toxicity than its analogue. Given that most of the literature data are contradictory with respect to the cytotoxicity of these drugs and DNA adduct formation, the present study aimed to determine some of the potential underlying mechanisms in view of their cellular uptakes. We evaluated the cytotoxicity, DNA cross-link formation, and cellular uptake of cisplatin and oxaliplatin in Colo320, HT-29, and Caco-2 colorectal adenocarcinoma cell lines. Our results showed higher cytotoxicity of oxaliplatin in Colo320 (P<0.05) and HT-29 cell lines and of cisplatin in Caco-2 (P<0.05). Oxaliplatin induced more DNA cross-links than cisplatin in a dose-dependent manner in Colo320 cells (P<0.0001); in HT-29 and Caco-2 cells, the induction of DNA damage was not dose dependent. Multiple accumulation of cisplatin versus oxaliplatin occurred in all the cell types, doses, and time points we tested. Oxaliplatin showed more potent biological activities versus cisplatin in terms of a significantly lower cellular uptake. In addition to their analogous mechanisms of action, these drugs might activate different signal transduction pathways, ultimately leading to apoptotic DNA fragmentation and cell death. DNA damage, although perhaps the most important, represents only one aspect of the multiple effects of platinum drugs.

Heterocycles 27. Microwave assisted synthesis and antitumour activity of novel phenothiazinyl-thiazolyl-hydrazine derivatives.

A series of new phenothiazinyl-thiazolyl-hydrazine derivatives were synthesized by Hantzsch cyclization of 1-(10-ethyl-10H-phenothiazin-3-yl)-methylidene-thiosemicarbazide with α-halocarbonyl derivatives. Comparison between classical and microwave assisted synthesis emphasizes the great advantages induced by microwaves irradiation which afforded high reaction yields in much shorter reaction time. Structural assignments were based on spectroscopic methods (high resolution NMR, FTIR, MS). The new compounds were tested in vitro for their antiproliferative activity against tumor cell lines using spectrometric methods. Most of the compounds exhibit cytotoxicity against hepatic and colon tumor cells in a dose-dependent mode and a relationship between the structure and their biological activity was observed.

Assessment of cytotoxicity, apoptosis and DNA damages in Colo320 colorectal cancer cells selected for oxaliplatin resistance.

Despite the notable efficacy of oxaliplatin in the treatment of colorectal cancers, the metastatic tumours ultimately become resistant to the drug. This study investigated whether the oxaliplatin-resistant cells display different behaviour to this drug versus the sensitive cells and if this difference may be further exploited into the clinical treatments improvement. In order to establish a stable cell line resistant to oxaliplatin, a human colorectal cancer cell line (Colo320) was exposed to increasing doses of the drug up to the clinically relevant plasma concentration. Four cell groups with different levels of chemoresistance were subjected to additional doses of oxaliplatin, and their cytotoxicity, apoptosis and DNA damage production were assessed. Cells selected for resistance to oxaliplatin reacted differently to the application of additional doses of the drug, displaying lower toxicity and cellular death and fewer DNA cross-links formation, in accordance with the extent of the oxaliplatin pretreatments. As the cross-links formation by oxaliplatin being the main cause for cytotoxicity of this drug and a correlation between cytotoxicity and clinical outcome being shown repeatedly, we consider that the evaluation of oxaliplatin-induced cytotoxicity, apoptosis and DNA damage could be a valuable tool to assess the tumour cells sensitivity and thus to predict the response to chemotherapy.

Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.

BACKGROUND AND PURPOSE: Ovarian cancers are composed of heterogeneous cell populations, including highly proliferative immature precursors and differentiated cells that may belong to different lineages. The main reason why epithelial ovarian cancer is difficult to treat is the unusual mechanism of dissemination that involves local invasion of pelvic and abdominal organs. But, unlike many other carcinomas, initial dissemination rarely requires blood or lymph vessels. Because it has been proven that aggregates of malignant cells within the ascites of patients diagnosed with ovarian cancer represent an impediment to cure such cancers, in the present study we adopted suspension culture combined with anti-cancer regimens as a laboratory strategy for research of the initial process of peritoneal micrometastasis. EXPERIMENTAL DESIGN: MLS human ovarian cancer cells were cultured in serum-free medium. Cells of passage eight were treated in combination with the anticancer agent doxorubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The acquired increased aggressiveness properties was confirmed by multidrug resistance assays and by their ability to grow in an anchorage-independent manner in vitro as tumor spheroids. RESULTS: Cells selected after chemotherapy had a increased proliferative potential, eliminated Rhodamine 123 in culture and also formed spheroids in suspension. CONCLUSIONS: Here we present direct evidence that the metastasis of human ovarian cancer may be a result of transformation and dysfunction of immature precursor cells in the ovary. Also, spheroid formation may represent a key component of chemotherapy recurrence and a better understanding of these 3D structures can contribute to the development of new treatments for metastatic carcinoma.

Latest advances in chronic rhinosinusitis with nasal polyps endotyping and biomarkers, and their significance for daily practice.

The term chronic rhinosinusitis (CRS) comprises of an assortment of diseases that share a common feature: inflammation of the sinonasal mucosa. The phenotype classification of CRS, based on the presence of polyps, has failed to offer a curative treatment for the disease, particularly in refractory cases. Chronic rhinosinusitis with nasal polyps (CRSwNP) remains a challenging entity. Researchers have made efforts trying to characterize subtypes of the disease according to the endotypes, which are delineated by different immunological pathways, using biomarkers. Even if the inflammatory processes controlling CRSwNP are not fully understood, data suggested that the disease associated with a type 2 inflammatory mechanisms can be also linked to the type 1 or type 3 pathomechanism, being highly heterogeneous. Biomarkers for CRSwNP are proposed, such as: eosinophil count, cytokines, metalloproteinases, bitter and sweet taste receptors, and the nasal microbiome. For endotyping to be clinically applicable and simply determined, biomarkers referring to the intrinsic biomolecular mechanism still need to be found. Precision medicine is becoming the new standard of care, but innovative therapies such as biologics may be rather challenging for the clinicians in their daily practice. This new approach to CRSwNP implies patient selection and a simple algorithm for deciding the right treatment, easy to implement and adjust. Our review points out the ongoing new research on the pathophysiology of CRSwNP, biomarkers and treatment opportunities. It allows clinicians to keep abreast of current evidence-based knowledge and to individualize the management of CRSwNP, especially in refractory cases.

Effects of 60Co gamma-rays on human osteoprogenitor cells.

BACKGROUND: Radiation therapy is one of the most efficient treatments of neoplastic diseases used worldwide. However, patients who undergo radiotherapy may develop side effects that can be life threatening because tissue complications caused by radiation-induced stem cell depletion may result in structural and functional alterations of the surrounding matrix. This treatment also damages the osteogenic activity of human bone marrow by suppressing osteoblasts, leading to post-irradiation sequelae. Even if widely used in oncology, there is still little information on the fate and potential therapeutic efficacy of electromagnetic rays. MATERIAL AND METHODS: We addressed this question using both human mesenchymal stem cells and osteoblasts. Monoclonal antibody characterization identified specific surface markers for stem cells (SSEA-4, CD29, CD105, Oct 3, Nanog and SOX2) and osteoblasts (Osteopontin and Osteonectin). The technique of anti-alkaline phosphatase FITC-staining demonstrated the presence of this specific ectoenzyme. Cells were cultured in complex osteogenic medium (DMEM, 15% fetal calf serum, non-essential amino acids, L-glutamine, dexametazone, ascorbic acid, insulin, TGF-beta, BMP-2 and beta-glycero-phosphate) after being irradiated at 0.5 Gy, 1 Gy, 2 Gy and 4 Gy using a Theratron 1000 60Co source. The viability of irradiated cells was assessed using Trypan Blue staining. The comparison between cell lineages after culture in osteogenic media regarding phenotypical characterization and the intensity of the mineralization process included histology stainings (Alizarin Red S, Alcian Blue and von Kossa), and the MTT-based proliferation assay. RESULTS: After irradiation, the proliferation and differentiation of osteoprogenitor cells is dose-dependent. CONCLUSIONS: This study is one among the first papers investigating the biophysics of low-dose gamma-irradiation on stem cell culture, focusing on the potential applications in radiation oncology and various palliative treatments.

Effects of silver nanoparticles functionalized with Cornus mas L. extract on architecture and apoptosis in rat testicle.

AIM: To assess ultrastructural changes, alterations in matrix metalloproteinase activity and apoptosis induced by silver nanoparticles (AgNPs) in the rat testicle. MATERIALS & METHODS: For 45 days, two groups of animals received different doses of AgNPs (0.8 and 1.5 mg/kg b.w.), and a control group was given the buffer used as vehicle for AgNPs. At 7 and 15 days post-treatment, transmission electron microscopy, TUNEL assay, evaluation of NFkB, pNFkB, p53, Bcl-2 and Nrf2 expressions were performed on the removed testes. RESULTS: Transmission electron microscopy revealed severe ultrastructural changes of interstitial tissue and seminiferous epithelium sustained by positive signal for apoptosis. The promatrix metalloproteinase-2 activity and NFkB, Bcl-2 expressions were increased, mainly at 7 days. CONCLUSION: AgNPs induced severe cell lesions identified even a long time after the exposure.

Targeting MAPK (p38, ERK, JNK) and inflammatory CK (GDF-15, GM-CSF) in UVB-Activated Human Skin Cells with Vitis vinifera Seed Extract.

Ultraviolet B radiation (UVB) activates mitogen-activated protein kinases (MAPK): p38, extracellular signal regulated (ERK), and c-Jun N-terminal (JNK) kinases in human skin cells. Human keratinocytes (KC) exposed to UVB secrete several cytokines (CK), among which the growth differentiation factor-15 (GDF-15) is augmented in inflammatory and aging processes and the granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in cell proliferation, differentiation, and survival, and both CK have implications in skin carcinogenesis. We assessed p38, ERK, JNK, GDF-15, and GM-CSF in UVB-exposed skin cells and a red grape (Vitis vinifera) seed extract's (GSE) capacities to regulate these pathways in UVB-exposed KC. Two concentrations of the GSE extract were selected: GSE1 (37.5 µgEqGA/mL) and GSE2 (75 µgEqGA/mL) and a UVB dose (100 mJ/cm2) within the physiological range. Molecules were assessed with ELISA, semiquantitative results being confirmed by Western blot. UVB triggered the signaling molecules' phosphorylation and the concentrations of CK. All molecules but GM-CSF increased early, at 2 h, from UVB exposure while GM-CSF increased later (at 8 h). MAPK and GDF-15 were regulated by GSE1; GM-CSF, by the higher concentration, GSE2. The amplitude and kinetics of the responses were diverse according to time point, molecules, and the extract's concentration. GSE exerted beneficial effects on MAPK and CK triggered by UVB in human skin cells: reduction of phosphorylation of the assessed signaling molecules and anti-inflammatory effects. Targeting MAPK and specific inflammatory mediators such as GDF-15 and GM-CSF with GSE in UVB-induced skin cells represents a novel and a promising starting point for future photoprotection strategies.

The effect of Sambucus nigra L. extract and phytosinthesized gold nanoparticles on diabetic rats.

Nanomaterials such as gold nanoparticles (NPs) conjugated with natural products have shown good results in lowering the glycated hemoglobin and have an anti-inflamatory effect. The aim of our study is to evaluate the antidiabetic effect of NPs functionalized with Sambucus nigra L. (SN) extract on experimental model of diabetes in rats. Diabetes was induced to 18 Wistar male rats (n=6) by a single intramuscular injection of streptozotocin (30mg/kg body weight - b.w.). SN extract (15mg/kg b.w.), NPs (0.3mg/kg b.w.) and vehicle (normal saline) were administered by gavage once a day, every morning, for 2 weeks. Other 18 animals were used as control groups and were treated with the same compounds, at the same time. Afterwards, blood, liver and muscle samples were taken to assess the oxidant/antioxidant status and the liver for the evaluation of metalloproteinases (MMP)-2 and 9 activities, COX-2 and NFKB expressions and for immunohistochemistry. Serum glycemia, cholesterol, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) were also measured. The administration of NPs extract increased the muscle and systemic GSH/GSSG ratio in the diabetic group vs. diabetic (p<0.03) or non-diabetic groups treated with vehicle (p<0.05) and decreased MDA levels compared to non-diabetic group (p<0.05). COX-2 expression (p<0.0001) and proMMP-2 activity (p<0.05) decreased after pretreatment with NPs in parallel with the reduction of Kupffer cells percent (<0.001). No morphological abnormalities were detected in histopathology. NPs present a great potential for further usage as adjuvants in the diabetic therapy due to the increase of antioxidant defence and reduction of MMPs activity and inflammation in liver tissue.

Vitis vinifera seeds extract for the modulation of cytosolic factors BAX-α and NF-kB involved in UVB-induced oxidative stress and apoptosis of human skin cells.

BACKGROUND AND AIMS: The depletion of the ozone layer allows overexposure of the skin to UV radiation, which is prolonged due to the increasing life expectancy, together with inappropriate life habits contribute to the increasing incidence of cutaneous malignancies. Plant extracts with antioxidant capacities are frequently employed as a means to protect skin against ultraviolet (UV) radiations, thus preventing skin cancers. In the present study we assessed a red grape seed extract (GSE) potential capacities to reduce ultraviolet B (UVB) radiation-induced reactive oxygen species (ROS) and subsequent apoptosis in a human keratinocytes cell line (HaCaT). We identified molecules and pathways modulated by the GSE through which this may exert its photoprotective effect. METHODS: The GSE was standardized according to its polyphenolic content and the most important biologically active compounds, such as epigallocatechin and epicatechin, catechin hydrate, procyanidin B and gallic acid were evidenced by high-performance liquid chromatography. According to the plant extract cytotoxicity on the HaCaT cell line, two concentrations were selected for testing from the non-toxic range: GSE1 (37.5 µgEqGA/ml) and GSE2 (75 µgEqGA/ml). The level of ROS was evaluated with CM-H2DCFDA assay, while apoptosis, Bax-α and NF-kß p65 proteins with ELISA and confirmed by western-blot. RESULTS: Both concentrations of the extract decreased the level of ROS in UVB-irradiated keratinocytes (p<0.001), whereas apoptosis and Bax-α pro-apoptotic protein were only reduced by the higher concentration (GSE2). The NF-kB p65 protein level registered increasing values in time after UVB exposure of the cells, while the tested plant extract re-established its level when its smaller concentration was used (GSE1). CONCLUSION: These results encourage further studies on this extract in order to identify other molecules and pathways through which this extract might exert its beneficial effects and also recommend its use as a potential photoprotective agent.

Active and inactive forms of matrix metalloproteinases 2 and 9 in cutaneous melanoma.

BACKGROUND: Cutaneous melanoma is the most aggressive type of skin cancer, with high implications on the morbidity and mortality of patients. Matrix metalloproteinases (MMP) 2 and 9 have been involved in melanoma progression because they degrade important components of the basement membrane. We studied the relationship between the levels of active and inactive MMP 2, MMP 9, and clinicopathological parameters. MATERIALS AND METHODS: Expression of both active and latent forms of MMP 2 and MMP 9 was evaluated by zymography in 21 melanoma tissue samples and 19 benign melanocytic nevi samples. RESULTS: In the melanoma group, inactive MMP 2 was detected in 100% of samples and active MMP 2 in 95%. Inactive MMP 9 was detected in 85% of samples and active MMP 9 in 38%. In the nevi group, 78.9% of samples expressed inactive MMP 2, 5.26% active MMP 2, 21% inactive MMP 9, and 0% active MMP 9. Both forms of MMP 2 and MMP 9 were found to be correlated with skin tumor malignancy. Expression of active and latent MMP 9 was higher in tumors >2 mm thick (P = 0.03, P = 0.014). A correlation was also found between positive lymph node metastasis, inactive MMP 9, and active MMP 9 expression (r = 0.59 P < 0.01, r = 0.668, P < 0.01). The amount of active and latent form of MMP 2 did not have an impact on lymph node metastasis. CONCLUSIONS: Our study demonstrates that active and latent MMP 2 and MMP 9 correlate with melanoma, and both forms of MMP 9 correlate with positive lymph node metastasis.

The effect of TSPP-mediated photodynamic therapy and Parecoxib in experimental tumours.

AIMS: The study investigated the effects of the combined treatment Parecoxib (Pcox) and 5,10,15,20-tetra-sulphonato-phenyl-porphyrin(TSPP)-mediated photodynamic therapy on Walker 256 carcinosarcoma. MAIN METHODS: Five groups of male Wistar rats were used: the control group, treated with TSPP, group 2, irradiated 24 h thereafter, group 3, treated with Pcox and irradiated 24 h thereafter, groups 4 and 5 treated with combined therapies, TSPP and Pcox before irradiation, and Pcox 24 h after TSPP and irradiation respectively. Tumour inflammation, growth and non-growth factors, apoptosis/necrosis rate and oxidative/nitrosative stress markers were investigated. KEY FINDINGS: Malondialdehyde levels and cyclooxygenase (COX)-2 expression increased significantly in the group treated with Pcox after TSPP-PDT when compared with TSPP + IR group (p < 0.05, p < 0.001 respectively), in correlation with a decrease in glutathione levels (p < 0.05). The quantification of apoptosis, based on the TUNEL-assay, and necrosis rate revealed an increase of apoptotic/necrotic index in the same group (p < 0.05). On the other hand, Pcox administered before irradiation showed a significant increase in both vascular endothelial growth factor (VEGF) and COX-2 levels (p < 0.05) and in nitric oxide production (p < 0.01), when compared with the control group. SIGNIFICANCE: The administration of Pcox after TSPP-mediated PDT showed promising antitumoural effects, leading to an increase in oxidative and nitrosative stress as well as apoptosis/necrosis rate in tumour tissue. These results show that combined regimens that involve selective COX-2 inhibitors administration after irradiation may improve the therapeutic effectiveness of PDT.

Metformin associated with photodynamic therapy--a novel oncological direction.

The aim of our study was to assess the effect of the combined treatment of Metformin (Metf) and 5, 10, 15, 20-tetra-sulfophenyl-porphyrin (TSPP)-mediated photodynamic therapy (PDT) on an in vivo tumour model. Wistar male rats were divided in 6 groups: group 1, treated with TSPP; groups 2 and 4 treated with TSPP and Metf, respectively, and irradiated 24h thereafter; group 3 was treated with Metf and the last two groups received the combined treatment, Metf administered prior (group 5) or after (group 6) irradiation. 72 h from the start of the treatment, tumour tissue was sampled for the investigation of oxidative and nitrosative stress. The apoptotic rate, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions and matrix metalloproteinases activities were also quantified. Malondialdehyde and glutathione levels were significantly elevated in the groups treated with combined therapy (p<0.05). Metf associated with TSPP-PDT reduced iNOS and COX-2 expressions and enhanced nitrotyrosine levels in both therapeutic regimens. Peroxynitrate formation and its cytotoxic effect on tumour cells were related to an elevated index of apoptosis and necrosis. Moreover, MMP-2 activity reached a minimum in the groups which received combined therapy. Our results confirmed that the association of Metf with PDT might prove a new and promising oncological approach.

Gallium phosphinoarylbisthiolato complexes counteract drug resistance of cancer cells.

In cancer therapy the platinum-based drugs are used frequently with a good clinical outcome, but besides unwanted side effects which occur, the tumour cells subjected to treatment are prone to develop tolerance or even multidrug resistance (MDR). Metal compounds with a central atom other than platinum are efficient in targeting the chemoresistant cells, therefore the biological outcome of two recently synthesized gallium phosphinoarylbisthiolato complexes was studied, having the formula [X][Ga{PPh(2-SC6H4)2-κ(3)S,S',P}{PPh(2-SC6H4)2-κ(2)S,S'}] where [X] is either the NEt3H (1) or PPh4 (2) cation. Compounds 1 and 2 display in vitro cytotoxicity against both platinum-sensitive and platinum-resistant cell lines (A2780 and A2780cis). Morphological and ultrastructural evidence points toward their capacity to impair tumour cells survival. This behaviour is based on malignant cells capacity to selectively intake gallium, and to bind to the cellular DNA. They are able to cause massive DNA damage in treated cancer cells, focusing on 7-methylguanine and 8-oxoguanine sites and oxidizing the pyrimidine bases; this leads to early apoptosis of a significant percent of treated cells. The intrinsic and extrinsic apoptotic pathways are influenced through the modulation of gene expression following the treatment with complexes 1 and 2, which accompanies the negative regulation of P-glycoprotein 1 (Pgp-1), an important cellular ABC-type transporter from the multidrug resistance (MDR) family. The studied Ga(III) compounds demonstrated the capacity to counteract the chemoresistance mechanisms in the tumours defiant to standard drug action. Compound 2 shows a good anticancer potential and it could represent an alternative to platinum-based drugs especially in the situation of standard treatment failure.

Grape seed extract as photochemopreventive agent against UVB-induced skin cancer.

BACKGROUND: In the recent years, the use of natural antioxidants as photochemoprotective agents against skin damages produced by ultraviolet radiation gained considerable attention. Our goal was to show that the hydroethanolic extract obtained from red grape seeds, Burgund Mare (BM) variety could have a protective effect on keratinocytes exposed to UVB radiation. MATERIALS AND METHODS: HaCaT keratinocytes were treated with BM extract 30 min. before UVB exposure. The effect was evaluated by assessing cell viability with MTT; the generation of lipid peroxides with malondialdehide (MDA) assay; DNA damage using comet assay; the quantification of DNA photolesions by ELISA and apoptosis by immunocytochemistry with AnnexinV. RESULTS: After irradiation with UVB, HaCaT cells pretreated with BM showed: increased cell viability compared to those exposed to UVB only; significantly lower lipid peroxides level; the lesion scores and DNA photolesions were significantly lower and a significant reduction of the cells undergoing apoptosis. CONCLUSIONS: These results recommend the use of the BM extract as photochemoprotective agent as such or in combination with sunscreens and/or other natural products with similar or complementary properties.