Biomarkers for the early detection of relapses in metastatic colorectal cancers.

PURPOSE: To assess prognostic/predictive value of carcinoembryonic antigen (CEA), transthyretin (TRT), αenolase (NNE), ß2-microglobulin (ß2-micro), B-cell activating factor (BAFF) and circulating tumor cells (CTCs) in metastatic colorectal cancer (mCRC) patients treated with chemotherapy with or without bevacizumab. METHODS: 72 histologically confirmed mCRC patients treated at Oncology Institute Cluj were included. Biomarker levels were measured through validated methods. A manual method was used for CTCs, involving hemolysis, cytospin centrifugation and immunocytochemical staining for pan-cytokeratin. Statistical endpoints were response, progression- free survival (PFS) and overall survival (OS). RESULTS: Initial chemotherapy was fluoropyrimidine/oxaliplatin-based in 93.1%; bevacizumab was added in 58.3% of the patients. Median PFS and OS were 16.4 and 24.4 months. Two-year OS for CR & PR vs SD vs PD were 90% vs 48% vs 12%, respectively (p<0.01). Two-year OS for chemo/ bevacizumab vs chemotherapy: 65% vs 42% (p=0.09). Baseline CEA ≥5 ng/ml had a negative prognostic impact on OS and PFS (p<0.01). High baseline CEA was predictive of improved OS when adding bevacizumab (2-year OS chemo/bevacizumab vs chemo: 60% vs 17%, p<0.01); adding bevacizumab in patients with normal CEA did not improve OS (p=0.29). Higher than cut-off values for TRT had a positive OS prognostic value (p<0.01); higher levels for NNE, ß2-microglobulin and BAFF had a negative impact (p<0.01). Two-year OS for baseline <1 CTC/ml vs ≥1 CTC/ ml was 74% vs 64% respectively (p=0.15). CONCLUSIONS: The evaluated biomarkers could be useful prognostic factors for survival. Baseline CEA also has predictive value, suggesting that patients with low levels do not benefit from bevacizumab. A non-statistically significant correlation was observed between the number of CTCs and outcome.

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.

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.

Synthesis of structural analogues of hexadecylphosphocholine and their antineoplastic, antimicrobial and amoebicidal activity.

Twelve derivatives of hexadecylphosphocholine (miltefosine) were synthesized to determine how the position and length of the alkyl chain within the molecule influence their biological activities. The prepared alkylphosphocholines have the same molecular formula as miltefosine. Activity of the compounds was studied against a spectrum of tumour cells, two species of protozoans, bacteria and yeast. Antitumour efficacy of some alkylphosphocholines measured up on MCF-7, A2780, HUT-78 and THP-1 cell lines was higher than that of miltefosine. The compounds showed antiprotozoal activity against Acanthamoeba lugdunensis and Acanthamoeba quina. Some of them also possess fungicidal activity against Candida albicans equal to miltefosine. No antibacterial activity was observed against Staphylococcus aureus and Escherichia coli. A difference in position of a long hydrocarbon chain within the structure with maximum efficacy was observed for antitumour, antiprotozoal and antifungal activity.

Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate.

BACKGROUND: The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol-gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO2). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation. RESULTS: The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO2 and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8-9 with an advantage for Ti SiO2 implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO2 implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO2 implants. CONCLUSIONS: The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO2 scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFß1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.

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.

Anticancer and antimicrobial activities of some antioxidant-rich cameroonian medicinal plants.

Traditional remedies have a long-standing history in Cameroon and continue to provide useful and applicable tools for treating ailments. Here, the anticancer, antimicrobial and antioxidant activities of ten antioxidant-rich Cameroonian medicinal plants and of some of their isolated compounds are evaluated.The plant extracts were prepared by maceration in organic solvents. Fractionation of plant extract was performed by column chromatography and the structures of isolated compounds (emodin, 3-geranyloxyemodin, 2-geranylemodin) were confirmed spectroscopically. The antioxidant activity (AOA) was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) bleaching method, the trolox equivalent antioxidant capacity (TEAC), and the hemoglobin ascorbate peroxidase activity inhibition (HAPX) assays. The anticancer activity was evaluated against A431 squamous epidermal carcinoma, WM35 melanoma, A2780 ovary carcinoma and cisplatin-resistant A2780cis cells, using a direct colorimetric assay. The total phenolic content in the extracts was determined spectrophotometrically by the Folin-Ciocalteu method. Rumex abyssinicus showed the best AOA among the three assays employed. The AOA of emodin was significantly higher than that of 3-geranyloxyemodin and 2-geranylemodin for both TEAC and HAPX methods. The lowest IC(50) values (i.e., highest cytotoxicity) were found for the extracts of Vismia laurentii, Psorospermum febrifugum, Pentadesma butyracea and Ficus asperifolia. The Ficus asperifolia and Psorospermum febrifugum extracts are selective against A2780cis ovary cells, a cell line which is resistant to the standard anticancer drug cisplatin. Emodin is more toxic compared to the whole extract, 3-geranyloxyemodin and 2-geranylemodin. Its selectivity against the platinum-resistant A2780cis cell line is highest. All of the extracts display antimicrobial activity, in some cases comparable to that of gentamycin.

Modulation of doxorubicin-induced oxidative stress by a grape (Vitis vinifera L.) seed extract in normal and tumor cells.

The major limitation of Doxorubicin (Dox) clinical use is the development of chronic and acute toxic side effects induced through the generation of reactive oxygen species. The present work was designated to investigate in vitro effects of a red grape-seed hydroethanolic extract Burgund Mare (BM), in associated administration with Dox (30 min before drug administration) in normal (Hfl-1) and tumor cell lines (HepG2 and Mls). The BM concentrations administered were below the level of the extract cytotoxiciy threshold (40 µg gallic acid [GA] Eq/mL; 37.5, 25.0, and 12.5 µg GA Eq/mL). The antioxidant capacity of the BM extract was assessed by measuring the acute toxicity at 24 h, lipid peroxides (LP), and protein oxidation. In normal cells, the product statistically decreased cytotoxicity and markedly inhibited LP and protein carbonyl (PC) formation, in a dose-dependent relationship. On contrary, in tumor cells, such treatment resulted in a reversed effect, cell death, malondialdehyde, and PC contents increasing with BM dose enhancement. BM extract treatment prior to subsequent administration of Dox afforded a differential protection against Dox-negative toxic side effects in normal cells without weakening (even enhancing) Dox's antitumor activity.

Photoprotective effect of Calluna vulgaris extract against UVB-induced phototoxicity in human immortalized keratinocytes.

There is an increasing interest in the use of natural antioxidants as photoprotective agents against skin damages produced by ultraviolet radiation. The aim of our study was to investigate the protective effect of a Calluna vulgaris extract in human keratinocytes (HaCaT) exposed to ultraviolet B (UVB) radiation. HaCaT cells were treated with C. vulgaris extract 30 minutes prior to irradiation with UVB. The protective effect was evaluated by assessing cell viability using tetrasolium salt (MTT) assay; the generation of lipid peroxides was evaluated using malondialdehide assay (MDA); and DNA damage was evaluated using the comet assay and the quantification by ELISA of specific DNA photolesions [i.e., cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs)]. After irradiation with cytotoxic doses of UVB (300 and 500 mJ/cm(2)), HaCaT cells pretreated with C. vulgaris extract (50 µg GAE/ml) showed significantly increased viability compared to control cells exposed to UVB only. Irradiation alone increased MDA levels in a dose-dependent fashion. Pretreatment with 12 µg GAE/ml extract lowered MDA levels both at 100 mJ/cm(2) (ρ<0.01) and 300 mJ/cm(2) (ρ<0.001). Treatment with C. vulgaris extract before exposure to UVB also reduced DNA damage: Lesion scores in a comet assay were significantly reduced at UVB doses of 50 mJ/cm2 (ρ<0.01) and 100 mJ/cm(2) (ρ<0.05), while CPDs and 6-4PPs (via ELISA) were significantly lower after irradiation with 100 mJ/cm(2) in the protected cells (ρ<0.05 for CPDs and ρ<0.001 for 6-4PPs). These results recommend the use of the C. vulgaris extract as photoprotective agent, in combination with sunscreens and/or other natural products with similar or complementary properties.