Menadione and protocatechuic acid: A drug combination with antitumor effects in murine osteosarcoma cells.

Osteosarcoma (OS) is a primary malignant bone tumor that has an abnormal expression of oncogenesis and tumor suppressors and causes dysregulation of various signaling pathways. Thus, novel therapeutic strategies for OS are needed to overcome the resistance of traditional treatments. This study evaluated the cytotoxic and anticancer effects of the association between menadione (MEN) and protocatechuic acid (PCA) in murine OS cells (UMR-106). The concentrations were 3.12 µM of isolated MEN, 500 µM of isolated PCA, and their associations. We performed cell viability assays, morphology modification analysis, cell migration by the wound-healing method, apoptosis by flow cytometry, reactive oxygen species (ROS) production, gene expression of NOX by RT-qPCR, and degradation of MMP-2 and 9 by zymography. Our results showed that the association of MEN+PCA was more effective in OS cells than the compounds alone. The association decreased cell viability, delayed cell migration, and decreased the expression of NOX-2 and ROS. In addition, the MEN+PCA association induced a slight increase in the apoptotic process. In summary, the association can enhance the compound's antitumor effects and establish a higher selectivity for tumor cells, possibly caused by significant mitochondrial damage and antioxidant properties.

Effects of Qualea grandiflora Extract on the Expression of MMP-14 and HIF-1α in Cultured Fibroblasts and Preosteoblasts

Abstract Background: Qualea grandiflora (QG) (Vochysiaceae), also known as "pau-ferro", "pau-terra" or "pau-de-tucano", is a very common deciduous tree in the Brazilian Cerrado used in traditional medicine to treat inflammations, ulcers, diarrhea, and infections. There are reports in the scientific literature that demonstrate the medicinal effects of the bark and leaf of the QG. However, studies involving this plant are rather imited. Aim of the study: To perform the phytochemical analysis of the QG hydroalcoholic extract (HAE) of leaves, and to investigate it effects on fibroblast and preosteoblasts. Methods: Phytochemical analysis was done by HPLC-DAD. Murine NIH/3T3 fibroblasts and MC3T3-E1 preosteoblasts cell lines (ATCC) were used for the experiments. Cell viability was assessed by the MTT colorimetric assay and the expression of MMP-14 and HIF-1α by immunofluorescence. Results and conclusion: The following compounds were identified by HPLC-DAD, such as quinic acid, ethyl galate, ellagic acid derivatives as O-methylellagic acid O-galloyl, O-methylellagic acid O-deoxyhexoside, galloyl derivatives, flavonol glycoside as kaempferol-O-deoxyhexoside, quercetin-O-deoxyhexoside, myricetin-O-deoxyhexoside and the pentacyclic triterpene arjunglucoside. Cell viability results demonstrated no cytotoxic effects in the studied concentrations. We found in QG HAE some compounds with therapeutic properties that can increase the expression of MMP-14 and HIF-1α, in fibroblasts and preosteoblasts. These data suggest that QG HAE has an action on these two molecules widely involved in physiological conditions, such as collagen remodeling, bone development and growth and pathological processes as HIF signaling in cancer metastasis.

The cytotoxic effect of TiF4 and NaF on fibroblasts is influenced by the experimental model, fluoride concentration and exposure time.

OBJECTIVE: Titanium tetrafluoride (TiF4) has shown promising effect in preventing tooth lesions. Therefore, we compared the cytotoxicity of TiF4 with sodium fluoride (NaF) (already applied in Dentistry) considering different fluoride concentrations, pH values and experimental models. MATERIALS AND METHODS: Step 1) NIH/3T3 fibroblasts were exposed to mediums containing NaF or TiF4 (from 0.15 to 2.45% F), both at native and adjusted pH, for 6 h. Step 2) NIH/3T3 were exposed to NaF or TiF4 varnishes with 0.95, 1.95 or 2.45% F (native pH), for 6, 12 or 24 h. We applied MTT (1st and 2nd steps) and Hoescht/PI stain (2nd step) assays. Step 3) NIH/3T3 were exposed to NaF or TiF4 varnish (2.45% F), at native pH, for 6 or 12 h. The cell stiffness was measured by atomic force microscopy (AFM). RESULTS: Step 1) All cells exposed to NaF or TiF4 mediums died, regardless of the F concentration and pH. Step 2) Both varnishes, at 1.90 and 2.45% F, reduced cell viability by similar extents (33-86% at 6 h, 35-93% at 12 h, and 87-98% at 24 h) compared with control, regardless of the type of fluoride. Varnishes with 0.95% F did not differ from control. Step 3) TiF4 and NaF reduced cell stiffness to a similar extent, but only TiF4 differed from control at 6 h. CONCLUSIONS: Based on the results of the 3 experimental steps, we conclude that TiF4 and NaF have similar cytotoxicity. The cytotoxicity was dependent on F concentration and exposure time. This result gives support for testing the effect of TiF4 varnish in vivo.

Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts.

Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm2 or 3.8J/cm2, respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm2 promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica / Leukotriene-induced intracellular mechanisms during osteogenic differentiation

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.(AU)

Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNFα in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.(AU)

"Aroeira" (Myracrodruon urundeuva) methanol extract: the relationship between chemical compounds and cellular effects.

CONTEXT: "Aroeira" [Myracrodruon urundeuva Allemão (Anacardiaceae)] is a tree whose leaves have been studied for therapeutic purposes in medicine and dentistry. OBJECTIVE: The study chemically identifies the leaf extract of aroeira and determines its effect on human gingival fibroblasts. MATERIALS AND METHODS: An 80% methanol leave extract was obtained by maceration and chemically identified through flow-injection analysis-electrospray ionization-ion trap-tandem mass spectrometry (FIA-ESI-IT-MSn). Cytotoxicity of the aroeira's methanol extract was evaluated in lineage of fibroblasts. Adherent cells were treated with different concentrations of aroeira's methanol extract in the medium: 0.1, 1, 10, 100 and 1000 µg/mL. Control cells were cultivated in the medium only. Analyses were done at 24, 48, 72 and 96 h of culture by neutral red assay; and at 24, 48 and 96 h by crystal violet assay. RESULTS: FIA-ESI-IT-MS analysis determined the presence of compounds, for the first time in the species: quercetin-O-glucuronide and quercetin-O-deoxyhexose-O-glucose in the extract. On one hand, neutral red and crystal violet assay showed a reduction (to 50% up until 100%) of cellular viability of groups of 100 and 1000 µg/mL compared with control at 96 h (p < 0.05). On the other hand, lower concentrations (0.1; 1 and 10 µg/mL) of the extract were similar to that of the control at 96 h (p < 0.05), in general. CONCLUSIONS: In view of the results, we can conclude that the extract of aroeira presents tannins and flavonoids. Furthermore, the extract is capable of modulating the viability of human gingival fibroblasts according to its concentration.