A novel and easy protocol to obtain 6-alkoxy-Δ4,6-diene-3-one derivatives from available sterols.

Herein we report an unprecedented and efficient methodology for accessing 6-alkoxy-Δ4,6-diene-3-one derivatives. Such scaffolds were serendipitously obtained in the course of the study of the reaction of Δ4-3-keto steroids with catalytic amounts of iodine in refluxing methanol. A series of 6-methoxy and 6-ethoxy- Δ4,6-diene-3-ones were prepared from easily-available sterols in a two-step sequence; first, oxidation of sterols furnished the Δ4-3-keto steroids, which were then refluxed with ethanol or methanol with I2 as catalyst to obtain a series of ten derivatives. Furthermore, this protocol was also effective for the introduction of a larger carbon chain at C-6. Druglikeliness properties of synthesized compounds were predicted using the SwissADME tool.

Preparation and cytotoxic evaluation of new steroidal oximes and aza-homosteroids from diosgenin and cholesterol.

Using cholesterol and diosgenin as starting materials, we have designed a straightforward methodology to prepare in a reduced number of steps a novel series of steroidal oximes and their aza-homolactam analogs with four types of side chains: cholestane, spirostane, 22-oxocholestane and 22,26-epoxycholestene. The products were evaluated for their cytotoxic activity against the MCF-7 breast cancer cell line. Moreover, the selectivity of the most active compounds was determined against peripheral blood lymphocytes. Compounds 5, 8 and 13 were found to be the most active derivatives, exhibiting IC50 values in the low micromolar range (7.9-9.5 µM) and excellent selectivities (IC50 > 100 µM) against the non-tumor cell line.

In search of cytotoxic selectivity on cancer cells with biogenically synthesized Ag/AgCl nanoparticles.

Green synthesis may be a useful approach to achieve selective cytotoxicity of silver nanoparticles on cancer cells and healthy cells. In this study, the concomitant biosynthesis of silver (Ag)/silver chloride (AgCl) nanoparticles from pineapple peel extracts and their behavior on the breast cancer cell line MCF-7 is shown. Bioreactions were monitored at different temperatures. Fourier-transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis), thermogravimetric analysis (TGA), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM) techniques were used to characterize nanoparticle development. The breast cancer cell line MCF-7 was used as a test model to study the cytotoxic behavior of Ag/AgCl nanoparticles and, as a counterpart, the nanoparticles were also tested on mononuclear cells. Ag/AgCl nanoparticles with spherical and triangular morphology were obtained. The size of the nanoparticles (10-70 nm) and the size distribution depended on the reaction temperature. A dose close to 20 µg/mL of Ag/AgCl nanoparticles considerably decreased the cell viability of the MCF-7 line. The best cytotoxicity effects on cancer cells were obtained with nanoparticles at 60 and 80 °C where cell viability was reduced up to 80% at a concentration of 50 µg/mL. A significant preference was observed in the cytotoxic effect of Ag/AgCl nanoparticles against cancer cells in comparison to monocytes.

Addition of proteinase K during the culture alter the physiology of Bacillus thuringiensis culture and the cry1Ac, nprX, nprA, and spo0A gene transcription.

Bacillus thuringiensis is the major bioinsecticide worldwide produced due to the Cry protein activity. Several studies have been done to improve the cost-productivity relation. The neutral protease A (NprA) is the major extracellular protein massively produced during the stationary phase by this bacterium, contributing to the Cry proteins' degradation. Also, the deletion of aprA and nprA genes enhanced the yield of Cry protein, stabilizing it. Therefore, to increase Cry production, one possibility is to degrade the NprA protease in the culture media. In the present study, proteinase K was used to hydrolyze the NprA to increase Cry production. Proteinase K was added during the exponential growth of B. thuringiensis culture. The bacilli and endospores were measured along all culture, while the Cry protein was measured at the end of the culture. The addition of PK affects the bacilli and spore kinetics positively but negatively to the Cry protein (there is no Cry protein detection). Therefore, the gene expression of the cry1Ac, nprX, nprA, and spo0A was measured. The expression of each gene was followed along all culture. Results demonstrated that PK alters both the transcriptional levels and the expression order of the genes.

Parasporin A13-2 of Bacillus thuringiensis Isolates from the Papaloapan Region (Mexico) Induce a Cytotoxic Effect by Late Apoptosis against Breast Cancer Cells.

The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% cell viability at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect normal cells. According to microscopy and flow cytometry, A13-2 parasporin leads to the death of MCF-7 cells by late apoptosis together with necrosis and without allowing the triggering of the survival mechanisms. When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis preferentially kills MCF- 7 (cancer cells) over HEK 293 and PBMC cell lines (normal cells), thus representing a promising alternative for the treatment of cancer breast.

Fibrillar Collagen Type I Participates in the Survival and Aggregation of Primary Hepatocytes Cultured on Soft Hydrogels.

Liver is an essential organ that carries out multiple functions such as glycogen storage, the synthesis of plasma proteins, and the detoxification of xenobiotics. Hepatocytes are the parenchyma that sustain almost all the functions supported by this organ. Hepatocytes and non-parenchymal cells respond to the mechanical alterations that occur in the extracellular matrix (ECM) caused by organogenesis and regenerating processes. Rearrangements of the ECM modify the composition and mechanical properties that result in specific dedifferentiation programs inside the hepatic cells. Quiescent hepatocytes are embedded in the soft ECM, which contains an important concentration of fibrillar collagens in combination with a basement membrane-associated matrix (BM). This work aims to evaluate the role of fibrillar collagens and BM on actin cytoskeleton organization and the function of rat primary hepatocytes cultured on soft elastic polyacrylamide hydrogels (PAA HGs). We used rat tail collagen type I and Matrigel® as references of fibrillar collagens and BM respectively and mixed different percentages of collagen type I in combination with BM. We also used peptides obtained from decellularized liver matrices (dECM). Remarkably, hepatocytes showed a poor adhesion in the absence of collagen on soft PAA HGs. We demonstrated that collagen type I inhibited apoptosis and activated extracellular signal-regulated kinases 1/2 (ERK1/2) in primary hepatocytes cultured on soft hydrogels. Epidermal growth factor (EGF) was not able to rescue cell viability in conjugated BM but affected cell aggregation in soft PAA HGs conjugated with combinations of different proportions of collagen and BM. Interestingly, actin cytoskeleton was localized and preserved close to plasma membrane (cortical actin) and proximal to intercellular ducts (canaliculi-like structures) in soft conditions; however, albumin protein expression was not preserved, even though primary hepatocytes did not remodel their actin cytoskeleton significantly in soft conditions. This investigation highlights the important role of fibrillar collagens on soft hydrogels for the maintenance of survival and aggregation of the hepatocytes. Data suggest evaluating the conditions that allow the establishment of optimal biomimetic environments for physiology and cell biology studies, where the phenotype of primary cells may be preserved for longer periods of time.

Fructose-1,6-bisphosphate reduces the mortality in Candida albicans bloodstream infection and prevents the septic-induced platelet decrease.

Due to the fact that an increased number of patients have experienced bloodstream infections caused by Candida species and the high mortality of this infection, there is a need for a strategy to reduce this scenery. One possible strategy is the use of new drugs, such as fructose-1,6-bisphosphate (FBP), which is a high-energy glycolytic metabolite and has shown to have therapeutic effects in several pathological conditions such as ischemia, shock, toxic injuries, and bacterial sepsis. The aim of this manuscript was to determine the role of FBP in experimental Candida albicans bloodstream infection. We used mice that were divided into three experimental groups: sham (not induced), bloodstream infection (induced with intratracheal instillation of C. albicans) and FBP (bloodstream infection plus FBP 500 mg/kg i.p.). Blood was taken for assessment of complete hematological profile and cytokine assay (IL-6 and MCP-1). Results of the study demonstrated that mortality decreased significantly in groups that received FBP. All cytokine and hematological indexes of FBP group were similar to bloodstream infection group with exception of platelets count. FBP significantly prevented the decrease in platelets. Taken together, our results demonstrate that FBP prevented the mortality in C. albicans bloodstream infection.

Fructose-1,6-bisphosphate protects against Zymosan-induced acute lung injury in mice.

It has been previously showed that fructose-1,6-bisphosphate (FBP) has anti-inflammatory and immunomodulatory effects on several experimental inflammation models. However, the effects and mechanism of FBP on Zymosan-induced acute lung injury (ALI) in mice had not been tested. In this study, our aim was to assess the anti-inflammatory activities of FBP on Zymosan-induced ALI. We found that in vivo treatment with FBP (500 mg/kg i.p.) markedly decreased the nitric oxide (NO) levels in the lungs and significantly reduced bronchoalveolar lavage fluid total cell and neutrophil counts and protein exudation after Zymosan challenge. Furthermore, FBP inhibited inducible nitric oxide synthase (iNOS) activities in RAW macrophages. Meanwhile, FBP did not inhibit the cyclooxigenase 2, interleukin-6, and nuclear factor kappa B transcription. Taken together, these results suggest that FBP shows anti-inflammatory effects through inhibiting lung edema, NO, and iNOS activities.

TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells.

BACKGROUND AND PURPOSE: The TP53 induced glycolysis and apoptosis regulator (TIGAR) functions to lower fructose-2,6-bisphosphate (Fru-2,6-P(2)) levels in cells, consequently decreasing glycolysis and leading to the scavenging of reactive oxygen species (ROS), which correlate with a higher resistance to cell death. The decrease in intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic lesions. Given these good prospects of TIGAR for metabolic regulation and p53-response modulation, we analyzed the effects of TIGAR knockdown in U87MG and T98G glioblastoma-derived cell lines. METHODS/RESULTS: After TIGAR-knockdown in glioblastoma cell lines, different metabolic parameters were assayed, showing an increase in Fru-2,6-P(2), lactate and ROS levels, with a concomitant decrease in reduced glutathione (GSH) levels. In addition, cell growth was inhibited without evidence of apoptotic or autophagic cell death. In contrast, a clear senescent phenotype was observed. We also found that TIGAR protein levels were increased shortly after irradiation. In addition, avoiding radiotherapy-triggered TIGAR induction by gene silencing resulted in the loss of capacity of glioblastoma cells to form colonies in culture and the delay of DNA repair mechanisms, based in γ-H2AX foci, leading cells to undergo morphological changes compatible with a senescent phenotype. Thus, the results obtained raised the possibility to consider TIGAR as a therapeutic target to increase radiotherapy effects. CONCLUSION: TIGAR abrogation provides a novel adjunctive therapeutic strategy against glial tumors by increasing radiation-induced cell impairment, thus allowing the use of lower radiotherapeutic doses.

Steroidogenic impairment due to reduced ovarian transcription of cytochrome P450 side-chain-cleavage (P450scc) and steroidogenic acute regulatory protein (StAR) during experimental nephrotic syndrome.

The nephrotic syndrome is a renal disease characterized by proteinuria, hypoproteinemia, edema and hyperlipidemia. It has been reported that female nephrotic rats are characterized by loss of the oestrus cycle, follicle atresia, low gonadotropin and steroid concentrations; particularly, undetectable estradiol levels. Therefore, to determine the mechanisms involved in the ovarian steroidogenesis impairment, in this present study we evaluated the ovarian expression of the essential steroidogenesis components: cytochrome P450 side cholesterol chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). The experiments were conducted in the rat experimental model of nephrosis induced by puromycin aminonucleoside (PAN) and in control groups. The evaluation of the expression of P450scc and StAR mRNA were performed during the acute phase of nephrosis as well as after the exogenous administration of 1 or 4 doses of human chorionic gonadotrophin (hCG), or a daily dose of FSH or FSH+hCG for 10 days. In addition, serum hormone concentrations, intra-ovarian steroid content, and the reproductive capacity were determined. The results revealed a decreased expression of mRNA of P450scc enzyme and StAR during nephrosis, and eventhough they increased after gonadotropins treatment, they did not conduce to a normal cycling rat period or fertility recovery. This study demonstrates that the mechanism by which ovarian steroid biosynthesis is altered during acute nephrosis involves damage at the P450scc and StAR mRNA synthesis and processing.