In VitroandIn VivoAnticancer Effects of Sterol Fraction from Red AlgaePorphyra dentata

Porphyra dentata, an edible red macroalgae, is used as a folk medicine in Asia. This study evaluatedin vitroandin vivothe protective effect of a sterol fraction fromP. dentataagainst breast cancer linked to tumor-induced myeloid derived-suppressor cells (MDSCs). A sterol fraction containing cholesterol,β-sitosterol, and campesterol was prepared by solvent fractionation of methanol extract ofP. dentata in silicagel column chromatography. This sterol fractionin vitrosignificantly inhibited cell growth and induced apoptosis in 4T1 cancer cells. Intraperitoneal injection of this sterol fraction at 10 and 25 mg/kg body weight into 4T1 cell-implanted tumor BALB/c mice significantly inhibited the growth of tumor nodules and increased the survival rate of mice. This sterol fraction significantly decreased the reactive oxygen species (ROS) and arginase activity of MDSCs in tumor-bearing mice. Therefore, the sterol fraction fromP. dentatashowed potential for protecting an organism from 4T1 cell-based tumor genesis.

Two Clinical Isolates of Candida glabrata Exhibiting Reduced Sensitivity to Amphotericin B Both Harbor Mutations inERG2

ABSTRACTTwo novel isolates ofCandida glabrataexhibiting reduced sensitivity to amphotericin B (MIC, 8 μg ml−1) were found to beERG2mutants, wherein Δ8-sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr121in ERG2; the corresponding residue (Thr119) inSaccharomyces cerevisiaeis essential for sterol Δ8-Δ7 isomerization. This constitutes the first report ofC. glabrataharboring mutations inERG2and exhibiting reduced sensitivity to amphotericin B.

Identification and Characterization of Four Azole-Resistant erg3 Mutants of Candida albicans

ABSTRACT Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol Δ5,6-desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ≥256, 16, 16, 8, and 1 μg ml−1, respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 μg ml−1); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 μg ml−1). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.

A Clinical Isolate of Candida albicans with Mutations in ERG11 (Encoding Sterol 14α-Demethylase) and ERG5 (Encoding C22 Desaturase) Is Cross Resistant to Azoles and Amphotericin B

ABSTRACT A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 μg ml−1, respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 μg ml−1 for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14α-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 μg ml−1). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.

Total, Free and Conjugated Sterolic Forms in Three Microalgae Used in Mariculture

Total, free and conjugated forms (steryl esters, steryl glycosides and acyl steryl glycosides) of sterols from three microalgae that are extensively used in mariculture (Tetraselmis chuii, Nannochloropsis salina and Skeletonema costatum) were examined. The results revealed that cholesterol is the only common fraction detected in all investigated species and distributed in free and all conjugated forms. However, the total sterol content of T. chuii was about 325 μg/g dry wt, most of it was concentrated amongst 24-methylcholesta-5,24-diene-3β-ol and 24-methylcholest-5-en-3β-ol. On the other hand, the majority of the fractions were distributed in the free form. The total sterol content of N. salina was about 180 μg/g dry wt, cholesterol was the major fraction that was detected. Nevertheless, the dominant distribution forms were esterified. While in S. costatum, the total sterol content was 76 μg/g dry wt, approximately most fractions are quantitatively alike and dominated in the free form. Furthermore, our study shows clearly that most sterols are not distributed regularly within each form, a result that encouraged us to suggest a distribution of specific sterol fraction as a free or conjugated can be used as a serving tool in chemotaxonomic studies.

GC/MS Analysis of Some Bioactive Constituents from Carthamus lanatus L.

Sterols, triterpenes, volatiles, polar and other constituents in aerial parts of Carthamus lanatus were analyzed by gas chromatography-mass spectrometry. Over 90 compounds were identified most of them new for the species. Sitosterol and stigmasterol were the most abundant of 10 sterols identified in the sterol fraction. Taraxasterol, α- and β-amyrine prevailed in the triterpene fraction. Volatiles, sterols and a fraction of the dichloromethane extract showed strong cytotoxicity (Artemia salina assay).

Studies to Investigate the Absorption and Excretion of Shea Oleine Sterols in Rat and Man

Shea oleine (SU), an oil fraction derived from the nut of the African tree, Butyrospermum parkii, is used as a frying oil and, after hardening (SH), in margarine and toffee fat. Both SU and SH contain a high level (approximately 8%) of 4,4-dimethylsterols (4,4-DMS), mostly as esters of cinnamic acid. As part of a series of studies evaluating SU, investigations to study rat and human dietary utilization were carried out. These comprised fecal fat analysis of groups of Wistar rats and a small number (four subjects) of human volunteers. Groups of rats were administered SU in a semisynthetic diet over 3 weeks at up to 20% in the diet (approximately 10 g/kg/day). In the human study, four male volunteers consumed a single 25-g portion of SU (approximately 0.4 g/kg) and ate no other vegetable fat during the course of the study. No preferential absorption of any of the 4,4-DMS occurred in the rat or man. Apparent absorption of the most prominent sterol fraction in the unsaponifiable material, 4,4-DMS, as estimated by its disappearance from feces, was similar in both species (27% to 52% in the rat compared with 13% to 49% in human subjects). Both rats and humans showed a similar profile of dietary and fecal 4,4-DMS fraction sterol components.