Oxidized Cholesterol Derivatives in Fraction B Prepared from Gulf Catfish (Arius bilineatus, Val.) Skin Regulate Calcium Response and Neutrophil Extracellular Traps (NETs) Formation.

In this study, we present in vitro actions of pure commercial preparations of oxidized and/or dehydrated metabolites of cholesterol (OS) identified in the lipid fraction of Fraction B (FB) prepared from a catfish skin preparation on calcium transients and on the formation of human neutrophil extracellular traps (NETs). These investigations are part of an ongoing effort to understand the important roles these compounds play as components of FB when FB is applied to accelerate the healing of wounds and the healing of highly infected non-healing diabetic foot ulcers, without the use of antibiotics. Our aim was to determine potential therapeutic interventions for various disease states. Our results reveal interesting findings, demonstrating specific actions of the individual compounds. Compounds 7α-hydroxy-cholesterol (S3), Cholestane-3,5,6-triol (S5), 5-cholesten-3ß-ol-7-one (S8) and Cholesta-3,5 dien-7-one (S10) are inhibitory, while Cholesterol 5ß,6ß-epoxide (S4) and 5α-cholestane-3,6-dione (S11) activate the response for calcium influx in human neutrophils. A somewhat similar response is observed in dHL60 cell lines, where S3, S5, S7, S8, and cholesta-2,4-diene (S14) inhibit the calcium influx, although S4, S10, and S11 activate the response in this cell line. Furthermore, we observed a relationship between actions against NETosis and calcium transients. Interestingly, relative to the vehicle control, S3, Cholesta-3,5 diene (S9), and S14 appeared to significantly stimulate DNA release (NETosis), while S2, 7α-hydroxy-cholesterol (S6) and cholesta-3,5 dien-7-one (S10) caused lesser stimulation. We provide the IC50 activities for each compound tested in each assay. Calcium influx and NETs formation (NETosis) correlate with diseases exacerbation. These findings offer valuable insights into the potential therapeutic applications of individual OS for various diseases, highlighting their importance in future interventions.

Oxysterols in catfish skin secretions (Arius bilineatus, Val.) exhibit anti-cancer properties.

The edible catfish Arius bilineatus, (Valenciennes) elaborates a proteinaceous gel-like material through its epidermis when threatened or injured. Our on-going studies on this gel have shown it to be a complex mixture of several biologically active molecules. Anti-cancer studies on lipid fractions isolated from the gel-like materials showed them to be active against several cancer cell lines. This prompted us to investigate further the lipid composition of the catfish epidermal gel secretions (EGS). Analysis of the lipid fraction of EGS resulted in identification of 12 oxysterols including cholesterol and 2 deoxygenated steroids i.e., 7α-hydroxy cholesterol, 7ß-hydroxycholesterol, 5,6 epoxycholesterol, 3ß-hydroxycholest-5-ene-7-one and cholesta-3,5-dien-7-one. Progesterone, cholest-3,5-diene, cholesta-2,4-diene, cholest-3,5,6-triol and 4-cholesten-3-one were found as minor components, and were identified through their MS, 1HNMR and FTIR spectral data and were compared with those of the standards. Cholest-3,6-dione, cholesta-4,6-diene-3-one, cholesta-2,4-diene, and cholesta-5,20(22)-dien-3-ol were found only in trace amounts and were identified by GC/MS/MS spectral data. Since cholesterol is the major component of EGS, the identified oxysterols (OS) are presumably cholesterol oxidation products. Many of the identified OS are known important biological molecules that play vital physiological role in the producer and recipient organisms. We report herein the effects of these sterols on three human cancer cell lines in vitro, i.e., K-562 (CML cell line), MDA MB-231 (estrogen positive breast cancer cell line) and MCF-7 (estrogen negative breast cancer cell line). Interestingly significant (p < 0.05) dose differences were observed between tested OS on cell types used. The presence of these sterols in EGS may help explain some aspects of the physiological activities of fraction B (FB) prepared from EGS, such as enhanced wound and diabetic ulcer healing, anti-inflammatory action and cytotoxic activities reported in our previous studies. The anti-proliferative actions of some of these oxysterols especially the cholesterol 3,5,6-triol (#5) as established on selected cancer cell lines in this study support our previous studies and make them candidates for research for human application.

Fraction B From Catfish Epidermal Secretions Kills Pancreatic Cancer Cells, Inhibits CD44 Expression and Stemness, and Alters Cancer Cell Metabolism.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in western countries. The successful treatment of PDAC remains limited. We investigated the effect of Fraction B, which is a fraction purified from catfish (Arius bilineatus, Val.) skin secretions containing proteins and lipids, on PDAC biology both in-vivo and in-vitro. We report here that Fraction B potently suppressed the proliferation of both human and mouse pancreatic cancer cells in vitro and significantly reduced the growth of their relevant xenograft (Panc02) and orthotopic tumors (human Panc-1 cells) (p < 0.05). The Reverse Phase Protein Array (RPPA) data obtained from the tumor tissues derived from orthotopic tumor bearing mice treated with Fraction B showed that Fraction B altered the cancer stem cells related pathways and regulated glucose and glutamine metabolism. The down-regulation of the cancer stem cell marker CD44 expression was further confirmed in Panc-1 cells. CBC and blood chemistry analyses showed no systemic toxicity in Fraction B treated Panc-1 tumor bearing mice compared to that of control group. Our data support that Fraction B is a potential candidate for PDAC treatment.

Potential Mechanism of Dermal Wound Treatment With Preparations From the Skin Gel of Arabian Gulf Catfish: A Unique Furan Fatty Acid (F6) and Cholesta-3,5-Diene (S5) Recruit Neutrophils and Fibroblasts to Promote Wound Healing.

Preparations from Arabian Gulf catfish (Arius bilineatus, Val) epidermal gel secretion (PCEGS) effectively heal chronic wounds in diabetic patients. However, specific lipid components of PCEGS that are responsible for various aspects of wound healing are unknown. Here, we report for the first time that, i) a unique preparation containing only proteins and lipids (Fraction B, FB), derived from the PCEGS accelerated the healing of experimental dermal wounds in female rats (transdermal punch biopsy) in vivo. Histological analyses showed that topical treatment of these wounds with FB promoted the migration of fibroblasts, facilitated the production of extracellular matrix (collagen, fibronectin), induced capillary formation and recruitment of immune cells, and accelerated overall wound healing by day 4 (tested at 1, 2, 3, 4, and 10 days; n=15 for vehicle; n=15 for FB treatment), ii) the lipids responsible for different stages of wound healing were separated into a protein-free bioactive lipid fraction, Ft, which contained a few common long-chain fatty acids, a unique furan fatty acid (F6) and a cholesterol metabolite, cholesta-3,5-diene (S5). Ft (the partially purified lipid fraction of PCEGS), and F6 and S5 present in Ft, proved to be bioactive for wound healing in human dermal fibroblasts. Ft increased the production and extracellular deposition of collagen and fibronectin, ex vivo, iii) Ft and its subcomponents, pure F6 and S5, also promoted human dermal fibroblast migration into the scratch wound gaps, ex vivo, iv) Ft, F6, and S5 promoted the recruitment of neutrophils (Green fluorescence protein labeled) to the site of injury in the transected tailfins of transgenic zebrafish, in vivo, v) Ft, but not F6 or S5, promoted the regeneration of tissues at the wound site in the transgenic zebrafish tailfin, in vivo. Therefore, we conclude that lipid fraction Ft from PCEGS contains the components necessary to promote complete wound healing, and F6 and S5 are responsible for promoting fibroblast and neutrophil recruitment to the site of wounds.

Furanoic Lipid F-6, A Novel Anti-Cancer Compound that Kills Cancer Cells by Suppressing Proliferation and Inducing Apoptosis.

Identifying novel anti-cancer drugs is important for devising better cancer treatment options. In a series of studies designed to identify novel therapeutic compounds, we recently showed that a C-20 fatty acid (12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid, a furanoic acid or F-6) present in the lipid fraction of the secretions of the Arabian Gulf catfish skin (Arius bilineatus Val.; AGCS) robustly induces neutrophil extracellular trap formation. Here, we demonstrate that a lipid mix (Ft-3) extracted from AGCS and F-6, a component of Ft-3, dose dependently kill two cancer cell lines (leukemic K-562 and breast MDA MB-231). Pure F-6 is approximately 3.5 to 16 times more effective than Ft-3 in killing these cancer cells, respectively. Multiplex assays and network analyses show that F-6 promotes the activation of MAPKs such as Erk, JNK, and p38, and specifically suppresses JNK-mediated c-Jun activation necessary for AP-1-mediated cell survival pathways. In both cell lines, F-6 suppresses PI3K-Akt-mTOR pathway specific proteins, indicating that cell proliferation and Akt-mediated protection of mitochondrial stability are compromised by this treatment. Western blot analyses of cleaved caspase 3 (cCasp3) and poly ADP ribose polymerase (PARP) confirmed that F-6 dose-dependently induced apoptosis in both of these cell lines. In 14-day cell recovery experiments, cells treated with increasing doses of F-6 and Ft-3 fail to recover after subsequent drug washout. In summary, this study demonstrates that C-20 furanoic acid F-6, suppresses cancer cell proliferation and promotes apoptotic cell death in leukemic and breast cancer cells, and prevents cell recovery. Therefore, F-6 is a potential anti-cancer drug candidate.

Furanoid F-Acid F6 Uniquely Induces NETosis Compared to C16 and C18 Fatty Acids in Human Neutrophils.

Various biomolecules induce neutrophil extracellular trap (NET) formation or NETosis. However, the effect of fatty acids on NETosis has not been clearly established. In this study, we focused on the NETosis-inducing ability of several lipid molecules. We extracted the lipid molecules present in Arabian Gulf catfish (Arius bilineatus, Val) skin gel, which has multiple therapeutic activities. Gas chromatography⁻mass spectrometry (GC-MS) analysis of the lipid fraction-3 from the gel with NETosis-inducing activity contained fatty acids including a furanoid F-acid (F6; 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid) and common long-chain fatty acids such as palmitic acid (PA; C16:0), palmitoleic acid (PO; C16:1), stearic acid (SA; C18:0), and oleic acid (OA; C18:1). Using pure molecules, we show that all of these fatty acids induce NETosis to different degrees in a dose-dependent fashion. Notably, F6 induces a unique form of NETosis that is rapid and induces reactive oxygen species (ROS) production by both NADPH oxidase (NOX) and mitochondria. F6 also induces citrullination of histone. By contrast, the common fatty acids (PA, PO, SA, and OA) only induce NOX-dependent NETosis. The activation of the kinases such as ERK (extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) is important for long-chain fatty acid-induced NETosis, whereas, in F-acid-induced NETosis, Akt is additionally needed. Nevertheless, NETosis induced by all of these compounds requires the final chromatin decondensation step of transcriptional firing. These findings are useful for understanding F-acid- and other fatty acid-induced NETosis and to establish the active ingredients with therapeutic potential for regulating diseases involving NET formation.

Tryptophan oxidative metabolism catalyzed by geobacillus stearothermophilus: a thermophile isolated from kuwait soil contaminated with petroleum hydrocarbons.

Tryptophan metabolism has been extensively studied in humans as well as in soil. Its metabolism takes place mainly through kynurenine pathway yielding hydroxylated, deaminated and many other products of physiological significance. However, tryptophan metabolism has not been studied in an isolated thermophilic bacterium. Geobacillus stearothermophilus is a local thermophile isolated from Kuwait desert soil contaminated with petroleum hydrocarbons. The bacterium grows well at 65 °C in 0.05 M phosphate buffer (pH 7), when supplied with organic compounds as a carbon source and has a good potential for transformation of steroids and related molecules. In the present study, we used tryptophan ethyl ester as a carbon source for the bacterium to study the catabolism of the amino acid at pH 5 and pH 7. In this endeavor, we have resolved twenty one transformation products of tryptophan by GC/LC and have identified them through their mass spectral fragmentation.

Transformation of chenodeoxycholic acid by thermophilic Geobacillus stearothermophilus.

We performed a series of experiments with Geobacillus stearothermophilus, a thermophile isolated from oil-contaminated soil in the Kuwaiti desert. The organism has a good potential for the transformation of a broad spectrum of organic molecules such as steroids, amino acids, and aromatic hydrocarbons. In the present study, we tested its potential for the transformation of a bile component, chenodeoxycholic acid (CDCA). Five transformed products, namely, cholic acid, methylcholate, methylchenodeoxycholate, 3α-hydroxy-7-oxo-5ß-cholanic acid, and 7α-hydroxy-3-oxo-5ß-cholanic acid, were the major transformation products catalyzed by G. stearothermophilus. Under aerobic conditions, no evidence of side chain degradation, ring cleavage, or dehydrogenation was found among the metabolites of CDCA. CDCA transformation by a thermophile is reported for the first time.

Antioxidant activity of biotransformed sex hormones facilitated by Bacillus stearothermophilus.

Bacillus stearothermophilus, a thermophilic bacterium isolated from Kuwaiti desert, when incubated with exogenous progesterone for 10 days at 65 degrees C produced two monohydroxylated, two dihydroxy isomers of progesterone and a B-Seco compound. These metabolites were purified by TLC and HPLC followed by their identification through (1)H, (13)C NMR and other spectroscopic data. Microbial hydroxylation of 17beta-estradiol resulted in the production of estrone. The effect of some inducers resulted in the production of two metabolites from 17beta-estradiol one of which was identified as 3,6beta,17beta-trihydroxyestra-1,3,5,14(10)-tetrene and the other metabolite remained unidentified. The transformation products were identified through their spectral data and comparison with reference to compounds. Antioxidant activities of progesterone transformed mixture and purified metabolites of 17beta-estradiol were studied by linoleic acid/beta-carotene assay. An enhanced antioxidant activity for progesterone transformation products was observed, when compared to progesterone. A comparison of antioxidant activity of progesterone and 17beta-estradiol transformation products is reported.

Modification of progesterone and testosterone by a food-borne thermophile Geobacillus kaustophilus.

The present work was carried out to study structural modification of steroids by Geobacillus kaustophilus, a bacterial thermophile present in milk and the environment. Incubation of progesterone and testosterone with G. kaustophilus at 65 degrees C resulted in oxygenated steroid nuclei. The oxygenation of the steroid molecule was stereo specific. Seven metabolites of progesterone horizontal line 6beta/6alpha-hydroxytestosterone, 20-hydroxyprogesterone, 6beta-/6alpha-20-dihydroxyprogesterone, 5alpha-pregnane-3,6,20-trione, and 3beta-hydroxy-5alpha-pregnane-6,20-dione horizontal line were identified. Four compounds horizontal line namely, 66-/6--hydroxytestosterone and 6beta/6alpha-hydroxyandrostenedione horizontal line and androst-4-en-3,17-dione were identified as testosterone metabolites. This shows that G. kaustophilus is capable of modifying steroid nuclei at elevated temperatures. G. kaustophilus is a stable thermophile first isolated from milk. Our results show that endogenous steroids present in milk can be modified by G. kaustophilus, causing detrimental effect on human health.

Antioxidant activity of biotransformed sex hormones facilitated by Bacillus stearothermophilus.

Bacillus stearothermophilus, a thermophilic bacterium isolated from Kuwaiti desert, when incubated with exogenous progesterone for 10 days at 65 degrees C produced two monohydroxylated, two dihydroxy isomers of progesterone and a B-Seco compound. These metabolites were purified by TLC and HPLC followed by their identification through (1)H, (13)C NMR and other spectroscopic data. Microbial hydroxylation of 17beta-estradiol resulted in the production of estrone. The effect of some inducers resulted in the production of two metabolites from 17beta-estradiol, one of which was identified as 3,6beta,17beta-trihydroxyestra-1,3,5,14(10)-tetrene and the other metabolite remains unidentified. The transformation products were identified through their spectral data and comparison with reference compounds. Antioxidant activities of progesterone transformed mixture and purified metabolites of 17beta-estradiol were studied by linoleic acid/beta-carotene assay. An enhanced antioxidant activity for progesterone transformation products was observed when compared to progesterone. A comparison of antioxidant activity of progesterone and 17beta-estradiol transformation products is reported.