Anti-Obesity Properties of Blackberries Fermented with L. plantarum JBMI F5 via Suppression of Adipogenesis Signaling Mechanisms.

Blackberries (Rubus fruticosus), which are known to include a variety of bioactive substances, have been extensively studied for their antioxidant properties. Blackberries possess multiple health beneficial effects, including anti-inflammation, anti-atherosclerosis, anti-tumor and immunomodulatory activity. However, the potential biological effects and precise molecular mechanisms of the fermented extracts remain largely unexplored. In this research, we demonstrate the effect of blackberries fermented with Lactobacillus for addressing obesity. We investigated the effect of blackberries fermented by Lactobacillus on mice fed a high-fat (60% kcal) diet for 12 weeks. Fermented blackberry administration reduced the body weight and epididymal fat caused by a high-fat diet compared to the obese group. The triglyceride and total cholesterol, which are blood lipid indicators, and the levels of leptin, which is an insulin resistance indicator, were significantly increased in the obese group but were significantly decreased in the fermented blackberries-treated group. Additionally, the expression of adipogenesis marker proteins, such as CEBPα, PPAR-γ and SREBP-1, was significantly increased in the obese group, whereas it was decreased in the fermented blackberries-treated group. These results suggest that fermented blackberries have a protective effect against high-fat-diet-induced obesity by inhibiting adipogenesis and are a potential candidate for the treatment of obesity.

Immunomodulatory effects of a mycelium extract of Cordyceps (Paecilomyces hepiali; CBG-CS-2): a randomized and double-blind clinical trial.

BACKGROUND: Cordyceps is a traditional Chinese herb that produces various biopharmaceutical effects, including immune-enhancing effects. In this study, we prepared a Cordyceps mycelium culture extract (Paecilomyces hepiali, CBG-CS-2) to confirm its efficacy in enhancing the immune system and to evaluate its safety in healthy adults. METHODS: Healthy adults were divided into the intervention group (n = 39), who were given 1.68 g/day of CBG-CS-2 in capsules, and the control group (n = 40) for 8 weeks. The activities of natural killer (NK) cells and serum levels of monocyte-derived mediators were assessed initially for a baseline measurement and after 8 wks. RESULTS: The CBG-CS-2 group showed a significant 38.8 ± 17.6% enhancement from the baseline of NK cell cytotoxic activity relative to the placebo group after the administration of the capsules for 8 wks. (P < 0.019). CONCLUSION: The results suggest that the immune system functions well with CBG-CS-2 supplementation, perhaps with less accompanying inflammation. Thus, CBG-CS-2 is safe and effective for enhancing cell-mediated immunity in healthy adults. TRIAL REGISTRATION: This study was registered at Clinical Trials.gov ( NCT 02814617 ).

The retinoic acid derivative, ABPN, inhibits pancreatic cancer through induction of Nrdp1.

Combination chemotherapy for the treatment of pancreatic cancer commonly employs gemcitabine with an EGFR inhibitor such as erlotinib. Here, we show that the retinoic acid derivative, ABPN, exhibits more potent anticancer effects than erlotinib, while exhibiting less toxicity toward noncancerous human control cells. Low micromolar concentrations of ABPN induced apoptosis in BxPC3 and HPAC pancreatic cancer cell lines, concomitant with a reduction in phosphorylated EGFR as well as decreased ErbB3, Met and BRUCE protein levels. The degradation of ErbB3 is a result of proteasomal degradation, possibly due to the ABPN-dependent upregulation of Nrdp1. Administration of ABPN showed significant reductions in tumor size when tested using a mouse xenograft model, with higher potency than erlotinib at the same concentration. Analysis of the tumors demonstrated that ABPN treatment suppressed ErbB3 and Met and induced Nrdp1 in vivo. The data suggest that ABPN may be more suitable in combination chemotherapy with gemcitabine than the more widely used EGFR inhibitor, erlotinib.

Fermented Aloe arborescens Miller Leaf Extract Suppresses Acute Alcoholic Liver Injury via Antioxidant and Anti-Inflammatory Effects in C57BL/6J Mice.

This study confirmed the change in functional composition and alcohol-induced acute liver injury in Aloe arborescens after fermentation. An acute liver injury was induced by administration of ethanol (3 g/kg/day) to C57BL/6J mice for 5 days. A fermented A. arborescens Miller leaf (FAAL) extract was orally administered 30 minutes before ethanol treatment. After fermentation, the emodin content was approximately 13 times higher than that of the raw material. FAAL extract significantly attenuated ethanol-induced aspartate aminotransferase, alanine aminotransferase, and triglyceride increases in serum and liver tissue. Histological analysis revealed that FAAL extract inhibits inflammatory cell infiltration and fat accumulation in liver tissues. The cytochrome P450 2E1, superoxide dismutase, and glutathione (GSH), which involved in alcohol-induced oxidative stress, were effectively regulated by FAAL extract in serum and liver tissues, except for GSH. FAAL also maintained the antioxidant defense system by upregulating heme oxygenase 1 and nuclear factor erythroid 2-related factor 2 protein expression. In addition, FAAL extract inhibited the decrease in alcohol dehydrogenase and aldehyde dehydrogenase activity, which promoted alcohol metabolism and prevented the activation of inflammatory response. Our results suggest that FAAL could be used as a potential therapeutic agent for ethanol-induced acute liver injury.

Protective Effects of Fermented Paprika (Capsicum annuum L.) on Sodium Iodate-Induced Retinal Damage.

Diseases of the outer retina, including age-related macular degeneration (AMD), are major cause of permanent visual damage. The pathogenesis of AMD involves oxidative stress and damage of the retinal pigment epithelium. Capsicum annuum L. (paprika) fruits have been known as a source of vitamins, carotenoids, phenolic compounds, and metabolites with a well-known antioxidant activity, which have positive effects on human health and protection against AMD and cataracts. In this study, we investigated whether paprika (fermented (FP), yellow, and orange colored) fermented with Lactobacillus (L.) plantarum could increase the protective effect of retinal degeneration using in vitro and in vivo models. FP significantly increased cell survival and reduced levels of lactate dehydrogenase as well as intracellular reactive oxygen species (ROS) increase in SI (sodium iodate, NaIO3)-treated human retinal pigment epithelial (ARPE-19) cells. We developed a model of retinal damage in C57BL/6 mice using SI (30 mg/kg) via intraperitoneal injection. Seven days after SI administration, deformation and a decrease in thickness were observed in the outer nuclear layer, but improved by FP treatment. FP administration protected the SI-mediated reduction of superoxide dismutase and glutathione levels in the serum and ocular tissues of mice. The overproduction of cleaved poly(ADP-Ribose) Polymerase (PARP)1, caspase-3 and -8 proteins were significantly protected by FP in SI-treated cells and ocular tissues. In addition, we evaluated the potentiating effects of FP on antioxidants and their underlying mechanisms in RAW 264.7 cells. Lipopolysaccharide (LPS)-induced nitrite increase was markedly blocked by FP treatment in RAW 264.7 cells. Furthermore, FP reduced LPS-induced inducible nitric oxide synthase and cyclooxygenase-2 activation. The FP also enhanced the inhibitory effects on mitogen activated kinase signaling protein activation in ARPE-19 and RAW 264.7 cells and ocular tissues. There was no significant difference in total phenol and flavonoid content in paprika by fermentation, but the vitamin C content was increased in orange colored paprika, and protective effect against oxidative stress-mediated retinal damage was enhanced after fermentation. These results suggest that FP may be a potential candidate to protect against retinal degenerative diseases through the regulation of oxidative stress.

Fermentation of Blackberry with L. plantarum JBMI F5 Enhance the Protection Effect on UVB-Mediated Photoaging in Human Foreskin Fibroblast and Hairless Mice through Regulation of MAPK/NF-κB Signaling.

Chronic and extensive exposure of ultraviolet (UV)-irradiation causes human skin sunburn, inflammation, or photoaging, which is associated with downregulated collagen synthesis. This study investigated the effects of fermented blackberry (Rubus fruticosus B., FBB) by Lactobacillus plantarum JBMI F5 (LP) on UVB-induced photoaging in human foreskin fibroblast (Hs68) as well as in SKH-1 hairless mice. FBB pretreatment inhibited UVB-mediated type-1 procollagen degradation, matrix metalloproteinase (MMP)-1 and MMP-2 protein expression, and suppressed nuclear factor-κB (NF-κB) activation as well as mitogen-activated protein kinase (MAPK) phosphorylation in Hs68. In addition, FBB administration diminished the wrinkle formation in dorsal skin and epidermal thickening in UVB-irradiated hairless mice. Moreover, UVB-induced Type-1 procollagen reduction and antioxidant enzyme inactivation were reversed by FBB administration. These results suggest that FBB may have antiphotoaging effects on UVB-induced wrinkle formation by maintaining the extracellular matrix density in the dermis, which occurs via regulation of reactive oxygen species and related MAPK and NF-κB signaling. Therefore, FBB can be a potential candidate for protecting skin aging against UV irradiation.

Hypocholesterolemic Effects of Probiotic Mixture on Diet-Induced Hypercholesterolemic Rats.

Growing evidence has indicated that supplementation with probiotics improves lipid metabolism. We aimed to investigate the beneficial effects of a probiotics mixture (PM) of three strains belonging to the species Bifidobacterium (B. longum, B. lactis, and B. breve) and two strains belonging to the species Lactobacillus (L. reuteri and L. plantarum) on cholesterol-lowering efficacy in hypercholesterolemic rats. A hypercholesterolemic rat model was established by feeding a high-cholesterol diet for eight weeks. To test the effects of PM on hypercholesterolemia, hypercholesterolemic rats were assigned to four groups, which were treated daily with low (1.65 × 108 cfu/kg), medium (5.5 × 108 cfu/kg), or high (1.65 × 1010 cfu/kg) doses of probiotic mixture or simvastatin for eight weeks. Significant reductions of serum total cholesterol (TC), triacylglycerol (TG), and low-density lipoprotein (LDL)-cholesterol levels, but increases of high-density lipoprotein (HDL)-cholesterol were observed after supplementation of PM in hypercholesterolemic rats. In PM-supplemented hypercholesterolemic rats, hepatic tissue contents of TC and TG also significantly decreased. Notably, the histological evaluation of liver tissues demonstrated that PM dramatically decreased lipid accumulation. For their underlying mechanisms, we demonstrated that PM reduced expressions of cholesterol synthesis-related proteins such as sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) in the liver. Taken together, these findings suggest that PM has beneficial effects against hypercholesterolemia. Accordingly, our PM might be utilized as a novel therapeutic agent for the management of hypercholesterolemia.

A newly synthesized photostable retinol derivative (retinyl N-formyl aspartamate) for photodamaged skin: profilometric evaluation of 24-week study.

BACKGROUND: Retinol (vitamin A) is used in the cosmetics industry as an antiwrinkle agent. However, its photoinstability and skin irritation potential make it challenging to use in general cosmetic formulations. OBJECTIVE: The aim of this study was to assess the efficacy of a newly synthesized photostable retinol derivative (retinyl N-formyl aspartamate) in patients with photodamaged skin. Retinyl N-formyl aspartamate is a newly synthesized retinol derivative with higher photostability, and a similar effect on collagenase expression level as retinol. METHODS: In all, 29 Korean women (age range: 31-54 years), who were not pregnant, nursing, or undergoing any concurrent therapy, were enrolled in this study. A total of 24 patients completed a 24-week trial of retinyl N-formyl aspartamate twice daily on the left half of the face and a placebo on the right half of the face. A clinical evaluation, photographs, and silicone replicas of both crow's-feet areas were taken at baseline and at weeks 12, 20, and 24. Skin replicas were then analyzed using an optical profilometry technique. The standard wrinkle and roughness features were then calculated and statistically analyzed. The tolerance profile of the product was also clinically evaluated during the study. RESULTS: The 24 women who completed this study showed more improvement on the left side of the crow's-feet area in terms of the signs of photodamage than on the right side according to both their own (P < .001) and the investigator's (P < .05) evaluations. These results were confirmed by skin replica analyses. The average roughness showed significant improvement (P < .001). The smoothness depth was improved, but this was not statistically significant. One patient noted burning and prickling sensations, and she withdrew during the study. LIMITATIONS: Pigmentation changes were not assessed, investigators were not blinded, and the study size was relatively small. CONCLUSION: In this small study retinyl N-formyl aspartamate applied on a photodamaged face twice daily was significantly more effective than a placebo without severe side effects.

In vitro Stimulation of NK Cells and Lymphocytes Using an Extract Prepared from Mycelial Culture of Ophiocordyceps sinensis.

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and used in traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild Ophiocordyceps is becoming increasingly rare in its natural habitat, and its price limits its use in clinical practice. Therefore, the development of a standardized alternative is a great focus of research to allow the use of Ophiocordyceps as a medicine. To develop an alternative for wild Ophiocordyceps, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vitro immune-modulating effect of CBG-CS-2 on natural killer cells and B and T lymphocytes. CBG-CS-2 stimulated splenocyte proliferation and enhanced Th1-type cytokine expression in the mouse splenocytes. Importantly, in vitro CBG-CS-2 treatment enhanced the killing activity of the NK-92MI natural killer cell line. These results indicate that the mycelial culture extract prepared from Ophiocordyceps exhibits immune-modulating activity, as was observed in vivo and this suggests its possible use in the treatment of diseases caused by abnormal immune function.

Antitumor activity of spinasterol isolated from Pueraria roots.

We purified phytoestrogens from Pueraria root (Pueraria mirifica from Thailand and Pueraria lobata from Korea), which is used as a rejuvenating folk medicine in Thailand and China. Dried, powdered plant material was extracted with 100% ethanol and further separated by concentration, filtration, and thin layer silica gel chromatography. Using the fractions obtained during separation, we first investigated their cytotoxicity in several cancer cell lines from various tissues. The ethanol-extracted components (PE1, PE4) had significant antiproliferative effects on breast cancer cell lines, including MCF-7, ZR-75-1, MDA-MB-231, SK-BR-3, and Hs578T. Second, we compared these results with the cytotoxic effects of known flavonoids, sterols, and coumarins from Pueraria root. The known compounds were not as effective, and occurred in a different polarity region on HPLC. Third, further separation resulted in the isolation of eight different components (Sub PE-A to -H). One of these, PE-D, affected the growth of some breast cancer cell lines (MCF-7, MDA- MB-231) in a dose- and time-dependent manner, as well as the growth of ovarian (2774) and cervical cancer cells (HeLa). Finally, a transfection assay showed that this component had an estrogenic effect similar to 17beta - estradiol, which activates both estrogen receptor alpha (ERalpha) and ERbeta. The NMR analysis determined that spinasterol (stigmasta-7, 22-dien-3beta-ol) is an active cytotoxic component of Pueraria root.

Anti-inflammatory effects of Cordyceps mycelium (Paecilomyces hepiali, CBG-CS-2) in Raw264.7 murine macrophages.

Cordyceps (CS) is a traditional Chinese herb with various biological effects that include immune modulation. CBG-CS-2 is a strain, Paecilomyces hepiali, of the Cordyceps spp. The anti-inflammatory effects of CBG-CS-2 were investigated. The water-soluble fraction of CBG-CS-2 has high anti-inflammatory activity in LPS-induced Raw264.7 macrophages. We tested the role of CBG-CS-2 on the anti-inflammation cascade in LPS-stimulated Raw264.7 cells. CBG-CS-2 significantly decreased NO production, iNOS expression, and pro-inflammatory cytokine secretion in a dose-dependent manner. To investigate the mechanism by which CBG-CS-2 inhibits NO, iNOS, and pro-inflammatory cytokines, we examined the activities of NF-κB and AP-1 in LPS-activated macrophages. The results demonstrate that CBG-CS-2 suppresses the production and expression of NO, iNOS, and pro-inflammatory cytokines in LPS-activated macrophages via inhibition of NF-κB and AP-1, which may play an important role in inflammation. These findings suggest that CBG-CS-2 has modulatory effects on the inflammatory system in macrophages, and that it can serve as a useful anti-inflammatory dietary supplement or drug.

Immune-Modulating Activity of Extract Prepared from Mycelial Culture of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild O. sinensis is becoming increasingly rare in its natural habitats, and its price is out of reach for clinical practice. For these reasons, development of a standardized alternative is a great focus of research to allow the use of O. sinensis as a medicine. To develop an alternative for wild O. sinensis, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vivo immune-modulating effect of CBG-CS-2 in mice. Oral administration of CBG-CS-2 supported splenocyte stimulation and enhanced Th1-type cytokine expression from the splenocytes. Importantly, the same treatment significantly enhanced the natural killer cell activity of the splenocytes. Finally, oral administration of CBG-CS-2 enhanced the potential for inflammatory responses. Together, these findings indicate that the mycelial culture extract prepared from O. sinensis exhibited immune-modulating activity and suggest its possible use in the treatment of diseases caused by abnormal immune function.

In vitro antitumor potential of 4-BPRE, a butyryl aminophenyl ester of retinoic acid: role of the butyryl group.

Retinoic acid (RA) and sodium butyrate (NaB) have been implicated in the regulation of growth and differentiation in various cancer cells. To produce an agent with the properties of both RA and NaB, a butyryl aminophenyl ester of RA (4-BPRE) was synthesized. The agent was compared with an aminophenyl ester devoid of the butyryl group (4-APRE) for antitumor potential in vitro. Like RA, 4-hydroxyphenyl retinamide (4-HPR) and 4-APRE, 4-BPRE was an active ligand for all three subtypes of RAR, but not for RXR, as determined by transcription assays in COS-1 cells. In addition, regardless of the butyryl group, 4-BPRE actively suppressed c-Jun transcriptional activity, which may result in reduced expression of matrix metalloproteinases (MMP-1 and MMP-2), and effectively inhibited HCT116 cell invasion into Matrigel. In these respects, 4-BPRE is similar to 4-APRE, and even to RA and 4-HPR. However, our results showed that in HCT116 colon and A549 lung cancer cells, 4-BPRE was much more cytotoxic than RA and 4-APRE, and was also more cytotoxic than 4-HPR, which is the most cytotoxic retinoid derivative under clinical investigation. Subsequent assays using DAPI staining, DNA fragmentation, and FACS analysis suggested that the cytotoxic effect of 4-BPRE is mediated by apoptosis in HCT116 cells. Moreover, 4-BPRE inhibited histone deacetylase (HDAC) activity to some degree, although inhibition was less than that induced by the known HDAC inhibitors TSA and NaB. These results suggest that 4-BPRE could be a promising antitumor retinoid with both NaB activity and RA function.

Utilization of the recombinant human beta-carotene-15,15'-monooxygenase gene in Escherichia coli and mammalian cells.

In animals, beta-carotene 15,15'-monooxygenase (BCMO) is the key enzyme involved in the metabolism of plant beta-carotene to retinal. In the present study, we utilized beta-carotene-producing Escherichia coli to screen for mutants with higher BCMO activity which was monitored by color changes derived from beta-carotene cleavage. Recombinant wild-type and T381L mutant BCMO proteins were purified to near homogeneity in E. coli, and their enzymatic activities were determined by HPLC analysis. The catalytic efficiency for beta-carotene and retinal production of the mutant were 1.5-fold and 1.7-fold higher than those of wild-type, respectively. Further BCMO function in mammalian cells was analyzed by a retinoic acid receptor reporter assay, which responds to the metabolic conversion of beta-carotene to retinoic acid in vivo. Overall, these tools can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from beta-carotene.

Additional sex comb-like 1 (ASXL1), in cooperation with SRC-1, acts as a ligand-dependent coactivator for retinoic acid receptor.

Additional sex comb-like 1 (ASXL1, 170 kDa), a mammalian homolog of Drosophila ASX, was identified as a protein that interacts with retinoic acid receptor (RAR) in the presence of retinoic acid (RA). Systematic binding assays showed that the C-terminal nuclear receptor box (LVMQLL) of ASXL1 and the activation function-2 activation domain (AF-2 AD) core of the RAR are critical for ligand-dependent interaction. The interaction was confirmed using in vitro glutathione S-transferase pulldown and in vivo immunoprecipitation (IP) assays. Confocal microscopy revealed that ASXL1 localizes in the nucleus. In addition to the intrinsic transactivation function of ASXL1, its cotransfection together with an RA-responsive luciferase reporter increased the RAR activity. This ASXL1 activity appears to be mediated through the functional cooperation with SRC-1, as shown by GST pulldown, IP, chromatin IP, and transcription assays. In the presence of ASXL1, more acetylated histone H3 was accumulated on the RA-responsive promoter in response to RA. Finally, stable expression of ASXL1 increased the expression of endogenous RA-regulated genes and enhanced the antiproliferative potential of RA. Overall, these results suggest that ASXL1 is a novel coactivator of RAR that cooperates with SRC-1 and implicates it as a potential antitumor target of RA in RA-resistant cancer cells.

Synthesis of new glycyrrhetinic acid (GA) derivatives and their effects on tyrosinase activity.

To synthesize glycyrrhetinic acid (GA) derivatives (3, 4, 5, 10, 13, 14, 15, and 16), we first removed the ketonic group in the C-11 position, and the carboxylic function at the C-30 position was kept intact, reduced to an alcohol, or transformed to an aldehyde corresponding derivatives 10 and 13. Glycyrrhetinic acid (GA) derivatives (3, 4, 5, 15, and 16) were coupled with 4-amino piperpyridine derivatives (12 and 14) and 4-fluorobenzyl bromide at C-30 carboxylic acid position of glycyrrhetinic acid. In subsequent tyrosinase assays, we found that GA derivatives 4, 5, and 16 were not active at early time points, but strongly inhibited tyrosinase activity at late time points. Of the GA derivatives examined, derivative 5 was most active, with an IC(50) value of 50 microM after 2 h reaction. IC(50) values of derivatives 4 and 16 were 120 and 170 microM, respectively. Further kinetic data indicated that these derivatives are slow-binding inhibitors of tyrosinase. The time-dependent inhibition was reversed when vitamin C or kojic acid was used, that is, both compounds showed active inhibition at early time points. These results suggest that GA derivatives are much more stable than vitamin C or kojic acid, although their intrinsic inhibitory potentials are relatively low. Higher stability and activity suggest that GA derivative 5 might be a useful candidate for skin whitening.

Synthesis and biological activity of novel retinamide and retinoate derivatives.

Retinoic acid and its amide derivative, N-(4-hydroxyphenyl)retinamide (4-HPR), have been proposed as chemopreventative and chemotherapeutic agents. However, their low cytotoxic activity and water solubility limit their clinical use. In this study, we synthesized novel retinoid derivatives with improved cytotoxicity against cancer cells and increased hygroscopicity. Our syntheses were preceded by selective O-acylation and N-acylation, which led to the production of retinoate and retinamide derivatives, respectively, in one pot directly from aminophenol derivatives and retinoic acid without protection. Transcription assays in COS-1 cells indicated that the N-acylated derivatives (2A-5A) and 4-HPR (1A) were much weaker ligands for all three subtypes of retinoic acid receptor (RAR) than all-trans retinoic acid (ATRA), although they showed some selectivity for RARbeta and RARgamma. In contrast, the O-acylated retinoate derivatives (1B-5B) activated all three RAR isotypes without specificity to an extent similar to ATRA. The cytotoxicity was determined using an MTT assay with HCT116 colon cancer cells, and the IC(50) of N-acylated retinamide derivative 4A and O-acylated retinoate derivative 5B was 1.67 microM and 0.65 microM, respectively, which are about five and 13-fold better than that of 4-HPR (8.21 microM), a prototype N-acylated derivative. When retinoate derivative 5B was coupled to organic acid salts, the resulting salt derivatives 5C and 5D had RAR activation and cytotoxicity similar to those of 5B. These data may delineate the relationship between the structure and function of retinoate and retinamide derivatives.

Potent effect of 5-HPBR, a butanoate derivative of 4-HPR, on cell growth and apoptosis in cancer cells.

Fenretinide, 4-(N-hydroxyphenyl) retinamide (4-HPR), has demonstrated anticancer activity associated with a favorable toxicity profile and is now being investigated in several clinical trials. However, its plasma levels in patients have been far lower than the effective concentration required to induce apoptosis (usually 10 microM). This result has led to the synthesis of derivatives with better efficacy. Sodium butyrate's potential as an anticancer agent prompted us to synthesize a butanoate derivative of 4-HPR, 5-hydroxyphenyl butanoate retinamide (5-HPBR) and compare it to the parent compound for antitumor potential in vitro. The cytotoxicity of 5-HPBR was 2- to 6-fold greater than that of 4-HPR against cancer cell lines derived from various tissues. In premalignant bronchial cells (BEAS2B), 5-HPBR exhibited about a 10-fold stronger cytotoxicity than did 4-HPR. Normal CHANG liver cells were unaffected by either 4-HPR or 5-HPBR. Subsequent assays using DNA fragmentation, DAPI staining, FACS and Western blotting suggested that the potent inhibitory effect of 5-HPBR is mediated by apoptosis; the exact mechanism appears to differ among cancer cell types. In transcription assays with COS-1 cells, 5-HPBR selectively activated RARbeta and RARgamma but was a weaker ligand for all 3 subtypes of RAR than either all-trans retinoic acid or 4-HPR. Overall, these data suggest that 4-BHPR may be a promising retinoid with enhanced antitumor activity and reduced toxicity.

Novel retinoic acid derivative ABPN has potent inhibitory activity on cell growth and apoptosis in cancer cells.

Retinoids are natural and synthetic derivatives of vitamin A that have great promise for cancer therapy and chemoprevention. Of the retinoids developed so far, 4-(N-hydroxyphenyl)retinamide (4-HPR or fenretinide) appears to have the best therapeutic potential in vitro and in vivo and is currently being tested in clinical trials for cancer prevention and therapy. To develop other potentially potent antitumor agents, we synthesized 85 retinoid derivatives. In an initial screening of these synthetic retinoids using the HCT116 colon cancer cell line, we found that 4-amino-2-(butyrylamino)phenyl(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate (ABPN or CBG41) induced the greatest growth inhibition, with an IC(50) value of 0.6 microM. Subsequent studies in other cancer cell lines indicated that ABPN was much more growth-inhibitory than all-trans retinoic acid or 4-HPR. Compared to 4-HPR, ABPN induced 5.5- to 70.0-fold more growth inhibition in most cancer cells, with the exception of gynecologic cancer cells. In these cells, the antiproliferative effect was only 1.5- to 2.8-fold more than 4-HPR. We examined the molecular mechanism underlying the difference in growth inhibition between 4-HPR and ABPN. DAPI staining, DNA fragmentation, FACS and Western blotting analyses suggest that ABPN induced apoptosis by activating caspase-3 and -8, which may result in increased PARP cleavage. Unlike 4-HPR, ABPN activated all 3 RAR isotypes to an extent similar to AtRA. In addition, ABPN significantly inhibited AP-1 transcriptional activity and thus greatly suppressed the expression of the matrix metalloproteinase -1, -2 and -3 genes, which are involved in tumor invasion. These results suggest that ABPN may be a promising retinoid derivative offering not only enhanced cytotoxicity, but also increased inhibition of tumor invasiveness.

Potent cytotoxic effects of novel retinamide derivatives in ovarian cancer cells.

4-(N-Hydroxyphenyl)retinamide (also known as 4-HPR or fenretinide), a synthetic amide of all-trans retinoic acid (RA), has been implicated as a promising anticancer agent associated with reducing the toxicity related to RA. However, the low plasma levels of 4-HPR in patients limited clinical trials, leading to a search for derivatives with better efficacy. In this study, we synthesized a series of 4-HPR derivatives in good yields by introducing acetate (compound 1). propionate (2). pyruvate (3). butyrate (4). or stearate (5). to the 4-hydroxylphenyl moiety of 4-HPR. In our initial proliferation assays, we identified compound 3 as the most cytotoxic of the series against four ovarian cancer cell lines (OVCAR-3, PA-1, 2774, and SKOV-3). Dose-response curves yielded IC(50) values of 3.75-7.75 microM for AtRA, 2.80-5.50 microM for 9-cis RA, 0.65-4.05 microM for 4-HPR, and 0.25-0.75 microM for compound 3, depending on the cell type treated. Nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) and DNA fragmentation assays clearly indicated that the antiproliferative effect of compound 3 was mediated by apoptosis. In contrast to natural retinoids, both 4-HPR and compound 3 activated two (RARbeta and RARgamma) of the three retinoic acid receptor (RAR) subtypes tested, but did not activate any of the three retinoid X receptors (RXRs), as determined by transcription assays in OVCAR-3 cells. However, like natural retinoids, 4-HPR and compound 3 actively suppressed c-Jun transcriptional activity. Thus, compound 3 not only showed more potent antiproliferative activity than any other retinoid derivatives tested, but also effectively inhibited the c-Jun activity that has been implicated in tumor promotion and invasion. These results, together with compound 3's selectivity for RAR subtypes, suggest that compound 3 could be an effective anticancer drug for ovarian cancer, with less toxicity than RA.