A cottonseed oil-enriched diet improves liver and plasma lipid levels in a male mouse model of fatty liver.

A high-fat (HF) diet causes fatty liver, hyperlipidemia, and hypercholesterolemia, and cottonseed oil (CSO) has been shown to improve liver and plasma lipids in human and mouse models. The purpose of this study was to determine the effect of CSO vs. olive oil (OO)-enriched diets on lipid levels in a HF-diet model of fatty liver disease. We placed mice on a HF diet to induce obesity and fatty liver, after which mice were placed on CSO or OO diets, with chow and HF (5.1 kcal/g) groups as control. When CSO- and OO-fed mice were given isocaloric diets with the HF group, there were no differences in body weight, plasma, or hepatic lipids. However, when the CSO and OO diets were reduced in calories (4.0 kcal/g), CSO and OO groups reduced body weight. The CSO group had lower plasma total cholesterol (-56 ± 6%, P < 0.01), free cholesterol (-53 ± 7%, P < 0.01), triglycerides (-61 ± 14%, P < 0.01), and LDL (-42 ± 16%, P = 0.01) vs. HF group whereas the OO diet lowered LDL (-18 ± 12%, P = 0.05) vs. HF. Furthermore, the CSO diet decreased hepatic total cholesterol (-40 ± 12%, P < 0.01), free cholesterol (-23 ± 11%, P = 0.04), and triglycerides (-47 ± 12%, P = 0.02). There were no significant changes in lipogenesis and fatty acid oxidation among the groups. However, the CSO group increased lipid oxidative gene expression in liver and dihydrosterculic acid increased PPARα target genes with in vitro models. Taken together, consuming a reduced calorie diet enriched in CSO reduces liver and plasma lipid profiles in an obese model of fatty liver.

Production of tocotrienols in seeds of cotton (Gossypium hirsutum L.) enhances oxidative stability and offers nutraceutical potential.

Upland cotton (Gossypium hirsutum L.) is an economically important multi-purpose crop cultivated globally for fibre, seed oil and protein. Cottonseed oil also is naturally rich in vitamin E components (collectively known as tocochromanols), with α- and γ-tocopherols comprising nearly all of the vitamin E components. By contrast, cottonseeds have little or no tocotrienols, tocochromanols with a wide range of health benefits. Here, we generated transgenic cotton lines expressing the barley (Hordeum vulgare) homogentisate geranylgeranyl transferase coding sequence under the control of the Brassica napus seed-specific promoter, napin. Transgenic cottonseeds had ~twofold to threefold increases in the accumulation of total vitamin E (tocopherols + tocotrienols), with more than 60% γ-tocotrienol. Matrix assisted laser desorption ionization-mass spectrometry imaging showed that γ-tocotrienol was localized throughout the transgenic embryos. In contrast, the native tocopherols were distributed unequally in both transgenic and non-transgenic embryos. α- Tocopherol was restricted mostly to cotyledon tissues and γ-tocopherol was more enriched in the embryonic axis tissues. Production of tocotrienols in cotton embryos had no negative impact on plant performance or yield of other important seed constituents including fibre, oil and protein. Advanced generations of two transgenic events were field grown, and extracts of transgenic seeds showed increased antioxidant activity relative to extracts from non-transgenic seeds. Furthermore, refined cottonseed oil from the two transgenic events showed 30% improvement in oxidative stability relative to the non-transgenic cottonseed oil. Taken together, these materials may provide new opportunities for cottonseed co-products with enhanced vitamin E profile for improved shelf life and nutrition.

Dihydrosterculic acid from cottonseed oil suppresses desaturase activity and improves liver metabolomic profiles of high-fat-fed mice.

Polyunsaturated fatty acid (PUFA)-rich diets are thought to provide beneficial effects toward metabolic health in part through their bioactive properties. We hypothesized that increasing PUFA intake in mice would increase peroxisome proliferator activated receptor delta (PPARδ) expression and activity, and we sought to examine the effect of different PUFA-enriched oils on muscle PPARδ expression. One of the oils we tested was cottonseed oil (CSO) which is primarily linoleic acid (53%) and palmitic acid (24%). Mice fed a CSO-enriched diet (50% energy from fat) displayed no change in muscle PPARδ expression; however, in the liver, it was consistently elevated along with its transcriptional coactivator Pgc-1. Male mice were fed chow or CSO-, saturated fat (SFA)-, or linoleic acid (18:2)-enriched diets that were matched for macronutrient content for 4 weeks. There were no differences in food intake, body weight, fasting glucose, glucose tolerance, or energy expenditure between chow- and CSO-fed mice, whereas SFA-fed mice had increased fat mass and 18:2-fed mice were less glucose tolerant. Metabolomic analyses revealed that the livers of CSO-fed mice closely matched those of chow-fed but significantly differed from SFA- and 18:2-enriched groups. Fatty acid composition of the diets and livers revealed an impairment in desaturase activity and the presence of dihydrosterculic acid (DHSA) in the CSO-fed mice. The effect of DHSA on PPARδ and stearoyl-CoA desaturase-1 expression mimicked that of the CSO-fed mice. Taken together, these data suggest that DHSA from CSO may be an effective means to increase PPARδ expression with concomitant suppression of liver stearoyl-CoA desaturase-1 activity.

Di(phenylpropylamino)gossypol: a derivative of the dimeric natural product gossypol.

Di(phenylpropylamino)gossypol [systematic name: 2,2'-bis{1,6-dihydroxy-5-isopropyl-8-[(3-phenylpropylamino)methylidene]naphthalen-7-one}, C48H52N2O6, was formed by reaction of the dimeric natural product gossypol with 3-phenylpropylamine. The structure of this compound has its two naphthalene ring systems oriented approximately perpendicular to each other, and the two pendant phenylpropyl groups have different conformations. One of these side groups is considerably disordered at room temperature but less so at 120 K. The enantiomeric molecules form centrosymmetric dimers that are supported by intermolecular hydrogen bonds and by hydrophobic interactions between a pair of naphthalene rings. Two additional hydrogen bonds tie the dimer pairs into layers. Unlike gossypol and many gossypol Schiff base derivatives, the title compound crystallizes without the inclusion of solvent, which appears to occur because of the size and flexibility of its phenylpropyl pendent groups.

Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi.

Racemic gossypol and its related derivatives gossypolone and apogossypolone demonstrated significant growth inhibition against a diverse collection of filamentous fungi that included Aspergillus flavus, Aspergillus parasiticus, Aspergillus alliaceus, Aspergillus fumigatus, Fusarium graminearum, Fusarium moniliforme, Penicillium chrysogenum, Penicillium corylophilum, and Stachybotrys atra. The compounds were tested in a Czapek agar medium at a concentration of 100 µg/mL. Racemic gossypol and apogossypolone inhibited growth by up to 95%, whereas gossypolone effected 100% growth inhibition in all fungal isolates tested except A. flavus. Growth inhibition was variable during the observed time period for all tested fungi capable of growth in these treatment conditions. Gossypolone demonstrated significant aflatoxin biosynthesis inhibition in A. flavus AF13 (B(1), 76% inhibition). Apogossypolone was the most potent aflatoxin inhibitor, showing greater than 90% inhibition against A. flavus and greater than 65% inhibition against A. parasiticus (B(1), 67%; G(1), 68%). Gossypol was an ineffectual inhibitor of aflatoxin biosynthesis in both A. flavus and A. parasiticus. Both gossypol and apogossypolone demonstrated significant inhibition of ochratoxin A production (47%; 91%, respectively) in cultures of A. alliaceus.

Characterization of D-glucaric acid using NMR, X-ray crystal structure, and MM3 molecular modeling analyses.

D-Glucaric acid was characterized in solution by comparing NMR spectra from the isotopically unlabeled molecule with those from D-glucaric acid labeled with deuterium or carbon-13 atoms. The NMR studies provided unequivocal assignments for all carbon atoms and non-hydroxyl protons of the molecule. The crystal structure of D-glucaric acid was obtained by X-ray diffraction techniques and the structure was a close match to the low energy conformation generated from a Monte-Carlo-based searching protocol employing the MM3 molecular mechanics program. The molecule adopts a bent structure in both the crystalline and computationally generated lowest-energy structure, a conformation that is devoid of destabilizing eclipsed 1,3-hydroxyl interactions.

Monte Carlo-based searching as a tool to study carbohydrate structure.

A torsion angle-based Monte Carlo searching routine was developed and applied to several carbohydrate modeling problems. The routine was developed as a Unix shell script that calls several programs, which allows it to be interfaced with multiple potential functions and various utilities for evaluating conformers. In its current form, the program operates with several versions of the MM3 and MM4 molecular mechanics programs and has a module to calculate hydrogen-hydrogen coupling constants. The routine was used to study the low-energy exo-cyclic substituents of ß-D-glucopyranose and the conformers of D-glucaramide, both of which had been previously studied with MM3 by full conformational searches. For these molecules, the program found all previously reported low-energy structures. The routine was also used to find favorable conformers of 2,3,4,5-tetra-O-acetyl-N,N'-dimethyl-D-glucaramide and D-glucitol, the latter of which is believed to have many low-energy forms. Finally, the technique was used to study the inter-ring conformations of ß-gentiobiose, a ß-(1→6)-linked disaccharide of D-glucopyranose. The program easily found conformers in the 10 previously identified low-energy regions for this disaccharide. In 6 of the 10 local regions, the same previously identified low-energy structures were found. In the remaining four regions, the search identified structures with slightly lower energies than those previously reported. The approach should be useful for extending modeling studies on acyclic monosaccharides and possibly oligosaccharides.

6,6'-Dimethoxygossypolone.

6,6'-Dimethoxygossypolone (systematic name: 7,7'-dihydroxy-5,5'-diisopropyl-6,6'-dimethoxy-3,3'-dimethyl-1,1',4,4'-tetraoxo-2,2'-binaphthalene-8,8'-dicarbaldehyde), C(32)H(30)O(10), is a dimeric molecule formed by oxidation of 6,6'-dimethoxygossypol. When crystallized from acetone, 6,6'-dimethoxygossypolone has monoclinic (P2(1)/c) symmetry, and there are two molecules within the asymmetric unit. Of the four independent quinoid rings, three display flattened boat conformations and one displays a flattened chair/half-chair conformation. The angles between the planes of the two bridged naphthoquinone structures are fairly acute, with values of about 68 and 69°. The structure has several intramolecular O-H...O and C-H...O hydrogen bonds and several weak intermolecular C-H...O hydrogen bonds, but no intermolecular O-H...O hydrogen bonds.

Induction of apoptosis by (-)-gossypol-enriched cottonseed oil in human breast cancer cells.

Induction of apoptosis is one of the mechanisms of chemotherapeutic agents against breast cancer. In addition, recent studies have shown that diets containing polyphenolic components possess anticancer activities either in vitro or in vivo by inhibiting cell proliferation and inducing apoptosis. The aim of our study was to explore the effects of (-)-gossypol-enriched cottonseed oil [(-)-GPCSO], a polyphenolic compound, on the proliferation of the breast cancer cell line MCF-7 as well as primary cultured human breast cancer epithelial cells (PCHBCEC). We investigated whether the mechanism of the effects of (-)-GPCSO was mediated via the induction of cell apoptosis and the regulation of Bcl-2 gene expression at both the mRNA and protein levels. Our results showed that (-)-GPCSO inhibited the proliferation of MCF-7 and PCHBCEC in a dose-dependent manner. (-)-GPCSO (0.1 and 0.2%) induced DNA fragmentation in both MCF-7 cells and PCHBCEC. (-)-GPCSO suppressed the expression of Bcl-2 at both the mRNA and protein levels in MCF-7 cells and PCHBCEC in a dose-dependent fashion. Our results suggest that the growth inhibitory effect of (-)-GPCSO on MCF-7 and PCHBCEC is due, at least partially, to the induction of cell apoptosis, which is mediated by down-regulation of Bcl-2 expression at both the mRNA and protein levels. It might be possible for (-)-GPCSO to be developed as a novel chemotherapeutic agent for breast cancer patients.

Experimental and theoretical electron density distribution of alpha,alpha-trehalose dihydrate.

alpha,alpha-Trehalose is of interest because of its cryoprotective and antidessicant properties, and because it possesses various technical anomalies such as (13)C NMR spectra that give misleading indications of intramolecular structural symmetry. It is a non-reducing disaccharide, with the glycosidic oxygen atom shared by the anomeric carbon atoms of the two glucose rings, and is therefore subject to a proposed 'overlapping'exo-anomeric effect. We report here a study of the electron density of trehalose with X-ray diffraction and quantum mechanics calculations, similar to a recent study of sucrose, also a non-reducing molecule. In particular we studied the electron density around the glycosidic linkage and the hydrogen bonding with both deformation density and Atoms in Molecules (AIM) analyses. A total of 129,952 single crystal X-ray intensity measurements were collected on alpha,alpha-trehalose dihydrate to a resolution of sintheta/lambda=1.18A(-1) at 100K and refined with an aspherical multipole model to a final agreement factor of R(1)=0.0160. Wavefunctions were calculated at three levels of theory. Redistribution of electron density due to anomeric effects was reduced in trehalose, compared to sucrose. Five new C-Hcdots, three dots, centeredO hydrogen bonds were confirmed with bond critical points and bond paths from AIM analyses, as were the previously proposed O-Hcdots, three dots, centeredO hydrogen bonds.

(-)-Gossypol reduces invasiveness in metastatic prostate cancer cells.

BACKGROUND: Acquisition of metastatic ability by prostatic cancer cells is the most lethal aspect of prostatic cancer progression. (-)-Gossypol, a polyphenolic compound present in cottonseeds, possesses anti-proliferative and proapoptotic effects in various cancer cells. MATERIALS AND METHODS: In this study, the differences between MAT-LyLu, rat prostate cancer cells, with a novel isolated subline from metastasized tumors in the lungs of MAT-LyLu-bearing Copenhagen rats (MLL cells) were compared with respect to cell growth and invasion. The effects of (-)-gossypol on cell viability, colony formation, invasive ability and cell migration in MAT-LyLu and MLL cells were also evaluated. RESULTS: Results showed that MLL cells displayed higher growth ability, colony formation and aggressive penetration than those of MAT-LyLu cells. MLL cells possess lower protein expression of Bcl-xL and nm23-H1 than those of MAT-LyLu cells, implying differences in invasive ability. Moreover, (-)-gossypol treatment induced a dose-dependent inhibition of invasive activity and cell viability and reduced Bcl-2 and Bcl-xL proteins but induced nm23-H1 protein in both cell lines. CONCLUSION: These findings illustrated that (-)-gossypol reduced in vitro invasion of both the parental MAT-LyLu cells and the isolated MLL cells, suggesting that (-)-gossypol might serve as a chemotherapeutic and/or chemopreventive agent.

Effect of racemic, (+)- and (-)-gossypol on survival and development of Heliothis virescens larvae.

Gossypol is a constituent of the lysigenous foliar glands of cotton plants and is also found in glands in cottonseed. Gossypol exists as enantiomers because of restricted rotation around the binaphthyl bond. The biological activities of the enantiomers differ. For example, (+)-gossypol can be fed safely to nonruminants such as chickens, but (-)-gossypol cannot. Most commercial cottonseed contain a (+)- to (-)-gossypol ratio of approximately 3:2. Conventional breeding techniques can be used to develop cottonseed that contains >95% (+)-gossypol. Notably, gossypol protects the plant from insect herbivores. Herein, we report the effect of various forms of gossypol on Heliothis virescens (Fabricius) larvae. Three levels (0.16, 0.24, and 0.32%) of racemic, (+)-, and (-)-gossypol were added to artificial rearing diets and were fed to H. virescens larvae. All 0.24 and 0.32% gossypol diets significantly lengthened days-to-pupation and decreased pupal weight compared with the control. Percent survival was significantly less for larvae reared on diets containing 0.24% of all three forms of gossypol as compared with the control diet. (+)-Gossypol was superior or equivalent to racemic gossypol as measured by the three parameters studied. Higher concentrations of all gossypol forms were required to reduce survival and pupal weights and increase days-to-pupation for larvae of H. virescens larvae compared with the concentration needed to affect larvae of Helicoverpa zea (Boddie), which was studied previously. These results indicate that current efforts to breed cotton lines containing mostly (+)-gossypol in seed should not significantly impair the plant's natural defenses against insects.

HPLC preparation of the chiral forms of 6-methoxy-gossypol and 6,6'-dimethoxy-gossypol.

A concentrated mixture of gossypol, 6-methoxy-gossypol, and 6,6'-dimethoxy-gossypol was extracted from the root bark of St. Vincent Sea Island cotton with acetone. This extract was derivatized with R-(-)-2-amino-1-propanol to form diastereomeric gossypol Schiff's bases. Analytical-scale reverse-phase chromatography of these Schiff's bases produced six peaks, indicating separation of the enantiomeric forms of the three gossypol compounds. The elution order of the peaks was found to vary with the polarity of the mobile phase. The chromatography was scaled to a preparative level and was used to isolate each compound. After hydrolysis of the separated Schiff's bases, the original compounds were recovered by precipitation from solutions of diethyl ether, acetic acid, and water. Fifty injections yielded approximately 500 mg of each methoxy-gossypol enantiomer and 300 mg of each dimethoxy-gossypol enantiomer. Each compound was characterized for carbon and hydrogen content, optical rotation, UV-vis light absorption, and melting point. Standard curves were developed and were used to measure the concentration of each gossypol form in the root bark and dehulled seed of St. Vincent Sea Island cotton. In seed tissue, 48% of the gossypol compounds were methylated, and the (-)-optical form was found to be in a slight excess to the (+)-optical form (53-54%) for all three compounds. In root bark, 71% of the gossypol compounds were methylated, and the (+)-optical form was in excess to the (-)-optical form for all three compounds. However, in this tissue the extent of enantiomeric excess decreased with the degree of methylation, with 77% of the gossypol existing in the (+)-optical form and 59% of the 6,6'-dimethoxy-gossypol existing in the (+)-optical form.

Aspergillus flavus hydrolases: their roles in pathogenesis and substrate utilization.

Aspergillus flavus is a fungus that principally obtains resources for growth in a saprophytic mode. Yet, it also possesses the characteristics of an opportunistic pathogen with a wide, non-specific host range (plants, animals, and insects). It has attained a high level of agricultural significance due to production of the carcinogen aflatoxin, which significantly reduces the value of contaminated crops. To access a large variety of nutrient substrates and penetrate host tissues, A. flavus possesses the capacity to produce numerous extracellular hydrolases. Most work on A. flavus hydrolases has focused on the serine and metalloproteinases, pectinase P2c, and amylase. Many hydrolases are presumed to function in polymer degradation and nutrient capture, but the regulation of hydrolase secretion is complex and substrate dependent. Proteinases are employed not only to help access protein substrates, such as elastin that is found in mammals and insects, but may also play roles in fungal defense and virulence. Secretion of the endopolygalacturonase P2c is strongly correlated with isolate virulence (against plants) and maceration of cotton boll tissues. In some hosts, secretion of alpha-amylase is critical for starch digestion and may play a critical role in induction of aflatoxin biosynthesis. Despite a significant body of work, much remains to be learned about hydrolase production and utilization by A. flavus. This information may be critical for the formulation of successful strategies to control aflatoxin contamination in affected commodities.

Modulation of multidrug resistance gene expression in human breast cancer cells by (-)-gossypol-enriched cottonseed oil.

BACKGROUND: Multidrug resistance (MDR) is a major impediment to successful cancer chemotherapy. P-glycoprotein (P-gp), the product of the multidrug resistance 1 (MDR1) gene, acts as an efflux pump and prevents sufficient intracellular accumulation of several anticancer agents, thus, playing a major role in MDR. Tamoxifen (Tam), ICI 182 780 (ICI) and Adriamycin (Adr) alone or with (-)-gossypol-enriched cottonseed oil [(-)-GPCSO] possible effects on cell growth inhibition and regulation of MDR1, mRNA and P-gp expression were examined in both an MDR human breast cancer cell line, MCF-7/Adr cells, and primary cultured human breast cancer epithelial cells (PCHBCEC). MATERIALS AND METHODS: Cells were treated with 0.05% of (-)-GPCSO either in the absence or presence of either 0.1 microM Tam, ICI or Adr for 24 h. RESULTS: Using the non-radioactive cell proliferation MTS assay, none of these chemotherapeutic agents alone inhibited MCF-7/Adr cell and PCHBCEC proliferation; meanwhile, the combination of 0.1 microM Tam, ICI or Adr with 0.05% (-)-GPCSO significantly reduced MCF-7/Adr cell growth by approximately 34%, 32% and 23%, respectively, of that of the vehicle-treated cells. For PCHBCEC, the combination of 0.05% (-)-GPCSO with 0.1 microM of Tam, ICI and Adr reduced cell growth to about 94%, 90%, and 71% respectively, of the vehicle treated PCHBCEC. Furthermore, (-)-GPCSO inhibited MDR1/P-gp expression in both MCF- 7/Adr and PCHBCEC in a dose-dependent manner. Our results provide insight into the MDR-reversing potential of (-)-GPCSO in human breast cancer cells resistant to current chemotherapeutic agents.

Gossypol inhibits calcineurin phosphatase activity at multiple sites.

Calcineurin, the Ca2+/calmodulin-dependant serine/threonine phosphatase is the target for the immunosuppressant drugs FK506 and cyclosporine-A. These established calcineurin inhibitors each require an immunophilin protein cofactor. Gossypol, a polyphenol produced by the cotton plant, inhibits calcineurin (IC50=15 microM), in a noncompetitive, reversible manner, and is independent of any cofactor. We found that gossypol acts by at least two mechanisms to inhibit calcineurin phosphatase activity. A calmodulin-independent form of calcineurin was less sensitive to inhibition by gossypol than native calcineurin (IC50=41 and 18 microM, respectively) indicating that gossypol may interfere with calmodulin binding. A fluorescence polarization based assay demonstrated that 100 microM gossypol reduced the affinity of calmodulin for calcineurin (from K(d)=2.4 to 250 nM). Inhibition of calcineurin phosphatase activity by gossypol could not be overcome by adding excess calmodulin or by testing the inhibition toward a calmodulin-independent calcineurin indicating that gossypol acts at a site different from the calmodulin-binding site. Gossypol decreased the affinity of calcineurin for immunosuppressant/immunophilin complexes only in the presence of calmodulin, indicating that gossypol blocks the effects of calmodulin binding to calcineurin. In addition, gossypol had a stimulatory effect on native calcineurin in the absence of calmodulin, possibly indicating a calmodulin mimetic effect. Gossypol exists in two enantiomeric forms which are reported to have different potency for cell toxicity. (+) and (-) gossypol had equivalent potency for inhibition of native and calmodulin-independent calcineurin phosphatase activity, and for inhibition of calmodulin binding. The inhibition of calcineurin by gossypol via multiple binding sites without stereo-specificity indicates that gossypol is not a specific calcineurin inhibitor.

Effects of serum on (-)-gossypol-suppressed growth in human prostate cancer cells.

BACKGROUND: Gossypol, a natural polyphenolic compound present in cottonseeds, possesses antiproliferative and pro-apoptotic effects in in vivo and in vitro models. There are two enantiomers, (+)-gossypol and (-)-gossypol, the latter being a more potent inhibitor of cancer cell growth. Here, the effect of bovine serum albumin (BSA) and dextran-coated charcoal-treated fetal bovine serum (DCC-FBS)-containing medium on the ability of (-)-gossypol to inhibit the growth of human prostate cancer cells was studied. MATERIALS AND METHODS: BSA- and DCC-FBS-supplemented medium were used to examine the influence of serum proteins on the antiproliferative effects of (-)-gossypol in DU-145 cells, a human prostate cancer cell line. The viability of the DU-145 cells was determined by CellTiter 96 Aqueous assay. The expressions of mRNA and protein for the cell cycle regulators, cyclin-D1, Rb, CDK, p21 and TGF-beta, were determined by RT-PCR and Western blot analyses, respectively. RESULTS: (-)-Gossypol caused growth suppression of the DU-145 cells. In comparison with BSA-supplemented medium, DCC-FBS blocked the antiproliferative effects of (-)-gossypol at 1 and 2.5 microM, but not at 5 microM. Furthermore, (-)-gossypol treatment down-regulated cyclin-D1, Rb, CDK4 and CDK6, and up-regulated p21 and TGF-beta1 at the mRNA and/or protein levels. CONCLUSION: The data suggested that (-)-gossypol-suppressed prostate cancer cell growth may be influenced through cell cycle regulators, which may lead to better prognosis. We further speculate that (-)-gossypol might serve as a chemotherapeutic agent for human prostate cancer patients.

Molecular mechanisms of (-)-gossypol-induced apoptosis in human prostate cancer cells.

BACKGROUND: Gossypol, a natural compound present in cottonseeds, displays antiproliferative and pro-apoptotic effects against various cancer cells. The (-)-gossypol enantiomer is a more potent inhibitor of cancer cell growth. Here, the molecular mechanisms of apoptosis induced by (-)-gossypol were studied in human prostate cancer cells. MATERIALS AND METHODS: After the prostate cancer cell DU-145 had been treated with (-)-gossypol, the trypan blue exclusion assay and DNA fragment end-labeling assay were used to stain the dead cells and to detect DNA laddering, respectively. The effects of (-)-gossypol on the expression of apoptotic-regulated gene markers in both death receptor- and mitochondria-mediated apoptotic pathways, such as the Bcl-2 family and caspase, etc., were detected by RT-PCR and Western blot analysis. To further investigate the apoptotic pathways induced by (-)-gossypol, different caspase inhibitors were used to block caspase activities and cell viability was detected by the CellTiter 96 AQueous assay in DU-145 cells. RESULTS: At a 5-10 microM dose-level, (-)-gossypol significantly enhanced apoptosis measured by DNA fragmentation. (-)-Gossypol caused apoptosis in DU-145 cells through the down-regulation of Bcl-2 and Bcl-xL and the up-regulation of Bax at the mRNA and protein levels. (-)-Gossypol also activated caspases-3, -8 and -9 and increased PARP [poly (ADP-ribose) polymerase] cleavage. Furthermore, (-)-gossypol-induced apoptosis might be due to an increase in CAD (caspase-activated deoxyribonuclease) proteins and a decrease in ICAD (inhibitor of CAD) proteins. By using caspase inhibitors, (-)-gossypol caused apoptosis via the caspase-dependent pathways. CONCLUSION: Our results indicated that the apoptotic processes caused by (-)-gossypol are mediated by the regulation of the Bcl-2 and caspase families in human prostate cancer cells. Our data also suggested that (-)-gossypol may have chemotheraputic benefits for prostate cancer patients.

Effect of racemic and (+)- and (-)-gossypol on the survival and development of Helicoverpa zea larvae.

Gossypol is a sesquiterpene that occurs naturally in seed and other parts of the cotton plant. Because of restricted rotation around the binaphthyl bond, it occurs naturally as enantiomeric mixtures with (+)-gossypol to (-)-gossypol ratios that vary between 97:3 and 31:69. Commercial cotton varieties (Gossypium hirsutum) normally exhibit an approximate 3:2 ratio. (+)-Gossypol is significantly less toxic than (-)-gossypol to nonruminant animals; thus, cottonseed containing high levels of (+)-gossypol might be safely fed to nonruminants. Gossypol, however, is an important component in the cotton plant's defense against insect herbivores, but it is not known how cotton plants that exhibit high levels of (+)-gossypol in the foliage might be affected by insect herbivory. To address this question, 1-d-old Helicoverpa zea larvae were fed diets with 0.16, 0.20, and 0.24% racemic, (+)-, and (-)-gossypol. Larval pupal weights, days-to-pupation, and survival were adversely affected by all gossypol diets compared with the control diet. Statistical differences were determined by comparing the compounds among themselves at the three levels and between the three compounds at the same level. When the compounds were compared among themselves, no large differences were observed in pupal weights or in days-to-pupation among any of the diets. Among the three compounds, at the 0.16% level, the diet containing racemic gossypol was the most effective at reducing survival. At the 0.20 and 0.24% levels of racemic (+)- and (-)-gossypol, survival was not statistically different. The overall results indicate that (+)-gossypol is as inhibitory to H. zea larvae as racemic or (-)-gossypol, and thus, cotton plants containing predominantly the (+)-enantiomer in foliage may maintain significant defense against insect herbivory.

Isolation of 6-methoxy gossypol and 6,6'-dimethoxy gossypol from Gossypium barbadense Sea Island cotton.

6-methoxy gossypol and 6,6'-dimethoxy gossypol were isolated from the seeds and root bark of a St. Vincent Sea Island variety of cotton (AZK-267, GRIN# PI 528406). Crude mixtures of gossypol and the methoxy compounds were obtained by extraction of the tissue with acetone and precipitation with acetic acid. After recrystallization, the preparations were treated with 3-amino-1-propanol to form gossypol Schiff's bases, which were separated by preparative reverse phase chromatography. The separated Schiff's bases were then hydrolyzed with acid, extracted into diethyl ether, concentrated, and precipitated with acetic acid. From the above procedure, both methoxy gossypol compounds were obtained as 1:1 molar acetic acid solvates. Each compound was prepared in sufficient amounts to determine its physical properties and begin testing for bioactivity. Light absorbance differed significantly for the di-3-amino-1-propanol derivatives of gossypol and the methoxy gossypol compounds at 254 nm. Relative response factors were developed, which can be used for determining or correcting analytical measurements of these methylated gossypol forms.