3,beta-Hydroxy-5agr-cholestan-6-one: A Possible Precursor of agr-Ecdysone Biosynthesis.

The conversion of cholesterol into 3beta-hydroxy-5alpha-cholestan-6-one has been demonstrated to occur in prothoracic glands of last instar larvae of the silkworm, Bombyx mori. Incubation of glands containing radiolabeled 3beta-hydroxy-5alpha-cholestan-6-one results in the disappearance of this sterol from the glands and the concomitant appearance of radiolabeled alpha-ecdysone in the medium. The observations suggest that the sterol is an intermediate in the synthesis of alpha-ecdysone.

2-Deoxy-alpha-ecdysone from ovaries and eggs of the silkworm, Bombyx mori.

From ovaries dissected from developing and emerged adults of the silkworm, Bombyx mori, two substances having high molting hormone activity were isolated. One of these was identified as 2-deoxy-alpha-ecdysone by means of high-pressure liquid chromatography and mass spectrometry. Although this compound had previously been isolated from the fern Blechnum minus and postulated to be the precursor of alpha-ecdysone, it had not been obtained from insect material. The compound is also contained in the form of a conjugate in ovaries as well as in diapausing silkworm embryos.

Biosynthesis of agr-Ecdysone by Prothoracic Glands in vitro.

An in vitro study in which isolated prothoracic glands of the Bombyx silkworm were cultured has provided definite evidence that the prothoracic gland is the site where molting hormone is synthesized. The hormone behaved very similarly to free ecdysone on thin-layer chromatography. Analysis by liquid chromatography and mass fragmentography revealed that the hormone is identical with alpha-ecdysone.

C26 sterol in a human urine.

A new C26 sterol, 22-trans-27-norcholesta-5,22-dien-3 beta-ol, was found in the urine of a 6-year-old girl, with a clinical diagnosis of congenital adrenal hyperplasia of the salt losing type, accompanied by symptoms of mixed sex anatomy and skin pigmentation. The structure of the sterol was determined by comparison with the synthetic compound. The sterol was also detected in ther serum. This appears to be the first case in which a C26 sterol has occurred in mammalia.

24-hydroxylation of 25-hydroxyvitamin D3: is it required for embryonic development in chicks?

As shown previously, laying hens given 1,25-dihydroxyvitamin D3 as their sole source of vitamin D produce fertile eggs having normal shells, but only 35 to 55 percent of the embryos are normal. Giving these hens additional 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 at 1.25 nanomoles per day resulted in 90 to 100 percent normal embryos, and hence, hatchability. Since 24,24-difluoro-25-hydroxyvitamin D3 cannot be 24-hydroxylated, 24-hydroxylation is not required for this function of 25-hydroxyvitamin D3.

Induction of macrophage differentiation of human normal and leukemic myeloid stem cells by 1,25-dihydroxyvitamin D3 and its fluorinated analogues.

This study shows for the first time that 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] and two fluorinated analogues of 1,25-(OH)2D3 [24,24-F2-1,25-(OH)2D3 and 26, 26, 26, 27, 27, 27-F6-1,25-(OH)2D3] induce macrophage differentiation of human normal and leukemic myeloid stem cells. The addition of either 1,25-(OH)2D3 or one of the two fluorinated analogues of 1,25-(OH)2D3 at concentrations as low as 10(-9) M to culture plates containing normal human marrow cells stimulated myeloid stem cells to preferentially differentiate to colonies of monocytes and macrophages. Over 80% of the normal human myeloid colonies were composed of only monocytes and macrophages in culture plates containing 10(-7) M 1,25-(OH)2D3 or one of the fluorinated analogues. In contrast, control plates not containing 1,25-(OH)2D3 had less than 35% macrophage colonies. Likewise, 1,25-(OH)2D3 and the two vitamin D analogues induced macrophage differentiation of leukemic colony-forming cells taken from patients. In plates containing 10(-7) M 1,25-(OH)2D3 or one of the analogues at 10(-8) M, 80% of chronic myelogenous leukemia and approximately 50% of acute nonlymphocytic leukemia colony-forming cells differentiated to macrophage-like cells. In contrast, control plates had about 30 and 20% macrophage colonies in cultures from chronic myelogenous leukemia and acute nonlymphocytic leukemia patients, respectively. Our findings suggest that 1,25-(OH)2D3 may play a role in hematopoiesis and that the compound or a related analogue may possibly have a therapeutic role in some leukemias.

Inhibition of HT-29 human colon cancer growth under the renal capsule of severe combined immunodeficient mice by an analogue of 1,25-dihydroxyvitamin D3, DD-003.

An analogue of 1,25-dihydroxyvitamin D3, 22(S)-24-homo-26,26,26,27,27,27-hexafluoro-1 alpha,22,25-trihydroxyvitamin D3 (DD-003), showed 10-fold greater inhibiting effect than 1,25-dihydroxyvitamin D3 on the growth of HT-29 human colonic adenocarcinoma cells in culture. To examine the anticancer activity of DD-003 in vivo, a fibrin clot of HT-29 cells was prepared with fibrinogen and thrombin and implanted under the renal capsule of the severe combined immunodeficient mouse. Starting 7 days after implantation of HT-29 tumor, mice were given 3 micrograms/kg body weight of DD-003 or the vehicle i.p. every other day for 5 times. The HT-29 tumor grew rapidly in control mice; malignant growth was clearly observed with mitosis, massive tumor angiogenesis, and invasion into normal kidney tissue. Tumors in DD-003 treated mice were smaller with less invasion compared to the control. Administration of DD-003 inhibited growth of HT-29 tumor by 63%. Serum calcium concentrations and body weights of the treated mice were similar to those of the control. DD-003 inhibited growth of HT-29 tumor in a dose-dependent manner over the range of 0.1-10 micrograms/kg body weight, with no increase of serum calcium concentration observed at any dose level. When DD-003 was withdrawn after 2 weeks of treatment, tumor growth resumed. Since chemosensitivity tested by the subrenal capsule assay correlates well with clinical response, DD-003 may be clinically applicable in procedures such as postsurgical chemotherapy of colon cancer.

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7.

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.

Inhibition of vitamin D3-induced cell differentiation by interferon-gamma in HL-60 cells determined by a nitroblue tetrazolium reduction test.

The combined effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and interferon-gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) on cell differentiation in HL-60 human promyelocytic leukemia cells were examined by a nitroblue tetrazolium (NBT) reduction test. 1,25(OH)2D3 at the concentrations of 7-70 nM induced NBT-positive cells, which was used as a criterion of cell differentiation. IFN-gamma itself showed little effect on induction of NBT-positive cells or on cell growth at a concentration up to 1000 U/ml. However, in a combination of 1,25(OH)2D3 with IFN-gamma (100 and 300 U/ml), cell differentiation was strongly inhibited and was accompanied by growth inhibition. Treatment with a combination of 1,25(OH)2D3 and TNF-alpha or IFN-gamma and TNF-alpha showed an additive effect on cell differentiation. IFN-gamma seems to act as a specific inhibitor for 1,25(OH)2D3-induced cell differentiation. To elucidate the cause of the inhibition of cell differentiation by IFN-gamma, the ability of the cells to produce superoxide (O2-) was examined after culture for 5 days in the presence of 1,25(OH)2D3 and IFN-gamma. The results indicated that the inhibition of IFN-gamma was caused by a reduction in the ability of the cells to produce O2- in response to stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA).

Inhibitory effect of 15-oxygenated sterols on cholesterol synthesis from 24,25-dihydrolanosterol.

Several 15-oxygenated sterols were examined as to their inhibitory activity toward cholesterol synthesis from [24,25-3H]-24,25-dihydrolanosterol in the 10,000 X g supernatant fraction of a rat liver homogenate. At 40 microM, three 15 alpha-hydroxylated compounds, 14 alpha-ethylcholest-7-ene-3 beta,15 alpha-diol, 14 alpha-methylcholest-7-ene-3 beta,15 alpha-diol, and lanost-7-ene-3 beta,15 alpha-diol, were found to be extremely potent inhibitors (more than 90% inhibition) of dihydrolanosterol metabolism. The inhibitory effect of the C-15 substituents appeared to be in the order of: 15 alpha-hydroxyl greater than 15-ketone greater than 15 beta-hydroxyl.

Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells.

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1 alpha,25-(OH)2D3 > or = 1 alpha,25-(OH)2-26,27-CH3)2D3 > 1 alpha,25-(OH)2-26,27-(C2H5)2D3 >> 1 alpha,25-(OH)2-26,27-(C3H7)2D3. These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1 alpha,25-(OH)2D3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1 alpha-OH group from 1 alpha,25-(OH)2D3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1 alpha-OH groups in the 1 alpha,25-(OH)2D3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1 alpha,25-(OH)2D3 receptor of HL-60 cells, suggesting a 1 alpha,25-(OH)2D3 receptor-mediated induction mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Sitosterol-stimulative production of plasminogen activator in cultured endothelial cells from bovine carotid artery.

The endothelial cell is a rich source of plasminogen activator that is associated with fibrinolytic activity in blood vessel. Addition of sitosterol to the culture medium of endothelial cells from bovine carotid artery gave rise to a marked increment in the activity of plasminogen activator. Removal of sitosterol from the culture medium resulted in a decrease of plasminogen activator activity back to normal levels. Enhancement of plasminogen activator activity in cultured endothelial cells was not observed by cholesterol, 5-androsten-3 beta-ol and others.

Effect of 22R-hydroxycholesterol on the action of sphingomyelinase from Bacillus cereus toward bovine erythrocytes.

The incorporation of 22R-hydroxycholesterol [(22R)-5-cholestene-3 beta,22-diol] into the bovine erythrocyte membranes remarkably enhanced the degradation of sphingomyelin in erythrocyte membranes by the action of sphingomyelinase from Bacillus cereus, causing much faster hemolysis of erythrocytes. The stimulative effect of 22R-hydroxycholesterol on the breakdown of sphingomyelin was maximal in the presence of Mg2+. On the other hand, in spite of the presence of 22R-hydroxycholesterol, the breakdown of sphingomyelin was inhibited by increasing concentrations of Ca2+. Also, the incorporation of 22R-hydroxycholesterol into the erythrocyte membranes facilitated the specific adsorption of the enzyme onto the surface of the erythrocyte membranes. The specific adsorption of sphingomyelinase amounted to 20-40% of the total activity in the presence of Mg2+ and the absence of divalent metal ions. In the presence of Ca2+, the incorporation of 22R-hydroxycholesterol enhanced the enzyme adsorption, exceeding more than 90% of the total activity. Therefore, the incorporation of 22R-hydroxycholesterol into bovine erythrocyte membranes remarkably accelerates the breakdown of sphingomyelin in the presence of Mg2+, and the specific adsorption of sphingomyelinase onto erythrocytes in the presence of Ca2+.

Structural requirement of sterol nucleus for the silkworm growth and development.

Several cholesterol analogs structurally modified in nuclear substitutions were tested for sustaining the growth of the silkworm Bombyx mori. 5 alpha-Cholest-7-en-3 beta-o1, 5,7-cholestadien-3 beta-o1 and cholesteryl acetate can replace cholesterol as sterol source for B. mori. Considerably good growth was also obtained with 5 alpha-cholest-14-en-3 beta-o1 and 5 alpha-cholesta-6,8(14)-dien-3 beta-o1. Other sterols tested were either partially effective or ineffective as nutrients.

Structural requirement of sterol side chain for the silkworm growth and development.

Several cholesterol analogs with modifications in the side chain were added to the artificial diet of the silkworm, and their effects on insect growth and development were determined. It was found that slight deviations of the cholesterol's side chain induced pronounced growth-retarding effects, suggesting an important functional role of the isooctane side chain of cholesterol.

Elimination of C-6-hydrogen during the formation of ecdysteroids from cholesterol in Locusta migratoria ovaries.

Being administered to Locusta migratoria adult females, [6-3H, 4-14C]cholesterol was incorporated into ecdysone and 2-deoxyecdysone. The ratio of 3H/14C of the two ecdysteroids isolated from newly laid eggs revealed that C-6-hydrogen of cholesterol was eliminated during the conversion to ecdysteroids in the ovaries of the insects. Thus, a hypothetical mechanism involving migration of the C-6-hydrogen to the C-5 position in the formation of A/B cis junction turned out to be less likely.

Identification of 23-demethylacanthasterol in an asteroid, Acanthaster planci and its synthesis.

23-Demethylacanthasterol was identified in an asteroid, Acanthaster planci, by GC-MS analysis and direct comparison with the synthetic sample prepared from 23-demethylgorgosterol. The sterol composition of A. planci is also described.

Sex pheromones in marine polychaetes: steroids from ripe Nereis succinea.

A number of neutral marine steroids such as desmosterol, campesterol, brassicasterol, gorgosterol, and other trace steroids were isolated from the coelomic fluid of ripe Nereis succinea and checked for biological activity as sex pheromones on swarming specimens of Platynereis dumerilii and Nereis succinea. No significant influence of synthetic gorgosterol or a natural extract of gorgosterol or the other identified steroids on the swarming behavior was observed.

Effects of novel 26,27-dialkyl analogs of 1 alpha,25-dihydroxyvitamin D3 on differentiation-inducing activity of human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture.

Monocytic differentiation-inducing activity of steroidal side chain-lengthened 26,27-dialkyl analogs of 1 alpha,25-dihydroxyvitamin D3 was examined in human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture. The order of in vitro potency for reducing nitroblue tetrazolium was 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 greater than or equal to 1 alpha,25-dihydroxy-26,27-diethylvitamin D3 much greater than 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 under serum-free culture conditions. Analysis by sucrose density-gradient centrifugation or polyethylene glycol precipitation technique showed that the potency order for differentiation-inducing activity correlated well with binding affinity of these analogs for vitamin D3 receptor of HL-60 cells. Under serum-supplemented culture conditions, the lack of correlation between biologic activity and analog-binding affinity for receptor was caused by differences in binding affinity of these analogs for serum vitamin D-binding proteins. These results suggest that serum vitamin D-binding proteins apparently modulate monocytic differentiation of HL-60 cells by these analogs under serum-supplemented culture conditions.

Effect of hexafluoro-1,25-dihydroxyvitamin D3 and sodium butyrate combination on differentiation and proliferation of HL-60 leukemia cells.

We have investigated the combined effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and its fluoroanalog 26,26,26,27,27,27-hexafluoro-1,25-(OH)2D3 [F6-1,25-(OH)2D3] with sodium butyrate (NaB) on growth and differentiation of HL-60 human promyelocytic leukemia cells. F6-1,25-(OH)2D3 was 10-fold more active than 1,25-(OH)2D3 for induction of cell differentiation in HL-60 cells. Exposure to suboptimal concentration of F6-1,25-(OH)2D3 and NaB had synergistic effects compared to that of F6-1,25-(OH)2D3 or NaB alone and in the presence of 0.1-0.3 mM NaB, the dosage of F6-1,25-(OH)2D3 required to inhibit cell growth and colony formation and to induce cell differentiation was significantly reduced. The mechanism for the synergistic effect is probably that NaB increases cytoplasm content and nuclear binding of 1,25-(OH)2D3.