Suppressive effects of thyroxine on glucocorticoid (gc)-induced metabolic changes and cataract formation on developing chick embryos.

To prevent cataracts induced by glucocorticoids (GC) as a systemic disease, the suppression of oxidative stress caused by GC in the hepatic metabolism is of significant interest. In this study, to elucidate the formative mechanism of GC-induced cataracts, we examined the preventive effect and then analysed the mechanisms of thyroxine on GC-induced cataract formation. Fifteen day old chick embryos were administered with 0.25 micromol hydrocortisone succinate sodium (HC), and then 12-30 nmol of thyroxine 4 hr after HC administration. At the indicated time after HC treatment, we examined the incidence of cataract formation, the levels of serum glucose and lipids, lenticular and hepatic glutathione (GSH), and lipid peroxide (LPO) in the lens, blood and liver. Almost all lenses (96%) removed 48 hr after HC administration were opaque. Thyroxine prevented HC-induced cataract formation effectively, and suppressed the elevations of serum glucose and LPO in the lens, blood and liver. The treatment prevented the decreased lenticular GSH level at 48 hr, but the hepatic GSH level at 24 hr remained lowered in contrast to the results of previous studies using insulin. Moreover, thyroxine did not decrease the elevated serum lipid level (triglyceride and non-esterified fatty acid) caused by HC. Under thyroxine treatment, in constant to insulin, acceleration of GSH-GSSG cycle rather than GSH de novo synthesis keeps a certain level of hepatic GSH necessary for diminishing the elevation of LPO as a risk factor of GC-induced cataract formation. The regulation of metabolic changes ensured the maintenance of hepatic GSH level, which is necessary to reduce oxidative stress produced by GC and to protect the lens from oxidative stress leading to opacification.

Stimulation of melanogenesis in murine melanoma cells by 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB).

The effects of 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB), one of the benzimidazole derivatives designed for a nucleic acid analogue, on melanogenesis of murine B16-F10 melanoma cell lines were investigated. MPB (40 microM) induced a striking dendricity in B16 melanoma cells within 12 h and maximal dendricity between 48 and 72 h. The stimulation of melanin synthesis was observed after only 2 days of treatment together with a dose-dependent growth inhibition. Moreover, MPB increased the activity of tyrosinase through the expression of tyrosinase mRNA without increasing the intracellular cyclic AMP content. MPB-induced melanogenesis was inhibited by novel protein kinase A inhibitors, KT-5720 and H-85. These findings indicate that MPB stimulated B16 cells to terminally differentiate and may be a useful drug in studying the regulation of melanogenesis.

Steroid-induced short term diabetes in chick embryo: reversible effects of insulin on metabolic changes and cataract formation.

PURPOSE: To determine the reversible effect of insulin on glucocorticoid (GC)-induced cataract formation in relation to systemic metabolic changes in the developing chick embryo. METHODS: Hydrocortisone sodium succinate (HC; 0.25 micromoles) was administered to 15-day-old embryos followed by administration of long-acting recombinant human insulin, 4 and 28 hours later. At the indicated time after HC administration, the incidence of cataractous lenses and any changes in the components of the lenses, liver, and blood were determined. RESULTS: At 48 hours after HC administration, the following observations were made: opacification of lenses; an elevation of glucose and lipids in the blood and lenses; an increase in lipid peroxide (LPO) in the blood, liver, and lenses; a decrease in glutathione (GSH) in the lens and liver (at 24 hours after HC administration); and a depletion of adenosine triphosphate (ATP) in the liver. These changes in response to HC administration were reversed by a double application of insulin. CONCLUSIONS: Insulin antagonizes GC-induced gluconeogenesis, stimulates glycolysis, and ultimately leads to recovery of decreased activity in the citric acid cycle. The restoration of ATP by the recovered citric acid cycle may facilitate de novo synthesis of GSH, which in turn may diminish GC-induced elevation of LPO in the liver. Thus, the metabolic changes in response to HC-accelerated gluconeogenesis in the liver, which can be reversed by insulin, are likely to produce oxidative stress that leads to cataract formation. GC-induced metabolic changes in the liver, which are antagonized by insulin, may relate to production of one of the risk factors for cataract formation.

ProMMP-9 (92 kDa gelatinase) in vitreous fluid of patients with proliferative diabetic retinopathy.

Matrix metalloproteinases (MMPs) are implicated in tissue destruction during various pathophysiologic conditions. The vitreous body is a gel-like extracellular matrix that undergoes liquefaction during aging and pathological processes. To investigate the pathogenic role of MMPs in proliferative diabetic retinopathy (PDR), we studied 73 eyes from PDR patients and 25 eyes from patients with non-diabetic ocular diseases. Vitreous MMPs were measured by zymography. Retinopathy was assessed by ophthalmoscopy and PDR was classified into 3 stages, 'naked', 'active', and 'quiescent'. Although proMMP-9 was expressed in only 8% (2/25) of non-diabetic patients, it was expressed in more than 80% (38/47) of 'active' PDR patients and still expressed in 60% (9/15) of those with 'quiescent' PDR. Vascular endothelial growth factor (VEGF) in vitreous fluids was undetectable (<0.16 ng/ml) in most of the non-diabetic patients, and was maximally elevated in the 'active' PDR patients (mean=2.20 ng/ml, range; 0.16-7.61), declining in patients with 'quiescent' PDR (1.04 ng/ml, 0.16-3.77). These results suggest that MMP-9 is one of the noteworthy factors in relation to the progress of PDR, as well as angiogenic cytokines such as VEGF.

The assembly of progesterone receptor-hsp90 complexes using purified proteins.

The progesterone receptor can be reconstituted into hsp90-containing complexes in vitro, and the resulting complexes are needed to maintain hormone binding activity. This process requires ATP/Mg2+, K+, and several axillary proteins. We have developed a defined system for the assembly of progesterone receptor complexes using purified proteins. Five proteins are needed to form complexes that are capable of maintaining hormone binding activity. These include hsp70 and its co-chaperone, hsp40, the hsp70/hsp90-binding protein, Hop, hsp90, and the hsp90-binding protein, p23. The proteins Hip and FKBP52 were not required for this in vitro process even though they have been observed in receptor complexes. Each of the five proteins showed a characteristic concentration dependence. Similar concentrations of hsp70, hsp90, and p23 were needed for optimal assembly, but hsp40 and Hop were effective at about 1/10 the concentration of the other proteins, suggesting that these two proteins act catalytically or are needed at levels similar to the receptor concentration. ATP was required for the functioning of both hsp70 and hsp90. The binding of hsp70 to the receptor requires hsp40 and about 10 microM ATP; however, hsp90 binding appears to occur subsequent to hsp70 binding and is optimal with 1 mM ATP. A three-step model is presented to describe the assembly process.

Hepatocyte growth factor in vitreous fluid of patients with proliferative diabetic retinopathy and other retinal disorders.

OBJECTIVE: To determine whether hepatocyte growth factor (HGF) is elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR). RESEARCH DESIGN AND METHODS: Vitreous fluid samples were obtained at the time of vitreoretinal surgery from 73 eyes of PDR patients and from 17 eyes of nondiabetic patients (control subjects) who had macular hole, rhegmatogenous retinal detachment, or epiretinal membrane (9, 4, and 4 eyes, respectively) but no associated proliferative vitreoretinopathy Stages of PDR were classified as active or quiescent. Concentrations of HGF and vascular endothelial growth factor (VEGF) in vitreous fluid were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Intravitreous concentrations of HGF (median [range]) were significantly higher in diabetic patients with PDR (6.00 ng/ml [0.75-22.21) than in control patients (2.86 ng/ml [0.75-5.801). Intravitreous concentrations of VEGF were also higher in diabetic patients with PDR (1.62 ng/ml [0.15-7.91) than in control patients (0.16 ng/ml [0.160.29]). Both VEGF and HGF concentrations were significantly higher in patients with active retinopathy than in those with quiescent retinopathy However, vitreous concentrations of HGF were unrelated to those of VEGE CONCLUSIONS: We found that levels of HGF in vitreous fluid of PDR patients are significantly higher than in nondiabetic patients and that the levels of HGF are elevated in the active PDR stage. This suggests that HGF stimulates or perpetuates neovascularization in PDR.

Comparison of lens biochemistry and structure between BSO-treated and glucocorticoid-treated developing chick embryos.

In this paper various changes in glutathione level, which were influenced by balance of its synthesis, degradation, transport and utilization, were analysed in chick embryos administered with glucocorticoid (GC) or buthionine sulfoximine (BSO; an inhibitor of glutathione synthesis). When BSO (30 mumol egg-1) was administered twice to chick embryos on day 14 and 15, the GSH in both the lens and the liver decreased to 15-20% and 30-40% of the age-matched control level, respectively, between 24 and 48 hr after the second treatment, then began to recover. Although this decline in the GSH level in these tissues was greater and more prolonged in embryos treated with BSO than with GC, the former embryos maintained lens transparency even up to 144 hr by a visual examination. However, histological changes in the lens occurred after 96 hr and more significantly 144 hr after second administration of BSO. The changes mainly consisted of pale epithelial cells on the anterior peripheral surface of the lens, irregular height of the epithelial cells at the equator, clefts between the epithelium and the cortex and swelling of almost all the cortical fibers. These observations may suggest that BSO treatment could produce the beginning of a cataract. Embryos with GC-cataract revealed the following changes at 48 hr: loss of transparency, elevation of LPO (TBA-reacting substance) in the lens, the blood and the liver. These were not observed in BSO-treated embryos during the experimental period. The GC-cataract may well depend on the generation of LPO. BSO cataract, having a distinct mechanism compared to that caused by GC, develops more slowly in GSH-depleted lenses. The BSO-treated chick embryos will be a useful model to screen the risk factors which accelerate cataract formation.

Pyrroloquinoline quinone (PQQ) inhibits the expression of tyrosinase mRNA by alpha-melanocyte stimulating hormone in murine B16 melanoma cells.

The biological functions of pyrroloquinoline quinone (PQQ), a bacterial redox coenzyme and potent radical scavenger, have not been elucidated in mammals. In this paper, we studied the effects of PQQ on tyrosinase activity and subsequent melanogenesis in murine B16-F10 melanoma and found that alpha-Melanocyte stimulating hormone (MSH)-induced melanogenesis was inhibited by 6.3 to 25 microM PQQ in a dose-dependent manner. Moreover, PQQ inhibited MSH-induced tyrosinase activity by suppressing tyrosinase mRNA expressed by MSH. However, PQQ had no effect on MSH-stimulated cyclic adenosine 3', 5'-monophosphate (cAMP) production. These observations suggest that PQQ inhibits the expression of tyrosinase mRNA at a post receptor level and that PQQ may be useful in investigating hormone actions mediated by cAMP.

Preventive effect of SA3443, a novel cyclic disulfide, on glucocorticoid-induced cataract formation of developing chick embryo.

The preventive effect of SA3443 [(4R)-hexahydro-7,7-dimethyl-6-oxo-1,2,5-dithiazocine-4-carboxylic acid] against glucocorticoid-induced cataract of developing chick embryos was studied. When hydrocortisone succinate sodium (HC: 0.25 mumol per egg) was administered to 15-day-old embryos, almost all lenses became opaque (stage I:O%, II: 2.5 +/- 4.6%, III: 5 +/- 5.4%, IV-V 92.5 +/- 7.1%) at 48 hr after the treatment. However, a double application of SA3443 (10 mumol per egg) at 3 and 10 hr after HC treatment effectively prevented the cataract formation (stage I: 52.8 +/- 13.7%, II: 11.6 +/- 6.3%, III: 22.9 +/- 8.9%, IV-V: 13.9 +/- 11.0%) and diminished the decline in glutathione in the lens at 48 hr and in the liver at 24 hr after HC administration. The cleavage of the cyclic disulfide bond of SA3443 did not occur in the lens homogenate but in the liver homogenate. These results suggest that the appearance of sulfhydryl residue in the liver may contribute to the anticataract effects by representing radical scavenger activities.

[Synthetic estrogens--new pharmacological actions and mechanisms].

Many constitutional analogues of estrogen have been reported. In this review, some new synthetic estrogens possessing a different action from natural estrogens are described. Aminoestrogens inhibit platelet aggregation. Estradiol-chlorambucil conjugate (KM2210) decreases the level of estrogen receptor mRNA in the human breast cancer cell MCF-7. The representative synthetic estrogen, diethylstilbestrol, and its derivatives show an inhibitory action against H(+)- and Ca(2+)-ATPase. Moreover, the effect of the diastereometric [1,2-bis(2,6-dihalo-3-hydroxyphenyl)ethylenediamine ] sulfatoplatinum (II) complexes, derivatives of hexesterol-platinum complexes, on estrogenic action and the growth-inhibitory action of hormone dependent breast cancer, are strongly dependent on the halogen atoms and on their position in the aromatic ring. These drugs are expected to be applicable for clinical use.

Mode of action of estra-1,3,5(10)-triene-3, 17 beta-diol, 3-benzoate, 17[4-[4-[bis(2-chloroethyl)amino]phenyl]-1-oxobutoxy] acetate] (KM2210) on MCF-7 human breast tumours transplanted into nude mice.

KM2210 is a benzoate of an estradiol-chlorambucil conjugate with three active metabolites, KM2202, estradiol (E2) and chlorambucil (CBL). The mode of action of this compound was assessed using MCF-7 human breast tumours transplanted into nude mice. The growth of MCF-7 in nude mice was inhibited by KM2210 and enhanced by E2, although the serum levels of E2 in nude mice treated with KM2210 and E2 were similar. The antitumour activity of CBL was completely blocked by extrinsic E2, while KM2210 suppressed the growth of MCF-7 in spite of the presence of E2 in the serum of tumour-bearing nude mice. KM2210 and KM2202 suppressed the expression of cytosol estrogen receptor (ER) of MCF-7 cells detected by the dextran-coated charcoal and fluorescent E2 staining method, although CBL did not affect the ER expression of MCF-7 cells. This inhibitory effect of KM2210 on ER was also corroborated by the fact that the pretreatment with KM2210 prevented the E2-stimulated growth of MCF-7 in nude mice. These results indicated that one of the effects induced by KM2210 is the blockage of ER expression in combination with the alkylating antitumour activity of CBL. KM2210 is thought to be a promising agent with unique modes of action for ER-positive breast carcinomas.

Growth-inhibitory action of an estrogen-chlorambucil conjugate (KM2210) in human breast cancer cell line MCF-7: its relation to reduction of estrogen receptor and transforming growth factor-alpha secretion.

We investigated the effects of a benzoate of an estradiol-chlorambucil conjugate (KM2210) and chlorambucil on growth, estrogen receptor, and secretion of transforming growth factor (TGF)-alpha in the hormone-dependent human breast cancer cell line MCF-7. In the presence of 10(-10)-10(-6) M KM2210, the estrogen-induced growth of MCF-7 was completely inhibited. Inhibited growth of MCF-7 treated with 10(-8) or 10(-6) M KM2210 for 4 days was not rescued by removal of the drug and the addition of estradiol. By treatment of MCF-7 with KM2210 for 4 days, estrogen receptor-binding sites were decreased at 10(-8) M and were not detected at 10(-6) M but were unaltered by 10(-8) M chlorambucil. Moreover, estrogen receptor immunoreactivity and the level of estrogen receptor mRNA were decreased through treatment with 10(-6) M KM2210 for 4 days. These suppressions occurred prior to the onset of inhibitory action on MCF-7 growth. Secretion of TGF-alpha from MCF-7 was decreased by 4 days of treatment with 10(-8) and 10(-6) M KM2210 but not with chlorambucil. The addition of exogenous TGF-alpha generally restored the growth of MCF-7 treated with 10(-8) M KM2210. We concluded that KM2210 has irreversible or at least long-standing inhibitory effect on estrogen-dependent growth of MCF-7. It is conceivable that the decrease of estrogen receptor renders the cell unable to respond to estrogen with increased TGF-alpha secretion and succeeding cell growth.

A case of asymptomatic cortisol producing adrenal adenoma.

We describe a man without the clinical findings of Cushing's syndrome, but who harbored an incidentally found cortisol-producing adrenal adenoma. On adrenal 131I-adsterol imaging, there was good uptake to the nodule, but no visualization of the contralateral adrenal. No abnormalities were found in the basal plasma cortisol, ACTH, urinary free cortisol and 17OHCS. However, dynamic hormone assessment revealed the existence of abnormal cortisol secretion: no suppression to dexamethasone, incomplete response to human corticotropin-releasing hormone, and lack of diurnal variation in plasma cortisol. Left adrenalectomy was performed with the diagnosis of cortisol-producing adrenal tumor. The pathological finding was an adrenal adenoma, and the perifusion of the excised tissues revealed a negligible response of the tumor tissue to ACTH though the residual normal cortex responded. Postoperative course was uneventful without replacement therapy with cortisol. It is suggested that the tumor autonomously produced a small amount of cortisol not only insufficient to provide clinical Cushing's syndrome, but also to provide typical suppression of hypothalamo-pituitary corticotroph-adrenal system.

Genesis of breast cancer in Japanese: a possible relationship between sex hormone binding globulin (SHBG) and serum lipid components.

We investigated changes in the amount of sex hormone binding globulin (SHBG) and the percentage of free estradiol (% FE2) during normal menstrual cycles in the sera of 8 young Japanese controls, and also the relation between the lipid components of serum and the serum level of SHBG in 250 normal Japanese controls aged 18-77 years. The amount of SHBG measured by IRMA was significantly higher in the luteal phase than in the follicular phase, whereas the % FE2 values remained constant throughout the menstrual cycle in 8 healthy young controls. These results suggest that the estrogen status in serum is constantly controlled by SHBG. The amount of SHBG was significantly correlated with the serum level of triglycerides (TG) in 250 normal Japanese controls. From these results, we postulate that the decrease in serum SHBG, probably caused by increased level of serum TG, leads to elevation of % FE2 and later to a status with a high risk of breast cancer.

The effect of PGF2 alpha on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1.

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.

Purification and partial sequencing of inhibitory factor on renal membrane adenylate cyclase in pancreatic cancer extract: identity with histones H1b or H1d.

Inhibitory activity on renal membrane adenylate cyclase (AC) has previously been found in the extract of a pancreatic cancer associated with humoral hypercalcemia of malignancy (HHM). AC inhibitor was purified employing inhibition of AC activity of renal membrane stimulated by forskolin as its index. N-terminal 9 residues and a digested fragment of purified protein (14 residues) were completely consistent with that of histones H1b and H1d. Not only histone H1 but also histones H2A, H2B and H3 from calf thymus inhibited AC activity. These results indicate that the AC inhibitor in the pancreatic cancer extract is histone H1b or H1d and histones H2A, H2B and H3 also have an AC inhibitory activity.

Inhibition of estradiol uptake and transforming growth factor alpha secretion in human breast cancer cell line MCF-7 by an alkyl-lysophospholipid.

We investigated the effect of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), an alkyl-lysophospholipid, on the uptake of estrogen, the secretion of transforming growth factor (TGF) alpha and the content of progesterone receptors (PRs) in the hormone-dependent breast cancer cell line, MCF-7. The uptake of labeled estradiol by MCF-7 was dose dependently decreased by 12 h pretreatment with 10-25 micrograms/ml ET-18-OCH3, and this suppression occurred prior to the onset of the inhibitory action of ET-18-OCH3 on MCF-7 growth. Scatchard analysis demonstrated that ET-18-OCH3 reduced the number of estrogen receptors in MCF-7 without affecting their affinity. Both the secretion of TGF-alpha from MCF-7 into the conditioned medium and the PR content of MCF-7 were decreased by 48 h treatment with 10 micrograms/ml ET-18-OCH3. The estradiol uptake, the TGF-alpha secretion, and the PR content were not affected by platelet-activating factor, lyso-PAF, and palmitoyl-lysophosphatidylcholine, all at 10 micrograms/ml. These results suggest that the reduction of estrogen receptor level induced by ET-18-OCH3 resulted in decreases in both the secretion of TGF-alpha and the content of PR in MCF-7, and these effects are specific to ET-18-OCH3. We concluded that these effects of ET-18-OCH3 may lead, at least partly, to its antitumor action in hormone-dependent breast cancer cell lines.

Inhibition of renal membrane adenylate cyclase by extract of pancreatic cancer associated with humoral hypercalcemia of malignancy.

The parathyroid hormone (PTH)-like activity, defined by the stimulation of cAMP production in MC3T3E1 cells, in the extract of a pancreatic cancer associated with humoral hypercalcemia of malignancy (HHM) was eluted in two peaks (I and II) by reverse phase HPLC. Both peaks dose-dependently inhibited the binding of human (h) PTH(1-34) to canine renal membrane in an essentially similar fashion to hPTH(1-34) or PTH-related protein (rP). In the renal membrane, neither of these peaks stimulated adenylate cyclase (AC) but rather dose-dependently inhibited AC activity stimulated by hPTH(1-34), PTH-rP(1-34) or forskolin. In rat renal cortical slices, however, both peaks could exhibit their own stimulatory effect and did not inhibit PTH or forskolin-stimulated cAMP production. It has been concluded that a factor which inhibits AC activity, probably reflecting the direct action at catalytic site, can occasionally be produced with PTH-like factor. Although PTH-like and AC-inhibiting activities were very close on reverse phase HPLC, currently the interrelation between these two activities is not clear. It may be important to be aware of the presence of such a factor in the evaluation of the bioassay data employing a broken cell preparation, which is often used to assess the PTH-like activity of tumor products.

Increase of transferrin binding induced by an alkyl-lysophospholipid in breast cancer cells.

The effect of ET-18-OCH3 on transferrin (Tf) binding in human breast cancer cell lines, MCF-7, ZR-75-1 and BT-20, and mouse fibroblast, Balb/c 3T3, was investigated. Pretreatment with 3-25 micrograms/ml ET-18-OCH3 increased the Tf binding at 37 degrees C and 4 degrees C in all cell lines tested. In MCF-7, both the affinity and the number of Tf binding sites were increased by ET-18-OCH3. These observations are of interest with respect to the action of ET-18-OCH3 on cell surface receptors.

Relationship between the levels of serum thyroid hormones or estrogen status and the risk of breast cancer genesis in Japanese women.

For the purpose of investigating a possible correlation between the genesis of breast cancer and the levels of serum thyroid hormones or the estrogen status, which is one of the potential risk factors for breast cancer in Japanese women, we measured the percentage of free estradiol (E2) and the amounts of sex hormone-binding globulin (SHBG) and thyroid hormones in serum samples from Japanese patients with breast cancer (N = 39) and normal controls (N = 36). The patients were found to have significantly higher free E2 and significantly lower SHBG than controls. Moreover, the serum levels of free triiodothyronine (FT3) and free thyroxine (FT4) were lower in the patients than in controls, while the serum levels of TSH and TBG in the patients were not significantly different from those in controls. The percentage of free E2 in serum was not significantly correlated with the level of any one of FT3, FT4, TSH, and TBG either in the patients or in controls regardless of menstrual status. These results suggest the possibility that the reduction in the serum FT3 and FT4 levels, which is independent of changes in the serum level of free E2, may be one of the risk factors for breast cancer in Japanese women.