Biomedical subjects

Osamu Wada

Publications and source records attributed to Osamu Wada.

17 recordsLinked to original sources

PubMed2006

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine 3beta-Sulfooxy-12alpha-hydroxy-5beta-cholanoic acid is an abundant nonamidated sulfate.

We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3beta,12alpha-dihydroxy-5beta-cholanoic acid 3-sulfate.

Adult↗

PubMed2006

Involvement of a cellular ubiquitin-protein ligase E6AP in the ubiquitin-mediated degradation of extensive substrates of high-risk human papillomavirus E6.

Human scribble (hScrib), which was identified as substrate of human papillomavirus (HPV) E6 for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP, is a human homolog of Drosophila neoplastic tumor suppressor scribble, in which mutation causes loss of polarity and overgrowth of epithelia. Drosophila discs large (Dlg) is one of neoplastic tumor suppressors, which genetically links to scribble. E6 also targets human Dlg (hDlg) for ubiquitin-mediated degradation. Ubiquitin-protein ligase involved in this process has not been identified thus far. Here we investigated mechanism underlying degradation of three target proteins of E6, hScrib, hDlg, and p53 by using eighteen HPV 16 E6 mutants with single amino acid substitution. In vitro degradation ability of each E6 mutant was equivalent for these tumor suppressors. We investigated whether E6AP is involved in ubiquitin-mediated degradation of hDlg. In vitro binding assay revealed that hDlg formed ternary complex with E6-E6AP complex. The ability of E6 mutants to degrade these tumor suppressors was correlated with their ability to interact with E6AP. Furthermore, hDlg was targeted for in vitro ubiquitination in the presence of both E6 and E6AP. These data revealed that E6AP is extensively involved in the ubiquitin-mediated degradation of E6-dependent substrates as a cellular E3 ubiquitin-protein ligase.

Adaptor Proteins, Signal Transducing↗

PubMed2005

An hGCN5/TRRAP histone acetyltransferase complex co-activates BRCA1 transactivation function through histone modification.

It is well established that genetic mutations that impair BRCA1 function predispose women to early onset of breast and ovarian cancer. However, the co-regulatory factors that support normal BRCA1 functions remain to be identified. Using a biochemical approach to search for such co-regulatory factors, we identified hGCN5, TRRAP, and hMSH2/6 as BRCA1-interacting proteins. Genetic mutations in the C-terminal transactivation domain of BRCA1, as found in breast cancer patients (Chapman, M. S., and Verma, I. M. (1996) Nature 382, 678-679), caused the loss of physical interaction between BRCA1 and TRRAP and significantly reduced the co-activation of BRCA1 transactivation function by hGCN5/TRRAP. The reported transcriptional squelching between BRCA1 and estrogen receptor alpha (Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F., Auborn, K. J., Goldberg, I. D., and Rosen, E. M. (1999) Science 284, 1354-1356) was rescued by the overexpression of TRRAP or hGCN5. Histone acetyltransferase hGCN5 activity appeared to be indispensable for coregulator complex function in both BRCA1-mediated gene regulation and DNA repair. Biochemical purification of the hGCN5/TRRAP-containing complex suggested that hGCN5/TRRAP formed a complex with hMSH2/hMSH6, presumably as a novel subclass of hGCN5/TRRAP-containing known TFTC (TBP-free TAF-containing)-type histone acetyltransferase complex (hTFTC, hPCAF, and hSTAGA) (Yanagisawa, J., Kitagawa, H., Yanagida, M., Wada, O., Ogawa, S., Nakagomi, M., Oishi, H., Yamamoto, Y., Nagasawa, H., McMahon, S. B., Cole, M. D., Tora, L., Takahashi, N., and Kato, S. (2002) Mol. Cell 9, 553-562). Unlike other subclasses, the isolated complex harbored a previously unknown combination of components including hMSH2 and hMSH6, major components of the BRCA1 genome surveillance repair complex (BASC). Thus, our results suggested that the multiple BRCA1 functions require a novel hGCN5/TRRAP histone acetyltransferase complex subclass.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Cross-Talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia.

Recent studies have shown the involvement of Fas/Fas ligand (FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-gamma, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 microm, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-gamma, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.

Animals↗

PubMed2004

BRCA1 function mediates a TRAP/DRIP complex through direct interaction with TRAP220.

Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene mutated in a high percentage of hereditary breast and ovarian cancers. The multifunctional BRCA1 protein acts on cell cycle control, exerting several highly specialized DNA repair processes through diverse domains. Gene regulation through its C-terminal domain (BRCT) is indispensable for BRCA1-mediated tumor suppression, suggesting the possibility that the BRCT domain interacts with co-regulator complexes. Using a biochemical approach with HeLa S3 nuclear extracts, we isolated BRCT-associated complexes and identified one of the purified components as TRAP220. We then performed interaction studies in vivo (co-immunoprecipitation) and in vitro (glutathione S-transferase pull-down assays) and showed that BRCT directly interacted with TRAP220. This in vitro interaction was completely abolished by BRCT point mutations typical of those found in patients with BRCA1 that lack transactivation function. BRCA1 transactivation function was dependent on TRAP220 expression level in a transient expression assay. Moreover, a cell survival assay showed that antisense TRAP220 expression to disrupt endogenous TRAP220 expression significantly reduced the survival rate potentiated by BRCA1 after DNA damage. These results suggested that a TRAP220 complex play an important role as putative co-activator complexes in BRCA1-mediated tumor suppression.

BRCA1 Protein↗

PubMed2004

Excessive zinc intake elevates systemic blood pressure levels in normotensive rats--potential role of superoxide-induced oxidative stress.

OBJECTIVES: The present study was designed to examine whether or not excessive Zn intake affects systemic blood pressure (BP) levels in a normotensive state. METHODS: Systolic BP (SBP) and mean arterial pressure (MAP) before and after administration of the nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME) or the exogenous superoxide scavenger, tempol and the activity of the endogenous superoxide scavenger, Cu/Zn-superoxide dismutase (SOD) and levels of endothelial type (e)NOS mRNA and protein in the thoracic aorta were analyzed in male Sprague-Dawley rats fed a standard diet containing 0.005% Zn or a high Zn diet containing 0.5% Zn for 8 weeks. RESULTS: SBP and MAP levels observed at the end of dietary conditioning were significantly elevated in rats fed a high Zn diet relative to rats fed a standard diet. Administration of L-NAME caused an increase in MAP levels in rats fed a standard and a high Zn diet, demonstrating the involvement of the vasodilator, nitric oxide (NO) in the regulation of systemic BP in the two groups of rats. However, the expression of eNOS mRNA and protein in the thoracic aorta was not significantly different between rats fed a standard and a high Zn diet. On the other hand, administration of tempol led to a decrease in MAP levels in rats fed a standard and a high Zn diet, indicating the participation of the oxygen free radical, superoxide in the modification of systemic BP in the two groups of rats. As reported recently, the mechanism involved is due likely to a decrease in the action of the vasodilator, NO through the formation of peroxynitrite based on the non-enzymatic reaction of superoxide and NO. In addition, tempol treatment dramatically restored MAP levels in rats fed a high Zn diet to levels comparable with those observed in rats fed a standard diet, indicating that an elevation in systemic BP levels seen in rats fed a high Zn versus a standard diet is presumably brought by a reduction in the action of the vasodilator, NO resulting from an increase in the action of superoxide. The activity of Cu/Zn-SOD in the thoracic aorta was significantly reduced in rats fed a high Zn diet relative to rats fed a standard diet, appearing to at least in part, play a role in an increase in the action of superoxide in the vessel wall of rats fed a high Zn diet. CONCLUSIONS: Excessive Zn intake may be a factor to elevate systemic BP levels in a normotensive state presumably through the oxidative stress caused by superoxide.

Animals↗

PubMed2004

Silicosis and smoking strongly increase lung cancer risk in silica-exposed workers.

It remains controversial whether silica is a human lung carcinogen. In this study, we estimated the relative risks of lung cancer due to silica and silicosis by meta-analysis. We collected papers published from 1966-2001 which epidemiologically reported on the relationship between silica/silicosis and lung cancer. We removed papers which did not exclude the effects of asbestos and radioactive materials including radon. We selected the most recent one if some papers were based on the same cohort. Based on the selected papers, we summarized the lung cancer risks from silica, silicosis and non-silicosis with exposure to silica, by meta-analysis using a random effects model. The pooled relative risks were 1.32 (95% confidence interval (CI), 1.23-1.41) for silica, 2.37 (95% CI, 1.98-2.84) for silicosis and 0.96 (95% CI, 0.81-1.15) for non-silicosis with exposure to silica. Since some papers on silica did not exclude silicosis, the risk due to silica itself may be smaller than 1.32. It was less possible that silica exposure directly increases lung cancer risk. On the other hand, the relative risk, 2.37 for silicosis suggested that silicosis increases lung cancer risk. Meta-analysis also revealed that cigarette smoking strongly increased the lung cancer risk in silicotic patients (relative risk, 4.47; 95% CI, 3.17-6.30). Thus, the present study suggested the great importance of preventing silicosis and smoking cessation in reducing lung cancer incidence in silica-exposed workers.

Carcinogens↗

PubMed2004

[Zinc (Zn)].

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Acrodermatitis↗

PubMed2003

Both N- and C-terminal transactivation functions of DNA-bound ERalpha are blocked by a novel synthetic estrogen ligand.

Estrogen receptors (ERs) play a central role in the diverse actions of estrogen. A number of synthetic ER ligands have been generated that can modulate various ER functions. Here we show that TAS-108, representing a novel class of synthetic ER ligands, blocked both ER transactivation functions without inhibiting DNA-binding activity. A transient expression assay showed that similar to ICI182,780, TAS-108 exhibited pure antagonistic activity as it blocked both the N-terminal AF-1 and C-terminal AF-2 transactivation functions. However, unlike ICI182,780, TAS-108 promoted the recruitment of the SMRT co-repressor that abolished ER transactivation function without inhibition of the ability of ERalpha to bind to its target DNA. Both TAS-108 and ICI182,780 acted as antagonists for the transactivation functions of the D351Y mutant, derived from tamoxifen-resistant breast cancer cells, while estrogen and known selective estrogen receptor modulators (SERMs), 4-OH tamoxifen and raloxifene, stimulated D351Y-mediated transcription. Thus, our findings indicated that TAS-108 acts as a novel estrogen antagonist that recruits co-repressors to ERs without AF-1 activation or prevention of DNA binding. Therefore, TAS-108 may be effective against tamoxifen-resistant breast cancer via a different mechanism than that for ICI182,780.

Animals↗

PubMed2003

Full activation of estrogen receptor alpha activation function-1 induces proliferation of breast cancer cells.

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) alpha and beta, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptor-mediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ERalpha. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ERalpha to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ERalpha was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ERalpha and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ERalpha. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen.

3T3 Cells↗

PubMed2003

Dietary Zn deficiency does not influence systemic blood pressure and vascular nitric oxide signaling in normotensive rats.

Because zinc (Zn) is an important component for cell protection against certain oxygen species, it has been suggested that Zn deficiency impairs the potent oxidant defense capacity, which is constitutively provided in the vascular system. However, the influence of dietary Zn deficiency on systemic blood pressure and vascular system is controversial and unclear. We therefore examine the effect of dietary Zn deficiency on systemic blood pressure, a potent superoxide scavenger, aortic Cu/Zn superoxide dismutase (SOD) activity, a most representative synthase of the endothelium-derived relaxing factor, and aortic endothelial nitric oxide synthase (eNOS) expression. Furthermore, the direct effects of intravenous administration of NOS inhibitor, Nomega-nitro-L-arginine methyl ester (LNAME), and a SOD mimetic compound, tempol, in normotensives were tested in Wistar-Kyoto (WKY) rats. A Zn-deficient diet (4 wk) contributed to growth retardation, the decrease in thymus weight, and the lower levels of serum Zn compared with the standard diet group. However, no significant difference in conscious systolic and diastolic blood pressure was found in the Zn-deficiency group. The administration of L-NAME caused an increase in the mean arterial pressure (MAP) levels in the two groups of rats and the involvement of the vasodilator nitric oxide (NO) in the regulation of systemic BP in the normotensive state. On the other hand, administration of the superoxide scavenger, tempol, led to a decrease in MAP levels in the two groups of rats, indicating the participation of the oxygen free radical, superoxide, in the maintenance of the systemic BP in a normotensive state. There were no significant differences between the Zn-deficient diet group and the standard diet group in the normotensive state. eNOS expression and Cu/Zn SOD activity in the aorta were also intact in Zn-deficient normotensive rats. These findings suggest that the 4 wk of Zn deficiency was inadequate to alter systemic blood pressure and focal NO signaling in the normotensive state. Long-term Zn deficiency affects the neuronal, immune, and hematopoietic systems, which contribute to systemic and/or local circulation. However, Zn deficiency alone does not cause hypertension and local vascular dysfunction in the normotensive state.

Animals↗

PubMed2002

Decreased expression of brain nitric oxide synthase in macula densa cells and glomerular epithelial cells of rats with mercury chloride-induced acute renal failure.

To elucidate the mechanisms responsible for the development of HgCl(2)-induced acute renal failure (ARF), we examined the expression of brain type (b) nitric oxide synthase (NOS), which is involved in the generation of the vasodilator nitric oxide (NO), in the renal cortex of rats at 20 h after exposure to 7.5 mg/kg HgCl(2). Both blood urea nitrogen and serum creatinine were significantly increased in rats exposed to HgCl(2) relative to control rats, indicating the induction of ARF resulting from HgCl(2) exposure. Histopathological analysis demonstrated that, in addition to necrosis of proximal tubule epithelial cells, necrosis of macula densa cells and swelling of glomerular epithelial cells were observed in the renal cortex of rats with HgCl(2)-induced ARF. Consequently, the number of pars maculata segments was decreased by 42% in rats with HgCl(2)-induced ARF compared to control rats. The primary sites of bNOS mRNA and protein expression were macula densa cells and glomerular epithelial cells in the renal cortex of control rats and rats with HgCl(2)-induced ARF. The abundance of the bNOS mRNA and protein was significantly decreased in rats with HgCl(2)-induced ARF relative to control rats. These observations suggest that the production of the vasodilator NO derived from bNOS is decreased at the glomerulus level in the HgCl(2)-induced ARF setting. Thus, the reduction in bNOS expression may in part contribute to the progression of HgCl(2)-induced ARF through the deterioration of glomerular hemodynamics. In addition, the decrease in bNOS expression may be primarily the result of cell injury caused by the cytotoxic effect of HgCl(2).

Acute Kidney Injury↗

PubMed2002

Nuclear receptor function requires a TFTC-type histone acetyl transferase complex.

Nuclear receptors (NRs) regulate transcription in a ligand-dependent way through two types of coactivator complexes: the p160/CBP histone acetyl transferase (HAT) complex and the DRIP/TRAP/SMCC complex without HAT activity. Here we identified a large human (h) coactivator complex necessary for the estrogen receptor alpha (ERalpha) transactivation. This complex contains the GCN5 HAT, the c-Myc interacting protein TRRAP/PAF400, TAF(II)30, and other subunits. Similarly to known TFTC (TBP-free TAF(II)-containing)-type HAT complexes (hTFTC, hPCAF, and hSTAGA), TRRP directly interacted with liganded ER alpha, or other NRs. ER alpha transactivation was enhanced by the purified complex in vitro. Antisense TRRAP RNA inhibited estrogen-dependent cell growth of breast cancer cells. Thus, the isolated TFTC-type HAT complex acts as a third class of coactivator complex for NR function.

Acetyltransferases↗

PubMed2002

Human chorionic gonadotropin combined with progesterone for luteal support improves pregnancy rate in patients with low late-midluteal estradiol levels in IVF cycles.

PURPOSE: To investigate how late-midluteal estradiol levels relate to the pregnancy outcome in IVF cycles, and to assess whether human chorionic gonadotropin (hCG) for luteal support benefits the pregnancy outcome of patients with low late-midluteal estradiol levels. METHODS: The pregnancy rate of 436 women undergoing first IVF cycles with long protocol and luteal support with progesterone alone were analyzed. Unsuccessful women with low late-midluteal estradiol levels (< 100 pg/mL) proceeded with the exploratory second IVF cycles where they were randomly given with either progesterone alone (P protocol) or hCG +progesterone (P+hCG protocol) for luteal support. RESULTS: Pregnancy rate in women with low late-midluteal estradiol levels was significantly lower compared to that with medium (100-500 pg/mL) and high (> 500 pg/mL) levels (13.3, 26.8, and 36.3%, respectively). P+hCG protocol increased late-midluteal estradiol levels and produced a significantly higher pregnancy rate (31.7%) than P protocol (13.7%). CONCLUSIONS: hCG in combination with progesterone for luteal support was suggested to benefit women undergoing IVF with low late-midluteal estradiol levels.

Adult↗

PubMed2002

Increased renal vascular resistance in zinc-deficient rats: role of nitric oxide and superoxide.

1. Zinc deficiency (ZD) induces many kinds of pathological states. However, the effects of ZD on haemodynamics remain unclear. In the present study, we measured mean blood pressure (BP) and renal blood flow (RBF) under anaesthesia and calculated renal vascular resistance (RVR) from these parameters in rats maintained on a ZD diet (0.5 p.p.m. zinc) for 4 weeks. 2. Zinc deficiency did not change mean BP, but significantly reduced RBF and increased RVR (each P < 0.01). In addition, these effects of ZD were reversible. 3. Because Cu/Zn superoxide dismutase (SOD) is a zinc-containing enzyme and superoxide is a potent scavenger of nitric oxide (NO), a vasodilator, we hypothesized that one of the mechanisms by which ZD increases RVR is by decreasing NO bioavailability by the enhanced formation of superoxide due to low Cu/Zn SOD activity. To test this hypothesis, we observed the roles of NO and superoxide in the mechanism, after having confirmed the low activity of Cu/Zn SOD in the kidneys of ZD rats. 4. Administration of the SOD mimetic tempol (5 mg/kg per min) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that superoxide was responsible for the mechanism. Low doses of the NO donor sodium nitroprusside (SNP; 2.0 micro g/kg per min, continuous) decreased RVR to a significantly smaller extent in ZD rats compared with control, whereas a high dose of SNP (0.75 mg/kg, bolus) decreased RVR to a significantly greater extent in ZD rats compared with control, suggesting that the mechanism includes an inhibition of NO activity in ZD, which is most likely to be a scavenging of NO by the activated superoxide. 5. In summary, ZD may increase RVR. The mechanism probably includes changes in NO and superoxide activities.

Animals↗

PubMed2002

U-shaped effect of drinking and linear effect of smoking on risk for stomach cancer in Japan.

A case-control study was conducted to evaluate the relationship between smoking or drinking doses and risk for stomach cancer, and to clarify whether the relationship is dose-dependent or U-shaped. Smoking dose was categorized as 0, 1 - 399, 400 - 799, or 800 + cigarette-years, and drinking dose as 0, occasional/0.1 - 134.9, 135 - 1349.9, or 1350 + alcohol-years (ml of pure alcohol intake per day multiplied by years of drinking). Helicobacter pylori status was determined by serology for adjustment. Using logistic regression, the adjusted effects of smoking and drinking doses on risk for stomach cancer were calculated for both genders. Among male subjects, the odds ratios (95% confidence intervals (CIs)) were 1.29 (0.76, 2.18) for 1 - 399, 1.71 (1.05, 2.80) for 400 - 799 and 2.46 (1.49, 4.07) for 800 + cigarette-years compared with never-smokers, and 1.89 (0.97, 3.69) for never-drinkers, 2.82 (1.63, 4.86) for 135 - 1349.9 and 2.84 (1.97, 4.83) for 1350.0 +, compared with occasional/0.1 - 134.9 alcohol-years. Among female subjects, they were 0.44 (0.20, 1.00) for 1 - 399 and 2.471 (0.91, 6.68) for 400 + cigarette-years compared with never-smokers, and 1.54 (0.90, 2.63) for never-drinkers and 1.39 (0.66, 2.93) for 135.0 + alcohol-years. Smoking seems to exert a linear effect and drinking, a J- or U-shaped effect on risk for stomach cancer, although there might be a dip of risk in light smokers among female subjects.

Adult↗

PubMed2002

Cellular mechanisms of growth inhibition of human epithelial ovarian cancer cell line by LH-releasing hormone antagonist Cetrorelix.

We investigated the direct effects of LH-releasing hormone (LH-RH) antagonist, Cetrorelix, on the growth of HTOA human epithelial ovarian cancer cell line. RT-PCR revealed the expression of mRNA for LH-RH and its receptor in HTOA cells. Cetrorelix, at concentrations between 10(-9) and 10(-5) M, exerted a dose-dependent antiproliferative action on HTOA cells, as measured by 5-bromo-2'-deoxyuridine incorporation into DNA. Flow cytometric analysis indicated that Cetrorelix, at 10(-5) M, arrested cell cycle in HTOA cells, at G1 phase, after 24 h of treatment. Western blot analysis of cell cycle-regulatory proteins demonstrated that treatment with Cetrorelix (10(-5) M) for 24 h did not change the steady-state levels of cyclin D1, cyclin E, and cyclin-dependent kinase (Cdk)4 but decreased the levels of cyclin A and Cdk2. The protein levels of p21 (a Cdk inhibitor) and p53 (a suppressor of tumor cell growth and a positive regulator for p21 expression) were increased by Cetrorelix, but the levels of p27 (a Cdk inhibitor) did not change significantly. Flow cytometric analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick end labeling staining demonstrated that Cetrorelix (10(-5) M) induced apoptosis in HTOA cells. In conclusion, Cetrorelix directly inhibits the proliferation of human epithelial ovarian cancer cells through mechanisms mediated by LH-RH receptor and involving multiple events in cell cycle progression, including G1 phase cell cycle arrest coupled with down-regulation of cyclin A-Cdk2 complex levels, presumably attributable to an up-regulation of p53 and p21 protein levels and apoptosis.

Apoptosis↗