Elevated luteinizing hormone contributes to atherosclerosis formation by inhibiting nitric oxide synthesis via PI3K/Akt pathway.

BACKGROUND: The contentious effects of estrogen therapy on the risk of postmenopausal cardiovascular disease (CVD) indicate that this type of atherosclerosis is not solely induced by estrogen deficiency. Other sex hormones such as elevated luteinizing hormone (LH) may also affect CVD risk in this population. We therefore explored the relationship between LH and atherosclerosis in ovariectomized (OVX) female mice. METHODS: Aortic atherosclerotic lesions were assessed in OVX ApoE knock out (ApoE-/-) female mice administered with LH. Human umbilical vascular endothelial cells (HUVECs) were cultured as cell model. The influence of LH on NO release, phosphorylated endothelial nitric oxide synthase (eNOS) and Akt levels were evaluated. Immunoprecipitation and lentiviral particle transfection were applied to assess the role of Gαq on PI3K activity. RESULTS: LH increased the atherosclerotic lesion area and carotid artery intima-media thickness (IMT) in OVX ApoE-/- female mice. High levels of LH attenuated vasodilation induced by Ach and inhibited NO release from HUVECs. These effects were related to the findings that LH enhanced interaction between Gαq and p110α, which subsequently inhibited PI3K activity and suppressed the phosphorylation of Akt and eNOS. CONCLUSIONS: Elevated LH promotes atherosclerosis formation in OVX ApoE-/- female mice. This effect may be mediated by inhibiting endothelial NO synthesis via PI3K/Akt signaling pathway.

Differential effects of the persistent DDT metabolite methylsulfonyl-DDE in nonstimulated and LH-stimulated neonatal porcine Leydig cells.

3-Methylsulfonyl-DDE (MeSO2-DDE) is a potent adrenal toxicant formed from the persistent insecticide DDT. MeSO2-DDE is widely present in human plasma, milk and fat, and in tissues of marine mammals. In the present study, we investigated endocrine-disrupting properties of MeSO2-DDE in primary neonatal porcine Leydig cells. Unstimulated and LH-stimulated cells were exposed to MeSO2-DDE at concentrations ranging from 0.6 to 20 µM for 48 h. Cell viability, hormone secretion and expression of steroidogenesis related genes were recorded. Secretion of testosterone and estradiol was increased in a concentration-dependent fashion in unstimulated Leydig cells, while in LH-stimulated cells, secretion of testosterone, estradiol and progesterone was decreased. The expression of important steroidogenic genes was down-regulated both in unstimulated and LH-stimulated cells. Notably, no significant impairment of cell viability occurred at any exposure except the highest concentration (20 µM) in LH-stimulated cells. This indicated that the effects on hormone secretion and gene expression were not caused by cytotoxicity. We conclude that the adrenal toxicant MeSO2-DDE disrupts hormone secretion in a complex fashion in neonatal porcine Leydig cells. The different endocrine responses in unstimulated and LH-stimulated cells imply that the endocrine disruptive activity of MeSO2-DDE is determined by the physiological status of the Leydig cells.

Impact of infertility regimens on breast cancer cells: follicle-stimulating hormone and luteinizing hormone lack a direct effect on breast cell proliferation in vitro.

OBJECTIVE: To examine the impact of hormones used for controlled ovarian hyperstimulation (COH) on normal and malignant breast cell growth and proliferation. DESIGN: In vitro study of cultured normal and malignant breast cell lines. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): Normal and malignant breast cell lines cultured in two- and three-dimensional (2D and 3D) systems and treated with follicle-stimulating hormone (FSH), luteinizing hormone (LH), or FSH with LH or human chorionic gonadotropin (hCG). MAIN OUTCOME MEASURE(S): Effects of treatment on cell proliferation in 2D culture using the MTS assay and on colony growth in 3D culture. RESULT(S): Compared with untreated cells, normal MCF-10A cells showed a decrease in proliferation and colony size when exposed to a combination of FSH and hCG. The HCC 1937 cells treated with FSH and LH also showed a decrease in colony growth but no change in proliferation. None of the treatments had an effect on the proliferation or colony size of the MCF-7 cells. CONCLUSION(S): Follicle-stimulating hormone, LH, and hCG do not appear to cause an increase in cell proliferation or colony growth in either normal or malignant mammary epithelial cell lines. The potential risk for mammary cell transformation associated with these agents may be related to indirect endocrine effects on breast cell physiology.

Targeted destruction of androgen-sensitive and -insensitive prostate cancer cells and xenografts through luteinizing hormone receptors.

BACKGROUND: We have prepared a conjugate of a lytic peptide (hecate) and a 15-amino acid segment of the beta-chain of LH to test the concept that this conjugate will target cancer cells expressing LH receptors. METHODS: Hecate-betaLH was added in vitro to cultures of Chinese hamster ovary (CHO) cells with and without LH receptors and to prostate cancer cells in the presence or absence of steroids, follicle-stimulating hormone (FSH), epidermal growth factor (EGF), or betaLH. PC-3 xenografts were established in male athymic nude mice and treated once a week for 3 weeks with hecate-betaLH via the lateral tail vein. RESULTS: The conjugate showed concentration-dependent toxicity for the following prostate cancer cell lines: BRF 41 T>DU145>PC-3>LNCaP, according to their LH receptor capacities. Steroid removal reduced sensitivity to the drug in a reversible manner. Hecate-betaLH reduced the tumor burden in the nude mice from 60 to 12.5 mg/g body weight. CONCLUSIONS: We conclude that the hecate-betaLH conjugate selectively kills androgen-dependent and-independent prostate cancer cells both in vivo and in vitro; its toxicity depends on the number of LH receptor sites present.

Hormonotoxins: the role of positive charge of lysine residue on the immunological, biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates.

Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occurred through the epsilon-NH2 groups of alpha-subunit of oLH as judged from RP-HPLC analysis. A1:1 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.

Comparison of the cytotoxic effect of hormonotoxins prepared with the use of heterobifunctional cross-linking agents N-succinimidyl 3-(2-pyridyldithio)propionate and N-succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate.

With the aim of developing cytotoxic hybrid molecules which can be selectively targeted to specific cells in the gonads, a single chain ribosome-inactivating protein, gelonin, was conjugated to ovine luteinizing hormone (oLH) with the use of heterobifunctional cross-linking agents N-succinimidyl3-(2-pyridyldithio)-propionate (SPDP) and long-chain SPDP. Four hormonotoxins were synthesized having a variable spacer arm between oLH and gelonin. The spacer arms in C200A, C210A, C220A, and C230A were 13.6, 22.4, 22.4, and 31.2 A long, respectively. Extensive physiochemical and biochemical analysis revealed a 1:1 molar ratio of the ingredients in its oLH-S-S-gelonin conjugates. The linkage occurred through the epsilon-NH2 group of the alpha-subunit of oLH as judged from RP-HPLC analysis. The hormonotoxins retained substantial receptor binding ability, steriodogenic activity, and immunoreactivity of oLH and gelonin to their respective antibodies. Hormonotoxins bind to Leydig tumor cells via oLH, leaving gelonin free as judged by competitive displacement analysis. The hormonotoxins internalized to a sufficient degree to effectively inhibit protein synthesis. Upon comparison, immunoreactivity, receptor binding steroidogenic activity, and cytotoxicity of oLH-S-S-gelonin conjugates prepared with the use of only LC-SPDP (C230A, 31.2-A spacer arm) and by using both SPDP and LC-SPDP (C210A and C220A, 22.4-A spacer arm) were found to be comparable with that of conjugate prepared with SPDP alone (C200A, 13.6-A spacer arm). Therefore, it may be concluded that the cytotoxicity of oLH-based hormonotoxin remained unaffected with the use of long-chain spacer arms which are believed to be used generally to avoid steric hindrance.

In-vitro selective killing of gonadal cells by a hormonotoxin composed of ovine luteinizing hormone linked by a disulfide bond to the ribosome-inactivating protein, gelonin.

With the aim to synthesize a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, a single chain RIP was conjugated to oLH with the use of SPDP which generated oLH-S-S-gelonin hormonotoxin. Extensive physico-chemical, immunochemical and biochemical characterization reveal that 1:1 oLH-gelonin was linked through the alpha-subunit of the oLH. The hormonotoxin retained substantial receptor binding and steroidogenic activity in the leydig tumor cells. The competitive displacement analysis indicate that the binding occurs via the hormone. The presently described hormonotoxin exhibited higher receptor binding and cytotoxicity to the target cells than that of others reported so far.

The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions.

The surface of thin sections of aldehyde-fixed biological material shows a specimen-related relief of 2-6 nm with Lowicryl. Epon sections are about three times smoother. The relief is the consequence of thin-sectioning being in reality a cleavage. Epitopes are supposed to be laid open (or set free) because cleavage follows the interfaces between protein and Lowicryl. We have developed a simple theory on this basis and have theoretically estimated the efficiency of on-section labeling and compared it with experimental data. For randomly dispersed proteins in cytoplasm, Lowicryl sections will yield significant label only when the concentration of the antigen is about 10 microM or more. The complex situation of more compact proteins, as represented by fibers, sheets, and biological membranes is discussed and the difficulty of significant calculations is explained. Pre-embedding labeling and melted cryosections should give 10-30 times more label. The possible reasons for the observed much smaller gain of not more than two to three times are discussed.