Clinical Phenotypes of PCOS: a Cross-Sectional Study.

This cross-sectional study examines the Doi-Alshoumer PCOS clinical phenotype classification in relation to measured clinical and biochemical characteristics of women with polycystic ovary syndrome (PCOS). Two cohorts of women (Kuwait and Rotterdam) diagnosed with PCOS (FAI > 4.5%) were examined. These phenotypes were created using neuroendocrine dysfunction (IRMA LH/FSH ratio > 1 or LH > 6 IU/L) and menstrual cycle status (oligo/amenorrhea) to create three phenotypes: (A) neuroendocrine dysfunction and oligo/amenorrhea, (B) without neuroendocrine dysfunction but with oligo/amenorrhea, and (C) without neuroendocrine dysfunction and with regular cycles. These phenotypes were compared in terms of hormonal, biochemical, and anthropometric measures. The three suggested phenotypes (A, B, and C) were shown to be sufficiently distinct in terms of hormonal, biochemical, and anthropometric measures. Patients who were classified as phenotype A had neuroendocrine dysfunction, excess LH (and LH/FSH ratio), irregular cycles, excess A4, infertility, excess T, highest FAI and E2, and excess 17αOHPG when compared to the other phenotypes. Patients classified as phenotype B had irregular cycles, no neuroendocrine dysfunction, obesity, acanthosis nigricans, and insulin resistance. Lastly, patients classified as phenotype C had regular cycles, acne, hirsutism, excess P4, and the highest P4 to E2 molar ratio. The differences across phenotypes suggested distinct phenotypic expression of this syndrome, and the biochemical and clinical correlates of each phenotype are likely to be useful in the management of women with PCOS. These phenotypic criteria are distinct from criteria used for diagnosis.

Preclinical and Clinical Applications of Metabolomics and Proteomics in Glioblastoma Research.

Glioblastoma (GB) is a primary malignancy of the central nervous system that is classified by the WHO as a grade IV astrocytoma. Despite decades of research, several aspects about the biology of GB are still unclear. Its pathogenesis and resistance mechanisms are poorly understood, and methods to optimize patient diagnosis and prognosis remain a bottle neck owing to the heterogeneity of the malignancy. The field of omics has recently gained traction, as it can aid in understanding the dynamic spatiotemporal regulatory network of enzymes and metabolites that allows cancer cells to adjust to their surroundings to promote tumor development. In combination with other omics techniques, proteomic and metabolomic investigations, which are a potent means for examining a variety of metabolic enzymes as well as intermediate metabolites, might offer crucial information in this area. Therefore, this review intends to stress the major contribution these tools have made in GB clinical and preclinical research and highlights the crucial impacts made by the integrative "omics" approach in reducing some of the therapeutic challenges associated with GB research and treatment. Thus, our study can purvey the use of these powerful tools in research by serving as a hub that particularly summarizes studies employing metabolomics and proteomics in the realm of GB diagnosis, treatment, and prognosis.

Combined crizotinib and endocrine drugs inhibit proliferation, migration, and colony formation of breast cancer cells via downregulation of MET and estrogen receptor.

Hormone-dependent breast cancer is the most abundant molecular subtype of the disease. Despite the availability of endocrine treatments, the use of these drugs is limited by their serious adverse reactions and development of acquired resistance often mediated by growth factor receptors. The hepatocyte growth factor receptor, MET, is a receptor tyrosine kinase known for its oncogenic activity and mediating resistance to targeted therapies. Crizotinib is a small-molecule tyrosine kinase inhibitor of MET. In this study, the anticancer effects of combined crizotinib and endocrine drugs were investigated in breast cancer cells in vitro along with the molecular mechanisms associated with these effects. Results showed that crizotinib inhibited growth of MCF7 and T-47D breast cancer cells in a dose-dependent manner with IC50 values of 2.88 µM and 0.93 µM, respectively. Combined treatment of crizotinib and 4-hydroxytamoxifen resulted in synergistic growth inhibition of MCF7 and T-47D cells with combination index values of 0.39 and 0.8, respectively. The combined treatment significantly suppressed migration and colony formation of MCF7 and T-47D cells. Immunofluorescence showed a significant reduction of the expression of the nuclear protein Ki-67 with the combination of crizotinib and 4-hydroxytamoxifen in both cell lines. Western blotting indicated that the combination treatment reduced the levels of active and total MET, estrogen receptor α (ERα), total and active levels of AKT, ERK, c-SRC, NFĸB p65, GSK-3ß, and the anti-apoptotic BCL-2 protein. Findings from this study suggest a potential role of MET inhibitors in breast cancer treatment as monotherapy or combination with endocrine drugs.

ProBDNF is a Novel Mediator of the Interaction Between MDA-MB- 231 Breast Cancer Cells and Brain Microvascular Endothelial Cells.

AIM: The current work aims to assess the role of proBDNF/BDNF in the interaction between brain microvascular endothelial cells and the MDA-MB-231 breast cancer cell line that has been consistently reported to cause brain metastasis. BACKGROUND: Breast cancer brain metastasis (BM) is a significant health problem with limited therapeutic options. The development of BM is a multistep process that requires constant interaction with brain vasculature and the development of tumor blood supply. The benefits of anti-angiogenic modalities, based on antagonizing vascular endothelial growth factor in breast cancer metastasis, did not prove to be effective. Brain-derived neurotrophic factor (BDNF) is a neurotrophin with a reported angiogenic effect. There is a lack of data regarding the involvement of BDNF in metastatic breast cancer interaction with brain microvascular endothelial cells (HBEC-5i). METHODS: Using an adaptive transfer design, the cross-talk between HBEC-5i and MDAMB- 231 cell was investigated. HBEC-5i were treated with MDA-MB-231-conditioned media, and the involvement of BDNF/proBDNF in the interaction was assessed using both release and inhibitor-based assays in migration and in vitro tube formation assay. RESULTS: MDA-MB-231 and HBEC-5i released total BDNF (250 vs. 80 pg/ml, respectively). MDA-MB-231 conditioned media inhibited the migration of HBEC-5i by more than 80% (p<0.05) and tube formation by 75% (p<0.05). Neutralizing mature BDNF did not alter the MDA-MB-231 induced anti-angiogenic effect, which was completely blunted by antagonizing proBDNF. MDA-MB-231 released proBDNF (131.5 pg/ml), and more than 60% of total BDNF released was in the pro-form. CONCLUSION: proBDNF is a novel mediator of breast cancer-induced anti-angiogenic effect in brain endothelial cells.

Phenethyisoquinoline alkaloids from the leaves of Androcymbium palaestinum.

Thirteen compounds were isolated from the methanolic extract of the leaves of Androcymbium palaestinum Baker (Colchicaceae). Of these, three were new, two were new natural products, and eight were known. The new isolated compounds were (+)-1-demethylandrocine (5), (-)-andropalaestine (8), and (+)-2-demethyl-ß-lumicolchicone (10), while the new natural products were (+)-O-methylkreysigine-N-oxide (3) and (+)-O,O-dimethylautumnaline (9). Moreover, two known compounds are reported for the first time from this species, specifically (-)-colchicine (11) and (-)-3-demethyldemecolcine (13). The structures of the isolated compounds were elucidated using a series of spectroscopic and spectrometric techniques, principally HRESIMS, 1D-NMR (1H and 13C NMR) and 2D-NMR (COSY, edited-HSQC, and HMBC). ECD spectroscopy was used for assigning the absolute configurations of compounds 3, 5, and 10. The cytotoxic activities of the isolated compounds were evaluated using the MDA-MB-435 (melanoma), MDA-MB-231 (breast), and OVCAR3 (ovary) cancer cell lines. Compound 11 was the most potent against all tested cell lines, with IC50 values of 12, 95 and 23 nM, respectively.

Glycosylated Hemoglobin A1c Is Associated with Anthropometric Measurements and Tumor Characteristics in Breast Cancer Patients.

PURPOSE: To investigate the association between glycosylated hemoglobin A1c (HbA1c) with anthropometric measurements and clinicopathologic characteristics of breast cancer patients. Such data are lacking in Arabian countries. PATIENTS AND METHODS: A cross-sectional study was conducted at the Outpatient Oncology Unit at King Hussein Medical Center at the Royal Medical Services (RMS) and 223 breast cancer patients were included. Blood levels of HbA1c were measured and patients were classified into normal/non-diabetic (HbA1c <5.7%), prediabetic (HbA1c 5.7-6.4%), and diabetic (HbA1c ≥6.5%). RESULTS: The average age of patients was 49.9±10.3 years. Most patients had waist circumference equal to or more than 80 cm (91.9%) and more than half (55.2%) had waist-hip ratio equal to or more than 0.85. Mean body mass index (BMI) was 29.9±5.7 kg/m2. The mean level of HbA1c was 6.2±1.4% (range 4.7% to 12.6%). HbA1c levels revealed that most patients in this study classified as prediabetics (44.4%). There was a significant positive correlation between HbA1c levels and each of patient's age (r=0.267, p<0.001), waist circumference (r=0.180, p=0.008), and waist-hip ratio (r=0.278, p<0.001). Compared with premenopausal breast cancer patients, postmenopausal patients had significantly higher HbA1c blood levels (t=-3.542, p=0.003). HbA1c was significantly associated with stage (p=0.044) and grade (p=0.016) of carcinoma in premenopausal breast cancer patients. Among postmenopausal cases, HbA1c was significantly associated with molecular subtype of the disease (p=0.039). CONCLUSION: The majority of Jordanian breast cancer patients in this study are prediabetic, obese, and had visceral obesity. HbA1c levels are increased among older patients and those who have greater waist circumference and waist-hip ratio. HbA1c is associated with advanced stage and grade of breast carcinoma in premenopausal patients and with molecular subtype in postmenopausal cases. These findings urge the need to screen breast cancer patients for glycemic status upon disease presentation and to further consider treatments to control hyperglycemia in order to reduce the impact of metabolic derangements on disease prognosis and outcomes.

Silymarin Ameliorates Diabetes-Induced Proangiogenic Response in Brain Endothelial Cells through a GSK-3ß Inhibition-Induced Reduction of VEGF Release.

Diabetes mellitus (DM) is a major risk factor for cardiovascular disease. Additionally, it was found to induce a dysfunctional angiogenic response in the brain that was attributed to oxidative stress. Milk thistle seed extract (silymarin) has potent antioxidant properties, though its potential use in ameliorating diabetes-induced aberrant brain angiogenesis is unknown. Glycogen synthase kinase-3ß is a regulator of angiogenesis that is upregulated by diabetes. Its involvement in diabetes-induced angiogenesis is unknown. To evaluate the potential of silymarin to ameliorate diabetes-induced aberrant angiogenesis, human brain endothelial cells (HBEC-5i) were treated with 50 µg/mL advanced glycation end (AGE) products in the presence or absence of silymarin (50, 100 µM). The angiogenic potential of HBEC-5i was evaluated in terms of migration and in vitro tube formation capacities. The involvement of GSK-3ß was also evaluated. AGE significantly increased the migration and tube formation rates of HBEC-5i by about onefold (p = 0.0001). Silymarin reduced AGE-induced migration in a dose-dependent manner where 50 µM reduced migration by about 50%, whereas the 100 µM completely inhibited AGE-induced migration. Similarly, silymarin 50 µg/mL blunted AGE-induced tube formation (p = 0.001). This effect was mediated through a GSK-3ß-dependent inhibition of VEGF release. In conclusion, silymarin inhibits AGE-induced aberrant angiogenesis in a GSK-3ß-mediated inhibition of VEGF release.

Brain-Derived Neurotrophic Factor Knockdown Blocks the Angiogenic and Protective Effects of Angiotensin Modulation After Experimental Stroke.

Angiotensin type 1 receptor blockers (ARBs) have been shown to be neuroprotective and neurorestorative in experimental stroke. The mechanisms proposed include anti-inflammatory, antiapoptotic effects, as well as stimulation of endogenous trophic factors leading to angiogenesis and neuroplasticity. We aimed to investigate the involvement of the neurotrophin, brain-derived neurotrophic factor (BDNF), in ARB-mediated functional recovery after stroke. To achieve this aim, Wistar rats received bilateral intracerebroventricular (ICV) injections of short hairpin RNA (shRNA) lentiviral particles or nontargeting control (NTC) vector, to knock down BDNF in both hemispheres. After 14 days, rats were subjected to 90-min middle cerebral artery occlusion (MCAO) and received the ARB, candesartan, 1 mg/kg, or saline IV at reperfusion (one dose), then followed for another 14 days using a battery of behavioral tests. BDNF protein expression was successfully reduced by about 70 % in both hemispheres at 14 days after bilateral shRNA lentiviral particle injection. The NTC group that received candesartan showed better functional outcome as well as increased vascular density and synaptogenesis as compared to saline treatment. BDNF knockdown abrogated the beneficial effects of candesartan on neurobehavioral outcome, vascular density, and synaptogenesis. In conclusion, BDNF is directly involved in candesartan-mediated functional recovery, angiogenesis, and synaptogenesis.

Compound 21 is pro-angiogenic in the brain and results in sustained recovery after ischemic stroke.

INTRODUCTION: Angiotensin II type 2 receptor (AT2R) stimulation is neuroprotective after experimental stroke. However, the therapeutic utility of AT2R stimulation has been hampered by the lack of a specific agonist with favourable bioavailability. Compound 21 (C21) - the first non-peptide AT2R agonist - offers a potential option to enhance stroke recovery. This study aimed to investigate the effect of C21 administration on early and late stroke outcomes, and the molecular mediators involved. METHODS: Rats were subjected to 3 h or 90 min of middle cerebral artery occlusion (MCAO) and randomized to intraperitoneal C21 (0.03 mg/kg) or saline at reperfusion. Animals were sacrificed at 24 h or 7 days and brains were collected for molecular analysis and immunostaining, respectively. Functional outcome at days 1, 4 and 7 was assessed blindly. C21 angiogenic potential was assessed in vitro. RESULTS: After 3 h of MCAO, C21 treatment reduced infarct size and improved behavioural outcome at 24 h without affecting blood pressure. Co-administration of the AT2R antagonist (PD123319) blocked these effects. On the molecular level, C21 decreased brain haemoglobin content, down-regulated apoptotic and oxidative markers, and increased pro-survival molecules in the brain. After 90 min of MCAO, C21 treatment resulted in sustained functional improvement at 7 days, together with increased vascular density in the ischemic penumbra. In vitro, C21 showed a pro-angiogenic effect that was blocked with brain-derived neurotrophic factor neutralization. CONCLUSION: These findings demonstrate that a single dose of C21 is neurovascular-protective and improves stroke outcome possibly through increasing neurotrophin activity, mitigating brain inflammation, and promoting antioxidant and pro-angiogenic effects.

Clinically relevant doses of candesartan inhibit growth of prostate tumor xenografts in vivo through modulation of tumor angiogenesis.

Angiotensin II receptor type 1 blockers (ARBs), widely used antihypertensive drugs, have also been investigated for their anticancer effects. The effect of ARBs on prostate cancer in experimental models compared with meta-analysis data from clinical trials is conflicting. Whereas this discrepancy might be due to the use of supratherapeutic doses of ARBs in cellular and animal models as compared with the clinical doses used in human trials, further investigation of the effects of clinical doses of ARBs on prostate cancer in experimental models is warranted. In the current study, we sought to determine the effects of candesartan on prostate cancer cellular function in vitro and tumor growth in vivo, and characterize the underlying mechanisms. Our analysis indicated that clinically relevant doses of candesartan significantly inhibited growth of PC3 cell tumor xenografts in mice. Interestingly, the same concentrations of candesartan actually promoted prostate cancer cellular function in vitro, through a modest but significant inhibition in apoptosis. Inhibition of tumor growth by candesartan was associated with a decrease in vascular endothelial growth factor (VEGF) expression in tumors and inhibition of tumor angiogenesis, but normalization of tumor vasculature. Although candesartan did not impair PC3 cell viability, it inhibited endothelial-barrier disruption by tumor-derived factors. Furthermore, candesartan significantly inhibited expression of VEGF in PC3 and DU145 cell lines independent of angiotensin II type 2 receptor, but potentially via angiotensin II type 1 receptor inhibition. Our findings clearly demonstrate the therapeutic potential of candesartan for prostate cancer and establish a link between ARBs, VEGF expression, and prostate tumor angiogenesis.

AT1 receptor antagonism is proangiogenic in the brain: BDNF a novel mediator.

Candesartan is an angiotensin II type 1 receptor blocker (ARB) that has been to shown to limit ischemic stroke and improve stroke outcome. In experimental stroke, candesartan induces a proangiogenic effect that is partly attributable to vascular endothelial growth factor. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that has been reported to have angiogenic effects and play an important role in recovery after stroke. The purpose of this investigation was to determine the role of BDNF in the proangiogenic effect of candesartan in the brain under hypertensive conditions. Accordingly, spontaneously hypertensive rats were treated with candesartan, and brain tissue samples were collected for quantification of BDNF expression. In addition, human cerebromicrovascular endothelial cells were treated with either low-dose (1 ƒM) or high-dose (1 µM) angiotensin II alone or in combination with candesartan (0.16 µM) to assess the effect of candesartan treatment and BDNF involvement in the behavior of endothelial cells. Candesartan significantly increased the expression of BDNF in the SHR (P < 0.05). In addition, candesartan reversed the antiangiogenic effect of the 1-µM dose of AngII (P = 0.0001). The observed effects of candesartan were ablated by neutralizing the effects of BDNF. Treatment with the AT2 antagonist PD-123319 significantly reduced tube-like formation in endothelial cells. AT2 stimulation induced the BDNF expression and migration (P < 0.05). In conclusion, candesartan exerts a proangiogenic effect on brain microvascular endothelial cells treated with angiotensin II. This response is attributable to increased BDNF expression and is mediated through stimulation of the AT2 receptor.

Diabetes exacerbates retinal oxidative stress, inflammation, and microvascular degeneration in spontaneously hypertensive rats.

PURPOSE: Hypertension and diabetes are known risk factors for retinal microvascular damage. However, the combined effects of diabetes with early and established stages of hypertension on retinal microvascular degeneration remain incompletely understood. METHODS: Male spontaneously hypertensive rats (SHR) were compared to SHR with streptozotocin-induced diabetes (SHR+D) for 6 or 10 weeks and Wistar rats as controls. RESULTS: Hypertension alone (the SHR group) or in combination with diabetes (the SHR+D group) for 6 weeks induced additive increases in total retinal cell death, compared to the Wistar controls. This increase was associated with significant increases in phosphorylated-Jun N-terminal kinase (pJNK) activation, phosphorylated-Akt inhibition, plasma and retinal lipid peroxides, and soluble intracellular adhesion molecule-1 (sICAM-1) levels. After 10 weeks, a similar trend was still observed in retinal nitrotyrosine, nuclear factor kappaB p65, and tumor necrosis factor-α expression, associated with exacerbated pJNK activation and formation of acellular capillaries. CONCLUSIONS: In conclusion, combining diabetes and hypertension-potentiated retinal oxidative/inflammatory stress promoted imbalance between the JNK stress and survival Akt pathways resulting in accelerated retinal cell death and acellular capillary formation.

Vascular protection by angiotensin receptor antagonism involves differential VEGF expression in both hemispheres after experimental stroke.

We identified that the angiotensin receptor antagonist, candesartan, has profound neurovascular protective properties when administered after ischemic stroke and was associated with a proangiogenic state at least partly explained by vascular endothelial growth factor A (VEGFA). However, the spatial distribution of vascular endothelial growth factor (VEGF) isoforms and their receptors remained unknown. Protein analysis identified a significant increase in vascular endothelial grow factor B (VEGFB) in the cerebrospinal fluid (CSF) and the ischemic hemispheres (with increased VEGF receptor 1 activation) of treated animals (p<0.05) which was co-occurring with an increase in protein kinase B (Akt) phosphorylation (p<0.05). An increase in VEGFA protein in the contralesional hemisphere corresponded to a significant increase in vascular density at seven days (p<0.01) after stroke onset. Vascular restoration by candesartan after stroke maybe related to differential regional upregulation of VEGFB and VEGFA, promoting a "prosurvival state" in the ischemic hemisphere and angiogenesis in the contralesional side, respectively. These vascular changes in both hemispheres after effective treatment are likely to contribute to enhanced recovery after stroke.