Novel primary amine diazeniumdiolates-Chemical and biological characterization.

Hit, Lead & Candidate Discovery Diazeniumdiolates, also known as NONOates, are extensively used in biochemical, physiological, and pharmacological studies due to their ability to release nitric oxide (NO. ) and/or their congeneric nitroxyl (HNO). The purpose of this work was to synthesize a series of primary amine-based diazeniumdiolates as HNO/NO donors and to determine their efficacy as anticancer and antifungal agents in vivo. The seven compounds (3a-3g) were successfully synthesized and characterized, one of which had been previously reported in the literature (3g). Two compounds showed anti-proliferative effects against ovarian (ES2 and SKOV3) and AML monocyte-derived cancer cells (THP-1) when tested with standard MTT assays. Compounds 3a and 3g demonstrated reduced ovarian cancer cell proliferation when treated at doses from 0.033 to 1.0 mg/mL at the 24 hr time point. These compounds also exhibited moderate and selective antifungal activity against Fusarium oxysporum f.sp. lycopersici, one cause of opportunistic infections of immunocompromised patients, inhibiting the growth of the fungi at LD50 at 10 mg/mL. A third compound (3e) did not exhibit similar activities, possibly due to the alkyl chain. Our results suggest that the primary amine diazeniumdiolates may offer a versatile platform for the development of HNO/NO donors for biomedical applications.

Dietary supplementation of Ashwagandha (Withania somnifera, Dunal) enhances NK cell function in ovarian tumors in the laying hen model of spontaneous ovarian cancer.

PROBLEM: Ovarian cancer (OVCA) disseminates in a distinct pattern through peritoneal metastasis and little is known about the immunosuppression in the tumor microenvironment. Our goal was to determine changes in NK cell population during OVCA development and the effects of Ashwagandha (Withania somnifera, Dunal) supplementation on NK cell localization in laying hens with OVCA. METHODS: Frequency of NK cells in ovarian tumors at early and late stages in 3- to 4-year-old hens (exploratory study) as well as in hens supplemented with dietary Ashwagandha root powder for 90 days (prospective study) was examined. RESULTS: The population of stromal NK cells but not the intratumoral NK cells increased with OVCA development and progression. Ashwagandha supplementation decreased the incidence and progression of OVCA. Both the stromal and intratumoral NK cell population increased significantly (P < 0.0001) in Ashwagandha supplementated hens. CONCLUSION: The results of this study suggest that the population of stromal and tumorinfiltrating NK cells is increased by dietary Ashwagandha supplementation. Thus, Ashwagandha may enhance antitumor function of NK cells. This study may be useful for a clinical study to determine the effects of dietary Ashwagandha on NK cell immune function in patients with ovarian cancer.

Immune cells in the normal ovary and spontaneous ovarian tumors in the laying hen (Gallus domesticus) model of human ovarian cancer.

BACKGROUND: Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. METHODS: Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. RESULTS: T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. CONCLUSIONS: The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Contrast-enhanced sonography depicts spontaneous ovarian cancer at early stages in a preclinical animal model.

OBJECTIVE: Our goal was to examine the feasibility of using laying hens, a preclinical model of human spontaneous ovarian cancer, in determining the kinetics of an ultrasound contrast agent indicative of ovarian tumor-associated neoangiogenesis in early-stage ovarian cancer. METHODS: Three-year-old White Leghorn laying hens with decreased ovarian function were scanned before and after intravenous injection of a human serum albumin-perflutren contrast agent at a dose of 5 µL/kg body weight. Gray scale morphologic characteristics, Doppler indices, the arrival time, peak intensity, and wash-out of the contrast agent were recorded and archived on still images and video clips. Hens were euthanized thereafter; sonographic predictions were compared at gross examination; and ovarian tissues were collected. Archived clips were analyzed to determine contrast parameters and Doppler intensities of vessels. A time-intensity curve per hen was drawn, and the area under the curve was derived. Tumor types and the density of ovarian microvessels were determined by histologic examination and immunohistochemistry and compared to sonographic predictions. RESULTS: The contrast agent significantly (P < .05) enhanced the visualization of microvessels, which was confirmed by immunohistochemistry. Contrast parameters, including the time of wash-out and area under the curve, were significantly different (P < .05) between ovaries of normal hens and hens with ovarian cancer and correctly detected cancer at earlier stages than the time of peak intensity. CONCLUSIONS: The laying hen may be a useful animal model for determining ovarian tumor-associated vascular kinetics diagnostic of early-stage ovarian cancer using a contrast agent. This model may also be useful for testing the efficacy of different contrast agents in a preclinical setting.

Sphingosine-1 phosphate receptor (S1p1), a critical receptor controlling human lymphocyte trafficking, is expressed in hen and human ovaries and ovarian tumors.

BACKGROUND: Sphingosine-1 receptor 1 (S1P1) plays a major role in regulating lymphocyte egress from peripheral lymph tissue. Lymphocyte trafficking is potentially a critical response to tumors and to tumor vaccines. Also, the receptor has been shown to influence metastasis. However, there is little information on its expression in the aged ovary or ovarian tumors. As a basis for further studies in the laying hen model of spontaneous ovarian cancer, the objective of this study was to determine if S1P1 is expressed in hens, and if the morphological distribution of S1P1 is similar in hen and human ovary and ovarian tumors. METHODS: S1P1 mRNA was ascertained in hen tissue by RT-PCR using hen specific primers. S1P1 protein expression and localization was evaluated in hen and human tissue with a human S1P1 antibody by Western blot and immunohistochemistry. RESULTS: S1P1 mRNA was expressed in all hen tissues examined. Protein was detected in human and hen ovary and ovarian tumors at 47, 72 and 108 kDa in Western blots. S1P1 was similarly expressed on endothelial cells, lymphocytes and surface epithelial cells in normal ovaries and tumor-containing ovaries of the hen. In addition, S1P1 distribution was heterogeneous in both hen and human ovarian tumors by immunohistochemistry. CONCLUSION: The results show that S1P1 is expressed in the hen and human ovary as well as in ovarian tumors. These findings support the use of the hen in further studies of the role of S1P1 in metastasis and immune cell trafficking in ovarian tumor development.

Differential expression of aldehyde dehydrogenase 1a1 (ALDH1) in normal ovary and serous ovarian tumors.

BACKGROUND: We showed there are specific ALDH1 autoantibodies in ovarian autoimmune disease and ovarian cancer, suggesting a role for ALDH1 in ovarian pathology. However, there is little information on the ovarian expression of ALDH1. Therefore, we compared ALDH1 expression in normal ovary and benign and malignant ovarian tumors to determine if ALDH1 expression is altered in ovarian cancer. Since there is also recent interest in ALDH1 as a cancer stem cell (CSC) marker, we assessed co-expression of ALDH1 with CSC markers in order to determine if ALDH1 is a potential CSC marker in ovarian cancer. METHODS: mRNA and protein expression were compared in normal human ovary and serous ovarian tumors using quantitative Reverse-Transcriptase PCR, Western blot (WB) and semi-quantitative immunohistochemistry (IHC). ALDH1 enzyme activity was confirmed in primary ovarian cells by flow cytometry (FC) using ALDEFLUOR assay. RESULTS: ALDH1 mRNA expression was significantly reduced (p < 0.01; n = 5) in malignant tumors compared to normal ovaries and benign tumors. The proportion of ALDH1+ cells was significantly lower in malignant tumors (17.1 ± 7.61%; n = 5) compared to normal ovaries (37.4 ± 5.4%; p < 0.01; n = 5) and benign tumors (31.03 ± 6.68%; p < 0.05; n = 5). ALDH1+ cells occurred in the stroma and surface epithelium in normal ovary and benign tumors, although surface epithelial expression varied more in benign tumors. Localization of ALDH1 was heterogeneous in malignant tumor cells and little ALDH1 expression occurred in poorly differentiated malignant tumors. In benign tumors the distribution of ALDH1 had features of both normal ovary and malignant tumors. ALDH1 protein expression assessed by IHC, WB and FC was positively correlated (p < 0.01). ALDH1 did not appear to be co-expressed with the CSC markers CD44, CD117 and CD133 by IHC. CONCLUSIONS: Total ALDH1 expression is significantly reduced in malignant ovarian tumors while it is relatively unchanged in benign tumors compared to normal ovary. Thus, ALDH1 expression in the ovary does not appear to be similar to breast, lung or colon cancer suggesting possible functional differences in these cancers. SIGNIFICANCE: These observations suggest that reduced ALDH1 expression is associated with malignant transformation in ovarian cancer and provides a basis for further study of the mechanism of ALDH1 in this process.

Detection of tumor-associated neoangiogenesis by Doppler ultrasonography during early-stage ovarian cancer in laying hens: a preclinical model of human spontaneous ovarian cancer.

OBJECTIVE: Tumor-associated neoangiogenesis (TAN) is one of the earliest events in ovarian tumor growth and represents a potential target for early detection of ovarian cancer (OVCA). Because it is difficult to identify patients with early-stage OVCA, the goal of this study was to explore a spontaneous animal model of in vivo ovarian TAN associated with early-stage OVCA detectable by Doppler ultrasonography (DUS). METHODS: White Leghorn laying hens were scanned transvaginally at 15-week intervals up to 45 weeks. Gray scale ovarian morphologic characteristics and Doppler indices were recorded. Hens were euthanized at diagnosis for ultrasonographic morphologic/vascular abnormalities or at the end of the study (those that remained normal). Ovarian morphologic and histologic characteristics were evaluated. Vascular endothelial growth factor (VEGF) and alpha(v)beta(3)-integrin expression was assessed by immunohistochemical analysis. Doppler ultrasonographic observations were compared with histologic and immunohisto-chemical findings to determine the ability of DUS to detect ovarian TAN. RESULTS: Significant changes in ovarian blood flow parameters were observed during transformation from normal to tumor development in the ovary (P < .05). Tumor-related changes in ovarian vascularity were identified by DUS before the tumor became detectable by gray scale imaging. Increased expression of VEGF and alpha(v)beta(3)-integrins was associated with tumor development. Ovarian TAN preceded tumor progression in hens. CONCLUSIONS: The results suggest that ovarian TAN may be an effective target for the detection of early-stage OVCA. The laying hen may also be useful for studying the detection and inhibition of ovarian TAN using various means, including the efficacy of contrast agents, targeted molecular imaging, and antiangiogenic therapies.

Prevalence of antitumor antibodies in laying hen model of human ovarian cancer.

Antitumor antibodies are associated with tumors in human cancers. There is relatively little information on the timing and progression of antibody response to tumors. The objective of the study was to determine if spontaneous ovarian cancer in the egg-laying hen is associated with antitumor antibodies. Antibodies were detected by immunoassay and immunoblotting using proteins from normal ovary and ovarian tumors. Candidate antigens were identified by mass spectrometry of immunoreactive spots cut from 2-dimensional gels and Western blot. Antitumor (serum reacting against tumor ovarian extract) and antiovarian (serum reacting against normal ovarian extract) antibodies were significantly associated with ovarian cancer (67%; P

Histopathology of ovarian tumors in laying hens: a preclinical model of human ovarian cancer.

The high mortality rate due to ovarian cancer (OVCA) is attributed to the lack of an effective early detection method. Because of the nonspecificity of symptoms at early stage, most of the OVCA cases are detected at late stages. This makes the access to women with early-stage disease problematic and presents a barrier to development and validation of tests for detection of early stage of OVCA in humans. Animal models are used to elucidate disease etiologies and pathogenesis that are difficult to study in humans. Laying hen is the only available animal that develops OVCA spontaneously; however, detailed information on ovarian tumor histology is not available. The goal of this study was to determine the histological features of malignant ovarian tumors in laying hens. A total of 155 young and old (1-5 years of age) laying hens (Gallus domesticus) were selected randomly and evaluated grossly and microscopically for the presence of ovarian tumors. Histological classification of tumors with their stages and grades was determined with reference to those for humans. Similar to humans, all 4 types including serous, endometrioid, mucinous, and clear cell or mixed carcinomas were observed in hen ovarian tumors. Some early neoplastic as well as putative ovarian lesions were also observed. Similarities in histology, metastasis, and stages of hen OVCA to those of humans demonstrate the feasibility of the hen model for additional delineation of the mechanism underlying ovarian carcinogenesis, preclinical testing of new agents for the prevention, and therapy of this disease.

Detection of ovarian tumors in chicken by sonography: a step toward early diagnosis in humans?

OBJECTIVE: Animal models of spontaneous ovarian cancer are important for understanding early tumor development. Ovarian imaging may play an important role in following changes in tumor development. Laying hens are the only animals that develop spontaneous ovarian cancer similar to humans. The aim of this study was to determine the feasibility of detecting ovarian tumors in laying hens using sonography. METHODS: Ovaries of commercial strains of White Leghorn laying hens (n = 29, 2.5-3.0 years old) were examined by transvaginal 2-dimensional gray scale and color Doppler sonography. Sonographic evaluations were compared with ovarian anatomy and histologic features. RESULTS: Results of in vivo sonography and ovarian anatomic and histologic examinations were consistent. The presence of gross ovarian tumors was correctly detected in all hens by sonography. The resistive and pulsatility index values associated with ovarian tumors were lower than for normal ovaries (P < .001) suggesting that blood flow velocity was increased in ovarian tumors. Values associated with abnormal ovarian histologic findings but no gross tumors were intermediate. CONCLUSIONS: Transvaginal sonography can be used to determine ovarian status in hens. It offers the ability to make repeated examinations on the same hen to monitor early changes in the ovary associated with ovarian cancer.

Anti-tumor and anti-ovarian autoantibodies in women with ovarian cancer.

PROBLEM: There is a lack of validated marker(s) for the diagnosis of early-stage ovarian cancer (OVCA). The objective was to determine if women with OVCA had antibodies, to assess their potential as markers of ovarian cancer. The secondary objective was to compare the prevalence of antibodies to proteins from normal ovary and ovarian tumors to determine if antibodies primarily recognize tumor antigens, as many antigens are common to tumor and normal ovary. METHOD OF STUDY: Serum samples from patients with OVCA, borderline or benign ovarian tumors, endometrial cancer and healthy women were examined for anti-ovarian and anti-tumor antibodies by immunoassay. Immunoreactive proteins were characterized by one- and two-dimensional Western blot. RESULTS: Ovarian (81%, P < or = 0.001) and anti-tumor (69%, P < or = 0.001) autoantibodies in OVCA were significantly different from those of control sera. A majority of OVCA serum samples reacted with proteins at about 50 kDa from normal ovary or ovarian tumors in one-dimensional Western blot. While there were similar reactions in two-dimensional Western blots, there are differences between reactions to normal and tumor antigens and between reactions to autologous and allogeneic tumors. CONCLUSION: Serum autoantibodies are significantly associated with OVCA. Anti-tumor antibodies may provide a useful marker for the detection of ovarian cancer.