Aging in females is not only associated with the changes in hormonal status but is also responsible for dysregulation of immune functions in various organs including ovaries. The goal of this study ...was to determine whether the expression of interleukin 16 (IL-16), a proinflammatory and chemoattractant cytokine, changes during ovarian aging, to determine factors involved in such changes in IL-16 expression, and to examine if changes in IL-16 expression during aging predisposes the ovary to pathologies. Ovarian tissues from premenopausal women (30-50 years old), women at early menopause (55-59 years old), and late menopause (60-85 years old) were used. In addition, tumor tissues from patients with ovarian high-grade serous carcinoma at early stage (n=5) were also used as reference tissue for comparing the expression of several selected markers in aging ovaries. The expression of IL-16, frequency of macrophages (a source of IL-16) and expression of microRNA (miR) 125a-5p (a regulator of IL-16 gene) were performed by immunohistochemistry, immunoblotting, and gene expression assays. In addition, we examined changes in nuclear expression of IL-16 expression with regards to exposure to follicle-stimulating hormone (FSH) by in vitro cell culture assays with human ovarian cancer cells. The frequencies of IL-16 expressing cells were significantly higher in ovarian stroma in women at early and late menopause as compared with premenopausal women (P<0.0001). Similar patterns were also observed for macrophages. Expression of miR-125a-5p decreased significantly (P<0.001) with the increase in IL-16 expression during aging. Furthermore, expression of nuclear IL-16 increased remarkably upon exposure to FSH. Consequently, ovarian aging is associated with increased expression of IL-16 including its nuclear fraction. Therefore, persistent high levels of FSH in postmenopausal women may be a factor for enhanced expression of IL-16. Effects of increased nuclear fraction of IL-16 need to be examined.
Chronic inflammation fundamentally influences cancer risk and development. A mechanism of chronic inflammation is the formation of inflammasome complexes which results in the sustained secretion of ...the pro-inflammatory cytokines IL1β and IL18. Inflammasome expression and actions vary among cancers. There is no information on inflammasome expression in ovarian cancer (OvCa). To determine if ovarian tumors express inflammasome components, mRNA and protein expression of NLRP3 (nucleotide-binding domain, leucine-rich repeat family, pyrin domain containing 3), caspase-1, IL1β, and IL18 expression in hen and human OvCa was assessed. Chicken (hen) OvCa a valid model of spontaneous human OvCa. Hens were selected into study groups with or without tumors using ultrasonography; tumors were confirmed by histology, increased cellular proliferation, and expression of immune cell marker mRNA. mRNA expression was higher for hallmarks of inflammasome activity (caspase-1, 5.9x increase, p = 0.04; IL1β, 4x increase, p = 0.04; and IL18, 7.8x increase, p = 0.0003) in hen OvCa compared to normal ovary. NLRP3, caspase-8 and caspase-11 mRNA did not differ significantly between tumor and non-tumor containing ovaries. Similar results occurred for human OvCa. Protein expression by immunohistochemistry paralleled mRNA expression and was qualitatively higher in tumors. Increased protein expression of caspase-1, IL1β, and IL18 occurred in surface epithelium, tumor cells, and immune cells. The aryl hydrocarbon receptor (AHR), a potential tumor suppressor and NLRP3 regulator, was higher in hen (2.4x increase, p = 0.002) and human tumors (1.8x increase, p = 0.038), suggesting a role in OvCa. Collectively, the results indicate that inflammasome expression is associated with hen and human OvCa, although the NLR sensor type remains to be determined.
Ovarian high grade serous carcinoma (HGSC) is a lethal form of ovarian cancer (OVCA). In most cases it is detected at late stages as the symptoms are non-specific during early stages. Emerging ...information suggests that the oviductal fimbria is a site of origin of ovarian HGSC. Currently available tests cannot detect ovarian HGSC at early stage. The lack of a preclinical model with oviductal fimbria that develops spontaneous ovarian HGSC is a significant barrier to developing an early detection test for this disease. The goal of this study was to examine if the oviductal fimbria in hens is a site of origin of HGSC and whether it expresses several putative markers expressed in ovarian HGSC in patients. A total of 135 laying hens (4 years old) were selected from a flock using transvaginal ultrasound (TVUS) imaging, followed by euthanasia and gross examination for the presence of solid masses and ascites. Histological types of carcinomas were determined by hematoxylin and eosin staining. Expression of WT-1, mutant p53, CA-125, PAX2 and Ki67 in normal or malignant fimbriae or ovaries were examined using immunohistochemistry, immunoblotting and gene expression assays. This study detected tumors in oviductal fimbriae in hens and routine staining revealed ovarian HGSC-like microscopic features in these tumors. These tumors showed similarities to ovarian HGSC in patients in expressing several markers. Compared with normal fimbriae, intensities of expression of WT-1, mutant p53, CA-125, and Ki67 were significantly (P<0.05) higher in fimbrial tumors. In contrast, expression of PAX2 decreased gradually as the tumor progressed to late stages. The patterns of expression of these markers were similar to those in ovarian HGSC patients. Thus, tumors of the oviductal fimbria in hens may offer a preclinical model to study different aspects of spontaneous ovarian HGSC in women including its early detection.
Ovarian high-grade serous carcinoma (HGSC) is a fatal malignancy of women. Alterations in the expression of nuclear proteins are early steps in malignant transformation; nucleolin is one such ...protein. Changes in nucleolin expression and circulatory levels during ovarian HGSC development are unknown. The study goal was to determine if tissue and circulatory levels of nucleolin change in response to malignant transformation leading to ovarian HGSC.
Sera, ovaries, and BRCA+ fimbria from healthy subjects, and sera and tumor tissues from patients (
= 10 each), and healthy hens and hens with HGSC were examined in exploratory and prospective studies for nucleolin expression by immunohistochemistry, immunoblotting, gene expression, and immunoassay, and analyzed by analysis of variance (ANOVA).
Compared with normal, nucleolin expression was higher in patients and hens with ovarian HGSC and in women with a risk of HGSC (
< 0.05). Compared with normal (1400 + 105 pg/mL,
= 8), serum nucleolin levels were 1.5 and 1.7-fold higher in patients with early- (
= 5) and late-stage (
= 5) HGSC, respectively. Additionally, serum nucleolin levels increased significantly (
< 0.05) prior to the formation of detectable masses.
This pilot study concluded that tissue and serum levels of nucleolin increase in association with malignant changes in ovaries and fimbriae leading to ovarian HGSC.
Emerging information suggests a potential role of medicinal cannabis in pain medication in addition to enhancing immune functions. Endometriosis is a disease of women of reproductive age associated ...with infertility and reproductive failure as well as chronic pain of varying degrees depending on the stage of the disease. Currently, opioids are being preferred over nonsteroidal anti-inflammatory drugs (NSAID) due to the latter’s side effects. However, as the opioids are becoming a source of addiction, additional pain medication is urgently needed. Cannabis offers an alternative therapy for treating the pain associated with endometriosis. Information on the use and effectiveness of cannabis against endometriotic pain is lacking. Moreover, expression of receptors for endocannabinoids by the ovarian endometriotic lesions is not known. The goal of this study was to examine whether cannabinoid receptors 1 and 2 (CB1 and CB2) are expressed by ovarian endometriotic lesions. Archived normal ovarian tissues, ovaries with endometriotic lesions, and normal endometrial tissues were examined for the presence of endometrial stromal cells using CD10 (a marker of endometrial stromal cells). Expression of CB1 and CB2 were determined by immunohistochemistry, immunoblotting, and gene expression studies. Intense expression for CB1 and CB2 was detected in the epithelial cells in ovarian endometriotic lesions. Compared with stroma in ovaries with endometriotic lesions, the expression of CB1 and CB2 was significantly higher in the epithelial cells in endometriotic lesions in the ovary (P<0.0001 and P<0.05, respectively). Immunoblotting and gene expression assays showed similar patterns for CB1 and CB2 protein and CNR1 (gene encoding CB1) and CNR2 (gene encoding CB2) gene expression. These results suggest that ovarian endometriotic lesions express CB1 and CB2 receptors, and these lesions may respond to cannabinoids as pain medication. These results will form a foundation for a clinical study with larger cohorts.
Understanding malignant transformation associated with ovarian cancer (OVCA) is important to establish early detection tests. This study examined whether expression of glucose-regulated protein 78 ...(GRP78, marker of cellular stress) increases during OVCA development, and whether GRP78 can be detected by targeted-transvaginal ultrasound (TVUS) imaging.
Normal ovaries (
= 10), benign (
= 10) and malignant ovarian tumors at early (
= 8) and late stages (
= 16), hens with and without ovarian tumors at early and late stages (
= 10, each) were examined for GRP78 expression during OVCA development by immunohistochemistry, immunoblotting, gene expression and immunoassay. Feasibility of GRP78-targeted TVUS imaging in detecting early OVCA was examined.
Compared with normal ovaries and benign tumors, intensity of GRP78 expression was higher (
< 0.0001) in OVCA patients. Compared with normal (9007.76 ± 816.54 pg/mL), serum GRP78 levels were significantly higher (
< 0.05) in patients with early (12,730.59 ± 817.35 pg/mL) and late-stage OVCA (13,930.12 ± 202.35) (
< 0.01). Compared with normal (222.62 ± 181.69 pg/mL), serum GRP78 levels increased (
< 0.05) in hens with early (590.19 ± 198.18 pg/mL) and late-stage OVCA (1261.38 ± 372.85) (
< 0.01). Compared with non-targeted, GRP78-targeted imaging enhanced signal intensity of TVUS (
< 0.0001).
Tissue and serum levels of GRP78 increase in association with OVCA. GRP78 offers a potential serum and imaging marker for early OVCA detection.
Objective Long-term unresolved inflammation has been suggested as a risk factor for the development of various malignancies. The goal of this study was to examine whether the expression of ...interleukin (IL)-16, a proinflammatory cytokine, changes in association with ovarian cancer (OVCA) development. Study Design In an exploratory study, changes in IL-16 expression in association with OVCA development and progression were determined using ovarian tissues and serum samples from healthy subjects (n = 10) and patients with benign (n = 10) and malignant ovarian tumors at early (n = 8) and late (n = 20) stages. In the prospective study, laying hens, a preclinical model of spontaneous OVCA, were monitored (n = 200) for 45 weeks with serum samples collected at 15-week interval. Changes in serum levels of IL-16 relative to OVCA development were examined. Results The frequency of IL-16–expressing cells increased significantly in patients with OVCA ( P < .001) compared to healthy subjects and patients with benign ovarian tumors. The concentration of serum IL-16 was higher in patients with benign tumors ( P < .05) than in healthy subjects and increased further in patients with early-stage ( P < .05) and late-stage ( P < .03) OVCA. Increase in tissue expression and serum levels of IL-16 in patients with early and late stages of OVCA were positively correlated with the increase in ovarian tumor–associated microvessels. Prospective monitoring showed that serum levels of IL-16 increase significantly ( P < .002) even before ovarian tumors become grossly detectable in hens. Conclusion This study showed that tissue expression and serum levels of IL-16 increase in association with malignant ovarian tumor development and progression.
Chronic inflammation and long-standing oxidative stress are potential predisposing factors for developing malignancies, including ovarian cancer (OVCA). Information on the association of ovarian ...chronic abnormal conditions, including polycystic ovarian syndrome (PCOS), with the development of OVCA is unknown. The goal of this study was to examine if polycystic ovarian conditions are associated with OVCA development. In the exploratory study, 3–4-year-old laying hens were randomly selected and examined for the presence of polycystic ovaries with cancer (PCOC). In the prospective study, hens were monitored by ultrasound scanning to detect the incidence of a polycystic ovaries and subsequent development of OVCA. Tissues from normal ovaries and PCOC were examined for macrophage infiltration, expression of interleukin-16, and superoxide dismutase 2. The exploratory study detected spontaneous PCOC at early and late stages in hens. PCOC in hens were accompanied with influx of macrophages (17.33 ± 2.26 in PCOC at the early stage and 24.24 ± 2.5 in PCOC at the late stage in 20 mm2 areas of tissue as compared with 6.77 ± 1.58 in normal hens). Expression of interleukin-16 was more than 2.5-fold higher and superoxide dismutase 2 was approximately 3-fold higher in PCOC hens than normal hens. The prospective study showed the development of OVCA in some hens with polycystic ovarian condition (PCO). PCOC development in hens was associated with chronic inflammation in the ovary. Laying hens may represent a potential model for the study of spontaneous PCOS and its long-term risk of PCOC development.
Epithelial ovarian cancer (OVCA), a fatal malignancy of women, disseminates locally. Although NK cells mount immune responses against OVCA, tumors inhibit NK cells, and the mechanism is not well ...understood. Cytokines stimulate NK cells; however, chronic stimulation exhausts them and induces expression of cytokine-inducible SH2-containing protein (CISH). Tumors produce anti-inflammatory cytokine interleukin (IL)-10 which may induce NK cell exhaustion. The goal of this study was to examine if CISH expression in NK cells increases during OVCA development and to determine the mechanism(s) of OVCA-induced CISH expression in NK cells. Normal ovaries (
= 7) were used for CISH, IL-10 and GRP78 expression. In tumor ovaries, CISH was examined in early and late stages (
= 14 each, all subtypes) while IL-10 and GRP78 expression were examined in early and late stage HGSC (
= 5 each). Compared to normal, the population of CISH-expressing NK cells increased and the intensity of IL-10 and GRP78 expression was significantly higher in OVCA (
< 0.05). CISH expression was positively correlated with IL-10 expression (r = 0.52, r = 0.65,
< 0.05 at early and late stages, respectively) while IL-10 expression was positively correlated with GRP78 expression (r = 0.43, r = 0.52,
< 0.05, respectively). These results suggest that OVCA development and progression are associated with increased CISH expression by NK cells which is correlated with tumor-induced persistent cellular stress.
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.
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.
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.
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.
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.
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