Triple Silencing of HSP27, cFLIP, and CLU Genes Promotes the Sensitivity of Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells.

Background: This study investigated how the expression of heat shock protein 27 (HSP27), cellular FLICE-like inhibitory protein (cFLIP), and clusterin (CLU) affects the progression of cancer cells and their susceptibility to doxazosin-induced apoptosis. By silencing each of these genes individually, their effect on prostate cancer cell viability after doxazosin treatment was investigated. Methods: PC-3 prostate cancer cells were cultured and then subjected to gene silencing using siRNA targeting HSP27, cFLIP, and CLU, either individually, in pairs, or all together. Cells were then treated with doxazosin at various concentrations and their viability was assessed by MTT assay. Results: The study found that silencing the CLU gene in PC-3 cells significantly reduced cell viability after treatment with 25 µM doxazosin. In addition, the dual silencing of cFLIP and CLU decreased cell viability at 10 µM doxazosin. Notably, silencing all three genes of HSP27, cFLIP, CLU was most effective and reduced cell viability even at a lower doxazosin concentration of 1 µM. Conclusions: Taken together, these findings suggest that the simultaneous silencing of HSP27, cFLIP, and CLU genes may be a potential strategy to promote apoptosis in prostate cancer cells, which could inform future research on treatments for malignant prostate cancer.

Nesfatin-1 regulates steroidogenesis in mouse Leydig cells.

Nesfatin-1 is a polypeptide hormone known to regulate appetite and energy metabolism and is derived from the precursor protein nucleobindin 2 (NUCB2). Recent studies have shown that nesfatin-1 is expressed in many peripheral tissues in mice, including the reproductive organs. However, its function and regulation in the testis remain unknown. In this study, we investigated the expression of Nucb2 mRNA and nesfatin-1 protein in mouse Leydig cells and the Leydig cell line, TM3 cells. We also examined whether Nucb2 mRNA expression is regulated by gonadotropins and whether exogenous nesfatin-1 affects steroidogenesis in primary Leydig cells isolated from the testis and TM3 cells. We found that Nucb2 mRNA and nesfatin-1 protein were present in primary Leydig cells and TM3 cells, and nesfatin-1 binding sites were also found in both cell types. Nucb2 mRNA expression in testis, primary Leydig cells, and TM3 cells was increased after treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin. After nesfatin-1 treatment, the expression of steroidogenesis-related enzyme genes Cyp17a1 and Hsd3b was upregulated in primary Leydig cells and TM3 cells. Our results suggest that NUCB2/nesfatin-1 expression in mouse Leydig cells may be regulated through the hypothalamic-pituitary-gonadal axis and that nesfatin-1 produced by Leydig cells may locally regulate steroidogenesis in an autocrine manner. This study provides insight into the regulation of NUCB2/nesfatin-1 expression in Leydig cells and the effect of nesfatin-1 on steroidogenesis, which may have implications for male reproductive health.

Estradiol and progesterone regulate NUCB2/nesfatin-1 expression and function in GH3 pituitary cells and THESC endometrial cells.

Estradiol (E2) and progesterone (P4) are essential sex steroid hormones that play critical roles in the pituitary gland and uterus. Recently, nesfatin-1, a polypeptide hormone that regulates appetite and energy homeostasis in the hypothalamus, was found to be expressed in the pituitary gland and uterus. In this study, we aimed to investigate the relationship between these two steroid hormones and the expression and function of nesfatin-1 in the pituitary gland and uterus using GH3 cells, a lacto-somatotroph cell line, and THESC cells, an endometrial stromal cell line. First, we verified the presence of nesfatin-1 and nesfatin-1 binding sites in GH3 and THESC cells. E2 increased the mRNA expression of NUCB2, the gene encoding the nesfatin-1 protein, in GH3 cells, while P4 had no significant effect. In THESC cells, NUCB2 mRNA expression was decreased by E2 but increased by P4. In addition, nesfatin-1 significantly increased growth hormone (GH) and prolactin (PRL) mRNA expression in GH3 cells, and E2 enhanced this effect. In THESC cells, nesfatin-1 significantly increased the mRNA expression of insulin-like growth factor binding protein 1 (IGFBP1) and PRL, which are decidualization marker genes, and P4 further enhanced this effect. These results suggest that nesfatin-1 may act as a local regulator of GH and PRL production in the pituitary gland and decidualization in the uterus, modulating its effects in response to E2 and P4.

NUCB2/nesfatin-1 suppresses the acrosome reaction in sperm within the mouse epididymis.

Nesfatin-1, a polypeptide hormone derived from the nucleobindin 2 (NUCB2) precursor protein, is known to regulate appetite and energy metabolism. Recent studies have also shown that NUCB2/nesfatin-1 is expressed in the reproductive organs of mice. However, the expression and potential role of NUCB2/nesfatin-1 in the mouse epididymis remain unclear. Therefore, we investigated the expression of NUCB2/nesfatin-1 in the mouse epididymis and its potential function. NUCB2/nesfatin-1 was detected in the epididymis by qRT-PCR and western blotting, and high expression levels were observed in epididymal epithelial cells by immunohistochemical staining. Pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) injections significantly increased NUCB2/nesfatin-1 expression in the epididymis. After castration, NUCB2/nesfatin-1 expression in the epididymis decreased, but was significantly increased by testosterone injection. Nesfatin-1-binding sites were found in the middle piece of testicular sperm, but were scarcely detected in the sperm head. By contrast, nesfatin-1 binding sites were identified on the sperm head within the epididymis. Furthermore, nesfatin-1 treatment inhibited the acrosome reaction in epididymal sperm. These results suggest that the nesfatin-1 protein produced in the epididymis binds to nesfatin-1 binding sites on the sperm head and plays a role in suppressing the acrosome reaction before ejaculation.

17Beta-Estradiol Regulates NUCB2/ Nesfatin-1 Expression in Mouse Oviduct.

NUCB2/nesfatin-1 known to regulate appetite and energy homeostasis is expressed not only in the hypothalamus, but also in various organs and tissues. Our previous reports also demonstrated that NUCB2/nesfatin-1 was expressed in the reproductive organs, including the ovaries, uterus, and testes of mice. However, it is yet known whether NUCB2/nesfatin-1 is expressed in the oviduct and how its expression is regulated. Therefore, we investigated the expression of NUCB2/nesfatin-1 in the oviduct and its expression is regulated by gonadotropin. Immunohistochemical staining results showed that nesfatin-1 protein was localized in epithelial cells of the oviduct. As a result of quantitative real-time PCR (qRT-PCR) and Western blot, NUCB2/nesfatin-1 was detected strongly in the oviducts. During the estrus cycle, NUCB2/nesfatin-1 expression in the oviducts was markedly higher in the proestrus stage than in other estrus stages. In order to elucidate whether the expression of NUCB2 mRNA is controlled by the gonadotropins, we injected PMSG and hCG and measured NUCB2 mRNA level in the oviduct after injection. Its level was increased in the oviduct after PMSG injection, but no significant change after hCG injection. In addition, NUCB2 mRNA levels were markedly reduced after ovariectomy, while recovered after 17ß-estradiol (E2) injection, but not by progesterone (P4). This study demonstrated that NUCB2/nesfatin-1 is highly expressed in the oviduct of mouse and its expression is regulated by E2 secreted by the ovaries. These results suggest that NUCB2/nesfatin-1 expressed by the oviduct may affect the function of the oviduct regulated by the ovaries.

Red ginseng extract regulates differentiation of monocytes to macrophage and inflammatory signalings in human monocytes.

This study was aimed to investigate the effect of red ginseng extract (RGE) on monocyte to macrophage differentiation and inflammatory signalings in THP-1 human monocytes. In HPLC analysis, RGE contained saponin level of 516 µg/mg (extract) with 14 ginsenosides. RGE effectively suppressed the monocyte-to-macrophage differentiation induced by phorbol 12-myristate 13-acetated (PMA) by inhibiting the THP-1 cell adhesion. This result is evidenced by the down-regulation of cluster of differentiation molecule ß (CD11ß) and CD36. RGE significantly reduced translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (78%), while cytosolic NF-κB was increased (53%), compared with LPS group. In addition, RGE significantly increased the protein abundance of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its target protein, hemoxygenase-1 (HO-1), but, Kelch-like ECH-associated protein 1 (KEAP1), a negative regulator of Nrf2, was greatly decreased by RGE. Furthermore, RGE effectively mediated the regulation of Nrf2 level in nucleus and cytoplasm of THP-1.

Gonadotropin regulates NUCB2/nesfatin-1 expression in the mouse ovary and uterus.

NUCB2/nesfatin-1 is expressed in the hypothalamus and regulates food intake and energy metabolism. Recent studies showed that NUCB2/nesfatin-1 also plays a role in other organs. However, its expression pattern and function in female reproductive organs are unclear. Therefore, we investigated NUCB2/nesfatin-1 expression in the ovary and uterus of mice and determined whether it is regulated by gonadotropins and sex steroid hormones. NUCB2 mRNA and nesfatin-1 protein were detected in the ovary and uterus of mice. NUCB2/nesfatin-1 expression in both organs was highest in the estrus period of the estrus cycle. Administration of pregnant mare serum gonadotropin (PMSG) dose-dependently increased mRNA expression of NUCB2 in the ovary and uterus of mice. On the other hand, mRNA expression of NUCB2 in the uterus was dramatically decreased after ovariectomy and was not increased upon administration of PMSG. Injection of 17ß-estradiol upregulated mRNA expression of NUCB2 in the uterus of ovariectomized mice, whereas injection of progesterone did not. These results suggest that NUCB2/nesfatin-1 expression in the ovary and uterus of mice is regulated through the hypothalamus-pituitary-ovary axis and that NUCB2/nesfatin-1 is a local regulator of ovarian steroidogenesis and uterine function.

Tissue-Specific Localization NUCB2/nesfatin-1 in the Liver and Heart of Mouse Fetus.

NUCB2/nesfatin-1 is first known to be expressed in the hypothalamus while controlling appetite and energy metabolism. However, recent studies have shown that NUCB2/nesfatin-1 was expressed in the various organs as well as the hypothalamus. Our previous reports also demonstrated that NUCB2/nesfatin-1 was expressed in the ovary, testis, pituitary gland, lung, kidney, and stomach of fetal and adult mice. However, the role of NUCB2/nesfatin-1 in mouse fetus remains unknown. Thus, the aim of this study was to investigate whether NUCB2/nestatin-1 is expressed in mouse fetus at the developmental stage in which organogenesis begins. To do this, we performed in situ hybridization (ISH) and immunohistochemistry (IHC) staining to examine the distribution of NUCB2 mRNA and nesfatin-1 protein in the mouse fetal organs during early developmental stages, especially at embryonic day (E) 10.5. As a result of ISH, NUCB2 mRNA positive signals were more frequent in the liver, but there were relatively few positive signals in heart. On the other hand, no positive signals were detected in other organs. These ISH results were validated by IHC staining and qRT-PCR analysis. Expression of nesfatin-1 protein detected by IHC staining was similar to that of NUCB2 mRNA detected by ISH in the liver and heart. In addition, the levels of NUCB2 mRNA expression analyzed by qRT-PCR were significantly increased in the liver and heart compared to other organs of the mouse fetus at E13.5, whereas its level was extensively decreased in the liver, but increased in the lung, stomach, and kidney of the mouse fetus at E17.5. These results suggest that NUCB2/nesfatin-1 may play an important role in liver and heart development and physiological functions in the developmental process of mouse fetus. Further studies are needed on the function of NUCB2/nesfatin-1, which is highly expressed in the various organs, including liver and heart during mouse development.