Ethanol Extracts of Cornus alba Improve Benign Prostatic Hyperplasia by Inhibiting Prostate Cell Proliferation through Modulating 5 Alpha-Reductase/Androgen Receptor Axis-Mediated Signaling.

PURPOSE: The aim of this study was to investigate the efficacy of ethanol extracts of Cornus alba (ECA) against benign prostatic hyperplasia (BPH) in vitro and in vivo. MATERIALS AND METHODS: The prostate stromal cells (WPMY-1) and epithelial cells (RWPE-1) were used to examine the action mechanism of ECA in BPH in vitro. ECA efficacy was evaluated in vivo using a testosterone propionate (TP)-induced BPH rat model. RESULTS: Treatment with ECA inhibited the proliferation of prostate cells by inducing G1-phase cell cycle arrest through the regulation of positive and negative proteins. Treatment of prostate cells with ECA resulted in alterations in the mitogen-activated protein kinases and protein kinase B signaling pathways. The transcriptional binding activity of the NF-κB motif was suppressed in both ECA-treated prostate cells. In addition, treatment with ECA altered the level of BPH-associated axis markers (5α-reductase, fibroblast growth factor-2, androgen receptor, epidermal growth factor, Bcl-2, and Bax) in both cell lines. Finally, the administration of ECA attenuated the enlargement of prostatic tissues in the TP-induced BPH rat model, accompanied by histology, immunoblot, and serum dihydrotestosterone levels. CONCLUSIONS: These results demonstrated that ECA exerted beneficial effects on BPH both in vitro and in vivo and might provide valuable information in the development of preventive or therapeutic agents for improving BPH.

Corrigendum: Korean Ginseng Berry Extract Enhances the Male Steroidogenesis Enzymes In Vitro and In Vivo.

This corrects the article on p. 446 in vol. 41, PMID: 36649918.

Perceptual and quantitative analysis of discoloration of orthodontic elastomeric chains by food.

BACKGROUND: The objectives of this study were to use a digital camera to measure the discoloration of orthodontic elastomeric chains in various immersion solutions over different time periods and to determine the valid acceptability tolerances for color changes in orthodontic elastomeric chains by surveying digital photographs. METHODS: Orthodontic elastomeric chains were applied to the maxillary anterior teeth of nine typodont models. The models were divided into three groups and immersed in the curry, coffee, and wine solutions. The digital images of the elastomeric modules were captured and processed using commercial software after 30 min of immersion, thrice a day, for 28 days. The differences in color changes ([Formula: see text]), depending on the type of immersion solution and period, were analyzed using a repeated-measures analysis of variance (ANOVA) test. A web-based survey questionnaire was randomly distributed to 50 respondents for a visual analysis of the elastomeric chain discoloration. The relationship between the surveying score and [Formula: see text] value was analyzed using Spearman's correlation coefficient. The perceptibility and acceptability of elastomeric chain discoloration ([Formula: see text]) based on the type of immersion solutions and periods were analyzed using a regression model. RESULTS: Significant differences were observed in the discoloration of the elastomeric power chain depending on the immersion solution and period. The amount of discoloration was highest in curry, followed by coffee and wine (P < 0.05). The mean discoloration ([Formula: see text]) continued to increase over the entire immersion period. There was a significant correlation between visual scoring and discoloration ([Formula: see text]) over the entire period, especially in the early stages compared to the later stages (r = 0.918, P < 0.05). In 50% of the respondents, the predicted clinically unacceptable discoloration was between 4.46 and 9.98 and in 90% of the respondents, it was between 16.52 and 19.85. CONCLUSIONS: The amount of discoloration was the highest for curry, followed by coffee and wine, and continued to gradually increase during the observation period. Significant differences were found between the color measurements obtained and the visual assessment by observers. The observers varied in their tolerance for perceptibility and acceptability for elastomeric chain discoloration based on the type of dietary media.

Korean Ginseng Berry Extract Enhances the Male Steroidogenesis Enzymes In Vitro and In Vivo.

PURPOSE: Testosterone hormonal replacement is the most commonly prescribed solution for men with reproductive issues; however, this treatment has various drawbacks. Hence, the identification of a natural product that promotes steroidogenesis is urgently needed. Ginseng is a popular traditional medicine. This study aimed to investigate steroidogenic effects of Korean ginseng berry extract (GBE; Panax ginseng C.A. Meyer) in vitro and in vivo. MATERIALS AND METHODS: In vitro model, mouse Leydig cells were treated with varying concentrations of GBE, and the levels of steroidogenesis-related genes and proteins and testosterone were measured using western blotting, qRT-PCR, and enzyme-linked immunosorbent assay (ELISA). Similarly, in an in vivo model using lipopolysaccharide-injected C57BL/6J mice, expression of steroidogenesis-related genes and proteins and testosterone levels were analyzed. Additionally, sleep deprivation was used to simulate common life stressors related to late-onset hypogonadism (LOH) and the natural effects of aging. Mice were fed sham or GBE before being subjected to paradoxical sleep deprivation. RESULTS: In vitro, GBE induced steroidogenic effects by increasing the levels of enzymes associated with steroidogenesis, steroidogenic acute regulatory protein (STAR), CYP11A1, and CYP17A1. In vivo, GBE significantly increased mRNA and protein levels of steroidogenic enzymes. Furthermore, the synthetic testosterone levels in mouse Leydig cell supernatants and blood sera were increased. In the sleep deprivation study, mice fed GBE showed increased testosterone production and survival under such stressful conditions. CONCLUSIONS: GBE increased mRNA and protein levels of steroidogenesis-related enzymes STAR, CYP11A1, and CYP17A1. These key enzymes induced the increased production of testosterone both in vivo and in vitro. Thus, GBE might be a promising therapeutic or additive nutritional agent for improving men's health by increasing steroidogenesis or improving LOH.

Inhibitory Activities of Dimeric Ellagitannins Isolated from Cornus alba on Benign Prostatic Hypertrophy.

Benign prostatic hypertrophy (BPH) is an intractable chronic inflammatory disease. We studied the efficacy of two ellagitannins, namely camptothin B (1) and cornusiin A (2) that were isolated from Cornus alba (CA) for the treatment of BPH, which is a common health issue in older men. The ellagitannins (1 and 2) were evaluated on its inhibitory activities of the enzyme 5α-reductase and tumor necrosis factor (TNF)-α, its interleukin (IL)-1ß, IL-6, and IL-8 production, and its anti-proliferation and apoptosis induction in prostate cells that show hypertrophy (RWPE-1 cell). In inhibition of 5α-reductase, the ellagitannins (1 and 2) showed potential effects, compared to the positive control, finasteride. In the case of IL-1ß, IL-6, IL-8, and TNF-α, 1 and 2 showed good inhibitory effects as compared to the control group treated with LPS. The ellagitannins (1 and 2) were also shown to inhibit proliferation of, and induce apoptosis in, the RWPE-1 cell. These results suggest that the ellagitannins (1 and 2) may be good candidates for the treatment of BPH.

Peanut Sprout Extracts Cultivated with Fermented Sawdust Medium Inhibits Benign Prostatic Hyperplasia In Vitro and In Vivo.

PURPOSE: In this study, we tested whether the resveratrol-enriched peanut sprout extracts cultivated with fermented sawdust medium (PSEFS) could suppress benign prostatic hyperplasia (BPH) in vitro and in vivo. MATERIALS AND METHODS: The mode of action of PSEFS was estimated by employing high-performance liquid chromatography analysis, MTT assay, cell counting, cell cycle analysis, immunoblots, and immunoprecipitation and electrophoretic mobility shift assay. In vivo efficacy of PSEFS was analyzed in BPH animal model via immunostaining and enzyme-linked immunosorbent assay. RESULTS: We selected the Yesan peanut sprout variety, which contains the highest level of resveratrol. The resveratrol levels in PSEFS were higher than those obtained with hydroponic technology. PSEFS treatment induced cell cycle arrest at the G1-phase by downregulating CDK4 and cyclin D1 via p21WAF1 induction in the RWPE-1 and WPMY prostate cells, thereby decreasing their proliferation. Treatment with PSEFS decreased ERK1/2 phosphorylation and increased JNK phosphorylation. The levels of DNA-bound transcription factors associated with proliferation (nuclear factor-κB, Sp-1, and AP-1) decreased upon PSEFS treatment in both prostate cells. Additionally, the levels of the molecular markers of BPH development (5α-reductase, androgen receptor, fibroblast growth factor, Bcl-2, and Bax) also changed by the addition of PSEFS. Finally, in a testosterone propionate-induced BPH model in rats, PSEFS administration attenuated the size, weight, and thickness of prostate tissues with no signs of death. CONCLUSIONS: These results showed that PSEFS inhibited BPH both in vitro and in vivo and might be useful in the development of a potential BPH therapy.

Steroidogenic effects of Taraxacum officinale extract on the levels of steroidogenic enzymes in mouse Leydig cells.

In this study, we investigated the steroidogenic effect of Taraxacum officinale extract on mouse TM3 Leydig cells, which produce male hormones by increasing the levels of steroidogenic enzymes. Steroidogenic enzymes are involved in the production of testosterone in the testis. To date, the steroidogenic effect of T. officinale has not been reported. Therefore, we examined the steroidogenic effects of T. officinale extract (TOE) on mouse Leydig cells in vitro. Traditionally, plants have been used for the treatment of various kinds of ailments. For many years, some medicinal plants have been used to regulate steroidogenesis or late-onset hypogonadism (LOH). In particular, plants belonging to the genus Taraxacum have anti-inflammatory, anti-nociceptive, anti-oxidant, and anti-cancer properties. In this study, we determined whether the TOE exerts steroidogenic effects by increasing the levels of enzymes associated with steroidogenesis, such as the steroidogenic acute regulatory protein (STAR), CYP11A1, and translocator protein (TSPO) in the mitochondria and CYP17A1 in the smooth endoplasmic reticulum, in mouse Leydig cells. Our results showed that the TOE significantly increased the mRNA and protein levels of steroidogenic enzymes, thereby increasing the testosterone levels in mouse Leydig cells. Thus, our results indicate that the TOE increases the levels of steroidogenic enzymes, and further studies are required to establish the potential of this plant in regulating steroidogenesis and improving LOH.

Epigenetic suppression of the anti-aging gene KLOTHO in human prostate cancer cell lines.

KLOTHO was originally identified as an aging-suppressor gene that causes a human aging-like phenotype when tested in KLOTHO-deficient-mice. Recent evidence suggests that KLOTHO functions as a tumor suppressor by inhibiting Wnt signaling. KLOTHO gene silencing, including DNA methylation, has been observed in some human cancers. Aberrant activation of Wnt signaling plays a significant role in aging, and its silencing may be related to prostate cancer and other types of cancers. Thus, we investigated whether the expression of the anti-aging gene KLOTHO was associated with epigenetic changes in prostate cancer cell lines. KLOTHO mRNA was detected in the 22Rv1 cell line while it was not detected in DU145 and PC-3 cell lines. The restoration of KLOTHO mRNA in the DU145 and PC-3 cell lines was induced with a DNA methyltransferase inhibitor. Methylation-specific PCR was performed to determine the specific CpG sites in the KLOTHO promoter responsible for expression. In addition, the level of methylation was assessed in each CpG by performing bisulfite sequencing and quantitative pyrosequencing analysis. The results suggested a remarkable inverse relationship between KLOTHO expression and promoter methylation in prostate cancer cell lines.

The effects of Chamaecyparis obtusa essential oil on pain-related behavior and expression of pro-inflammatory cytokines in carrageenan-induced arthritis in rats.

Chamaecyparis obtusa essential oil (COE) has been widely used to treat allergic diseases and was suggested to exert anti-inflammatory, antioxidant, and antimicrobial effects. This study evaluated the effects of COE on pain-related behavior and pro-inflammatory cytokines in rats with carrageenan (CGN)-induced arthritis. Reduced dynamic weight load on inflamed joint in voluntarily walking rats was used as the behavior test for arthritic pain; 10% COE-treated group was significantly attenuated pain (6-8 h post-CGN injection) compared to VEH (mineral oil)-treated group. In addition, the protein levels of interleukin (IL)-1ß, tumor necrosis factor-α, IL-6 (6-8 h), and cyclooxygenase (COX)-2 (8 h) within the synovial membrane, as well as IL-1ß, COX-2 (6-8 h), and IL-6 (5-7 h) within the meniscus, of 10% COE-treated group were significantly reduced. The current results implicate that COE has anti-inflammatory and anti-nociceptive effects on arthritis in rats.

miR-29b attenuates tumorigenicity and stemness maintenance in human glioblastoma multiforme by directly targeting BCL2L2.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor and exhibits aggressive and invasive behavior. We previously identified four miRNAs-miR-29b, 494, 193a-3p, and 30e-with enhanced expression in GBM following treatment of ionizing radiation by miRNA microarray analysis. In this study, we found that only miR-29b inhibited tumor cell migration and invasion by reducing MMP-2 activity via phospho-AKT/ß-catenin signaling, and stimulated a more epithelial-like morphology. Moreover, miR-29b inhibits angiogenesis by attenuating tube formation and the expression of VEGF and Ang-2, and stemness maintenance in GBM cells, as demonstrated by decreasing neurosphere formation and cancer stem cell marker protein expression. These findings support the anti-tumor properties of miR-29b in human GBM cells. Furthermore, miR-29b expression was inversely proportional to that of BCL2L2 mRNA or protein in various cancer cell types. Interestingly, BCL2L2 mRNA is highly expressed in the mesenchymal type of GBM. To further elucidate the relationship between miR-29b and BCL2L2 in GBM, we performed co-transfection reporter assays and determined that miR-29b downregulates BCL2L2 expression by directly binding its 3'UTR. Finally, we confirmed that BCL2L2 repression is of central importance to miR-29b anti-tumor activity using functional assays to examine cell migration, invasion, angiogenesis, and stemness. From these data, we propose that miR-29b may be a useful therapeutic agent in GBM.

GABAergic excitation of vasopressin neurons: possible mechanism underlying sodium-dependent hypertension.

RATIONALE: Increased arginine-vasopressin (AVP) secretion is a key physiological response to hyperosmotic stress and may be part of the mechanism by which high-salt diets induce or exacerbate hypertension. OBJECTIVE: Using deoxycorticosterone acetate-salt hypertension model rats, we sought to test the hypothesis that changes in GABA(A) receptor-mediated inhibition in AVP-secreting magnocellular neurons contribute to the generation of Na(+)-dependent hypertension. METHODS AND RESULTS: In vitro gramicidin-perforated recordings in the paraventricular and supraoptic nuclei revealed that the GABAergic inhibition in AVP-secreting neurons was converted into excitation in this model, because of the depolarization of GABA equilibrium potential. Meanwhile, in vivo extracellular recordings in the supraoptic nuclei showed that the GABAergic baroreflexive inhibition of magnocellular neurons was transformed to excitation, so that baroreceptor activation may increase AVP release. The depolarizing GABA equilibrium potential shift in AVP-secreting neurons occurred progressively over weeks of deoxycorticosterone acetate-salt treatment along with gradual increases in plasma AVP and blood pressure. Furthermore, the shift was associated with changes in chloride transporter expression and partially reversed by bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor). Intracerebroventricular bumetanide administration during deoxycorticosterone acetate-salt treatment hindered the development of hypertension and rise in plasma AVP level. Muscimol (GABA(A) agonist) microinjection into the supraoptic nuclei in hypertensive rats increased blood pressure, which was prevented by previous intravenous V1a AVP antagonist injection. CONCLUSIONS: We conclude that the inhibitory-to-excitatory switch of GABAA receptor-mediated transmission in AVP neurons contributes to the generation of Na(+)-dependent hypertension by increasing AVP release. We speculate that normalizing the GABA equilibrium potential may have some utility in treating Na(+)-dependent hypertension.

Hoxc8 downregulates Mgl1 tumor suppressor gene expression and reduces its concomitant function on cell adhesion.

Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ~2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5' upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.

Potential anti-cancer activity of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), a histone deacetylase inhibitor, against breast cancer both in vitro and in vivo.

Histone deacetylase (HDAC) is an attractive target for cancer therapy because it plays a key role in gene expression and carcinogenesis. N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) is a novel synthetic HDAC inhibitor (HDACI) that shows better pharmacological properties than a known HDACI present in the human fibrosarcoma cell: suberoylanilide hydroxamic acid (SAHA). Here, we investigate the anti-cancer activity of HNHA against breast cancer both in vitro and in vivo. HNHA arrested the cell cycle at the G(1) /S phase via p21 induction, which led to profound inhibition of cancer cell growth in vitro. In addition, HNHA-treated cells showed markedly decreased levels of VEGF and HIF-1α than SAHA and fumagillin (FUMA) when accompanied by increased histone acetylation. HNHA significantly inhibited tumor growth in an in vivo mouse xenograft model. HNHA-treated mice survived significantly longer than SAHA- and FUMA-treated mice. Dynamic MRI showed significantly decreased blood flow in the HNHA-treated mice, implying that HNHA inhibits tumor neovascularization. This finding was accompanied by marked reductions of proangiogenic factors and significant induction of angiogenesis inhibitors in tumor tissues. We have shown that HNHA is an effective anti-tumor agent in breast cancer cells in vitro and in breast cancer xenografts in vivo. Collectively, these findings indicate that HNHA may be a potent anti-cancer agent against breast cancer due to its multi-faceted inhibition of HDAC activity, as well as anti-angiogenesis activity.

Mouse Homologue of the Schizophrenia Susceptibility Gene ZNF804A as a Target of Hoxc8.

Using a ChIP-cloning technique, we identified a Zinc finger protein 804a (Zfp804a) as one of the putative Hoxc8 downstream target genes. We confirmed binding of Hoxc8 to an intronic region of Zfp804a by ChIP-PCR in F9 cells as well as in mouse embryos. Hoxc8 upregulated Zfp804a mRNA levels and augmented minimal promoter activity in vitro. In E11.5 mouse embryos, Zfp804a and Hoxc8 were coexpressed. Recent genome-wide studies identified Zfp804a (or ZNF804A in humans) as a plausible marker for schizophrenia, leading us to hypothesize that this embryogenic regulatory control might also exert influence in development of complex traits such as psychosis.

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8.

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

In vitro osteoblast differentiation is negatively regulated by Hoxc8.

Hoxc8 has multiple roles in normal skeletal development. In this paper, a MC3T3-E1 subclone 4 osteogenic cell differentiation model was used to examine expression of Hoxc8 at multiple stages of osteogenesis. We found that Hoxc8 expression levels do not change in the early stage but increase in the middle stage and decrease in the late stage of osteogenesis. A knockdown of Hoxc8 by small-interfering RNA transfection in C2C12 cells indicated that Hoxc8 is a negative regulator of osteogenesis. Similarly, expression of Hoxc8 in C2C12 cells decreases alkaline phosphatase levels induced by bone morphogenetic protein-2 (BMP-2). The results of this study showed that Hoxc8 is involved in BMP-2-induced osteogenesis, and osteoblast differentiation in vitro is negatively regulated by Hoxc8, suggesting that Hoxc8 regulation is essential for osteoblast differentiation.