5-Demethylnobiletin Inhibits Cell Proliferation, Downregulates ID1 Expression, Modulates the NF-κB/TNF-α Pathway and Exerts Antileukemic Effects in AML Cells.

Acute myeloid leukemia (AML) is characterized by the dysregulation of hematopoietic cell proliferation, resulting in the accumulation of immature myeloid cells in bone marrow. 5-Demethylnobiletin (5-demethyl NOB), a citrus 5-hydroxylated polymethoxyflavone, has been reported to exhibit various bioactivities, such as antioxidant, anti-inflammatory and anticancer properties. In this study, we investigated the antileukemic effects of 5-demethyl NOB and its underlying molecular mechanisms in human AML cells. We found that 5-demethyl NOB (20−80 µM) significantly reduced human leukemia cell viability, and the following trend of effectiveness was observed: THP-1 ≈ U-937 > HEL > HL-60 > K562 cells. 5-Demethyl NOB (20 and 40 µM) modulated the cell cycle through the regulation of p21, cyclin E1 and cyclin A1 expression and induced S phase arrest. 5-Demethyl NOB also promoted leukemia cell apoptosis and differentiation. Microarray-based transcriptome, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) of differentially expressed genes (DEGs) analysis showed that the expression of inhibitor of differentiation/DNA binding 1 (ID1), a gene associated with the GO biological process (BP) cell population proliferation (GO: 0008283), was most strongly suppressed by 5-demethyl NOB (40 µM) in THP-1 cells. We further demonstrated that 5-demethyl NOB-induced ID1 reduction was associated with the inhibition of leukemia cell growth. Moreover, DEGs involved in the hallmark gene set NF-κB/TNF-α signaling pathway were markedly enriched and downregulated by 5-demethyl NOB. Finally, we demonstrated that 5-demethyl NOB (20 and 40 µM), combined with cytarabine, synergistically reduced THP-1 and U-937 cell viability. Our current findings support that 5-demethyl NOB dramatically suppresses leukemia cell proliferation and may serve as a potential phytochemical for human AML chemotherapy.

Roles of the proteasome and inhibitor of DNA binding 1 protein in myoblast differentiation.

This study was conducted to further understand the mechanism that controls myoblast differentiation, a key step in skeletal muscle formation. RNA sequencing of primary bovine myoblasts revealed many genes encoding the ubiquitin-proteasome system were up-regulated during myoblast differentiation. This up-regulation was accompanied by increased proteasomal activity. Treating myoblasts with the proteasome-specific inhibitor lactacystin impeded myoblast differentiation. Adenovirus-mediated overexpression of inhibitor of DNA binding 1 (ID1) protein inhibited myoblast differentiation too. Further experiments were conducted to determine whether the proteasome promotes myoblast differentiation by degrading ID1 protein. Both ID1 protein and mRNA expression decreased during myoblast differentiation. However, treating myoblasts with lactacystin reversed the decrease in ID1 protein but not in ID1 mRNA expression. Surprisingly, this reversal was not observed when myoblasts were also treated with the mRNA translation inhibitor cycloheximide. Direct incubation of ID1 protein with proteasomes from myoblasts did not show differentiation stage-associated degradation of ID1 protein. Furthermore, ubiquitinated ID1 protein was not detected in lactacystin-treated myoblasts. Overall, the results of this study suggest that, during myoblast differentiation, the proteasomal activity is up-regulated to further myoblast differentiation and that the increased proteasomal activity improves myoblast differentiation partly by inhibiting the synthesis, not the degradation, of ID1 protein.-Leng, X., Ji, X., Hou, Y., Settlage, R., Jiang, H. Roles of the proteasome and inhibitor of DNA binding 1 protein in myoblast differentiation.

Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells.

BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.

Novel bone morphogenetic protein receptor inhibitor JL5 suppresses tumor cell survival signaling and induces regression of human lung cancer.

BMP receptor inhibitors induce death of cancer cells through the downregulation of antiapoptotic proteins XIAP, pTAK1, and Id1-Id3. However, the current most potent BMP receptor inhibitor, DMH2, does not downregulate BMP signaling in vivo because of metabolic instability and poor pharmacokinetics. Here we identified the site of metabolic instability of DMH2 and designed a novel BMP receptor inhibitor, JL5. We show that JL5 has a greater volume of distribution and suppresses the expression of Id1 and pTak1 in tumor xenografts. Moreover, we demonstrate JL5-induced tumor cell death and tumor regression in xenograft mouse models without immune cells and humanized with adoptively transferred human immune cells. In humanized mice, JL5 additionally induces the infiltration of immune cells within the tumor microenvironment. Our studies show that the BMP signaling pathway is targetable in vivo and BMP receptor inhibitors can be developed as a therapeutic to treat cancer patients.

Soluble endoglin modulates the pro-inflammatory mediators NF-κB and IL-6 in cultured human endothelial cells.

AIMS: Endoglin is a transmembrane glycoprotein, that plays an important role in regulating endothelium. Proteolytic cleavage of membrane endoglin releases soluble endoglin (sEng), whose increased plasma levels have been detected in diseases related to the cardiovascular system. It was proposed that sEng might damage vascular endothelium, but detailed information about the potential mechanisms involved is not available. Thus, we hypothesized that sEng contributes to endothelial dysfunction, leading to a pro-inflammatory phenotype by a possible modulation of the TGF-ß and/or inflammatory pathways. MAIN METHODS: Human umbilical vein endothelial cells (HUVECs) and Human embryonic kidney cell line (HEK293T) were treated with different sEng concentration and time in order to reveal possible effect on biomarkers of inflammation and TGF-ß signaling. IL6 and NFκB reporter luciferase assays, quantitative real-time PCR analysis, Western blot analysis and immunofluorescence flow cytometry were used. KEY FINDINGS: sEng treatment results in activation of NF-κB/IL-6 expression, increased expression of membrane endoglin and reduced expression of Id-1. On the other hand, no significant effects on other markers of endothelial dysfunction and inflammation, including eNOS, peNOSS1177, VCAM-1, COX-1, COX-2 and ICAM-1 were detected. SIGNIFICANCE: As a conclusion, sEng treatment resulted in an activation of NF-κB, IL-6, suggesting activation of pro-inflammatory phenotype in endothelial cells. The precise mechanism of this activation and its consequence remains to be elucidated. A combined treatment of sEng with other cardiovascular risk factors will be necessary in order to reveal whether sEng is not only a biomarker of cardiovascular diseases, but also a protagonist of endothelial dysfunction.

Expression of Inhibitor of Differentiation-1 and Its Effects on Angiogenesis in Gastric Cancer.

OBJECTIVE: To explore the effects of recombinant human endostatin (endostar, ES) and cisplatin on the growth of gastric cancer-transplanted tumor in nude mice and the expression of microvessel density (MVD). METHODS: Human gastric cancer SGC-7901 cells were subcutaneously injected into the armpit of nude mice to prepare cancer-bearing nude mice. A total of 32 cancer-bearing nude mice were divided into four groups (each group with 8 mice). The four groups included control group and other three groups in which mice were treated with 5 mg/kg of ES (group E), 5 mg/kg of cisplatin (group Ci), and 5 mg/kg of ES combined with 5 mg/kg of cisplatin (group C), respectively. MVD was determined by immunohistochemistry, and the expressions of mRNA and protein of inhibitor of differentiation-1 (ID1) and vascular endothelial growth factor (VEGF) were detected with reverse transcription polymerase chain reaction (RT-PCR) and western blot, respectively. Apoptosis was observed with transmission electron microscope. RESULTS: Compared with control group, the sizes and weights of tumors were significantly decreased in other three groups (all p < 0.05). MVD was significantly lower in groups E, Ci, and C than in control group, and in groups E and C than in group Ci (all p < 0.05). Compared with control group, the expressions of mRNA and protein of ID1 and VEGF significantly decreased in groups E and C (all p < 0.05). There were no significant differences in the expressions of mRNA and protein of ID1 and VEGF between group Ci and control group. There was apoptosis in groups E and C, but no apoptosis was found in group Ci and control group. CONCLUSION: ES can inhibit the growth of gastric cancer cells through suppressing angiogenesis and promoting apoptosis of tumor cell. This study provides a new idea for the treatment of gastric cancer.

STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis.

Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways.

Inhibitor of DNA Binding 1 Is Induced during Kidney Ischemia-Reperfusion and Is Critical for the Induction of Hypoxia-Inducible Factor-1α.

In this study, rat models of acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) and HK-2 cell models of hypoxia-reoxygenation (H/R) were established to investigate the expression of inhibitor of DNA binding 1 (ID1) in AKI, and the regulation relationship between ID1 and hypoxia-inducible factor 1 alpha (HIF-1α). Through western blot, quantitative real-time PCR, immunohistochemistry, and other experiment methods, the induction of ID1 after renal I/R in vivo was observed, which was expressed mainly in renal tubular epithelial cells (TECs). ID1 expression was upregulated in in vitro H/R models at both the protein and mRNA levels. Via RNAi, it was found that ID1 induction was inhibited with silencing of HIF-1α. Moreover, the suppression of ID1 mRNA expression could lead to decreased expression and transcription of HIF-1α during hypoxia and reoxygenation. In addition, it was demonstrated that both ID1 and HIF-1α can regulate the transcription of twist. This study demonstrated that ID1 is induced in renal TECs during I/R and can regulate the transcription and expression of HIF-1α.

Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and Thymidylate Synthase Expression to Promote Esophageal Cancer Chemoresistance.

PURPOSE: Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, IGF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. EXPERIMENTAL DESIGN: In vitro and in vivo functional assays were performed to study the effects of Id1-E2F1-IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. RESULTS: Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. CONCLUSIONS: Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1-E2F1-IGF2 regulatory axis has important implications for cancer prognosis and treatment.

Overexpression of Id1 in transgenic mice promotes mammary basal stem cell activity and breast tumorigenesis.

Inhibitor of differentiation/DNA binding (Id)1 is a crucial regulator of mammary development and breast cancer progression. However, its effect on stemness and tumorigenesis in mammary epithelial cells remains undefined. Herein, we demonstrate that Id1 induces mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities in transgenic mice. MaSC-enriched basal cell expansion and increased self-renewal and in vivo regenerative capacity of MaSCs are observed in the mammary glands of MMTV-Id1 transgenic mice. Furthermore, MMTV-Id1 mice develop ductal hyperplasia and mammary tumors with highly expressed basal markers. Id1 also increases breast cancer stem cell (CSC) population and activity in human breast cancer lines. Moreover, the effects of Id1 on normal and malignant stem cell activities are mediated by the Wnt/c-Myc pathway. Collectively, these findings provide in vivo genetic evidence of Id1 functions as an oncogene in breast cancer and indicate that Id1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to breast tumor development.

Berberine suppresses Id-1 expression and inhibits the growth and development of lung metastases in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an invasive cancer with a high rate of recurrence and metastasis. Agents with anti-proliferative as well as anti-metastatic activity will be ideal for effective treatment. Here, we demonstrated that berberine, an isoquinoline alkaloid, harbored potent anti-metastatic and anti-proliferative activities in vivo. Using an orthotopic model of HCC (MHCC-97L), which spontaneously develops lung metastases (one of the most common sites of HCC metastasis), we found that berberine treatment (10mg/kg/2days) significantly reduced lung metastasis from the liver tumors by ~85% (quantitated by bioluminescence emitted from lung metastases). Histological examination also confirmed the reduced incidence and number of lung metastases in berberine-treated mice. Furthermore, berberine effectively suppressed extra-tumor invasion of the primary HCC implant into the surrounding normal liver tissue, illustrating its potent anti-metastatic action in vivo. Consistent with previous reports in other cancer, berberine's anti-tumor activity was accompanied by suppression of cellular proliferation, invasiveness and HIF-1α/VEGF signaling. Strikingly, further mechanistic investigation revealed that berberine exerted profound inhibitory effect on the expression of Id-1, which is a key regulator for HCC development and metastasis. Berberine could suppress the transcription level of Id-1 through inhibiting its promotor activity. Specific downregulation of Id-1 by knocking down its RNA transcripts in HCC cells inhibited cellular growth, invasion and VEGF secretion, demonstrating the functional relevance of Id-1 downregulation induced by berberine. Lastly, berberine's anti-proliferative and anti-invasive activities could be partially rescued by Id-1 overexpression in HCC models, revealing a novel anti-cancer/anti-invasive mechanism of berberine via Id-1 suppression.

Androgen deprivation therapy induces androgen receptor-dependent upregulation of Egr1 in prostate cancers.

Early growth response gene-1 (Egr1) has a crucial function in the development and progression of prostate cancer. However, whether Egr1 contributes to the transition of advanced androgen-independent prostate cancer (AIPC) from androgen-dependent prostate cancer (ADPC) remains largely unknown. To the best of our knowledge, through immunohistochemical staining methods, we were the first to identify that Egr1 is more highly expressed in AIPC clinical specimens than in androgen-dependent prostate cancer (ADPC). An in vitro study with quantitative RT-PCR and Western blot demonstrated that Egr1 also has a higher expression in androgen-independent PC3 cells than in the androgen-dependent LNCaP cells. Egr1 expression in LNCaP cells was significantly upregulated during the androgen deprivation treatment (ADT) and was re-downregulated through the addition of dihydrotestosterone. Although no variation in PC3 cells was identified, Egr1 responded to dihydrotestosterone and flutamide in the androgen receptor (AR)-transfected PC3 cells. Further investigation with Egr1 agonist and specific siRNA-targeting Egr1 revealed that Egr1 upregulation or downregulation was accompanied by a change in inhibitors of differentiation and DNA binding-1 (Id1) in the same direction in both LNCaP and PC3 cells. The variation is shown to be negatively regulated by androgen through AR during ADT. Our data suggested that upregulated Egr1 might partially contribute to the emergence of AIPC after prolonged ADT. This study also elucidated the potential mechanism underlying Id1 participation in the progression of prostate cancer. Understanding the key molecular events in the transition from ADPC to AIPC may provide new therapeutic intervention strategies for patients with AIPC.

Curcumin inhibits expression of inhibitor of DNA binding 1 in PC3 cells and xenografts.

Inhibitor of DNA binding 1 (Id1) plays an important role in genesis and metastatic progression of prostate cancer. We previously reported that down regulation of Id1 by small interfering RNA could inhibit the proliferation of PC3 cells and growth of its xenografted tumors. Curcumin, the active ingredient of turmeric, has shown anti-cancer properties via modulation of a number of different molecular regulators. Here we investigated whether Id1 might be involved in the anti-cancer effects of curcumin in vivo and in vitro. We firstly confirmed that curcumin inhibited cell viability in a dose-dependent fashion, and induced apoptosis in PC3 cells, associated with significant decrease in the mRNA and protein expression of Id1. Similar effects of curcumin were observed in tumors of the PC3 xenografted mouse model with introperitoneal injection of curcumin once a day for one month. Tumor growth in mice was obviously suppressed by curcumin during the period of 24 to 30 days. Both mRNA and protein levels of Id1 were significantly down-regulated in xenografted tumors. Our findings point to a novel molecular pathway for curcumin anti-cancer effects. Curcumin may be used as an Id1 inhibitor to modulate Id1 expression.

Inhibitor of differentiation 1 is a candidate prognostic marker in multicentric Castleman's disease.

Castleman's disease (CD) is a benign lymphoproliferative disorder characterized by dysfunctional lymphatic node hyperplasia. Lymphatic node hyperplasia is associated with elevated levels of inhibitor of differentiation 1 (ID1) in many human tumors. To assess the possible role of ID1 expression as a prognostic marker in multicentric CD (MCD), intra-lymph node ID1 expression was analyzed and related to clinical characteristics and outcomes in 48 patients. Furthermore, the correlation between ID1 and possible signaling molecules such as interleukin-6 (IL6), phosphorylated extracellular response kinase (p-ERK), and vascular endothelial growth factor C (VEGFC) was explored on six fresh MCD surgical specimens. Immunohistochemistry revealed that the patients with extensive ID1 expression had significantly poorer prognosis, compared to those with localized ID1. In addition, ID1 was positively associated with levels of IL6, p-ERK, and VEGFC. We conclude that ID1 may ultimately be a prognostic marker in MCD and that the IL6/ERK/VEGFC pathway is involved in the progress of this disease.

Regulation of Id1 expression by epigallocatechin­3­gallate and its effect on the proliferation and apoptosis of poorly differentiated AGS gastric cancer cells.

We investigated the inhibition of apoptosis and proliferation of poorly differentiated AGS gastric cancer cells by epigallocatechin-3-gallate (EGCG), to establish target genes for regulation by EGCG. The proliferation and apoptosis of AGS gastric cancer cells treated with EGCG were observed by cell counting kit (CCK)-8 and flow cytometry. Differential gene expression in AGS cells treated with EGCG was screened by gene expression microarrays. Id1 gene and protein expression were determined by quantitative PCR and western blot analysis. The effect of Id1 on EGCG-induced apoptosis and cell cycle arrest of AGS cells was verified with RNAi. The proliferation and apoptosis of AGS cells treated with siRNA­Id1 was observed by CCK-8 and flow cytometry. EGCG significantly promoted apoptosis and inhibited the proliferation of AGS cells. The Id1 gene was differentially expressed in AGS cells treated with EGCG, and Id1 mRNA and protein were downregulated in AGS cells treated with EGCG, confirmed by quantitative PCR and western blot analysis. Id1 mRNA and protein were also downregulated in AGS cells treated with siRNA-Id1. The apoptosis and proliferation of AGS cells treated with siRNA-Id1 were similar to those in cells treated with EGCG. EGCG induces apoptosis and inhibits proliferation of poorly differentiated AGS gastric cancer cells, and Id1 may be one of the target genes regulated by EGCG in cancer inhibition.

Gene expression profile reveals abnormalities of multiple signaling pathways in mesenchymal stem cell derived from patients with systemic lupus erythematosus.

We aimed to compare bone-marrow-derived mesenchymal stem cells (BMMSCs) between systemic lupus erythematosus (SLE) and normal controls by means of cDNA microarray, immunohistochemistry, immunofluorescence, and immunoblotting. Our results showed there were a total of 1, 905 genes which were differentially expressed by BMMSCs derived from SLE patients, of which, 652 genes were upregulated and 1, 253 were downregulated. Gene ontology (GO) analysis showed that the majority of these genes were related to cell cycle and protein binding. Pathway analysis exhibited that differentially regulated signal pathways involved actin cytoskeleton, focal adhesion, tight junction, and TGF-ß pathway. The high protein level of BMP-5 and low expression of Id-1 indicated that there might be dysregulation in BMP/TGF-ß signaling pathway. The expression of Id-1 in SLE BMMSCs was reversely correlated with serum TNF-α levels. The protein level of cyclin E decreased in the cell cycling regulation pathway. Moreover, the MAPK signaling pathway was activated in BMMSCs from SLE patients via phosphorylation of ERK1/2 and SAPK/JNK. The actin distribution pattern of BMMSCs from SLE patients was also found disordered. Our results suggested that there were distinguished differences of BMMSCs between SLE patients and normal controls.

Stalk cell phenotype depends on integration of Notch and Smad1/5 signaling cascades.

Gradients of vascular endothelial growth factor (VEGF) induce single endothelial cells to become leading tip cells of emerging angiogenic sprouts. Tip cells then suppress tip-cell features in adjacent stalk cells via Dll4/Notch-mediated lateral inhibition. We report here that Smad1/Smad5-mediated BMP signaling synergizes with Notch signaling during selection of tip and stalk cells. Endothelium-specific inactivation of Smad1/Smad5 in mouse embryos results in impaired Dll4/Notch signaling and increased numbers of tip-cell-like cells at the expense of stalk cells. Smad1/5 downregulation in cultured endothelial cells reduced the expression of several target genes of Notch and of other stalk-cell-enriched transcripts (Hes1, Hey1, Jagged1, VEGFR1, and Id1-3). Moreover, Id proteins act as competence factors for stalk cells and form complexes with Hes1, which augment Hes1 levels in the endothelium. Our findings provide in vivo evidence for a regulatory loop between BMP/TGFß-Smad1/5 and Notch signaling that orchestrates tip- versus stalk-cell selection and vessel plasticity.

Mono-(2-ethylhexyl) phthalate (MEHP) promotes invasion and migration of human testicular embryonal carcinoma cells.

Testicular dysgenesis syndrome refers to a collection of diseases in men, including testicular cancer, that arise as a result of abnormal testicular development. Phthalates are a class of chemicals used widely in the production of plastic products and other consumer goods. Unfortunately, phthalate exposure has been linked to reproductive dysfunction and has been shown to adversely affect normal germ cell development. In this study, we show that mono-(2-ethylhexyl) phthalate (MEHP) induces matrix metalloproteinase 2 (MMP2) expression in testicular embryonal carcinoma NT2/D1 cells but has no significant effect on MMP9 expression. NT2/D1 cells also have higher levels of MYC expression following MEHP treatment. It is widely recognized that activation of MMP2 and MYC is tightly associated with tumor metastasis and tumor progression. Gelatin zymographic analysis indicates that MEHP strongly activates MMP2 in NT2/D1 cells. Addition of the MMP2-specific inhibitor SB-3CT inhibited MEHP-enhanced cell invasion and migration, demonstrating that MMP2 plays a functional role in promoting testicular embryonal carcinoma progression in response to MEHP exposure. Furthermore, we investigated genome-wide gene expression profiles of NT2/D1 cells following MEHP exposure at 0, 3, and 24 h. Microarray analysis and semiquantitative RT-PCR revealed that MEHP exposure primarily influenced genes in cell adhesion and transcription in NT2/D1 cells. Gap junction protein-alpha 1, vinculin, and inhibitor of DNA-binding protein-1 were significantly down-regulated by MEHP treatment, while claudin-6 and beta 1-catenin expression levels were up-regulated. This study provides insight into mechanisms that may account for modulating testicular cancer progression following phthalate exposure.

Improvement of ovarian response and oocyte quality of aged female by administration of bone morphogenetic protein-6 in a mouse model.

BACKGROUND: Advancing female age remains a difficult problem in infertility treatment. Ovarian angiogenesis plays an important role in follicular development and the activation of ovarian angiogenesis has been emerged as a new strategy for the improvement of age-related decline of oocyte quality. BMP-6 affect gonadotropin signals in granulosa cells and it promotes normal fertility by enabling appropriate response to LH and normal oocyte quality. BMP-6 has a potential role in regulation of angiogenesis and regulates the expression of inhibitor of DNA-binding proteins (Ids). Ids involved in the control and timing of follicle selection and granulosa cells differentiation. Especially, Id-1 is well-characterized target of BMP-6 signaling. Therefore, this study investigated whether co-administration of BMP-6 during superovulation process improves ovarian response, oocyte quality and expression of Id-1 and vascular endothelial growth factor (VEGF) in the ovary of aged female using a mouse model. METHODS: Aged C57BL/6 female mice (26-31 weeks old) were superovulated by injection with 0.1 mL of 5 IU equine chorionic gonadotropin (eCG) containing recombinant mouse BMP-6 at various doses (0, 0.01, 0.1, 1, and 10 ng), followed by injection with 5 IU human chorionic gonadotropin (hCG) 48 h later. Then, the mice were immediately paired with an individual male. The aged control group was superovulated without BMP-6. Young mice of 6-9 weeks old were superovulated without BMP-6 as a positive control for superovulation and in vitro culture of embryos. Eighteen hours after hCG injection, zygotes were retrieved and cultured for 4 days. Both ovaries of each mouse were provided in the examination of ovarian expression of Id-1 and VEGF by reverse transcriptase-polymerase chain reaction, western blot, and immunohistochemistry. RESULTS: Administration of 0.1 ng BMP-6 significantly increased the number and blastocyst formation rate of oocytes ovulated and ovarian expression of Id-1 and VEGF compared to aged control mice. These increased levels were comparable to those of young control mice. CONCLUSIONS: This result suggests that BMP-6 during ovulation induction plays an important role in improvement of oocyte quality and ovarian response of aged female, possibly by regulating of ovarian Id-1 and VEGF expression.

Id1 and Id3 expression is associated with increasing grade of prostate cancer: Id3 preferentially regulates CDKN1B.

As transcriptional regulators of basic helix-oop-helix (bHLH) transcription and non-bHLH factors, the inhibitor of differentiation (Id1, Id2, Id3, and Id4) proteins play a critical role in coordinated regulation of cell growth, differentiation, tumorigenesis, and angiogenesis. Id1 regulates prostate cancer (PCa) cell proliferation, apoptosis, and androgen independence, but its clinical significance in PCa remains controversial. Moreover, there is lack of evidence on the expression of Id2 and Id3 in PCa progression. In this study we investigated the expression of Id2 and Id3 and reevaluated the expression of Id1 in PCa. We show that increased Id1 and Id3 protein expression is strongly associated with increasing grade of PCa. At the molecular level, we report that silencing either Id1 or Id3 attenuates cell cycle. Although structurally and mechanistically similar, our results show that both these proteins are noncompensatory at least in PCa progression. Moreover, through gene silencing approaches we show that Id1 and Id3 primarily attenuates CDKN1A (p21) and CDKN1B (p27), respectively. We also demonstrate that silencing Id3 alone significantly attenuates proliferation of PCa cells as compared with Id1. We propose that increased Id1 and Id3 expression attenuates all three cyclin-dependent kinase inhibitors (CDKN2B, -1A, and -1B) resulting in a more aggressive PCa phenotype.