Supplementary Figure 2 from Global Quantitative Phosphoproteome Analysis of Human Tumor Xenografts Treated with a CD44 Antagonist
Stefan WeigandFrank HertingDaniela MaiselAdam NoporaEdgar VoßChristoph SchaabMartin KlammerAndreas Tebbe
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<p>PDF file - 269K, Effect of RG7356 on p-MEK1/2 signal in MDA-MB 231 xenografts</p>Abstract The salmon gonadotropin‐releasing hormone (sGnRH) is considered to be involved in gonadal maturation via gonadotropin (GTH) secretion in salmonid fishes. However, there is no direct evidence for endogenous sGnRH‐stimulated GTH secretion in salmonids. In this study, to clarify whether endogenous sGnRH stimulates GTH secretion, we examined the effects of the mammalian GnRH (mGnRH) antagonist [Ac‐Δ 3 ‐Pro 1 , 4FD‐Phe 2 , D‐Trp 3,6 ]‐mGnRH on luteinizing hormone (LH) levels in 0‐year‐old masu salmon Oncorhynchus masou and sockeye salmon Oncorhynchus nerka . First, the effects of the GnRH antagonist on LH release were examined in 0‐year‐old precocious male masu salmon. GnRH antagonist treatment for 3 hr significantly inhibited an increase in plasma LH levels that was artificially induced by exogenous sGnRH administration, indicating that the GnRH antagonist is effective in inhibiting LH release from the pituitary. Subsequently, we examined the effect of the GnRH antagonist on LH synthesis in 0‐year‐old immature sockeye salmon that were pretreated with exogenous testosterone for 42 days to increase the pituitary LH contents; the testosterone treatment did not affect the plasma LH levels. GnRH antagonist treatment slightly but significantly inhibited an increase in the testosterone‐stimulated pituitary LH content levels. However, no significant differences in the plasma LH levels were observed between the GnRH antagonist‐treated and control groups. These results suggest that endogenous sGnRH is involved in LH secretion in salmonid fishes. J. Exp. Zool. 307A:535–541, 2007 . © 2007 Wiley‐Liss, Inc.
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The possibility of direct pituitary effects of sex steroids on gonadotropin gene expression and synthesis was studied in male rats. The animals were treated with a potent GnRH antagonist, Ac-D-pClPhe-D-pClPhe-D-Trp-Ser-Tyr-DArg- Leu-Arg-Pro-D-Ala-NH2CH3COOH (Org 30276; 0.5 mg/kg BW, sc, twice daily) for 10 days. Groups of the antagonisttreated rats were implanted at the beginning of the injections with Silastic capsules containing testosterone (T), 5α-dihydrotestosterone (DHT), or diethylstilbestrol (DES). Groups treated with the antagonist alone or vehicle served as controls. The antagonist treatment decreased unoccupied pituitary receptors of GnRH by 93% (P < 0.001), serum LH by 34% (P < 0.01), and serum FSH by 30% (P < 0.05), and serum T became undetectable (<0.10 nmol/liter). Compared to antagonist treatment alone, no further effects on serum or pituitary LH levels were found after steroid replacements. In contrast, the antagonist- induced decreases in serum and pituitary FSH (30% and 70%, respectively; P < 0.05-0.01) were totally reversed by the T and DHT implants, but not by DES. Pituitary levels of the LH β-subunit mRNA were decreased by 60% (P < 0.01) after antagonist treatment. Combination treatment with androgens had no further effect on this mRNA, whereas DES partially reversed this suppression (P < 0.05). In contrast, the pituitary mRNA level of the FSH β-subunit, which decreased with antagonist treatment by 90% (P < 0.01), returned to the control level with T and DHT replacements, but only partially with DES. The pituitary mRNA level of the common α-subunit was significantly suppressed only by combined antagonist plus DHT treatment (P < 0.01). However, combination of DES with the antagonist increased α-subunit mRNA levels 2.4-fold (P < 0.05) compared to antagonist treatment alone. It is concluded that the suppression of gonadotropin secretion by GnRH antagonist treatment is accompanied in male rats by a parallel reduction in mRNA levels of the gonadotropin β-subunits. Sex steroid replacement of the antagonist-treated animals selectively reverses some of the mRNA changes. Androgens (T and DHT) increase the mRNA of FSH β-subunit, but have no effect on the LH β- subunit. Estrogen increases the mRNA levels of common α- and LH β-subunits and slightly increases that of FSHβ. Since the action of endogenous GnRH was blocked in these in vitro experiments, the observations indicate that gonadal steroids are able to directly stimulate gonadotropin gene expression at the pituitary level. (Endocrinology126: 3204–3209, 1990)
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Abstract Background: Brain tumors contain stem-like cells are likely a therapeutic target in malignant glioma due to their therapeutic resistance and ability of tumor initiation. Several studies have suggested the presence of multiple types of brain tumor stem-like cells (BTSC). However, the molecular markers valuable for targeting these subpopulations of glioma cells are still unknown. CD44 is a cell surface protein linked to tumorigenesis in some cancers outside of the central nervous system (CNS). CD44 expression is recognized in various types of cancer stem cells. In particular, one of CD44 variant isoforms, CD44v6, is associated with malignant transformation of non-CNS tumor cells by inducing proliferation and migration. To date, expression and function of CD44 and CD44v6 in BTSC is not characterized yet. Here, we investigated the expression of CD44, including CD44v6, and their function in BTSC. Results: RT-PCR and Western blotting elucidated that a subset of GBM expresses high level of CD44. Immunostaining on glioma tissue microarray demonstrated that patients with high grade glioma expressing CD44 had poorer prognoses. Positive staining of CD44 on tumor cell surface was seen only in glioblastoma multiforme (GBM). FACS analysis of BTSC enriched from GBM specimens showed a subset of GBM expressed high level of CD44 in BTSC. CD44 neutralizing antibody inhibited cell growth of BTSC derived from CD44-high GBM, while BTSC from CD44-low GBM was not affected. Cultured BTSC from CD44-high GBM contains subpopulation expressing CD44v6. Knockdown of CD44v6 with siRNA inhibited in vitro growth of BTSC from CD44-high GBM but not from CD44-low GBM. This inhibition did not affect growth of cells derived from the same tumors that do not have stem-like characters. CD44 ligand osteopontin (OPN) activated AKT in BTSC from CD44-high GBM, but not in CD44-low samples. With CD44v6 knockdown, both phosphorylated AKT and S6R were under detectable level and OPN failed to activate AKT and S6R, while EGF substantially increased both molecules. When the PI3K/AKT pathway was inhibited by five different small molecules, a dose-dependent inhibition for neurosphere formation was observed for all inhibitors in BTSC from CD44-high GBM. In contrast, BTSC from CD44-low GBM were relatively resistant to the treatment. Conclusion: Our data indicate that a subset of GBM expresses high level of CD44 and CD44v6 in BTSC. GBM expressing high level of CD44 depends on CD44v6 on the growth of their BTSC. This action of CD44v6 is, at least in part, mediated through the AKT pathway. CD44 expression in high grade glioma corresponds to poorer prognosis. Immunoreactive site of CD44 on GBM cells correlates with pathological grade. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-103. doi:10.1158/1538-7445.AM2011-LB-103
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Groups of adult male rats were treated continuously for 30 days with either vehicle or the potent gonadotrophin-releasing hormone (GnRH) antagonist. (N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 )- GnRH (RS 68439; 35 micrograms/day). In addition, groups of vehicle- and antagonist-treated rats received s.c. testosterone implants sufficient to maintain serum testosterone concentrations 3.5- to 5-fold higher than those of vehicle-treated control rats. After 30 days of antagonist treatment serum LH, FSH and testosterone concentrations were at or below the detection limits of their respective assays and pituitary FSH content and GnRH receptor binding were reduced, relative to control animals, by 77 and 98% respectively. Testis weight in antagonist-treated rats was reduced by 75% and spermatogenesis was suppressed to an extent comparable to that observed in hypophysectomized rats. Testosterone, which caused a 40% reduction in serum FSH relative to control animals, prevented the antagonist-induced fall in both serum and pituitary FSH, but not GnRH receptors, below that observed in the vehicle plus testosterone-treated group. Furthermore, spermatogenesis in the antagonist plus testosterone-treated group was indistinguishable from that observed in control animals. It is concluded that testosterone is capable of maintaining serum and pituitary FSH levels in vivo, under conditions which presumably render the pituitary insensitive to hypothalamic GnRH.
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The diabetogenic activity of growth hormone (GH) was studied through the production of an insulin antagonist in the plasma of normal rat. This antagonist, called alpha2-inhibitor, has an inhibitory activity on the glucose uptake by isolated testis, epididymal fat and hemidiaphragm of normal rat, is GH-dependent and has been identified with a fraction of plasma alpha2-glycoproteins.
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Abstract CD44, an adhesion molecule that binds to the extracellular matrix, primarily to hyaluronan (HA), has been implicated in cancer cell migration, invasion, and metastasis. CD44 has also recently been recognized as a marker for stem cells of several types of cancer. However, the roles of CD44 in the development of bone metastasis are unclear. Here, we addressed this issue by using bone metastatic cancer cell lines, in which CD44 was stably knocked down. Tumor sphere formation and cell migration and invasion were significantly inhibited by CD44 knockdown. Furthermore, the downregulation of CD44 markedly suppressed tumorigenicity and bone metastases in nude mice. Of note, the number of osteoclasts decreased in the bone metastases. Microarray analysis revealed that the expression of HA synthase 2 was downregulated in CD44-knockdown cells. The localization of HA in the bone metastatic tumors was also markedly reduced. We then examined the roles of CD44–HA interaction in bone metastasis using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis. 4-MU decreased tumor sphere and osteoclast-like cell formation in vitro. Moreover, 4-MU inhibited bone metastases in vivo with reduced number of osteoclasts. These results collectively suggest that CD44 expression in cancer cells promotes bone metastases by enhancing tumorigenicity, cell migration and invasion, and HA production. Our results also suggest the possible involvement of CD44-expressing cancer stem cells in the development of bone metastases through interaction with HA. CD44–HA interaction could be a potential target for therapeutic intervention for bone metastases. Cancer Res; 73(13); 4112–22. ©2013 AACR.
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It is established that the blockade of the pituitary LHRH receptor by an LHRH antagonist will suppress pituitary LH secretion and reduce serum concentrations of gonadal steroids. Little is known, however, about the activity of the LHRH/LH pulse generator during this inhibitory period or during the recovery phase. To investigate this, a potent LHRH antagonist [N-Ac-D-pCl-Phe1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6, D-Ala10 LHRH was injected iv into sexually active rams and the changes in the blood plasma concentrations of LH, FSH, testosterone, and PRL were measured in samples collected every 15 min for 24-48 h. The treatment induced an immediate blockade of pulsatile LH secretion and a parallel decline in blood levels of testosterone. Plasma levels of FSH were not suppressed by treatment with the LHRH antagonist and there was no consistent effect on plasma levels of PRL. The duration of the inhibition of LH was dose dependent lasting 4.3 +/- 0.4 h, 18.0 +/- 1.0 h, and 31.8 +/- 1.3 h for the low (6 micrograms/kg), medium (36 micrograms/kg), and high (365 micrograms/kg) doses of LHRH antagonist, respectively. During the recovery period there was an approximate 2-fold increase in the frequency of LH pulses. These results suggest a compensatory response to the decline in the negative feedback effect of testosterone secretion. Even the lowest dose of antagonist elicited a decrease in the level of testosterone and an increase in LH pulse frequency. At this dose, the decline in testosterone was very transitory indicating an acute sensitivity of the hypothalamus to changes in the negative feedback signal. These results suggest that the suppression of LH and testosterone secretion in the ram by LHRH antagonist is associated with a compensatory increase in the activity of the LHRH pulse generator.
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To investigate the potential oncogene promoting recurrence of hepatocellular carcinoma (HCC) following liver transplantation (LT), throughput RNA sequencing was performed in a subgroup of HCC patients. The up-regulated FAM83D in HCC tissues was found and further verified in 150 patients by real-time PCR and immunohistochemistry. FAM83D overexpression significantly correlated with high HCC recurrence rate following LT and poor HCC characteristics such as high AFP, poor differentiation. Of cancer stem cells (CSCs) markers, CD44 expression was effectively suppressed when FAM83D was knocked down by siRNA. Meanwhile, the siRNA transfected cells suppressed formation of sphere and ability of self-renew. In a xenograft tumorigenesis model, FAM83D knockdown apparently inhibited tumor growth and metastasis. Microarray assays revealed that FAM83D promotes CD44 expression via activating the MAPK, TGF-β and Hippo signaling pathways. Furthermore, CD44 knockdown presented reverse effect on above signaling pathways, which suggested that FAM83D was a key activator of loop between CD44 and above signaling pathways. In conclusion, FAM83D promotes HCC recurrence by promoting CD44 expression and CD44+ CSCs malignancy. FAM83D provides a novel therapeutic approach against HCC recurrence after LT.
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Plasma ACTH increased after an intra‐third ventricular administration of noradrenaline (NA). An iv corticotrophin‐releasing factor (CRF) antagonist [alpha‐helical CRF(9–41)] injection did not affect ACTH secretion by itself, whereas it significantly reduced NA‐induced ACTH secretion. These results suggest that NA centrally stimulated ACTH secretion and that endogenous CRF is involved in this ACTH secretion.
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Alpha (finance)
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Thirty days of continuous treatment of adult male rats with 35 micrograms/day of the potent GnRH antagonist, (N-Ac-D-Nal (2)1, D-pCl-Phe2, D-Trp3, D-hArg (Et2)6, D-Ala10)-GnRH (RS-68439) reduced serum FSH to values below the limit of detection of the assay. Testosterone supplementation in the form of subcutaneous testosterone-filled silastic capsule implants present during an additional 30 days of GnRH antagonist administration restored serum FSH to values comparable to those observed after vehicle treatment. Pituitary FSH content, which was substantially reduced after GnRH antagonist treatment, was completely restored after concurrent testosterone supplementation. These results show that, under conditions of GnRH receptor blockade, testosterone is capable of stimulating pituitary and serum FSH in adult male rats.
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