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Objectives: To explore the mechanism of development and aggressiveness in gastric carcinomas by investigating the expression and role of CD97 and its cellular ligand CD55 in gastric carcinomas. Methods: Tumor and corresponding normal mucosal tissue, collected from 39 gastric carcinoma patients, were examined by immunohistochemistry and RT-PCR for the expression of CD97 and CD55. Results: CD97stalk was strongly stained on scattered tumor cells or small tumor cell clusters at the invasion front of gastric carcinomas. The expression of CD97stalk was frequently observed in tumors of stage I and T1 gastric carcinoma patients. The expression of CD97stalk between Stage I and Stage II, III, IV specimens showed significant difference ( P<0.05), between T1 and T2, T3, T4 specimens also showed significant difference ( P<0.05). Specimens with tumor invasion depth limited in mucosa of T1 specimens showed higher positive CD55 expression than specimens with the same tumor invasion depth in T2, T3, T4 specimens, the expression of CD55 between T1 and T2 T3, T4 specimens was significantly different ( P<0.05). There was strong correlation between the distribution patterns of CD97stalk and CD55 on tumor tissues ( r=0.73, P<0.05). Signet ring cell carcinomas frequently contained strong CD97stalk and CD55-staining. Conclusions: Our results suggest that CD97stalk is probably involved in the growth, invasion and aggressiveness of gastric carcinomas by binding its cellular ligand CD55. CD97stalk and CD55 could be useful as molecular markers for prognosis and therapy of gastric carcinoma patients. [ABSTRACT FROM AUTHOR]

Fifteen unmated Landrace sows with indwelling jugular vein catheters were used to monitor ovarian function by profiles of progesterone and 17β-oestradiol (Oe2) throughout the year. A biphasic pattern of acyclia was found. Prolonged periods of low progesterone and Oe2-concentrations occurred in two sows in February/March and in six sows in July until November. Seven of the 15 sows revealed acyclia at least once during the investigation. The progesterone concentrations during the luteal phases of cyclic animals were significantly lower in August than in November and January. These findings indicate similarities to the seasonal dependency of ovarian function which is well known in the wild pig. In cyclic sows, oestrous behaviour tended to be less pronounced in summer. Oe2 concentrations in August were also significantly lower than concentrations before ovulations between November and January. Zusammenfassung Nachweis saisonaler Schwankungen der Ovarfunktion bei ungedeckten Sauen durch Messung von Progesteron und 17bT-Oestradiol im Blutplasma Von 15 maturen, nicht gedeckten Sauen (DL) wurden über Jugularisdauerkatheter für 4-14 Monate Blutproben gewonnen und Progesteron und Oestradiol-17β gemessen. Azyklie wurde aufgrund der Hormonprofile in zwei Zeitabschnitten nachgewiesen: Phasen mit konstant niedrigen Konzentrationen von Progesteron und Oestradiol-17β traten bei 2 Sauen im Februar/März und bei 6 Sauen zwischen Juli und November auf. Insgesamt zeigten 7 der 15 Sauen azyklische Phasen. Bei den Sauen, die auch im Sommer zyklisch waren, wurden jedoch signifikant niedrigere Progesteronmaxima nach Ovulationen im August, verglichen mit Ovulationen im November und Januar, gemessen. Zudem waren die Maximalkonzentrationen von Oestradiol-17β vor Ovulationen im August ebenfalls signifikant niedriger als vor Ovulationen im November, Dezember und Januar. Oestrussymptome waren im Sommer weniger deutlich ausgeprägt als in den übrigen Jahreszeiten. Diese saisonalen Unterschiede im Zyklusgeschehen des Hausschweines zeigen somit Parallelen zur streng saisonalen Abhängigkeit des Reproduktionsgeschehens des Wildschweines. [ABSTRACT FROM AUTHOR]

Background: Vitamin C is one of the major extracellular nonenzymatic antioxidants involved in the biosynthesis of collagen. It promotes the growth of fibroblasts, wound healing processes, and enhances the survival and differentiation of osteoblasts. The potential effects of ascorbic acid on human dental pulp cells (DPC) and the cells of the apical papilla (CAP) used in actual regenerative endodontic procedures remain largely unknown. In this study, we investigated the possible employment of ascorbic acid in the differentiation and regenerative therapies of DPC and CAP., Methods: Nine extracted human wisdom teeth were selected for this study. Subpopulations of stem cells within DPC and CAP were sorted with the mesenchymal stem cell marker STRO-1, followed by treatments with different concentrations (0 mM, 0.1 mM, 0.5 mM, and 1.0 mM) of ascorbic acid (AA), RT-PCR, and Western blot analysis., Results: FACS analysis revealed the presence of cell subpopulations characterized by a strong expression of mesenchymal stem cell marker STRO-1 and dental stem cell markers CD105, CD44, CD146, CD90, and CD29. Treatment of the cells with defined amounts of AA revealed a markedly increased expression of proliferation marker Ki-67, especially in the concentration range between 0.1 mM and 0.5 mM. Further investigations demonstrated that treatment with AA led to significantly increased expression of common stem cell markers OCT4, Nanog, and Sox2. The most potent proliferative and expressional effects of AA were observed in the concentration of 0.1 mM., Conclusions: AA might be a novel and potent growth promoter of human dental cells. Increasing the properties of human dental pulp cells and the cells of the apical papilla using AA could be a useful factor for further clinical developments of regenerative endodontic procedures.

Introduction: Calcification is a highly relevant process in terms of development of cardiovascular diseases, and its prevention may be the key to prevent disease progression in patients. In this study we investigated the expression of osteocalcin (OC), osteopontin (OPN) and RUNX2 in patients' leukocytes and their possible role as diagnostic markers for cardiovascular diseases., Materials and Methods: Leucocytes from 38 patients were collected in the Department of Surgery of Martin-Luther-University Halle, including 8 patients without arteriosclerotic disease (PAD-) and 30 patients with symptomatic arteriosclerotic disease (PAD+). Patients' leucocytes, in vitro calcified human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) were subjected to qPCR analyses with TaqMan probes, which are specific for OC, OPN and RUNX2. Additionally, the interaction between monocytes and calcified HUVEC and VSMC was investigated in adhesion assays., Results: The leucocytes obtained from patients with symptomatic arteriosclerotic disease (PAD+) demonstrated decreased mRNA level expression of Osteocalcin, while OPN and RUNX2 were significantly upregulated in comparison to asymptomatic patients. The induction of calcification in HUVEC and VSMC cells led to an increased expression of OC, OPN and RUNX2. Immunocytochemistry of calcified HUVEC and VSMC revealed stronger expression of OC, OPN and RUNX2 in calcified cells., Conclusion: To conclude, these data demonstrate that symptomatic arteriosclerotic disease has a correlation with OC, OPN and RUNX2. The biological rationale of OC, OPN and RUNX-2 remains not yet entirely understood for atherosclerotic disease, which means it needs further investigation.

Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA-PKcs was observed. These data provide new insight into the mechanisms of hypoxia-associated radioresistance in thyroid-carcinoma.

The relaxin-like RXFP1 ligand-receptor system has important functions in tumor growth and tissue invasion. Recently, we have identified the secreted protein, CTRP8, a member of the C1q/tumor necrosis factor-related protein (CTRP) family, as a novel ligand of the relaxin receptor, RXFP1, with functions in brain cancer. Here, we review the role of CTRP members in cancers cells with particular emphasis on CTRP8 in glioblastoma.

Background: Expression of the small calcium-binding protein S100A4 is associated with poor prognosis in patients with thyroid cancer (TC). The authors have previously shown that S100A4 is a target for relaxin and insulin-like peptide 3 signaling in TC cells and that S100A4 is secreted from human TC cells. Although the pro-migratory role of intracellular S100A4 in binding to non-muscle myosin is well known, this study investigated here whether extracellular S100A4 contributes to TC migration., Methods: Human cell lines of follicular, papillary, and undifferentiated thyroid cancer, primary patient TC cells, and TC tissues were utilized to discover the presence of the receptor of advanced glycation end products (RAGE) in TC cells and TC tissues. Fluorescence imaging, protein pull-down assays, Western blot, siRNA protein silencing, small GTPase inhibitors, cell proliferation, and cell migration assays were used to investigate the interaction of extracellular S100A4 with RAGE in promoting a TC migratory response., Results: It was demonstrated that RAGE served as receptor for extracellular S100A4 mediating cell migration in TC cells. The RAGE-mediated increase in cell migration was dependent on the intracellular RAGE signaling partner diaphanous-1 (Dia-1) and involved the activation of the small GTPases Cdc42 and RhoA. Although extracellular S100A4 consistently activated ERK signaling in TC cells, it was shown that ERK signaling was not mediated by RAGE and not essential for the migratory response in TC cells., Conclusion: The data have identified the RAGE/Dia-1 signaling system as a mediator for the pro-migratory response of extracellular S100A4 in human TC. Thus, therapeutic targeting of the RAGE/Dia-1/small GTPases signaling may successfully reduce local invasion and metastasis in TC.

Background: Lactate dehydrogenase A (LDHA) and Pyruvate Kinase M2 (PKM2) are important enzymes of glycolysis. Both of them can be phosphorylated and therefore regulated by Fibroblast growth factor receptor 1 (FGFR1). While phosphorylation of LDHA at tyrosine10 leads to tetramerization and activation, phosphorylation of PKM2 at tyrosine105 promotes dimerization and inactivation. Dimeric PKM2 is found in the nucleus and regulates gene transcription. Up-regulation and phosphorylation of LDHA and PKM2 contribute to faster proliferation under hypoxic conditions and promote the Warburg effect., Methods: Using western blot and SYBR Green Real time PCR we investigated 77 thyroid tissues including 19 goiter tissues, 11 follicular adenomas, 16 follicular carcinomas, 15 papillary thyroid carcinomas, and 16 undifferentiated thyroid carcinomas for total expression of PKM2, LDHA and FGFR1. Additionally, phosphorylation status of PKM2 and LDHA was analysed. Inhibition of FGFR was performed on FTC133 cells with SU-5402 and Dovitinib., Results: All examined thyroid cancer subtypes overexpressed PKM2 as compared to goiter. LDHA was overexpressed in follicular and papillary thyroid cancer as compared to goiter. Elevated phosphorylation of LDHA and PKM2 was detectable in all analysed cancer subtypes. The highest relative phosphorylation levels of PKM2 and LDHA compared to overall expression were found in undifferentiated thyroid cancer. Inhibition of FGFR led to significantly decreased phosphorylation levels of PKM2 and LDHA., Conclusions: Our data shows that overexpression and increased phosphorylation of PKM2 and LHDA is a common finding in thyroid malignancies. Phospho-PKM2 and Phospho-LDHA could be valuable tumour markers for thyroglobulin negative thyroid cancer.

We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The aim of this study was to investigate the effect of short hairpin RNA (shRNA) targeting autotaxin (ATX) on the migratory and invasive capability of AGS human gastric cancer cells and the growth of xenografts in nude mice. pSUPER-ATX and pSUPER-mock (non-specific), which were constructed corresponding to the ATX-shRNA and negative control mock-shRNA synthesized based on gene sequence, were transfected with blank plasmid pSUPER-control into AGS human gastric cancer cells using Lipofectamine. At 24, 48 and 72 h post-transfection, the cells were harvested and analyzed. The endogenous ATX mRNA and protein of the different groups of AGS cells were detected by RT-PCR and western blot assays. Cell proliferation was measured by MTT assay. In vitro Transwell and Matrigel assays were used to detect the cell migratory and invasive capabilities. A tumor xenograft model was generated by subcutaneous injection of AGS cells into the dorsum of nude mice. The growth of xenograft tumors was monitored and measured; changes in tumor morphology and the organs of mice were observed by H&E staining. The expression of ATX, MMP-2 and MMP-9 protein in xenograft tumor tissues was detected by immunohistochemistry and western blotting. In vitro, both the mRNA and protein levels of ATX in the pSUPER-ATX group were significantly downregulated (P<0.01), and the cell proliferative, migratory and invasive capabilities were also significantly decreased. In vivo, no obvious damage to the organs was found. pSUPER-ATX significantly suppressed the tumor volume and weight from the 7th week after cell transplantation, compared to the pSUPER-mock, pSUPER-control and WT groups; the inhibition rates were approximately 50% (P<0.05). However, no significant differences in these parameters were found among the WT, pSUPER-mock and pSUPER-control groups. Furthermore, ATX, MMP-2 and MMP-9 were expressed at significantly lower levels in the pSUPER-ATX group compared to the levels in the other three groups (P<0.05), and a significant correlation between ATX and MMP-2 expression was found (r=0.869, P<0.01). The specific shRNA targeting ATX downregulated the endogenous ATX of AGS human gastric cancer cells, and inhibited AGS cell proliferative, migratory and invasive potentials. Moreover, shRNA targeting ATX inhibited the growth of human gastric cancer xenografts in nude mice.

CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi of p50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, and QPCT RNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.

IGF-1 regulates the metabolism of hard dental tissue through binding to the IGF-1 receptor on target cells. Furthermore, IGF-binding-protein-3 promotes the accessibility of IGF-1. The aim of this study was to investigate the expression of IGF-1, IGFBP-3 and IGF-1R in STRO-1-positive dental pulp stem cells (DPSCs) and fully impacted wisdom teeth in relation to tooth development. Third molars were surgically removed from 60 patients and classified into two groups: teeth showing ongoing development (group 1) and teeth that had completed root shaping (group 2). The transcript and protein levels of IGF-1, IGFBP-3 and IGF-1R were investigated using RT-PCR and immunohistochemistry. The expression of the same proteins was also analyzed in DPSCs. The teeth from group 1 showed significantly stronger expression of IGF-1 and IGF-1R. The major sources of all of the proteins investigated immunohistochemically in sections of wisdom teeth were odontoblasts, cementoblasts and cell colonies in the pulpal mesenchyme. These colonies were identified as stem cells in view of their positivity for STRO-1, and the cells were subsequently sorted by flow cytometry. These DPSCs demonstrated high levels of pluripotency markers and IGF-1 and IGF-1R. We conclude that members of the IGF-1 family are involved in the late stage of tooth development and the process of pulpal differentiation.

We report for the first time the distribution of human INSL5 and its cognate leucine rich G-protein coupled receptor RXFP4 in the large intestine and in neuroendocrine/carcinoid tissues. Immunoreactive INSL5 was uniquely expressed by enteroendocrine cells (EECs) located within the colonic mucosa, whereas colonocytes were immunopositive for RXFP4. INSL5+ and RXFP4+ cells were also detected in human neuroendocrine/carcinoid tissues. We employed a recently described Insl5 knockout mouse model and 2 mouse models of induced colitis to address the relevance of Insl5 in EEC development and in acute inflammation of the colon. We identified INSL5 as a specific marker for synaptophysin+ EECs in the mucosa of the normal human and mouse colon. Insl5 was not essential for the development of mouse synaptophysin+ EECs. The mouse models of chemically induced colitis (dextran sulfate sodium and dinitrobenzene-sulfonic acid) failed to show changes in the numbers of Insl5+ EECs at inflammatory sites during the acute phase of colitis. In conclusion, we showed that INSL5 is a novel marker of colorectal EECs and provide first evidence for the presence of a potentially autocrine/paracrine INSL5-RXFP4 signaling system in the normal human and mouse colon and in rare human neuroendocrine tumours.

Survivin is a novel apoptosis inhibitor. Its gene is related to the baculovirus gene, which is believed to play a crucial role in fetal development and in cancer. We attempted to determine the expression of survivin in both thyroid goiter and carcinoma tissues, and to evaluate its prognostic value in human thyroid disease. In the present study, we applied small interfering RNA (siRNA) directed against survivin to determine the effects of decreasing the high constitutive levels of this protein in the FTC-133 thyroid follicular cancer cell line. Using reverse transcription PCR and immunohistochemistry, we compared the expression of survivin with relevant clinical and pathological data of 90 postsurgical specimens from patients with primary thyroid carcinoma and patients with benign goiter (33 with papillary thyroid cancer, 24 with follicular thyroid cancer, 18 with undifferentiated thyroid cancer and 15 cases with goiter). For the siRNA treatment in a human follicular thyroid carcinoma cell line, fluorescein-labeled double-stranded ultrapure siRNAs were used. RT-PCR identified the survivin transcript in 67/75 (89.3%) tumor samples and in 4/15 benign goiter samples. Immunohistochemical analysis showed positive immunoreactivity in 65/75 (86.7%) carcinomas while no expression was noted in all of the 15 benign goiter tissues. Survivin mRNA and protein levels were significantly higher in cancer tissues compared to benign goiter tissues (P<0.001). Higher survivin expression was found in the tumor tissues of pT3/pT4 and in the tumors with lymph node metastasis (P<0.05). Tumors with distant metastasis demonstrated higher survivin expression compared to the tumors without distant metastasis. Additionally, the expression of survivin in undifferentiated carcinomas was higher than that in differentiated ones. There was no significant correlation between survivin expression and age, gender, histological subtype and pathological stage. Our additional studies demonstrated that siRNA directed against survivin markedly decreased the protein expression of survivin. In conclusion, we conclude that survivin expression indicates more aggressive behavior and metastatic ability in thyroid cancer cells in vivo. Survivin can be used as a diagnostic and therapeutic marker for thyroid carcinoma and an important target in the strategy of thyroid cancer therapy. Our results of siRNA silencing indicate that siRNA may have potential as a therapeutic modality in the treatment of human thyroid cancer.