Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC.Thiocoraline is a potent Notch pathway activator and an inhibitor of medullary thyroid cancer cell line (MTC-TT) cell proliferation at low nanomolar concentrations. These results provide evidence for the use of thiocoraline as a potential treatment for intractable MTC.

Although some of the fetal responses involving Epo were similar to adults, we did not find a hepcidin-Epo relationship like that of adults, where fetal liver is the site of both hepcidin and Epo production.

Twins, particularly monochorionic (MC) pairs, are at increased risk for fetal death. Whereas previous work has sought to understand the mechanisms for this increased mortality, most studies analyze viable twin pregnancies or liveborn twin cohorts. In the Wisconsin Stillbirth Service Program cohort of 3,137 stillbirths and second trimester miscarriages, we identified 175 twin pregnancies for a twinning rate of 56/1,000, which is approximately double the general population. The excess of twins among miscarriages and stillbirths was attributable to MC pairs as the incidence of dizygotic (DZ) twinning was not increased compared to livebirth data. The leading causes of fetal demise among twins were twin-twin transfusion, acardia, and twin-twin disruption. Maternal causes of death, primarily premature rupture of membranes, were moderately increased in both MC and DZ twins relative to singletons. Although deceased twins were smaller than expected for viable twins at comparable gestational ages, placenta weights of deceased MC pairs were large compared to combined fetal weight, which indicates placental inefficiency likely due to vascular shunting. Co-twin survival was much lower for MC than for DZ pairs. Therefore, earlier diagnosis and treatment of MC twinning complications may decrease prenatal mortality. K E Y W O R D S fetal demise, miscarriage, stillbirth, twin-twin transfusion

Introduction: Carcinoids are neuroendocrine (NE) tumors that secrete hormones causing the carcinoid syndrome. Metastatic carcinoids are not amenable to curative surgery. Our previous research has shown that Notch signaling has a tumor suppressor role in NE tumors. In the present study, we sought to examine Thailandepsin A (TDP-A), a newly discovered HDAC inhibitor as a Notch activating drug and carcinoid cancer agent. Methods: TDP-A is a bacterial natural product isolated from the fermentation broth of Burkholderia thailandensis E264. The cytotoxicity of TDP-A on human pancreatic (BON) carcinoid cells was measured by determining the IC50 value.The antiproliferative effect of TDP-A on BON cells growth was assessed by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay.The mechanism of growth inhibition was determined for cell cycle and apoptosis markers by Western blot and flow cytometry analyses. Expression of Notch isoforms 1-3 was assessed at the transcriptional level (real time RT-PCR) and protein level (Western blot) from parental and TDP-A treated BON cells. Functional analysis of Notch Intracellular Domain (NICD) was done by measuring the degree of luciferase activity by CBF1 binding assay. The gene expression of HES and HEY (Notch signaling mediators) was quantified by real time RT-PCR. Effect of TDP-A on NE markers -chromogranin A (CgA) and achaete-scute complex-like1 (ASCL1) - was assessed by Western blot analysis. Results: The IC50 value for TDP-A treatment of BON cells was determined to be 7 nM. TDP-A treatment decreased cell proliferation in a dose and time dependent manner. Western blot analysis and flow cytometry experiments indicated that the growth inhibition was due to cell cycle arrest (at G2/M phase) followed by apoptosis.Treatment of BON cells with TDP-A led to an induction of Notch isoforms in a dose-dependent manner. Functional Notch signaling was demonstrated by an increase in the CBF1 binding activity and upregualtion of transcriptional levels of HES and HEY families. More importantly, Notch activation led to a dose dependent reduction of NE markers ASCL1 and CgA. Conclusions: We demonstrated that TDP-A is a potent Notch pathway activator and an inhibitor of BON carcinoid cell proliferation at low nanomolar concentrations. TDP-A suppressed carcinoid cell growth by promoting cell cycle arrest and apoptosis and decreased NET marker expression (ASCL1 and CgA). These results indicate that TDP-A has therapeutic potential for carcinoid cancer. Citation Format: Renata Jaskula-Sztul, Ajitha Dammalapati, Colin Korlesky, Shaoqin Gong, Yi-Qiang Cheng, Herbert Chen. Thailandepsin A, a new HDAC inhibitor, reduces cellular proliferation and activates the Notch pathway in human carcinoids cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2013-1014

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