Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat’s digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively. HEMEC and HIMEC were stimulated with TNF-α/IL-1β with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial-leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways. BRE inhibited TNF-α/IL-1β-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations. BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1β activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer.

Aim To elucidate the signaling mechanisms involved in the protective effect of EUK-207 against irradiation-induced cellular damage and apoptosis in human intestinal microvasculature endothelial cells (HIMEC). Methods HIMECs were irradiated and treated with EUK-207. Using hydroethidine and DCF-DA fluorescent probe the intracellular superoxide and reactive oxygen species (ROS) were determined. By real-time PCR and western blotting caspase-3, Bcl2 and Bax genes and proteins were analyzed. Proliferation was determined by [3H]-thymidine uptake. Immunofluorescence staining was used for translocation of p65 NFκB subunit. Key finding Irradiation increased ROS production, apoptosis, Bax, Caspase3 and NFkB activity in HIMEC and inhibited cell survival/growth/proliferation. EUK-207 restored the endothelial functions, markedly inhibited the ROS, up-regulated the Bcl2 and down-regulated Bax and prevented NFκB caspase 3 activity in HIMEC. Significance HIMEC provide a novel model to define the effect of irradiation induced endothelial dysfunction. Our findings suggest that EUK-207 effectively inhibits the damaging effect of irradiation.

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BACKGROUND: Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries have been shown to prevent the carcinogenesis and tumorigenesis in esophagus and digestive tract. Angiogenesis has been implicated in the pathogenesis of inflammatory bowel disease (IBD). In this study, we aimed to determine the effects of black raspberry extract (BRE) on human primary microvascular endothelial cells isolated from intestine (HIMEC) and esophagus (HEMEC). METHODS: HIMEC and HEMEC monolayer were activated with either with VEGF or TNF‐α/IL‐1β with or without BRE. Effects of BRE on cells survival, apoptosis, proliferation, migration and tube formation were determined. COX‐2, ICAM‐1 and VCAM‐1 genes and proteins expression were assessed by qPCR and immunobloting and nuclear localization of p65 was observed by immunofluorescence staining. RESULTS: VEGF increased cell migration, proliferation and in vitro tube formation and BRE treatment of cells suppressed these effects in both HIMEC and HEMEC. TNF‐α/IL‐1β treatment translocated the p65 subunit of NFκB to nucleus, increased COX‐2, ICAM‐1 and VCAM‐1 gene and protein expression and the PGE2 activity in HIMEC and HEMEC. However, BRE treatment only was effective in inhibiting the COX2, NFκB and ICAM‐1 and VCAM‐1 in HEMEC, but exerted no inhibitory effect on HIMEC. CONCLUSIONS: Taken together, BRE is a potent anti‐inflammatory agent for HEMEC but not HIMEC. Grant Funding Source: MCW Digestive Disease

Background: Selinexor is a small-molecule therapeutic that inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) and subsequently cancer cell death while sparing normal cells. It has been previously demonstrated that the inhibitor of NFκB, IκB-, is localized to the cytoplasm by XPO1 in several cancer cell lines and that treatment of cancer cells with selinexor reduces NF-κB transcriptional activity. The mechanism of NF-κB inhibition by selinexor, however, is not fully understood. We hypothesized that nuclear retention of IκB- and down-regulation of IκB- kinase (IKK) in response to selinexor treatment would inhibit NF-κB transcriptional activity. Methods: U2OS (Osteosarcoma) cells were treated with selinexor in the presence or absence of TNF stimulation and whole protein lysates were analyzed by Western blotting. IκB- localization was evaluated by immunofluorescence microscopy and NF-κB transcriptional activity by ELISA (Thermo Scientific). Results: TNFα induced the phosphorylation of NF-κB p65 subunit on serine 536 and IκB- on serine 32/36 through IKK. This resulted in the dissociation of IκB-α from NF-κB and led to IκB-α degradation via the 26S proteasome. Free NF-κB could now migrate into the nucleus and initiate transcriptional activation supporting tumorigenicity and inflammation. The IKK kinase is a complex made of two kinases (IKKα and IKKβ) and one regulatory subunit, NEMO/IKKγ. We found that selinexor treatment blocked IKK activity through the down regulation of the IKK (IKKγ) gamma subunit protein levels. This inhibition was dose dependent and prevented IκB- phosphorylation, thereby protecting IκB- from degradation. The protection of intact IκB- from degradation and its forced nuclear accumulation through XPO1 inhibition enabled inhibition of NF-κB transcriptional activity even in the presence of TNFα. Selinexor did not alter the protein levels of IKK or IKK. Conclusions: Selinexor blocks TNFα induced degradation of IκB- by reducing the levels of IKK. In addition, selinexor increased nuclear IκB- levels through the inhibition of nuclear export. This blocked NF-κB activity and enhanced cancer cell death. We are currently investigating the beneficial effects of combining selinexor with proteasome inhibitors, which are known to prevent IκB- degradation. Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, TJ Unger, Benjamin Link, Maxwell Werman, Margaret Lee, Sharon Shacham, Yosef Landesman. Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2899.