Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression

AbstractBackgroundGlycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation.MethodsThe pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0–20 μM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1.ResultsAR-A014418 treatment had a significant dose-dependent growth reduction (P< 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1.ConclusionsThis study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics

Xanthohumol increases death receptor 5 expression and enhances apoptosis with the TNF-related apoptosis-inducing ligand in neuroblastoma cell lines.

High-risk neuroblastoma (NB) is lethal childhood cancer. Published data including ours have reported the anti-proliferative effect of Xanthohumol (XN), a prenylated chalcone, in various cancer types suggesting that XN could be a useful small molecule compound against cancer. The TNF-Related Apoptosis-Inducing Ligand (TRAIL) is an endogenous ligand that is expressed in various immune cells. TRAIL mediates apoptosis through binding of transmembrane receptors, death receptor 4 (DR4) and/or death receptor 5 (DR5). Cancer cells are frequently resistant to TRAIL-mediated apoptosis, and the cause of this may be decreased expression of death receptors. This study aimed to identify combination therapies that exploit XN for NB. First, the effect of XN on cellular proliferation in human NB cell lines NGP, SH-SY-5Y, and SK-N-AS were determined via MTT assay, colony forming assay, and real-time live cell imaging confluency. XN treatment causes a statistically significant decrease in the viability of NB cells with IC50 values of approximately 12 μM for all three cell lines. Inhibition of cell proliferation via apoptosis was evidenced by an increase in pro-apoptotic markers (cleaved PARP, cleaved caspase-3/-7, and Bax) and a decrease in an anti-apoptotic marker, Bcl-2. Importantly, XN treatment inhibited PI3K/Akt pathway and associated with increased expression of DR5 by both mRNA and protein levels. Furthermore, a statistically significant synergistic reduction was observed following combination treatment (50%) compared to either TRAIL (5%) or XN (15%) alone in SK-N-AS cells. Therefore, this study shows XN treatment reduces NB cell growth via apoptosis in a dose-dependent manner, and enhanced growth reduction was observed in combination with TRAIL. This is the first study to demonstrate that XN alters the expression of DR5 as well as the synergistic effect of XN on TRAIL in NB and provides a strong rationale for further preclinical analysis

Notch signaling in hepatocellular carcinoma: molecular targeting in an advanced disease

The use of alternative therapeutic approaches in advanced carcinogenesis is a growing investigative base. One such cancer, primary liver cancer, is one of the most commonly occurring cancers worldwide and often presents in late stage disease consequently preventing traditional curative modalities. As a result, hepatocellular carcinoma (HCC), representing the majority of primary liver cancer, is the third most common cause of cancer-related deaths globally. Survival rates are linked to stage of presentation as well as concomitant cirrhosis limiting the 5-year survival in these patients to &lt; 20%. Alternative strategies are in dire need as patients in this cohort have limited palliative options. Currently, sorafenib is the only approved systemic therapy; however, it has a limited survival advantage and low efficacy prompting the empirical need for further evaluation. Understanding of cancer therapy has led to an enhanced focus on the Notch pathway as a potential target for advanced HCC. Notch signaling is a critical component of development and cell fate and has been linked to various modalities including liver regeneration and as a key driver in carcinogenesis. In this review, we will provide a review of the current status of the Notch signaling in liver cancer and of Notch as an alternative potential strategy for advanced HCC

Antiproliferative and apoptotic effect of LY2090314, a GSK-3 inhibitor, in neuroblastoma in vitro

Abstract Background Neuroblastoma (NB) is a devastating disease. Despite recent advances in the treatment of NB, about 60% of high-risk NB will have relapse and therefore long-term event free survival is very minimal. We have reported that targeting glycogen synthase kinase-3 (GSK-3) may be a potential strategy to treat NB. Consequently, investigating LY2090314, a clinically relevant GSK-3 inhibitor, on NB cellular proliferation and may be beneficial for NB treatment. Methods The effect of LY2090314 was compared with a previously studied GSK-3 inhibitor, Tideglusib. Colorimetric, clonogenic, and live-cell image confluency assays were used to study the proliferative effect of LY2090314 on NB cell lines (NGP, SK-N-AS, and SH-SY-5Y). Western blotting and caspase glo assay were performed to determine the mechanistic function of LY2090314 in NB cell lines. Results LY2090314 treatment exhibited significant growth reduction starting at a 20 nM concentration in NGP, SK-N-AS, and SH-SY-5Y cells. Western blot analysis indicated that growth suppression was due to apoptosis as evidenced by an increase in pro-apoptotic markers cleaved PARP and cleaved caspase-3 and a reduction in the anti-apoptotic protein, survivin. Further, treatment significantly reduced the level of cyclin D1, a key regulatory protein of the cell cycle and apoptosis. Functionally, this was confirmed by an increase in caspase activity. LY2090314 treatment reduced the expression levels of phosphorylated GSK-3 proteins and increased the stability of β-catenin in these cells. Conclusions LY2090314 effectively reduces growth of both human MYCN amplified and non-amplified NB cell lines in vitro. To our knowledge, this is the first study to look at the effect of LY2090314 in NB cell lines. These results indicate that GSK-3 may be a therapeutic target for NB and provide rationale for further preclinical analysis using LY2090314

Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is “handcuffing,” in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded π protein with the seven 22-bp iterons of the γ origin of replication. Like other related Rep proteins, π exists as both monomers and dimers. However, the ability of π dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of π protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using π variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (π·P106L(∧)F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of π protein (π·M36A(∧)M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type π. The data suggest that π dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the γ ori system. Thus, dimers of π appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of π expression, in cis competition (for origin binding) with monomeric activator π, and handcuffing-mediated inhibition of replication in trans

XN inhibits Notch1 signaling pathway.

<p>A. Protein levels of intracellular domain (NICD), HES-1, an immediate downstream target of Notch1 signaling, cyclinD1, and survivin were analyzed by western blot after XN-treatment. Equal loading was confirmed by GAPDH. B. Notch1 inhibition by shRNA (seq #1 and seq #2) induced apoptosis similar to XN treatment. Functional Caspase assay confirmed the induction of apoptosis in Notch1 knock-down cell lines. A non-specific, no target, scrambled sequence (NS) as control was used. GAPDH was used as gel loading control. C. Effect of active Notch1 overexpression in XN treatment. Huh-7 and Hep3B cells were transfected with Notch1plasmid (pcDNA-Myc-ICN1) and treated with or without XN. Percent of growth was measured by MTT assay (n = 3; p = 0.05 compared to vector with XN10). D. Western analysis of (pcDNA-Myc-ICN1) or empty vector transfected Huh-7 and Hep3B cells treated with or without XN for the expression of the exogenous Notch1 protein. Myc-tag antibody was used to determine the level of exogenous Notch expression.</p