Fn14?Trail Effectively Inhibits Hepatocellular Carcinoma Growth

Background

New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL''s capacity to inhibit HCC growth was tested.

Results

Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL''s molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice.

Conclusions

In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein’s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies.     

Dietary Transforming Growth Factor-Beta 2 (TGF-��2) Supplementation Reduces Methotrexate-Induced Intestinal Mucosal Injury in a Rat

Background/Aims

Dietary supplementation with transforming growth factor-beta (TGF-β) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-β2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model.

Methods

Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-β2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-β rats were treated with diet enriched with TGF-β2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-β rats were treated with diet enriched with TGF-β2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, β-catenin, IL-1B and bax protein expression.

Results

Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-β2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-β2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-β2 also led to increased pERK, IL-1B and β-catenin protein levels in intestinal mucosa.

Conclusions

Treatment with TGF-β2 prevents mucosal-injury, enhances p-ERK and β-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.     

The cell adhesion nectin‐like molecules (Necl) 1 and 4 suppress the growth and tumorigenic ability of colon cancer cells

A key step in human colon cancer development includes the hyperactivation of Wnt/β‐catenin signaling and the induction of β‐catenin‐TCF target genes that participate in colon cancer progression. Recent studies identified members of the immunoglobulin‐like cell adhesion molecules (IgCAM) of the L1CAM family (L1 and Nr‐CAM) as targets of β‐catenin‐TCF signaling in colon cancer cells. L1 was detected at the invasive front of colon cancer tissue and confers metastasis when overexpressed in cells. In contrast to L1, we did not detect in colon cancer cells significant levels of another IgCAM family of molecules, the nectin‐like (Necl) receptors Necl1 and Necl4, while Necl4 was previously found in the normal small intestine and colon tissues. We studied the properties of colon cancer cells in which Necl4 and Necl1 were expressed either alone, or in combination, and found that such cells display a wide range of properties associated with tumor suppression. Expression of both Necl1 and Necl4 was the most efficient in suppressing the tumorigenicity of colon cancer cells. This was associated with enhanced rates of apoptosis and change in several apoptosis‐related markers. In contrast to its capacity to suppress tumorigenesis, Necl4 was unable to affect the highly malignant and metastatic capacities of colon cancer cells in which L1 was overexpressed. Our results suggest that various IgCAM receptor families play different roles in affecting the tumorigenic function of the same cells, and that Necl1 and Necl4 can fulfill a tumor suppressive role. J. Cell. Biochem. 108: 326–336, 2009. © 2009 Wiley‐Liss, Inc.     

A review of bird migration over Israel

The sheer number of migrating birds that pass over Israel give the country a unique situation in the study of the phenomenon, which was, not surprisingly, noticed even in biblical times. This paper is a review of current knowledge of bird migration in Israel. It includes a brief summary of migration in the Mediterranean basin and the Middle East and new information from an extensive migration survey done in the northern Arava Valley and Negev Highlands.     

Monoubiquitylation of alpha-synuclein by seven in absentia homolog (SIAH) promotes its aggregation in dopaminergic cells

alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for alpha-synuclein aggregation and Lewy body formation.     

Temporal robustness of linear relationships between production and transpiration

Seasonal dependence of biomass production on transpiration has been previously reported for a number of crops under salinity and drought. Linear yield (Y) to transpiration (T) relationships have been utilized in plant-growth and water-uptake models to estimate yield based on predicted transpiration values. The relationship is often employed for time steps that are very small compared with the whole season measurements, even though no empirical validation exists for such application. This work tests the hypothesis that linear Y-T relationships are valid throughout the life span of crops under varied natural conditions and levels of environmental stress. Effects of salinity and water supply on growth, water use and yields of tomatoes (Lycopersicon esculentum Mill.) were studied for two distinct conditions of potential transpiration. Linear relationships between relative Y and relative ET were found to be consistent throughout the life span of the crops for both growing seasons. Water-use efficiency increased together with plant growth as a result of changes in the plant's surface area to volume ratio. This empirical validation of linear Y-T relationships for short time periods is beneficial in confirming their usefulness in growth and water uptake models.     

Water use and yield of tomatoes under limited water and excess boron

The role of tripartite associations among Frankia, Alpova diplophloeus (an ectomycorrhizal fungus) and Alnus tenuifolia in growth, nitrogen fixation, ectomycorrhizal formation, and mineral acquisition of A. tenuifolia was investigated. Seedlings of A. tenuifolia were planted in pots containing a mixture of ground basalt–perlite, or perlite alone, which served as the control. The seedlings were inoculated with Frankia isolated from root nodules of alder, followed by spores of A. diplophloeus and grown for 5 months in a greenhouse. The seedlings grown in the pots with a mixture of ground basalt–perlite after dual inoculation with Frankia and A. diplophloeus had the heaviest shoots and root nodules in dry weight, and showed the greatest nitrogen-fixing ability measured by acetylene reduction. Ectomycorrhizae formed with A. diplophloeus increased when this fungus was inoculated together with Frankia. The mineral composition (P, K, Ca, Fe, Mg, Mn, Na, Si and Al) in the seedlings was also determined. The results of these experiments showed that the tripartite associations could improve the growth, nitrogen fixation and mineral acquisition (rock solubilization) of A. tenuifolia.     

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death,is necessary for developmental cell death in Drosophila midgut

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported¹ Dasari SK, Bialik S, Levin-Zaidman S, Levin-Salomon V, Merrill AH, Jr., Futerman AH, Kimchi A. Signalome-wide rnai screen identifies gba1 as a positive mediator of autophagic cell death. Cell Death Differ. 2017;24(7):1288-1302. https://doi.org/10.1038/cdd.2017.80. PMID:28574511[Crossref], [PubMed], [Web of Science ®] [Google Scholar] on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.