Dynamic Subunit Exchange and the Regulation of Microtubule Assembly by the Stress Response Protein Human αB Crystallin

Background

The small heat shock protein (sHSP), human αB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in αB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of αB crystallin complexes.

Methodology/Principal Findings

There is sequence homology between tubulin and the interactive domains in the β8-strand of the core domain and the C-terminal extension of αB crystallin. This study investigated the hypothesis that the formation of tubulin and αB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of αB crystallin. Low molar ratios of αB crystallin∶tubulin were favorable for microtubule assembly and high molar ratios of αB crystallin∶tubulin were unfavorable for microtubule assembly. Interactions between αB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE.

Conclusions/Significance

Subunits of αB crystallin that exchange dynamically with the αB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.     

VHL Type 2B Mutations Retain VBC Complex Form and Function

Background

von Hippel-Lindau disease is characterized by a spectrum of hypervascular tumors, including renal cell carcinoma, hemangioblastoma, and pheochromocytoma, which occur with VHL genotype-specific differences in penetrance. VHL loss causes a failure to regulate the hypoxia inducible factors (HIF-1α and HIF-2α), resulting in accumulation of both factors to high levels. Although HIF dysregulation is critical to VHL disease-associated renal tumorigenesis, increasing evidence points toward gradations of HIF dysregulation contributing to the degree of predisposition to renal cell carcinoma and other manifestations of the disease.

Methodology/Principal Findings

This investigation examined the ability of disease-specific VHL missense mutations to support the assembly of the VBC complex and to promote the ubiquitylation of HIF. Our interaction analysis supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF. We additionally demonstrated that Type 2B mutant pVHL forms a remnant VBC complex containing the active members ROC1 and Cullin-2 which retains the ability to ubiquitylate HIF-1α.

Conclusions

Our results suggest that subtypes of VHL mutations support an intermediate level of HIF regulation via a remnant VBC complex. These findings provide a mechanism for the graded HIF dysregulation and genetic predisposition for cancer development in VHL disease.     

The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

Background

IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium.

Methodology/Principal Findings

FcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains.

Conclusions/Significance

Our data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested.     

Transcriptional analysis of VEGF-D and TGFβ genes in MCF7 cells exposed to saponin isolated from Holothuria leucospilota (sea cucumber)

Background:

Marine natural products contain a wide range of bioactive compounds with therapeutic properties that have revealed crucial properties in the treatment of some diseases. Some of these compounds have recently received considerable attention for drug discovery. In this study we examined the anti-angiogenic effect of saponin isolated from Holothuria leucospilota (sea cucumber) through evaluation of vascular endothelial growth factor D (VEGF-D) and transforming growth factor-β (TGFβ) expression in a breast cancer cell line.

Methods:

To investigate the effect of SCS on VEGF-D and TGF-β expression in breast cancer cells, the cells were treated with various concentrations of sample. After 48 h the viability of the cells was evaluated by trypan blue staining, and VEGF-D and TGFβ mRNA expression was were evaluated by real time-PCR.

Results:

Our results revealed that SCS can suppress cell viability and VEGF-D and TGFβ mRNA expression in breast cancer cells. Sea cucumber saponin at a concentration of 12 μg/ml inhibited VEGF-D and TGFβ expression more than 90% compared with controls.

Conclusion:

Findings suggest that SCS could inhibit tumor growth via inhibition of angiogenesis.Key Words: Sea cucumber, Saponin, Angiogenesis, Anticancer     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Correlation of Immunoglobulin G Expression and Histological Subtype and Stage in Breast Cancer

Introduction

Recently, growing evidence indicates that immunoglobulins (Igs) are not only produced by mature B lymphocytes or plasma cells, but also by various normal cells types at immune privileged sites and neoplasm, including breast cancer. However, the association of breast cancer derived IgG with genesis and development of the disease has not yet been established.

Methods

In this study we examined the expression of IgG in 186 breast cancers, 20 benign breast lesions and 30 normal breast tissues. Both immunohistochemistry with antibodies to Igκ (immunoglobulin G κ light chain) and Igγ (immunoglobulin G heavy chain) and in situ hybridization with an antisense probe to IgG1 heavy chain constant region gene were performed. Various clinicopathological features were also analyzed.

Results

We found that IgG is specifically expressed in human breast cancer cells. Both infiltrating ductal carcinoma and infiltrating lobular carcinoma had significantly greater numbers of Igκ and Igγ positive cancer cells as compared with medullary carcinoma, carcinoma in situ, and benign lesions (all p<0.05). In addition, IgG expression was correlated with breast cancer histological subtypes (p<0.01) and AJCC stages (p<0.05), with more abundance of IgG expression in more malignant histological subtypes or in more advanced stage of the disease.

Conclusions

IgG expression in breast cancer cells is correlated with malignancy and AJCC stages of the cancers. This suggests that breast cancer derived IgG may be associated with genesis, development and prognosis of the cancer.     

Overexpression of CD151 Predicts Prognosis in Patients with Resected Gastric Cancer

Purpose

The tetraspanin CD151 acts as a promoter of metastasis and invasion in several tumors. However, the role of CD151 in human gastric cancer (HGC) remains unclear.

Methods

Twenty HGC specimens and matched nontumor samples, human gastric epithelial cells (HGEC), and four gastric cancer cell lines were used to analyze CD151 expression. Short hairpin RNA-mediated downregulation of CD151 expression in HGC cells was performed to examine the role of CD151 in the proliferation and metastasis/invasion of HGC cells in vivo and in vitro. The relationship of CD151 with integrin α3 in HGC cells was investigated by silencing integrin α3 followed by co-immunoprecipitation and immunofluorescence staining. Finally, the prognostic value of CD151 and integrin α3 was evaluated by immunohistochemistry in tissue microarrays of 76 HGC patients.

Results

CD151 was expressed at higher levels in HGC tissues and HGC cells than in nontumor tissues and HGEC cells. Down-regulation of CD151 by vshRNA-CD151 impaired metastasis and invasion of HGC-27 cells, but did not affect cell proliferation. CD151 formed a complex with integrin α3 in HGC cells. CD151-cDNA transfection rescued the metastatic potential and invasiveness of HGC-27-vshCD151 cells, but not those of HGC-27-vshintegrin α3 cells in vitro. Clinically, CD151 overexpression was significantly correlated with high TNM stage, depth of invasion and positive lymph node involvement (p<0.05), and high levels of integrin α3 were associated with large tumor size, high TNM stage, depth of invasion and lymph node involvement (p<0.05). Importantly, the postoperative 5-year overall survival of patients with CD151^low and/or integrin α3^low was higher than that of patients with CD151^high and/or integrin α3^high.

Conclusion

CD151 is positively associated with the invasiveness of HGC, and CD151 or the combination of CD151 and integrin α3 is a novel marker for predicting the prognosis of HGC patients and may be potential therapeutic targets.     

Membrane Bound Monomer of Staphylococcal α-Hemolysin Induces Caspase Activation and Apoptotic Cell Death despite Initiation of Membrane Repair Pathway

Background

Wild type Staphylococcal α-hemolysin (α-HL) assembly on target mammalian cells usually results in necrotic form of cell death; however, caspase activation also occurs. The pathways of caspase activation due to binding/partial assembly by α-HL are unknown till date.

Results

Cells treated with H35N (a mutant of α-HL that remains as membrane bound monomer), have been shown to accumulate hypodiploid nuclei, activate caspases and induce intrinsic mitochondrial apoptotic pathway. We have earlier shown that the binding and assembly of α-HL requires functional form of Caveolin-1 which is an integral part of caveolae. In this report, we show that the caveolae of mammalian cells, which undergo a continuous cycle of ‘kiss and run’ dynamics with the plasma membrane, have become immobile upon the binding of the monomer. The cells treated with H35N were unable to recover despite activation of membrane repair mechanism involving caspase-1 dependent activation of sterol regulatory element binding protein-1.

Conclusions

This is for the first time we show the range of cellular changes and responses that take place immediately after the binding of the monomeric form of staphylococcal α-hemolysin.     

Cell Surface Sialylation and Fucosylation Are Regulated by L1 via Phospholipase Cγ and Cooperate to Modulate Neurite Outgrowth,Cell Survival and Migration

Background

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown.

Methodology/Principal Findings

Using a panel of carbohydrate surface markers, we have shown that cell surface sialylation and fucosylation were downregulated in L1^−/y neurons versus L1^+/y neurons. Consistently, mRNA levels of sialyltransferase ST6Gal1, and fucosyltransferase FUT9 were significantly reduced in L1^−/y neurons. Moreover, treatment of L1^+/y neurons with L1 antibodies, triggering signal transduction downstream of L1, led to an increase in cell surface sialylation and fucosylation compared to rat IgG-treated cells. ShRNAs for both ST6Gal1 and FUT9 blocked L1 antibody-mediated enhancement of neurite outgrowth, cell survival and migration. A phospholipase Cγ (PLCγ) inhibitor and shRNA, as well as an Erk inhibitor, reduced ST6Gal1 and FUT9 mRNA levels and inhibited effects of L1 on neurite outgrowth and cell survival.

Conclusions

Neuronal surface sialylation and fucosylation are regulated via PLCγ by L1, modulating neurite outgrowth, cell survival and migration.     

Inhibition of β2-Microglobulin/Hemochromatosis Enhances Radiation Sensitivity by Induction of Iron Overload in Prostate Cancer Cells

Background

Bone metastasis is the most lethal form of several cancers. The β2-microglobulin (β2-M)/hemochromatosis (HFE) complex plays an important role in cancer development and bone metastasis. We demonstrated previously that overexpression of β2-M in prostate, breast, lung and renal cancer leads to increased bone metastasis in mouse models. Therefore, we hypothesized that β2-M is a rational target to treat prostate cancer bone metastasis.

Results

In this study, we demonstrate the role of β2-M and its binding partner, HFE, in modulating radiation sensitivity and chemo-sensitivity of prostate cancer. By genetic deletion of β2-M or HFE or using an anti-β2-M antibody (Ab), we demonstrate that prostate cancer cells are sensitive to radiation in vitro and in vivo. Inhibition of β2-M or HFE sensitized prostate cancer cells to radiation by increasing iron and reactive oxygen species and decreasing DNA repair and stress response proteins. Using xenograft mouse model, we demonstrate that anti-β2-M Ab sensitizes prostate cancer cells to radiation treatment. Additionally, anti-β2-M Ab was able to prevent tumor growth in an immunocompetent spontaneous prostate cancer mouse model. Since bone metastasis is lethal, we used a bone xenograft model to test the ability of anti-β2-M Ab and radiation to block tumor growth in the bone. Combination treatment significantly prevented tumor growth in the bone xenograft model by inhibiting β2-M and inducing iron overload. In addition to radiation sensitive effects, inhibition of β2-M sensitized prostate cancer cells to chemotherapeutic agents.

Conclusion

Since prostate cancer bone metastatic patients have high β2-M in the tumor tissue and in the secreted form, targeting β2-M with anti-β2-M Ab is a promising therapeutic agent. Additionally, inhibition of β2-M sensitizes cancer cells to clinically used therapies such as radiation by inducing iron overload and decreasing DNA repair enzymes.     

Antioxidant and Anti-Inflammatory Activities of Extracts from Cassia alata,Eleusine indica,Eremomastax speciosa,Carica papaya and Polyscias fulva Medicinal Plants Collected in Cameroon

Background

The vast majority of the population around the world has always used medicinal plants as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice.

Objective

The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of ethanol extracts of leaves of Cassia alata, Eleusine indica, Carica papaya, Eremomastax speciosa and the stem bark of Polyscias fulva, collected in Cameroon.

Methods

Chemiluminescence was used to analyze the antioxidant activities of plant extracts against hydrogen peroxide or superoxide anion. Comet assays were used to analyze the protection against antioxidant-induced DNA damage induced in white blood cells after treating with hydrogen peroxide. Flow cytometry was used to measure γδ T cells proliferation and anti-inflammatory activity of γδ T cells and of immature dendritic cells (imDC) in the presence of different concentrations of plant extracts.

Results

Ethanol extracts showed strong antioxidant properties against both hydrogen peroxide and superoxide anion. Cassia alata showed the highest antioxidant activity. The effect of plant extracts on γδ T cells and imDC was evidenced by the dose dependent reduction in TNF-α production in the presence of Cassia alata, Carica papaya, Eremomastax speciosa Eleusine indica, and Polyscias fulva. γδ T cells proliferation was affected to the greatest extent by Polyscias fulva.

Conclusion

These results clearly show the antioxidant capacity and anti-inflammatory activities of plant extracts collected in Cameroon. These properties of leaves and stem bark extracts may contribute to the value for these plants in traditional medicine and in general medical practice.     

The Role of Adhesion Molecules as Biomarkers for the Aggressive Prostate Cancer Phenotype

Background

Currently available methods for diagnosis and staging of prostate cancer lack the sensitivity to distinguish between patients with indolent prostate cancer and those requiring radical treatment. Alterations in key adherens (AJ) and tight junction (TJ) components have been hailed as potential biomarkers for prostate cancer progression but the majority of research has been carried out on individual molecules.

Objective

To elucidate a panel of biomarkers that may help distinguish dormant prostate cancer from aggressive metastatic disease.

Methods

We analysed the expression of 7 well known AJ and TJ components in cell lines derived from normal prostate epithelial tissue (PNT2), non-invasive (CAHPV-10) and invasive prostate cancer (LNCaP, DU145, PC-3) using gene expression, western blotting and immunofluorescence techniques.

Results

Claudin 7, α –catenin and β-catenin protein expression were not significantly different between CAHPV-10 cells and PNT2 cells. However, in PC-3 cells, protein levels for claudin 7, α –catenin were significantly down regulated (−1.5 fold, p = <.001) or undetectable respectively. Immunofluoresence showed β-catenin localisation in PC-3 cells to be cytoplasmic as opposed to membraneous.

Conclusion

These results suggest aberrant Claudin 7, α – and β-catenin expression and/or localisation patterns may be putative markers for distinguishing localised prostate cancer from aggressive metastatic disease when used collectively.     

β-TrCP Inhibition Reduces Prostate Cancer Cell Growth via Upregulation of the Aryl Hydrocarbon Receptor

Background

Prostate cancer is a common and heterogeneous disease, where androgen receptor (AR) signaling plays a pivotal role in development and progression. The initial treatment for advanced prostate cancer is suppression of androgen signaling. Later on, essentially all patients develop an androgen independent stage which does not respond to anti hormonal treatment. Thus, alternative strategies targeting novel molecular mechanisms are required. β-TrCP is an E3 ligase that targets various substrates essential for many aspects of tumorigenesis.

Methodology/Principal Findings

Here we show that β-TrCP depletion suppresses prostate cancer and identify a relevant growth control mechanism. shRNA targeted against β-TrCP reduced prostate cancer cell growth and cooperated with androgen ablation in vitro and in vivo. We found that β-TrCP inhibition leads to upregulation of the aryl hydrocarbon receptor (AhR) mediating the therapeutic effect. This phenomenon could be ligand independent, as the AhR ligand 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) did not alter prostate cancer cell growth. We detected high AhR expression and activation in basal cells and atrophic epithelial cells of human cancer bearing prostates. AhR expression and activation is also significantly higher in tumor cells compared to benign glandular epithelium.

Conclusions/Significance

Together these observations suggest that AhR activation may be a cancer counteracting mechanism in the prostate. We maintain that combining β-TrCP inhibition with androgen ablation could benefit advanced prostate cancer patients.     

Biomarkers of TGF-β Signaling Pathway and Prognosis of Pancreatic Cancer

Background

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

Method

We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

Results

The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

Conclusion

Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.     

The Vitamin D Receptor Agonist BXL-01-0029 as a Potential New Pharmacological Tool for the Treatment of Inflammatory Myopathies

Objective

This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects.

Methods

Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC₅₀ determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis.

Results

BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls.

Conclusions

Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.