Molecular Mechanisms Associated with the Inhibitory Role of Long Chain n-3 PUFA in Colorectal Cancer.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the world. Multiple evidence suggests that there is an association between excess fat consumption and the risk of CRC. The long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential for human health, and both in vitro and in vivo studies have shown that these fatty acids can prevent CRC development through various molecular mechanisms. These include the modulation of arachidonic acid (AA) derived prostaglandin synthesis, alteration of growth signaling pathways, arrest of the cell cycle, induction of cell apoptosis, suppression of angiogenesis and modulation of inflammatory response. Human clinical studies found that LC n-3 PUFA combined with chemotherapeutic agents can improve the efficacy of treatment and reduce the dosage of chemotherapy and associated side effects. In this review, we discuss comprehensively the anti-cancer effects of LC n-3 PUFA on CRC, with a main focus on the underlying molecular mechanisms.

Signaling pathways underlying TGF-ß mediated suppression of IL-12A gene expression in monocytes.

Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine essential for multiple biological processes, including the regulation of inflammatory and immune responses. One of the important functions of TGF-ß is the suppression of the proinflammatory cytokine interleukin-12 (IL-12), which is crucial for mounting an anti-tumorigenic response. Although the regulation of the IL-12p40 subunit (encoded by the IL-12B gene) of IL-12 has been extensively investigated, the knowledge of IL-12p35 (encoded by IL-12A gene) subunit regulation is relatively limited. This study investigates the molecular regulation of IL-12A by TGF-ß-activated signaling pathways in THP-1 monocytes. Our study identifies a complex regulation of IL-12A gene expression by TGF-ß, which involves multiple cellular signaling pathways, such as Smad2/3, NF-κB, p38 and JNK1/2. Pharmacological inhibition of NF-κB signaling decreased IL-12A expression, while blocking the Smad2/3 signaling pathway by overexpression of Smad7 and inhibiting JNK1/2 signaling with a pharmacological inhibitor, SP600125, increased its expression. The elucidated signaling pathways that regulate IL-12A gene expression potentially provide new therapeutic targets to increase IL-12 levels in the tumor microenvironment.

Interleukin-11/IL-11 Receptor Promotes Glioblastoma Cell Proliferation, Epithelial-Mesenchymal Transition, and Invasion.

Glioblastoma is highly proliferative and invasive. However, the regulatory cytokine networks that promote glioblastoma cell proliferation and invasion into other areas of the brain are not fully defined. In the present study, we define a critical role for the IL-11/IL-11Rα signalling axis in glioblastoma proliferation, epithelial to mesenchymal transition, and invasion. We identified enhanced IL-11/IL-11Rα expression correlated with reduced overall survival in glioblastoma patients using TCGA datasets. Proteomic analysis of glioblastoma cell lines overexpressing IL-11Rα displayed a proteome that favoured enhanced proliferation and invasion. These cells also displayed greater proliferation and migration, while the knockdown of IL-11Rα reversed these tumourigenic characteristics. In addition, these IL-11Rα overexpressing cells displayed enhanced invasion in transwell invasion assays and in 3D spheroid invasion assays, while knockdown of IL-11Rα resulted in reduced invasion. Furthermore, IL-11Rα-overexpressing cells displayed a more mesenchymal-like phenotype compared to parental cells and expressed greater levels of the mesenchymal marker Vimentin. Overall, our study identified that the IL-11/IL-11Rα pathway promotes glioblastoma cell proliferation, EMT, and invasion.

A New Systemic Disease Mouse Model for Glioblastoma Capable of Single-Tumour-Cell Detection.

BACKGROUND: Glioblastoma is characterised by extensive infiltration into the brain parenchyma, leading to inevitable tumor recurrence and therapeutic failure. Future treatments will need to target the specific biology of tumour recurrence, but our current understanding of the underlying mechanisms is limited. Significantly, there is a lack of available methods and models that are tailored to the examination of tumour recurrence. METHODS: NOD-SCID mice were orthotopically implanted with luciferase-labelled donor U87MG or MU20 glioblastoma cells. Four days later, an unlabelled recipient tumor was implanted on the contralateral side. The mice were euthanised at a humane end-point and tissue and blood samples were collected for ex vivo analyses. RESULTS: The ex vivo analyses of the firefly-labelled MU20 tumours displayed extensive invasion at the primary tumour margins, whereas the firefly-labelled U87MG tumours exhibited expansive phenotypes with no evident invasions at the tumour margins. Luciferase signals were detected in the contralateral unlabelled recipient tumours for both the U87MG and MU20 tumours compared to the non-implanted control brain. Remarkably, tumour cells were uniformly detected in all tissue samples of the supratentorial brain region compared to the control tissue, with single tumour cells detected in some tissue samples. Circulating tumour cells were also detected in the blood samples of most of the xenografted mice. Moreover, tumour cells were detected in the lungs of all of the mice, a probable event related to haematogenous dissemination. Similar results were obtained when the U87MG cells were alternatively labelled with gaussian luciferase. CONCLUSIONS: These findings describe a systemic disease model for glioblastoma which can be used to investigate recurrence biology and therapeutic efficacy towards recurrence.

A New Era of Integration between Multiomics and Spatio-Temporal Analysis for the Translation of EMT towards Clinical Applications in Cancer.

Epithelial-mesenchymal transition (EMT) is crucial to metastasis by increasing cancer cell migration and invasion. At the cellular level, EMT-related morphological and functional changes are well established. At the molecular level, critical signaling pathways able to drive EMT have been described. Yet, the translation of EMT into efficient diagnostic methods and anti-metastatic therapies is still missing. This highlights a gap in our understanding of the precise mechanisms governing EMT. Here, we discuss evidence suggesting that overcoming this limitation requires the integration of multiple omics, a hitherto neglected strategy in the EMT field. More specifically, this work summarizes results that were independently obtained through epigenomics/transcriptomics while comprehensively reviewing the achievements of proteomics in cancer research. Additionally, we prospect gains to be obtained by applying spatio-temporal multiomics in the investigation of EMT-driven metastasis. Along with the development of more sensitive technologies, the integration of currently available omics, and a look at dynamic alterations that regulate EMT at the subcellular level will lead to a deeper understanding of this process. Further, considering the significance of EMT to cancer progression, this integrative strategy may enable the development of new and improved biomarkers and therapeutics capable of increasing the survival and quality of life of cancer patients.

Dynamics of transforming growth factor ß signaling and therapeutic efficacy.

Transforming growth factor ß (TGFß) is a multifunctional cytokine, and its signalling responses are exerted via integrated intracellular pathways and complex regulatory mechanisms. Due to its high potency, TGFß signalling is tightly controlled under normal circumstances, while its dysregulation in cancer favours metastasis. The recognised potential of TGFß as a therapeutic target led to emerging development of anti-TGFß reagents with preclinical success, yet these therapeutics failed to recapitulate their efficacy in experimental settings. In this review, possible reasons for this inconsistency are discussed, addressing the knowledge gap between theoretical and actual behaviours of TGFß signalling. Previous studies on oncogenic cells have demonstrated the spatiotemporal heterogeneity of TGFß signalling intensity. Under feedback mechanisms and exosomal ligand recycling, cancer cells may achieve cyclic TGFß signalling to facilitate dissemination and colonisation. This challenges the current presumption of persistently high TGFß signalling in cancer, pointing to a new direction of research on TGFß-targeted therapeutics.

Label-free macrophage phenotype classification using machine learning methods.

Macrophages are heterogeneous innate immune cells that are functionally shaped by their surrounding microenvironment. Diverse macrophage populations have multifaceted differences related to their morphology, metabolism, expressed markers, and functions, where the identification of the different phenotypes is of an utmost importance in modelling immune response. While expressed markers are the most used signature to classify phenotypes, multiple reports indicate that macrophage morphology and autofluorescence are also valuable clues that can be used in the identification process. In this work, we investigated macrophage autofluorescence as a distinct feature for classifying six different macrophage phenotypes, namely: M0, M1, M2a, M2b, M2c, and M2d. The identification was based on extracted signals from multi-channel/multi-wavelength flow cytometer. To achieve the identification, we constructed a dataset containing 152,438 cell events each having a response vector of 45 optical signals fingerprint. Based on this dataset, we applied different supervised machine learning methods to detect phenotype specific fingerprint from the response vector, where the fully connected neural network architecture provided the highest classification accuracy of 75.8% for the six phenotypes compared simultaneously. Furthermore, by restricting the number of phenotypes in the experiment, the proposed framework produces higher classification accuracies, averaging 92.0%, 91.9%, 84.2%, and 80.4% for a pool of two, three, four, five phenotypes, respectively. These results indicate the potential of the intrinsic autofluorescence for classifying macrophage phenotypes, with the proposed method being quick, simple, and cost-effective way to accelerate the discovery of macrophage phenotypical diversity.

The Interleukin-11/IL-11 Receptor Promotes Glioblastoma Survival and Invasion under Glucose-Starved Conditions through Enhanced Glutaminolysis.

Glioblastoma cells adapt to changes in glucose availability through metabolic plasticity allowing for cell survival and continued progression in low-glucose concentrations. However, the regulatory cytokine networks that govern the ability to survive in glucose-starved conditions are not fully defined. In the present study, we define a critical role for the IL-11/IL-11Rα signalling axis in glioblastoma survival, proliferation and invasion when cells are starved of glucose. We identified enhanced IL-11/IL-11Rα expression correlated with reduced overall survival in glioblastoma patients. Glioblastoma cell lines over-expressing IL-11Rα displayed greater survival, proliferation, migration and invasion in glucose-free conditions compared to their low-IL-11Rα-expressing counterparts, while knockdown of IL-11Rα reversed these pro-tumorigenic characteristics. In addition, these IL-11Rα-over-expressing cells displayed enhanced glutamine oxidation and glutamate production compared to their low-IL-11Rα-expressing counterparts, while knockdown of IL-11Rα or the pharmacological inhibition of several members of the glutaminolysis pathway resulted in reduced survival (enhanced apoptosis) and reduced migration and invasion. Furthermore, IL-11Rα expression in glioblastoma patient samples correlated with enhanced gene expression of the glutaminolysis pathway genes GLUD1, GSS and c-Myc. Overall, our study identified that the IL-11/IL-11Rα pathway promotes glioblastoma cell survival and enhances cell migration and invasion in environments of glucose starvation via glutaminolysis.

The comparative anti-cancer effects of krill oil and oxaliplatin in an orthotopic mouse model of colorectal cancer.

BACKGROUND: Our in vitro studies demonstrated that krill oil (KO) has anti-cancer potential. This study aimed to compare the anti-cancer effects of KO with a commonly used chemotherapeutic drug, oxaliplatin and to identify the molecular mechanisms associated with KO supplementation in a mouse model of colorectal cancer (CRC). METHODS: Thirty-six male Balb/c mice were randomly divided into six groups. Five groups received standard chow diet supplemented with KO (150 g/kg)), corn oil (150 g/kg), KO combined with ½ dose of oxaliplatin (1.5 mg/kg body weight/3 times per week), corn oil combined with ½ dose of oxaliplatin (1.5 mg/kg body weight/3 times per week), or a full dose of oxaliplatin (3 mg/kg body weight/3 times per week). The control (sham) group received a standard chow diet. Treatments started three weeks before and continued for three weeks after orthotopic CRC induction. The number of metastases, tumour weight and volume were quantified ex-vivo. The expression of cytochrome c, cleaved caspase-9 and -3, DNA damage, PD-L1, PD-L2 and HSP-70 were determined. RESULTS: A significant reductions in the weight and volume of tumours were observed in mice treated with KO and KO plus a ½ dose of oxaliplatin compared to the sham group, similar to oxaliplatin-treated mice. KO, and KO plus ½ dose of oxaliplatin significantly increased the expression of cytochrome c, cleaved caspase-9 and -3, and DNA damage and decreased expression of PD-L1, PD-L2 and HSP-70 in tumour tissues compared to the sham group. CONCLUSIONS: The in vivo anti-cancer effects of KO are comparable with oxaliplatin. Thus, dietary KO supplementation has a great potential as a therapeutic/adjunctive agent for CRC treatment.

Extracellular Vesicles Secreted by Glioma Stem Cells Are Involved in Radiation Resistance and Glioma Progression.

Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs' specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.

Krill oil supplementation reduces the growth of CT-26 orthotopic tumours in Balb/c mice.

BACKGROUND: We have previously reported that the free fatty acid extract (FFAE) of krill oil (KO) significantly inhibits the proliferation and migration, and induces apoptosis of colorectal cancer (CRC) cells. This study aimed to investigate the in vivo efficacy of various doses of KO supplementation on the inhibition of CRC tumour growth, molecular markers of proliferation, angiogenesis, apoptosis, the epidermal growth factor receptor (EGFR) and its downstream molecular signalling. METHODS: Male Balb/c mice were randomly divided into four groups with five in each group. The control (untreated) group received standard chow diet; and other three groups received KO supplementation at 5%, 10%, and 15% of their daily dietary intake respectively for three weeks before and after the orthotopic implantation of CT-26 CRC cells in their caecum. The expression of cell proliferation marker Ki-67 and angiogenesis marker CD-31 were assessed by immunohistochemistry. The expression of EGFR, phosphorylated EGFR (pEGFR), protein kinase B (AKT), pAKT, extracellular signal-regulated kinase (ERK1/2), pERK1/2, cleaved caspase-7, cleaved poly (ADP-ribose) polymerase (PARP), and DNA/RNA damage were determined by western blot. RESULTS: KO supplementation reduced the CRC tumour growth in a dose-dependent manner; with 15% of KO being the most effective in reduction of tumour weight and volume (68.5% and 68.3% respectively, P < 0.001), inhibition of cell proliferation by 69.9% (P < 0.001) and microvessel density by 72.7% (P < 0.001). The suppressive effects of KO on EGFR and its downstream signalling, ERK1/2 and AKT, were consistent with our previous in vitro observations. Furthermore, KO exhibited pro-apoptotic effects on tumour cells as indicated by an increase in the expression of cleaved PARP by 3.9-fold and caspase-7 by 8.9-fold. CONCLUSIONS: This study has demonstrated that KO supplementation reduces CRC tumour growth by inhibiting cancer cell proliferation and blood vessel formation and inducing apoptosis of tumour cells. These anti-cancer effects are associated with the downregulation of the EGFR signalling pathway and activation of caspase-7, PARP cleavage, and DNA/RNA damage.

Cortical tension initiates the positive feedback loop between cadherin and F-actin.

Adherens junctions physically link two cells at their contact interface via extracellular binding between cadherin molecules and intracellular interactions between cadherins and the actin cytoskeleton. Cadherin and actomyosin cytoskeletal dynamics are regulated reciprocally by mechanical and chemical signals, which subsequently determine the strength of cell-cell adhesions and the emergent organization and stiffness of the tissues they form. However, an understanding of the integrated system is lacking. We present a new mechanistic computational model of intercellular junction maturation in a cell doublet to investigate the mechanochemical cross talk that regulates adherens junction formation and homeostasis. The model couples a two-dimensional lattice-based simulation of cadherin dynamics with a reaction-diffusion representation of the reorganising actomyosin network through its regulation by Rho signalling at the intracellular junction. We demonstrate that local immobilization of cadherin induces cluster formation in a cis-less-dependent manner. We then recapitulate the process of cell-cell contact formation. Our model suggests that cortical tension applied on the contact rim can explain the ring distribution of cadherin and actin filaments (F-actin) on the cell-cell contact of the cell doublet. Furthermore, we propose and test the hypothesis that cadherin and F-actin interact like a positive feedback loop, which is necessary for formation of the ring structure. Different patterns of cadherin distribution were observed as an emergent property of disturbances of this positive feedback loop. We discuss these findings in light of available experimental observations on underlying mechanisms related to cadherin/F-actin binding and the mechanical environment.

Blocking Muscarinic Receptor 3 Attenuates Tumor Growth and Decreases Immunosuppressive and Cholinergic Markers in an Orthotopic Mouse Model of Colorectal Cancer.

Tumor cells have evolved to express immunosuppressive molecules allowing their evasion from the host's immune system. These molecules include programmed death ligands 1 and 2 (PD-L1 and PD-L2). Cancer cells can also produce acetylcholine (ACh), which plays a role in tumor development. Moreover, tumor innervation can stimulate vascularization leading to tumor growth and metastasis. The effects of atropine and muscarinic receptor 3 (M3R) blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), on cancer growth and spread were evaluated in vitro using murine colon cancer cell line, CT-26, and in vivo in an orthotopic mouse model of colorectal cancer. In the in vitro model, atropine and 4-DAMP significantly inhibited CT-26 cell proliferation in a dose dependent manner and induced apoptosis. Atropine attenuated immunosuppressive markers and M3R via inhibition of EGFR/AKT/ERK signaling pathways. However, 4-DAMP showed no effect on the expression of PD-L1, PD-L2, and choline acetyltransferase (ChAT) on CT-26 cells but attenuated M3R by suppressing the phosphorylation of AKT and ERK. Blocking of M3R in vivo decreased tumor growth and expression of immunosuppressive, cholinergic, and angiogenic markers through inhibition of AKT and ERK, leading to an improved immune response against cancer. The expression of immunosuppressive and cholinergic markers may hold potential in determining prognosis and treatment regimens for colorectal cancer patients. This study's results demonstrate that blocking M3R has pronounced antitumor effects via several mechanisms, including inhibition of immunosuppressive molecules, enhancement of antitumor immune response, and suppression of tumor angiogenesis via suppression of the AKT/ERK signaling pathway. These findings suggest a crosstalk between the cholinergic and immune systems during cancer development. In addition, the cholinergic system influences cancer evasion from the host's immunity.

The effects of the dietary compound L-sulforaphane against respiratory pathogens.

L-sulforaphane (LSF) is an isothiocyanate derived from cruciferous vegetables that has long been known for its anticarcinogenic, antioxidant and anti-inflammatory effects. LSF also possesses antimicrobial properties, although the evidence for this is limited. Respiratory pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and respiratory syncytial virus (RSV), are leading global causes of illness and death among children aged under five years, particularly in resource-poor countries where access to vaccines are limited or, in the case of S. pyogenes and RSV, vaccines have not been licensed for use in humans. Therefore, alternative strategies to prevent and/or treat these common infectious diseases are urgently needed. This study was conducted to investigate the antimicrobial effects of LSF against common respiratory pathogens, S. pneumoniae (serotypes 1 and 6B), H. influenzae type B (HiB), non-typeable H. influenzae (NTHi), S. pyogenes and RSV in relevant human cell-based models. LSF significantly inhibited the growth of H. influenzae, but not S. pneumoniae or S. pyogenes. LSF did not improve opsonophagocytic capacity or killing by human phagocytic cell lines (HL-60s and THP-1 macrophages) for S. pneumoniae yet showed some improved killing for H. influenzae species in THP-1 macrophages. However, LSF significantly reduced RSV infection in human lung epithelial cells, associated with increased expression of cyclin D1 (CCND1) gene as well as the antioxidant genes, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX-1). Overall, LSF represents an exciting avenue for further antimicrobial research, particularly as a novel therapy against H. influenzae species and RSV.

Carfilzomib Promotes the Unfolded Protein Response and Apoptosis in Cetuximab-Resistant Colorectal Cancer.

Cetuximab is a common treatment option for patients with wild-type K-Ras colorectal carcinoma. However, patients often display intrinsic resistance or acquire resistance to cetuximab following treatment. Here we generate two human CRC cells with acquired resistance to cetuximab that are derived from cetuximab-sensitive parental cell lines. These cetuximab-resistant cells display greater in vitro proliferation, colony formation and migration, and in vivo tumour growth compared with their parental counterparts. To evaluate potential alternative therapeutics to cetuximab-acquired-resistant cells, we tested the efficacy of 38 current FDA-approved agents against our cetuximab-acquired-resistant clones. We identified carfilzomib, a selective proteosome inhibitor to be most effective against our cell lines. Carfilzomib displayed potent antiproliferative effects, induced the unfolded protein response as determined by enhanced CHOP expression and ATF6 activity, and enhanced apoptosis as determined by enhanced caspase-3/7 activity. Overall, our results indicate a potentially novel indication for carfilzomib: that of a potential alternative agent to treat cetuximab-resistant colorectal cancer.

Dinuclear orthometallated gold(I)-gold(III) anticancer complexes with potent in vivo activity through an ROS-dependent mechanism.

Increasingly explored over the last decade, gold complexes have shown great promise in the field of cancer therapeutics. A major obstacle to their clinical progression has been their lack of in vivo stability, particularly for gold(III) complexes, which often undergo a facile reduction in the presence of biomolecules such as glutathione. Herein, we report a new class of promising anticancer gold(I)-gold(III) complexes with the general formula [XAuI(µ-2-C6F4PPh2)(κ2-2-C6F4PPh2)AuIIIX] [X = Cl (1), Br (2), NO3 (3)] which feature two gold atoms in different oxidation states (I and III) in a single molecule. Interestingly, gold(I)-gold(III) complexes (1-3) are stable against glutathione reduction under physiological-like conditions. In addition, complexes 1-3 exhibit significant cytotoxicity (276-fold greater than cisplatin) toward the tested cancer cells compared to the noncancerous cells. Moreover, the gold(I)-gold(III) complexes do not interact with DNA-like cisplatin but target cellular thioredoxin reductase, an enzyme linked to the development of cisplatin drug resistance. Complexes 1-3 also showed potential to inhibit cancer and endothelial cell migration, as well as tube formation during angiogenesis. In vivo studies in a murine HeLa xenograft model further showed the gold compounds may inhibit tumor growth on par clinically used cisplatin, supporting the significant potential this new compound class has for further development as cancer therapeutic.

EGFRvIII Promotes Cell Survival during Endoplasmic Reticulum Stress through a Reticulocalbin 1-Dependent Mechanism.

Reticulocalbin 1 (RCN1) is an endoplasmic reticulum (ER)-residing protein, involved in promoting cell survival during pathophysiological conditions that lead to ER stress. However, the key upstream receptor tyrosine kinase that regulates RCN1 expression and its potential role in cell survival in the glioblastoma setting have not been determined. Here, we demonstrate that RCN1 expression significantly correlates with poor glioblastoma patient survival. We also demonstrate that glioblastoma cells with expression of EGFRvIII receptor also have high RCN1 expression. Over-expression of wildtype EGFR also correlated with high RCN1 expression, suggesting that EGFR and EGFRvIII regulate RCN1 expression. Importantly, cells that expressed EGFRvIII and subsequently showed high RCN1 expression displayed greater cell viability under ER stress compared to EGFRvIII negative glioblastoma cells. Consistently, we also demonstrated that RCN1 knockdown reduced cell viability and exogenous introduction of RCN1 enhanced cell viability following induction of ER stress. Mechanistically, we demonstrate that the EGFRvIII-RCN1-driven increase in cell survival is due to the inactivation of the ER stress markers ATF4 and ATF6, maintained expression of the anti-apoptotic protein Bcl-2 and reduced activity of caspase 3/7. Our current findings identify that EGFRvIII regulates RCN1 expression and that this novel association promotes cell survival in glioblastoma cells during ER stress.

Examination of Novel Immunomodulatory Effects of L-Sulforaphane.

The dietary isothiocyanate L-sulforaphane (LSF), derived from cruciferous vegetables, is reported to have several beneficial biological properties, including anti-inflammatory and immunomodulatory effects. However, there is limited data on how LSF modulates these effects in human immune cells. The present study was designed to investigate the immunomodulatory effects of LSF (10 µM and 50 µM) on peripheral blood mononuclear cell (PBMC) populations and cytokine secretion in healthy adult volunteers (n = 14), in the presence or absence of bacterial (lipopolysaccharide) and viral (imiquimod) toll-like receptor (TLRs) stimulations. Here, we found that LSF reduced pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and chemokines monocyte chemoattractant protein (MCP)-1 irrespective of TLR stimulations. This result was associated with LSF significantly reducing the proportion of natural killer (NK) cells and monocytes while increasing the proportions of dendritic cells (DCs), T cells and B cells. We found a novel effect of LSF in relation to reducing cluster of differentiation (CD) 14+ monocytes while simultaneously increasing monocyte-derived DCs (moDCs: lineage-Human Leukocyte Antigen-DR isotype (HLA-DR)+CD11blow-high CD11chigh). LSF was also shown to induce a 3.9-fold increase in the antioxidant response element (ARE) activity in a human monocyte cell line (THP-1). Our results provide important insights into the immunomodulatory effects of LSF, showing in human PBMCs an ability to drive differentiation of monocytes towards an immature monocyte-derived dendritic cell phenotype with potentially important biological functions. These findings provide insights into the potential role of LSF as a novel immunomodulatory drug candidate and supports the need for further preclinical and phase I clinical studies.

Elucidating the role of long intergenic non-coding RNA 339 in human endometrium and endometriosis.

Endometriosis is a complex disease, influenced by genetic factors. Genetic markers associated with endometriosis exist at chromosome 1p36.12 and lead to altered expression of the long intergenic non-coding RNA 339 (LINC00339), however, the role of LINC00339 in endometriosis pathophysiology remains unknown. The aim of this work was to characterize the expression patterns of LINC00339 mRNA in endometrium and endometriotic lesions in situ and to determine the functional role of LINC00339 in human endometrium. We employed RNA-sequencing (RNA-seq), quantitative RT-PCR and in situ hybridization to investigate the abundance of LINC00339 transcripts in endometrium and endometrial cell lines and to describe the pattern and localization of LINC00339 expression in endometrium and endometriotic lesions. LINC00339 mRNA expression was manipulated (overexpressed and silenced) in endometrial stromal cell lines and RNA-seq data from overexpression models were analysed using online bioinformatics platforms (STRING and Ingenuity Pathway Analysis) to determine functional processes. We demonstrated the expression of LINC00339 in endometriotic lesions for the first time; we found LINC00339 expression was restricted to the lesion foci and absent in surrounding non-lesion tissue. Furthermore, manipulation of LINC00339 expression in endometrial stromal cell lines significantly impacted the expression of genes involved in immune defence pathways. These studies identify a novel mechanism for LINC00339 activity in endometrium and endometriosis, paving the way for future work, which is essential for understanding the pathogenesis of endometriosis.

Krill oil extract inhibits the migration of human colorectal cancer cells and down-regulates EGFR signalling and PD-L1 expression.

BACKGROUND: The currently available treatments for colorectal cancer (CRC) are often associated with serious side-effects. Therefore, the development of a novel nutraceutical agent may provide an alternative complementary therapy for CRC. Overexpression of the epidermal growth factor receptor (EGFR) associates with a range of cancers while downregulation of EGFR signalling can inhibit cancer growth. Our previous studies have shown that the free fatty acid extract (FFAE) of krill oil exhibits anti-proliferative and pro-apoptotic properties. This study determines the effects of krill oil extract on the migration of human CRC cells, and its potential role in modulating EGFR signalling pathway and the expression of programmed death ligand 1 (PD-L1). METHODS: Human CRC cells, DLD-1 and HT-29 were treated with FFAE of KO at 0.03 and 0.12 µL/100 µL for 8 or 24 h. Cell migration was determined by Boyden chamber migration assay. The expression of EGFR, phosphorylated EGFR (pEGFR), protein kinase B (AKT), phosphorylated AKT (pAKT), extracellular signal regulated kinase (ERK1/2), phosphorylated ERK1/2 (pERK1/2) as well as PD-L1 were assessed by western blotting and immunohistochemistry. RESULTS: The FFAE of krill oil significantly inhibited cell migration compared to ethanol-treated (vehicle control) cells (P < 0.01 to P < 0.001). At the molecular level, krill oil extract reduced the expression of EGFR, pEGFR (P < 0.001 for both) and their downstream signalling, pERK1/2 and pAKT (P < 0.01 to P < 0.001) without altering total ERK 1/2 and AKT levels. In addition, the expression of PD-L1 was reduced by 67 to 72% (P < 0.001) following the treatment with krill oil extract. CONCLUSION: This study has demonstrated that krill oil may be a potential therapeutic/adjunctive agent for CRC attributed to its anti-migratory effects.. The potential anti-cancer properties of krill oil are likely to be associated with the downregulation of EGFR, pEGFR and their downstream pERK/ERK1/2 and pAKT/AKT signalling pathways along with the downregulation of PD-L1.