The role of RNA-modifying proteins in renal cell carcinoma.

Gene expression is one of the most critical cellular processes. It is controlled by complex mechanisms at the genomic, epigenomic, transcriptomic, and proteomic levels. Any aberration in these mechanisms can lead to dysregulated gene expression. One recently discovered process that controls gene expression includes chemical modifications of RNA molecules by RNA-modifying proteins, a field known as epitranscriptomics. Epitranscriptomics can regulate mRNA splicing, nuclear export, stabilization, translation, or induce degradation of target RNA molecules. Dysregulation in RNA-modifying proteins has been found to contribute to many pathological conditions, such as cancer, diabetes, obesity, cardiovascular diseases, and neurological diseases, among others. This article reviews the role of epitranscriptomics in the pathogenesis and progression of renal cell carcinoma. It summarizes the molecular function of RNA-modifying proteins in the pathogenesis of renal cell carcinoma.

Nucleic acid sensor STING drives remodeling and its inhibition enhances steroid responsiveness in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is progressive and irreversible chronic lung inflammatory disease. Cigarette smoke, the main cause of COPD, is often associated with double-stranded DNA release which potentially activates DNA-sensing pathways, such as STING. This study, therefore, analyzed the role of STING pathway in inducing pulmonary inflammation, steroid resistance, and remodeling in COPD. METHODS: Primary cultured lung fibroblasts were isolated from healthy non-smoker, healthy smoker, and smoker COPD individuals. The expression of STING pathway, remodeling, and steroid resistance signatures were investigated in these fibroblasts upon LPS stimulation and treatment with dexamethasone and/or STING inhibitor, at both mRNA and protein levels using qRT-PCR, western blot, and ELISA. RESULTS: At baseline, STING was elevated in healthy smoker fibroblasts and to a higher extent in smoker COPD fibroblasts when compared to healthy non-smoker fibroblasts. Upon using dexamethasone as monotherapy, STING activity was significantly inhibited in healthy non-smoker fibroblasts but showed resistance in COPD fibroblasts. Treating both healthy and COPD fibroblasts with STING inhibitor in combination with dexamethasone additively inhibited STING pathway in both groups. Moreover, STING stimulation triggered a significant increase in remodeling markers and a reduction in HDAC2 expression. Interestingly, treating COPD fibroblasts with the combination of STING inhibitor and dexamethasone alleviated remodeling and reversed steroid hyporesponsiveness through an upregulation of HDAC2. CONCLUSION: These findings support that STING pathway plays an important role in COPD pathogenesis, via inducing pulmonary inflammation, steroid resistance, and remodeling. This raises the possibility of using STING inhibitor as a potential therapeutic adjuvant in combination with common steroid treatment.

Potential anticancer properties of calotropis procera: An investigation on breast and colon cancer cells.

Calotropis procera is a perennial flowering plant of the Apocynaceae family, traditionally used in medicine to treat various ailments. Recent investigations have revealed its potential therapeutic activities such as anti-inflammatory, gastroprotective, analgesic, anti-obesity, and anti-diabetic properties. RP-HPLC qualitatively and quantitatively evaluated the phenolic acids and flavonoids in the ethanolic extract at two different wavelengths, 280 and 330 nm. In addition, total phenolic and flavonoid contents were measured via spectrophotometric determination in addition to the antioxidant activity. The antiproliferative effects of C. procera were investigated on two cancer cell lines: human colon (HCT-116) and breast (MCF-7) cancer. Several methods were utilised to analyse the effectiveness of the plant extract on the cytotoxicity, apoptosis, cell cycle progression, genes involved in the cell cycle, and protein expression profiles of HCT-116 and MCF-7 cells. These included the MTT assay, Annexin V-FITC/PI, analysis of the cell cycle, and Western blot. Results indicated that ferulic and caffeic acids were the major compounds at λmax 280 nm (1.374% and 0.561%, respectively), while the major compounds at λmax 325 nm were kaempferol and luteolin (1.036% and 0.512%, respectively). The ethanolic extract had significantly higher antioxidant activity (80 ± 2.3%) compared to ascorbic acid (90 ± 3.1%). C. procera extract exhibited dose-dependent cell growth inhibition, with an estimated IC50 of 50 µg/mL for MCF-7 and 55 µg/mL for HCT-116 cells at 24 h. Annexin V-FITC/PI confirmed the induction of apoptosis. Remarkably, cell cycle arrest occurred at the sub-G1 phase in MCF-7 cells, while in HCT-116 cells, it was observed at the G2-M phase. The sub-G1 arrest was associated with dysregulation of Akt, p-AKT, mTOR, and p-mTOR proteins, as confirmed by the Western blot analysis, while downregulation of CDK1, cyclin B1, and survivin caused G2-M arrest.

Integrated multi-omics analysis reveals unique signatures of paclitaxel-loaded poly(lactide-co-glycolide) nanoparticles treatment of head and neck cancer cells.

The use of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as carriers for chemotherapeutic drugs is regarded as an actively targeted nano-therapy for the specific delivery of anti-cancer drugs to target cells. However, the exact mechanism by which PLGA NPs boost anticancer cytotoxicity at the molecular level remains largely unclear. This study employed different molecular approaches to define the response of carcinoma FaDu cells to different types of treatment, specifically: paclitaxel (PTX) alone, drug free PLGA NPs, and PTX-loaded PTX-PLGA NPs. Functional cell assays revealed that PTX-PLGA NPs treated cells had a higher level of apoptosis than PTX alone, whereas the complementary, UHPLC-MS/MS (TIMS-TOF) based multi-omics analyses revealed that PTX-PLGA NPs treatment resulted in increased abundance of proteins associated with tubulin, as well as metabolites such as 5-thymidylic acid, PC(18:1(9Z)/18:1(9Z0), vitamin D, and sphinganine among others. The multi-omics analyses revealed new insights about the molecular mechanisms underlying the action of novel anticancer NP therapies. In particular, PTX-loaded NPs appeared to exacerbate specific changes induced by both PLGA-NPs and PTX as a free drug. Hence, the PTX-PLGA NPs' molecular mode of action, seen in greater detail, depends on this synergy that ultimately accelerates the apoptotic process, resulting in cancer cell death.

The Role of ATP-Binding Cassette Subfamily A in Colorectal Cancer Progression and Resistance.

Colorectal cancer (CRC) is one of the most common malignancies worldwide; it is the fourth leading cause of cancer-related deaths. CRC arises due to mutations that can affect oncogenes, tumour suppressor genes and DNA repair genes. The lack of novel diagnostic and therapeutic targets and the development of chemoresistance are some of the major issues when dealing with CRC. The overexpression of ATP-binding cassette (ABC) transporters is considered one facilitating mechanism for chemoresistance. Furthermore, ABC transporters have additional roles in cancer development beyond multidrug resistance. In CRC, lipid dysregulation has a key role in tumour development and progression, as cancer cells rely on lipids for energy and rapid cell proliferation. ABC subfamily A (ABCA) contains the largest members of ABC proteins, mainly known for their role in lipid transport, mostly membrane lipids such as cholesterol and phospholipids. Although the exact mechanism of action of these members is not confirmed, their expression is usually correlated with tumour progression and therapy resistance, probably due to their role in lipid homeostasis. CRC shows alteration in the expression of ABCA transporters, which is usually linked to poor prognosis and overall survival. Therefore, as lipid transporters, their role in CRC is investigated, and their diagnostic and prognostic potential is evaluated. This minireview presents evidence from various studies suggesting that ABCA transporters might have an active role in CRC and can be utilized as potential diagnostic and therapeutic targets.

Vitamin D Exerts Significant Antitumor Effects by Suppressing Vasculogenic Mimicry in Breast Cancer Cells.

Background: Numerous clinical and experimental observations have alluded to the substantial anti-neoplastic role of vitamin D in breast cancer (BC), primarily by inducing apoptosis and affecting metastasis. Tumor progression and resistance to chemotherapy have been linked to vasculogenic mimicry (VM), which represents the endothelial-independent formation of microvascular channels by cancer cells. However, the effect of vitamin D on VM formation in BC has not been thoroughly investigated. This study examined the impact of 1α,25-dihydroxyvitamin D3 (calcitriol), the active form of vitamin D, on the expression of major factors involved in BC migration, invasion, and VM formation. Experimental Methods: Publicly available transcriptomic datasets were used to profile the expression status of the key VM markers in vitamin D-treated BC cells. The in silico data were validated by examining the expression and activity of the key factors that are involved in tumor progression and MV formation in hormone-positive MCF-7 and aggressive triple-negative MDA-MB-231 BC cells after treatment with calcitriol. Results and Discussions: The bioinformatics analysis showed that tumor VM formation-enriched pathways were differentially downregulated in vitamin D-treated cells when compared with control counterparts. Treatment of BC cells with calcitriol resulted in increased expression of tissue inhibitors of metalloproteinases (TIMPs 1 and 2) and decreased content and gelatinolytic activity of matrix metalloproteinases (MMPs 2 and 9). Furthermore, calcitriol treatment reduced the expression of several pro-MV formation regulators including vascular endothelial growth factor (VEGF), tumor growth factor (TGF-ß1), and amphiregulin. Eventually, this process resulted in a profound reduction in cell migration and invasion following the treatment of BC cells with calcitriol when compared to the controls. Finally, the formation of VM was diminished in the aggressive triple-negative MDA-MB-231 cancer cell line after calcitriol treatment. Conclusion: Our findings demonstrate that vitamin D mediates its antitumor effects in BC cells by inhibiting and curtailing their potential for VM formation.

Synergistic Anti-Angiogenic Effect of Combined VEGFR Kinase Inhibitors, Lenvatinib, and Regorafenib: A Therapeutic Potential for Breast Cancer.

Background: Breast cancer currently affects more than two million women worldwide, and its incidence is steadily increasing. One of the most essential factors of invasion and metastasis of breast cancer cells is angiogenesis and non-angiogenic vascularization. Lenvatinib and Regorafenib share the same anti-angiogenic effect by inhibiting vascular endothelial growth factor receptors (VEGFRs subtypes 1 to 3) and have been approved for treating different types of cancer. Methods: We investigated Lenvatinib and Regorafenib effects on a well-established in-vitro model of breast cancer using MCF-7 (estrogen, progesterone receptor-positive, and HER2-negative), MDA-MB-231 (triple negative), as well as Human Umbilical Vascular Endothelial Cell line (HUVEC) cell lines. We performed the cell viability assay on four groups of cells, which included a control group, a Lenvatinib treated only group, a Regorafenib treated only group, and a group treated with a combination of both drugs at 24, 48, and 72 h. Data were analyzed as means ± standard deviation, and the drug−drug interactions with Compusyn software. Cellular migration assay, tube formation assay, and Western blots were conducted to determine the functional and the protein expression of downstream signals such as Caspase-9, anti-apoptotic Survivin, P-ERK, and total-ERK in the control and treatment groups. Results: MCF-7 cells showed a reduction in cell survival rates with higher dosing and longer incubation periods with each drug and with the combination of drugs. A synergistic interaction was identified (CI < 1) with both drugs on MCF7 at different dose combinations and at a higher dose in MDA-MB-231 cells. Furthermore, there was a marked decrease in the anti-angiogenic effect of both drugs in tube formation assay using MDA-MB-231 cells and survivin protein expression in MCF-7, and those antitumor markers showed a better outcome in drug combination than the use of each drug alone. Conclusion: Our result is the first to report the synergistic anti-angiogenic potential of combination therapy of Lenvatinib and Regorafenib. Therefore, it shows their therapeutic potential in breast cancer, including the aggressive types. Further studies are warranted to confirm and explore this therapeutic approach.

Metformin enhances LDL-cholesterol uptake by suppressing the expression of the pro-protein convertase subtilisin/kexin type 9 (PCSK9) in liver cells.

PURPOSE: Metformin (MF) intake associates with reduced levels of circulating low-density lipoprotein-cholesterol (LDL-C). This has been attributed to the activation of AMPK, which differentially regulates the expression of multiple genes involved in cholesterol synthesis and trafficking. However, the exact mechanism underlying the LDL-C lowering effect of MF remains ambiguous. METHODS: MF-treated Hep-G2 and HuH7 cells were evaluated for cell viability and the expression status of key lipid metabolism-related genes along with LDL-C uptake efficiency. RESULTS: MF treatment resulted in decreased expression and secretion of PCSK9, increased expression of LDLR and enhanced LDL-C uptake in hepatocytes. It also resulted in increased expression of activated AMPK (p-AMPK) and decreased expression of SREBP2 and HNF-1α proteins. Transcriptomic analysis of MF-treated Hep-G2 cells confirmed these findings and showed that other key lipid metabolism-related genes including those that encode apolipoproteins (APOB, APOC2, APOC3 and APOE), MTTP and LIPC are downregulated. Lastly, MF treatment associated with reduced HMG-CoA reductase expression and activity. CONCLUSIONS: These findings suggest that MF treatment reduces circulating LDL-C levels by suppressing PCSK9 expression and enhancing LDLR expression; hence the potential therapeutic utility of MF in hypercholesterolemia.

The role of disrupted iron homeostasis in the development and progression of arthropathy.

Arthropathy or joint disease leads to significant pain and disability irrespective of etiology. Clinical and experimental evidence point to the presence of considerable links between arthropathy and iron overload. Previous work has suggested that iron accumulation in the joints is often associated with increased oxidative stress, disrupted matrix metabolism, and cartilage degeneration. However, key issues regarding the role of iron overload in the pathogenesis of arthropathy remain ambiguous. For example, significant gaps in our knowledge of the primary cellular targets of iron overload-induced damage and the exact molecular mechanism through which disrupted iron homeostasis leads to joint damage still exist. The exact signaling pathway that links iron metabolism and cellular damage in arthropathy also remains largely unmapped. In this review, we focus on the relationship between iron overload and arthropathy with special emphasis on the adversarial relationship between iron that accumulates in the joints over time and cartilage homeostasis. A better understanding of the mechanisms and pathways underlying iron-induced cartilage degeneration may help in defining new prognostic markers and therapeutic targets in arthropathy.

Iron Overload Induces Oxidative Stress, Cell Cycle Arrest and Apoptosis in Chondrocytes.

Clinical and experimental evidence point to the presence of considerable links between arthropathy, osteoarthritis (OA) in particular, and iron overload possibly due to oxidative stress and tissue damage. However, the specific cellular targets of iron overload-related oxidative stress in OA remain ambiguous. We examined the effects of iron overload on chondrocyte health using the C-20/A4 chondrocyte cell line. Cells were treated with increasing concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro. Treated cells were assessed for cell viability, cycling, apoptosis, collagen II synthesis, and oxidative stress along with cellular iron content and the expression of key iron regulatory genes. FAC treatment resulted in an increase in ferritin expression and a significant decrease in the expression of hepcidin, ferroportin, transferrin receptors 1 (TfR1) and TfR2. Increased labile iron content was also evident, especially in cells treated with high FAC at 24 h. High doses of FAC treatment also induced higher levels of reactive oxygen species, reduced collagen II production, disrupted cell cycle and higher cell death as compared with untreated controls. In conclusion, findings presented here demonstrate that iron overload disrupts cellular iron homeostasis, which compromises the functional integrity of chondrocytes and leads to oxidative stress and apoptosis.

Fisetin Deters Cell Proliferation, Induces Apoptosis, Alleviates Oxidative Stress and Inflammation in Human Cancer Cells, HeLa.

BACKGROUND: Fisetin, a flavonol profusely found in vegetables and fruits, exhibited a myriad of properties in preclinical studies to impede cancer growth. PURPOSE: This study was proposed to delineate molecular mechanisms through analysing the modulated expression of various molecular targets in HeLa cells involved in proliferation, apoptosis and inflammation. METHODS: MTT assay, flow cytometry, nuclear morphology, DNA fragmentation and Annexin-Pi were performed to evaluate the anti-cancer potential of fisetin. Furthermore, qPCR and proteome profiler were performed to analyse the expression of variety of gene related to cell death, cell proliferation, oxidative stress and inflammation and cancer pathways. RESULTS: Fisetin demonstrated apoptotic inducing ability in HeLa cells, which was quite evident through nuclear morphology, DNA ladder pattern, decreased TMRE fluorescent intensity, cell cycle arrest at G2/M and increased early and late apoptosis. Furthermore, fisetin treatment modulated pro-apoptotic genes such as APAF1, Bad, Bax, Bid and BIK at both transcript and protein levels and anti-apoptotic gene Bcl-2, BIRC8, MCL-1, XIAP/BIRC4, Livin/BIRC7, clap-2/BIRC3, etc. at protein levels to mitigate cell proliferation and induce apoptosis. Interestingly, the aforementioned alterations consequently led to an elevated level of Caspase-3, Caspase-8 and Caspase-9, which was found to be consistent with the transcript and protein level expression. Moreover, fisetin downregulated the expression of AKT and MAPK pathways to avert proliferation and enhance apoptosis of cancer cells. Fisetin treatment also improves oxidative stress and alleviates inflammation by regulating JAK-STAT/NF-kB pathways. CONCLUSION: Together, these studies established that fisetin deters human cervical cancer cell proliferation, enhances apoptosis and ameliorates inflammation through regulating various signalling pathways that may be used as a therapeutic regime for better cancer management.

Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis.

Colorectal cancer (CRC) remains the third most common cause of cancer mortality worldwide. Precision medicine using OMICs guided by transcriptomic profiling has improved disease diagnosis and prognosis by identifying many CRC targets. One such target that has been actively pursued is an erbb2 receptor tyrosine kinase 2 (ERBB2) (Human Epidermal Growth Factor Receptor 2 (HER2)), which is overexpressed in around 3-5% of patients with CRC worldwide. Despite targeted therapies against HER2 showing significant improvement in disease outcomes in multiple clinical trials, to date, no HER2-based treatment has been clinically approved for CRC. In this study we performed whole transcriptome ribonucleic acid (RNA) sequencing on 11 HER2+ and 3 HER2- CRC patients with advanced stages II, III and IV of the disease. In addition, transcriptomic profiling was carried out on CRC cell lines (HCT116 and HT29) and normal colon cell lines (CCD841 and CCD33), ectopically overexpressing ERBB2. Our analysis revealed transcriptomic changes involving many genes in both CRC cell lines overexpressing ERBB2 and in HER2+ patients, compared to normal colon cell lines and HER2- patients, respectively. Gene Set Enrichment Analysis indicated a role for HER2 in regulating CRC pathogenesis, with Wnt/ß-catenin signaling being mediated via a HER2-dependent regulatory pathway impacting expression of the homeobox gene NK2 homeobox 5 (NKX2-5). Results from this study thus identified putative targets that are co-expressed with HER2 in CRC warranting further investigation into their role in CRC pathogenesis.

Histone Modification in NSCLC: Molecular Mechanisms and Therapeutic Targets.

Lung cancer is the leading cause of cancer mortality in both genders, with non-small cell lung cancer (NSCLC) accounting for about 85% of all lung cancers. At the time of diagnosis, the tumour is usually locally advanced or metastatic, shaping a poor disease outcome. NSCLC includes adenocarcinoma, squamous cell carcinoma, and large cell lung carcinoma. Searching for novel therapeutic targets is mandated due to the modest effect of platinum-based therapy as well as the targeted therapies developed in the last decade. The latter is mainly due to the lack of mutation detection in around half of all NSCLC cases. New therapeutic modalities are also required to enhance the effect of immunotherapy in NSCLC. Identifying the molecular signature of NSCLC subtypes, including genetics and epigenetic variation, is crucial for selecting the appropriate therapy or combination of therapies. Epigenetic dysregulation has a key role in the tumourigenicity, tumour heterogeneity, and tumour resistance to conventional anti-cancer therapy. Epigenomic modulation is a potential therapeutic strategy in NSCLC that was suggested a long time ago and recently starting to attract further attention. Histone acetylation and deacetylation are the most frequently studied patterns of epigenetic modification. Several histone deacetylase (HDAC) inhibitors (HDIs), such as vorinostat and panobinostat, have shown promise in preclinical and clinical investigations on NSCLC. However, further research on HDIs in NSCLC is needed to assess their anti-tumour impact. Another modification, histone methylation, is one of the most well recognized patterns of histone modification. It can either promote or inhibit transcription at different gene loci, thus playing a rather complex role in lung cancer. Some histone methylation modifiers have demonstrated altered activities, suggesting their oncogenic or tumour-suppressive roles. In this review, patterns of histone modifications in NSCLC will be discussed, focusing on the molecular mechanisms of epigenetic modifications in tumour progression and metastasis, as well as in developing drug resistance. Then, we will explore the therapeutic targets emerging from studying the NSCLC epigenome, referring to the completed and ongoing clinical trials on those medications.

Vitamin D-Mediated Anti-cancer Activity Involves Iron Homeostatic Balance Disruption and Oxidative Stress Induction in Breast Cancer.

Background: Vitamin D deficiency associates with high risk of breast cancer (BRCA) and increased cellular iron. Vitamin D exerts some of its anti-cancer effects by regulating the expression of key iron regulatory genes (IRGs). The association between vitamin D and cellular iron content in BRCA remains ambiguous. Herein, we addressed whether vitamin D signaling exerts a role in cellular iron homeostasis thereby affecting survival of breast cancer cells. Methods: Expression profile of IRGs in vitamin D-treated breast cancer cells was analyzed using publicly available transcriptomic datasets. After treatment of BRCA cell lines MCF-7 and MDA-MB-231 with the active form of vitamin D, labile iron content, IRGs protein levels, oxidative stress, and cell survival were evaluated. Results: Bioinformatics analysis revealed several IRGs as well as cellular stress relates genes were differentially expressed in BRCA cells. Vitamin D treatment resulted in cellular iron depletion and differentially affected the expression of key IRGs protein levels. Vitamin D treatment exerted oxidative stress induction and alteration in the cellular redox balance by increasing the synthesis of key stress-related markers. Collectively, these effects resulted in a significant decrease in BRCA cell survival. Conclusion: These findings suggest that vitamin D disrupts cellular iron homeostasis leading to oxidative stress induction and cell death.

Co-targeting BET bromodomain BRD4 and RAC1 suppresses growth, stemness and tumorigenesis by disrupting the c-MYC-G9a-FTH1axis and downregulating HDAC1 in molecular subtypes of breast cancer.

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient's samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1.

Copine 3 "CPNE3" is a novel regulator for insulin secretion and glucose uptake in pancreatic ß-cells.

Copine 3 (CPNE3) is a calcium-dependent phospholipid-binding protein that has been found to play an essential role in cancer progression and stages. However, its role in pancreatic ß-cell function has not been investigated. Therefore, we performed a serial of bioinformatics and functional experiments to explore the potential role of Cpne3 on insulin secretion and ß-cell function in human islets and INS-1 (832/13) cells. RNA sequencing and microarray data revealed that CPNE3 is highly expressed in human islets compared to other CPNE genes. In addition, expression of CPNE3 was inversely correlated with HbA1c and reduced in human islets from hyperglycemic donors. Silencing of Cpne3 in INS-1 cells impaired glucose-stimulated insulin secretion (GSIS), insulin content and glucose uptake efficiency without affecting cell viability or inducing apoptosis. Moreover, mRNA and protein expression of the key regulators in glucose sensing and insulin secretion (Insulin, GLUT2, NeuroD1, and INSR) were downregulated in Cpne3-silenced cells. Taken together, data from the present study provides a new understanding of the role of CPNE3 in maintaining normal ß-cell function, which might contribute to developing a novel target for future management of type 2 diabetes therapy.

Heme Oxygenase-1 (HMOX-1) and inhibitor of differentiation proteins (ID1, ID3) are key response mechanisms against iron-overload in pancreatic ß-cells.

Iron overload promotes the generation of reactive oxygen species (ROS). Pancreatic ß-cells can counter oxidative stress through multiple anti-oxidant responses. Herein, RNA-sequencing was used to describe the expression profile of iron regulatory genes in human islets with or without diabetes. Functional experiments including siRNA silencing, qPCR, western blotting, cell viability, ELISA and RNA-sequencing were performed as means of identifying the genetic signature of the protective response following iron overload-induced stress in human islets and INS-1. FTH1 and FTL genes were highly expressed in human islets and INS-1 cells, while hepcidin (HAMP) was low. FXN, DMT1 and FTHL1 genes were differentially expressed in diabetic islets compared to control. Silencing of Hamp in INS-1 cells impaired insulin secretion and influenced the expression of ß-cell key genes. RNA-sequencing analysis in iron overloaded INS-1 cells identified Id1 and Id3 as the top down-regulated genes, while Hmox1 was the top upregulated. Expression of ID1, ID3 and HMOX1 was validated at the protein level in INS-1 cells and human islets. Differentially expressed genes (DEGs) were enriched for TGF-ß, regulating stem cells, ferroptosis, and HIF-1 signaling. Hmox1-silenced cells treated with FAC elevated the expression of Id1 and Id3 expression than untreated cells. Our findings suggest that HMOX1, ID1 and ID3 define the response mechanism against iron-overload-induced stress in ß-cells.

The Coffee Diterpene, Kahweol, Ameliorates Pancreatic ß-Cell Function in Streptozotocin (STZ)-Treated Rat INS-1 Cells through NF-kB and p-AKT/Bcl-2 Pathways.

Kahweol is a diterpene molecule found in coffee that exhibits a wide range of biological activity, including anti-inflammatory and anticancer properties. However, the impact of kahweol on pancreatic ß-cells is not known. Herein, by using clonal rat INS-1 (832/13) cells, we performed several functional experiments including; cell viability, apoptosis analysis, insulin secretion and glucose uptake measurements, reactive oxygen species (ROS) production, as well as western blotting analysis to investigate the potential role of kahweol pre-treatment on damage induced by streptozotocin (STZ) treatment. INS-1 cells pre-incubated with different concentrations of kahweol (2.5 and 5 µM) for 24 h, then exposed to STZ (3 mmol/L) for 3 h reversed the STZ-induced effect on cell viability, apoptosis, insulin content, and secretion in addition to glucose uptake and ROS production. Furthermore, Western blot analysis showed that kahweol downregulated STZ-induced nuclear factor kappa B (NF-κB), and the antioxidant proteins, Heme Oxygenase-1 (HMOX-1), and Inhibitor of DNA binding and cell differentiation (Id) proteins (ID1, ID3) while upregulated protein expression of insulin (INS), p-AKT and B-cell lymphoma 2 (BCL-2). In conclusion, our study suggested that kahweol has anti-diabetic properties on pancreatic ß-cells by suppressing STZ induced apoptosis, increasing insulin secretion and glucose uptake. Targeting NF-κB, p-AKT, and BCL-2 in addition to antioxidant proteins ID1, ID3, and HMOX-1 are possible implicated mechanisms.

HER2 overexpression is a putative diagnostic and prognostic biomarker for late-stage colorectal cancer in North African patients.

Aim: Colorectal cancer (CRC) is one of the leading cancers in the world. Even though its mortality and pathophysiology are well documented in the US and the European countries, it is seldom studied in North African population. Recent studies have shown link of HER2 overexpression in oesophageal and gastric cancers. The aim of this study is to assess the HER2 protein and mRNA expression and its correlation with tumor pathogenesis in Libyan CRC patients.Methodology: A total of 17 FFPE tissue blocks were collected from patients with primary CRC. The HER2 protein expression was assessed by immunohistochemistry and the mRNA expression was assessed using qRT-PCR. Survival analysis of the role of HER2 overexpression on rectal adenocarcinoma was carried out on additional 165 patients.Results: From the CRC cohort, adenocarcinoma was found to be more frequent accounting for 88.2%, and 11.8% for mucinous adenocarcinomas. Almost 47% of the cases were positive for HER2 (score ≥ 2+) and about 50% adenocarcinoma cases with tumor grade II were positive for HER2. Moreover, 57.4% adenocarcinoma patients with grade-II tumor had undergone right hemicolectomy. Furthermore, significant correlation (p = 0.03) between the HER2 mRNA expression with the tumor grade was observed. In addition, poor overall all survival was observed with high HER2 expression in rectum adenocarcinoma.Conclusion: To our knowledge, this is the first study that HER2 overexpression correlates with more aggressive colorectal cancer in North African population. Our study shows that HER2 overexpression associates with right colon surgeries. Also, the correlation of mRNA and protein expression could warrant the implementation of a nationwide screening program for HER2 positivity in CRC patients. Taken together, stratifying patients according to HER2 expression can help in the diagnosis and prognosis of CRC patients from North African origin.