Oral Delivery of Psoralidin by Mucoadhesive Surface-Modified Bilosomes Showed Boosted Apoptotic and Necrotic Effects against Breast and Lung Cancer Cells.

This study aims to design and optimize chitosan-coated bilosomal formulations loaded with psoralidin (Ps-CS/BLs) with improved physicochemical properties, oral bioavailability, and boosted apoptotic and necrotic effects. In this regard, uncoated bilosomes loaded with Ps (Ps/BLs) were nanoformulated using the thin-film hydration technique using different molar ratios of phosphatidylcholine (PC), cholesterol (Ch), Span 60 (S60), and sodium deoxycholate (SDC) (1:0.4:0.2:0.125, 1:0.4:0.2:0.25, and 1:0.4:0.2:0.5, respectively). The best-optimized formulation with respect to size, PDI, zeta potential, and EE% was selected and then coated with chitosan at two different concentrations (0.125 and 0.25 w/v%), forming Ps-CS/BLs. The optimized Ps/BLs and Ps-CS/BLs showed a spherical shape and relatively homogenous size with negligible apparent agglomerations. Additionally, it was demonstrated that coating Ps/BLs with chitosan has significantly increased the particle size from 123.16 ± 6.90 in the case of Ps/BLs to 183.90 ± 15.93 nm in the case of Ps-CS/BLs. In addition, Ps-CS/BLs exhibited higher zeta potential (+30.78 ± 1.44 mV) as compared to Ps/BLs (-18.59 ± 2.13 mV). Furthermore, Ps-CS/BL showed enhanced entrapment efficiency (EE%) of 92.15 ± 7.20% as compared to Ps/BLs (68.90 ± 5.95%). Moreover, Ps-CS/BLs exhibited a more sustained release behavior of Ps compared to Ps/BLs over 48 h, and both formulations were best obeying the Higuchi diffusion model. More importantly, Ps-CS/BLs displayed the highest mucoadhesive efficiency% (74.89 ± 3.5%) as compared to Ps/BLs (26.78 ± 2.9%), indicating the ability of the designed nanoformulation to improve oral bioavailability and extend the residence time inside the gastrointestinal tract upon oral administration. Moreover, upon evaluating the apoptotic and necrotic effects of free Ps and Ps-CS/BLs on human breast cancer cell lines (MCF-7) and human lung adenocarcinoma cell lines (A549), there was a dramatic increase in the percentages of the apoptotic and necrotic cell compared to the control and free Ps. Our findings suggest the possible oral use of Ps-CS/BLs in hampering breast and lung cancers.

miR-516a-3P, a potential circulating biomarker in hepatocellular carcinoma, correlated with rs738409 polymorphism in PNPLA3.

Aim: The aim was to investigate the expression profile of miR-516a-3P and its correlation with the PNPLA3 rs738409 polymorphism in Egyptian hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) patients. Materials & methods: miR-516a-3P was quantified and rs738409 was genotyped by quantitative reverse transcription PCR. Results: miR-516a-3P was significantly upregulated in HCC patients compared with HCV patients (p = 0.001). Receiver operating characteristic curve analysis confirmed that miR-516a-3P discriminates HCC from HCV (p = 0.001). A significant (p = 0.015) correlation between miR-516a-3p level and PNPLA3 rs738409 genotypes was recorded in HCV patients, yet it was not recorded in either healthy individuals or HCC patients. miR-516a-3p level was significantly (p = 0.001) higher in HCV patients carrying the rs738409 GG genotype than in those carrying the CC genotype. Conclusion: miR-516a-3P is a potential biomarker in HCC. PNPLA3 rs738409 GG carriers affect miR-516a-3P expression in HCV, and this may highlight a new mechanism in liver disease.

In the past decade, miRNAs were established as biomarkers in various cancers, including liver cancer. Information about the deregulation of miR-516a-3p and its efficiency as a biomarker in hepatitis C virus (HCV) and liver cancer patients is lacking globally and in Egypt. This study proved for the first time that miR-516a-3p is differentially expressed in HCV and liver cancer patients and is statistically efficient in discriminating between them, so it might be used for early detection of HCC. Genetic variants that affect expression levels of genes are called expression quantitative trait loci (eQTL), and those acting on genes on other chromosomes are called trans-eQTL. The authors speculate that rs738409 is a genetic variant that might have a trans-eQTL effect on the miR-516a-3p gene in HCV patients.

PNPLA3 and IL 28B signature for predicting susceptibility to chronic hepatitis C infection and fibrosis progression.

BACKGROUND: Association studies identified genetic polymorphisms as predictive risk factors of rapid fibrosis progression in chronic hepatitis C (CHC). This study aims to assess the impact of IL28B rs8099917 polymorphism on CHC genotype 4 (G4) susceptibility and liver fibrosis progression individually; and in combination with PNPLA3 rs738409. PATIENTS AND METHODS: IL28B rs8099917 and PNPLA3 rs738409 were genotyped in 150 Egyptian CHC patients and 175 healthy controls using real-time PCR. RESULTS: IL28B rs8099917 genotype distribution significantly differs in healthy individuals versus CHC patients (p = .018); and in low versus advanced fibrosis IL28B (p = .013). The haplotype CC -GG (PNPLA3-IL28B) is considered a high-risk signature for susceptibility to CHC infection. Similarly, GG-GG (PNPLA3-IL28B) is considered a high-risk signature for higher degree of fibrosis. CONCLUSION: IL28B rs8099917 and PNPLA3 rs738409 introduce genetic signature to identify patients at higher risk for CHC susceptibility and fibrosis progression in CHC G4.

miR-744/eNOS/NO axis: A novel target to halt triple negative breast cancer progression.

BACKGROUND: Nitric oxide (NO) may have a dual role in cancer. At low concentrations, endogenous NO promotes tumor growth and proliferation. However, at very high concentrations, it mediates cancer cell apoptosis and inhibits cancer growth. High levels of NO have been observed in blood of breast cancer (BC) patients, which increases tumor blood flow and promotes angiogenesis. To date, the regulation of NO-synthesizing enzyme, eNOS, by miRNAs has not been adequately investigated in BC. Therefore, the main aim of this study is to unravel the possible regulation of eNOS by miRNAs in BC and to examine their influence on NO production and BC progression. METHODS: Expression profile of eNOS in Egyptian BC patients and MDA-MB-231 cell lines was investigated using qRT-PCR. In-silico analysis was performed to predict a putative upstream regulator of eNOS. miR-744-5p was selected and its expression was quantified in BC tissues using qRT-PCR. MDA-MB-231 cells were cultured and transfected with miR-744-5p using lipofection method. NO levels were determined using Griess Reagent. Cellular viability and colony-forming ability were assessed using MTT and colony-forming assays; respectively. RESULTS: eNOS and miR-744-5p were significantly up-regulated in BC tissues compared to paired normal tissues. In-silico analysis revealed that miR-744-5p putatively binds to eNOS transcript with high binding scores. Transfection of MDA-MB-231 cells with miR-744-5p mimics resulted in a significant up-regulation of eNOS and consequently NO levels. In addition, miR-744-5p transfection led to an increase in cellular viability and colony-forming ability of the MDA-MB-231. CONCLUSION: miR-744-5p acts as an upstream positive regulator of the NO synthesizing enzyme, eNOS which in turn elevates NO levels. Furthermore, miR-744-5p is a novel oncogenic miRNA in BC. Thus, targeting miR-744/eNOS/NO axis may act as a therapeutic tool in TNBC.

Targeting hydrogen sulphide signaling in breast cancer.

INTRODUCTION: Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer. OBJECTIVES: This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs. METHODS: Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively. RESULTS: The H2S synthesizing enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells. CONCLUSION: These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

miRNA-506-3p Directly Regulates rs10754339 (A/G) in the Immune Checkpoint Protein B7-H4 in Breast Cancer.

BACKGROUND: B7-H4 is a novel immune checkpoint protein that negatively regulates T cell activation and function. It is overexpressed in many malignant tumors, including Breast Cancer (BC). It was reported that the presence of the single nucleotide polymorphism rs10754339 (A/G) within the 3' UTR of the B7-H4 gene has a great influence on the risk and progression of BC as well as lymph node metastasis. On the other hand, mounting evidence demonstrated the potential of miR-506-3p to be employed in the diagnosis and treatment of a wide range of human malignancies. It is frequently down-regulated in BC despite its tumor suppressor role. Moreover, Myc, E2F and Rb proteins are key players in cell cycle regulation. In BC, the CDK-RB-E2F axis is extensively deregulated by several genetic mutations. Additionally, the potent proto-oncogene Myc is highly expressed in BC. AIM: The main aims of the study were to investigate the potential role of miR-506-3p in the regulation of B7-H4 SNP rs10754339 (A/G) in BC and to uncover the influence of miR-506-3p on cell cycle and tumor progression in BC cell lines. METHODS: This study employed different BC cell lines, including MDA-MB-231 and MCF7 cells. Several bioinformatics analysis was performed to identify the miRNA that could potentially target B7-H4 SNP rs10754339 (A/G). To confirm the binding Relative luciferase activity was measured using Dual Luciferase Reporter Assay. Transfection experiments were performed using miR-506-3p oligonucleotides using lipofection technique. Furthermore, vital cell cycle regulatory proteins such as cMyc, Rb, and E2F were transfected into BC cells using superfect. Finally, several functional analysis experiments were performed, such as MTT and wound healing assays. RESULTS: miR-506-3p down-regulates the disease-associated rs10754339 "G" allele in B7-H4 gene. It also inhibits cell cycle progression by simultaneously regulating important cell cycle proteins, including RB, E2F and c-Myc. Moreover, miR-506-3p decreased cellular viability and migration capacity of MDA-MB-231 TNBC cells and hormone receptor positive MCF-7 cells. CONCLUSION: miR-506-3p is a potential tumor suppressor miRNA in BC that has a potential role in regulating B7-H4 SNP rs10754339 (A/G).

Why natural killer cells in triple negative breast cancer?

The triple-negative subtype of breast cancer (TNBC) has the bleakest prognosis, owing to its lack of either hormone receptor as well as human epidermal growth factor receptor 2. Henceforth, immunotherapy has emerged as the front-runner for TNBC treatment, which avoids potentially damaging chemotherapeutics. However, despite its documented association with aggressive side effects and developed resistance, immune checkpoint blockade continues to dominate the TNBC immunotherapy scene. These immune checkpoint blockade drawbacks necessitate the exploration of other immunotherapeutic methods that would expand options for TNBC patients. One such method is the exploitation and recruitment of natural killer cells, which by harnessing the innate rather than adaptive immune system could potentially circumvent the downsides of immune checkpoint blockade. In this review, the authors will elucidate the advantageousness of natural killer cell-based immuno-oncology in TNBC as well as demonstrate the need to more extensively research such therapies in the future.

Epigallocatechin gallate (EGCG) and miR-548m reduce HCV entry through repression of CD81 receptor in HCV cell models.

Epigallocatechin gallate (EGCG) is the most abundant component in green tea extract, that has powerful antioxidant and antiviral effects. It has been previously reported to inhibit HCV entry via several mechanisms. Hence, this study aimed at further investigating the potential impact of EGCG on HCV entry through regulation of the expression of tetraspanin receptor CD81 by the novel predicted miR-548m. Liver biopsies were obtained from 29 HCV patients and 10 healthy controls for expression profiling. Huh7 cells were stimulated with EGCG and subsequently miR-548m expression was assessed. Naïve, HCV- ED43/JFH-1 and HCV-JFH-1 infected Huh7 cells were transfected by miR-548m mimics and inhibitors. Consequently, CD81 protein and mRNA levels were assessed using flow cytometry and qRT-PCR, respectively. Additionally, these cells were used to investigate HCV permissiveness into Huh7 cells using qRT-PCR for viral quantification. Direct binding confirmation of miR-548m to CD81 was done using luciferase reporter assay. In-silico analysis revealed miR-548m to have two potential binding sites in the 3'UTR of CD81 mRNA. EGCG boosted miR-548m expression in Huh7 cells. Additionally, miR-548m caused a downregulation of CD81 protein and mRNA levels as well as reduction in HCV infectivity of Huh7 cells. Luciferase binding assay confirmed the binding of miR-548m to CD81 mRNA at the two predicted binding sites. Intriguingly, miR-548m expression was not detected in healthy liver biopsies but was found in liver biopsies of HCV patients. This study shows that EGCG might act as an anti-HCV agent that reduces cellular infectivity via enhancing miR-548m expression and repressing CD81 receptor.

Long non-coding RNAs: Functional regulatory players in breast cancer.

Historically, the long-held protein-centered bias has denoted 98% of the human genome as 'Junk' DNA. However, the current work has shifted the perception of such 'junk' transcriptional products to functional regulatory molecules. The recent surveillance of the human transcriptome has highlighted the pivotal role of such non-coding RNA (ncRNA) molecules in diverse physiological and pathological conditions. Long non-coding RNA (lncRNA) is a recent class of ncRNA molecules that is still in its infancy stage. The main focus of this review is to unravel the importance of lncRNAs in the most prevalent malignancy among females which is Breast Cancer (BC). A specific focus on lncRNAs as prognostic markers among BC patients showing molecular subtype heterogeneity was also tackled in this review. Finally, the functional and the mechanistic roles of such booming ncRNA molecules in shaping the fate of the BC progression have been highlighted.

A novel role of sONE/NOS3/NO signaling cascade in mediating hydrogen sulphide bilateral effects on triple negative breast cancer progression.

Hydrogen sulphide (H2S) gas has been recognized as an intracellular mediator influencing an array of signaling pathways. Yet, the role of H2S in cancer progression has been controversial. This study aims to unravel the role of exogenous H2S in triple negative breast cancer (TNBC) and to further investigate any possible association of H2S mediated actions with the endogenous production of nitric oxide (NO) gas. A wide concentration range of NaHS (20-2000 µM) and a variable reaction time (2-72 h) were probed. A bell-shaped impact of H2S on TNBC cellular viability, proliferation, migration, invasion and colony forming ability was repeatedly observed in the aggressive TNBC cell lines, MDA-MB-231 but not in hormone receptor positive, MCF-7 cells. This bell-shaped effect was found to be shifted towards the left upon increasing the reaction time within the range of 2-24 h. However, this was totally opposed in case of continuous exposure (72 h) to exogenous H2S. An inverted bell-shaped effect of H2S on TNBC cellular growth, migration, proliferation and colony forming ability was shown. Moreover, this study provided the first evidence of a possible involvement of NO in mediating H2S actions in TNBC. Such intricate cross-talk was found to be orchestrated by the novel lncRNA, sONE and its down-stream target NOS3 building up a novel axis, sONE/NOS3/NO, that was shown to play a pivotal role in plotting the bilateral effect of H2S on TNBC progression. Finally, this study showed that low and continuous exposure of H2S serves as a novel, selective and effective strategy in harnessing TNBC oncogenic profile through cGMP dependent and independent pathways where alterations of cell cycle regulatory proteins such as TP53 and c-Myc was observed. Moreover, NaHS could repress TNBC migration and invasion capacities through repressing the intracellular adhesion molecule, ICAM-1. In conclusion, this study provides an insight about the role of exogenous H2S in TNBC cell lines highlighting a novel crosstalk between H2S and NO orchestrated by sONE/NOS3 axis.

MiR-615-5p depresses natural killer cells cytotoxicity through repressing IGF-1R in hepatocellular carcinoma patients.

miR-615-5p was characterized by our group as a tumour suppressor. IGF-1 R activates a downstream signalling pathway, well characterized in liver cells, however, its role in immunity especially Natural Killer cells (NKs) remains vague. This study aimed at investigating the regulatory role of miR-615-5p on IGF signalling and its impact on NKs cytotoxicity in HCC. Our results showed an upregulation in miR-615-5p and IGF-1 R in NKs of 130 HCC patients compared to 35 controls. Forcing the expression of miR-615-5p, repressed IGF-IR, attenuated NKs cytotoxicity, decreased CD56dim, increased CD56bright NK subsets and reduced the cytotoxic markers NKG2D, TNF-α and perforins. It repressed NKG2D ligand (ULBP2) in Huh-7 cells. In conclusion, miR-615-5p represses IGF-1 R in NKs and their target hepatocytes; however, it has a contradicting impact on HCC progression on both cell types. These findings might pave the way for better understanding the role of microRNAs in NKs function and HCC immune-pathogenesis.

MicroRNA-486-5p enhances hepatocellular carcinoma tumor suppression through repression of IGF-1R and its downstream mTOR, STAT3 and c-Myc.

The insulin-like growth factor (IGF)-axis has been paradigmatically involved in hepatocellular carcinoma (HCC) tumor initiation, progression and drug resistance. Consequently, members of the IGF-axis and most importantly, IGF-1 receptor (IGF-1R) have been considered as intriguing targets for HCC therapy. Few miRNAs have been recently reported to be associated with IGF-1R regulation. The present study aimed to investigate the role of microRNA (miRNA/miR)-486-5p in the regulation of IGF-1R and its downstream signaling cascades. miR-486-5p was markedly downregulated in hepatitis C virus-induced HCC tissues and Huh-7 cells. Forcing the expression of miR-486-5p in Huh-7 cells resulted in the repression of IGF-1R, mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3) and c-Myc mRNA levels. Ectopic expression of miR-486-5p in Huh-7 cells markedly repressed cellular viability, proliferation, migration and clonogenicity in a similar pattern to IGF-1R small interfering RNAs, and were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, BrdU incorporation, wound healing and colony forming assays, respectively. Overall, the study findings demonstrated that miR-486-5p acts as a tumor suppressor in HCC through the repression of essential members of the IGF-axis, including IGF-1R and its downstream mediators mTOR, STAT3 and c-Myc.

Contradicting interplay between insulin-like growth factor-1 and miR-486-5p in primary NK cells and hepatoma cell lines with a contemporary inhibitory impact on HCC tumor progression.

In this study, an impaired natural killer (NK) cell cytolytic activity in 135 hepatocellular carcinoma (HCC) patients parallel to a reduced expression level of insulin-like growth factor (IGF)-1 in NK cells of HCC patients has been revealed. Ectopic expression of miR-486-5p, a direct upstream regulator of IGF-1, restored the endogenous level of IGF-1 in NK cells of HCC patients, thus augmenting its cytolytic activity against Huh7 cells in an opposite manner to the IGF-1 siRNAs. Unorthodoxly, over-expression of miR-486-5p in target hepatocytes resulted in the repression of IGF-1, suppression of Huh7 cells proliferation and viability in a similar pattern to the IGF-1 siRNAs. Therefore, this study highlights a potential role of IGF-1 in modulating cytolytic potential of NK cells of HCC patients. miR-486-5p acts in a cell-specific manner, differentially modulating IGF-1 expression in NK cells and their target hepatocytes with a contemporary inhibitory impact on HCC progression.