GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia.

Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.

1H-NMR metabolomics analysis identifies hypoxanthine as a novel metastasis-associated metabolite in breast cancer.

Breast cancer is one of the leading causes of death in females, mainly because of metastasis. Oncometabolites, produced via metabolic reprogramming, can influence metastatic signaling cascades. Accordingly, and based on our previous results, we propose that metabolites from highly metastatic breast cancer cells behave differently from less-metastatic cells and may play a significant role in metastasis. For instance, we aim to identify these metabolites and their role in breast cancer metastasis. Less metastatic cells (MCF-7) were treated with metabolites secreted from highly metastatic cells (MDA-MB-231) and the gene expression of three epithelial-to-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin and vimentin were examined. Some metabolites secreted from MDA-MB-231 cells significantly induced EMT activity. Specifically, hypoxanthine demonstrated a significant EMT effect and increased the migration and invasion effects of MCF-7 cells through a hypoxia-associated mechanism. Hypoxanthine exhibited pro-angiogenic effects via increasing the VEGF and PDGF gene expression and affected lipid metabolism by increasing the gene expression of PCSK-9. Notably, knockdown of purine nucleoside phosphorylase, a gene encoding for an important enzyme in the biosynthesis of hypoxanthine, and inhibition of hypoxanthine uptake caused a significant decrease in hypoxanthine-associated EMT effects. Collectively for the first time, hypoxanthine was identified as a novel metastasis-associated metabolite in breast cancer cells and represents a promising target for diagnosis and therapy.

Peptide-functionalized graphene oxide quantum dots as colorectal cancer theranostics.

Colorectal cancer (CRC) accounts for approximately 10% of all new cancer cases worldwide with significant morbidity and mortality. The current imaging techniques are lacking diagnostic precision while traditional chemotherapeutic strategies are limited by their adverse side effects and poor response in advanced stages. Targeted nanoparticles (NPs) can specifically bind to surface antigens on cancer cells and provide effective delivery of diagnostic and chemotherapeutic agent. Placenta-specific protein 1 (PLAC-1) is overexpressed in CRC and can be used as a target for detection and treatment of the disease. The aim of this work was to develop a targeted nanotheranostic agent for early diagnosis and inhibition of the malignant progression and metastasis of CRC. Graphene oxide quantum dots (QD) were covalently labeled with a peptide (GILGFVFTL) having high affinity to PLAC-1. The covalent coupling between the QD and the peptide was confirmed using a series of physicochemical and morphological characterization techniques. Confocal microscopy was used to evaluate the uptake of QD and QD-P in HCT-29, HT-116 and LS-180 CRC cell lines. Selective targeting of antigen PLAC-1 overexpressed on HT-29 and HCT-116 cells was measured by immunofluorescence. Cell proliferation, cell invasion and extent of PLAC-1 expression in CRC cells after treatment with QD and QD-P were determined. The prepared QD-P showed a significant increase in targeting and specific uptake in cells expressing the antigen PLAC-1 compared to non-functionalized QD. Treatment with QD-P also increased the cell cytotoxicity, reduced the invasiveness of HT-29 and HCT-116 cells by 38% and 62%, respectively, and downregulated the expression of PLAC-1 by 53% and 33%, respectively. These results highlight the potential use of QD-P as a theranostic agent for the detection and treatment of CRC cells expressing the antigen PLAC-1.

Optimum inhibition of MCF-7 breast cancer cells by efficient targeting of the macropinocytosis using optimized paclitaxel-loaded nanoparticles.

AIMS: Breast cancer (BC) is the third leading cause of death among other cancer types. Worldwide, it is the most common harmful disease in women, representing 1/4 of all cancers. Treatment of BC remains an ongoing challenge to most researchers. Understanding how cancer cells differ from normal cells can enhance drug targeting and overall disease progression. Endocytosis is a major physiological process modified in cancer cells and affects the cellular uptake of chemotherapeutic agents. MCF-7 breast cancer cells exhibit constitutive macropinocytic activity in comparison to normal non-macropinocytic MCF-10A breast cells. Therefore, we hypothesized that blocking the macropinocytosis mechanism in MCF-7 cells may inhibit the cancer progression while maintaining the safety of normal cells. MAIN METHODS: Using nano-precipitation technique, paclitaxel-PLGA-NPs were successfully prepared in the size range and charge required to opt for macropinocytosis in MCF-7 cells. KEY FINDINGS: Uptake and endocytosis inhibitor assays indicated that the developed NPs acquired size and surface charges that efficiently target macropinocytosis of MCF-7 cells. Paclitaxel-loaded PLGA-NPs showed higher efficacy against MCF-7 cells, while providing no toxicity on normal MCF-10A cells. Metabolomics analysis indicated the nutrients deprivation because of occupying the macropinocytosis. However, treatment of fresh MCF-7 cancer cells by metabolites secreted from PLGA-NPs-treated MCF-7 cells showed a potential metastatic activity. Thus, co- administration with an anti-metastatic drug is advised. SIGNIFICANCE: Collectively, adjusting the size and surface characteristics of a drug can critically control its cellular uptake, affecting the efficacy of drugs and the microenvironment of cancer cells.

SARS-CoV-2 infection- induced growth factors play differential roles in COVID-19 pathogenesis.

AIMS: Biologically active molecules cytokines and growth factors (GFs) are critical regulators of tissue injury/repair and emerge as key players in COVID-19 pathophysiology. However, specific disease stage of GFs dysregulation and, whether these GFs have associations with thromboembolism and tissue injury/repair in COVID-19 remain vague. MAIN METHODS: GF profiling in hospitalized moderate (non-ICU) and critically ill (ICU) COVID-19 patients was performed through legendPlex assay. KEY FINDINGS: Investigation revealed profound elevation of VEGF, PDGFs, EGF, TGF-α, FGF-basic, and erythropoietin (EPO) in moderate cases and decline or trend of decline with disease advancement. We found strong positive correlations of plasma VEGF, PDGFs, and EPO with endothelial dysfunction markers P-selectin and sCD40L. Interestingly, the HGF and G-CSF were upregulated at the moderate stage and remained elevated at the severe stage of COVID-19. Moreover, strong negative correlations of PDGFs (r2 = 0.238, P = 0.006), EPO (r2 = 0.18, P = 0.01) and EGF (r2 = 0.172, P = 0.02) and positive correlation of angiopoietin-2 (r2 = 0.267, P = 0.003) with D-dimer, a marker of thromboembolism, was observed. Further, plasma PDGFs (r2 = 0.199, P = 0.01), EPO (r2 = 0.115, P = 0.02), and EGF (r2 = 0.108, P = 0.07) exhibited negative correlations with tissue injury marker, myoglobin. SIGNIFICANCE: Taken together, unlike cytokines, most of the assessed GFs were upregulated at the moderate stage of COVID-19. The induction of GFs likely occurs due to endothelial dysfunction and may counter the adverse effects of cytokine storms which is reflected by inverse correlations of PDGFs, EPO, and EGF with thromboembolism and tissue injury markers. The findings suggest that the assessed GFs play differential roles in the pathogenesis of COVID-19.

SARS-CoV-2 infection induces soluble platelet activation markers and PAI-1 in the early moderate stage of COVID-19.

INTRODUCTION: Coagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID-19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID-19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID-19 to understand the pathogenesis. METHODS: To understand this, hospitalized COVID-19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including prothrombin time (PT), activated partial thromboplastin time (APTT), D-Dimer, and tissue factor pathway inhibitor (TFPI). The flow cytometry-based multiplex assays were performed to assess FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and D-Dimer. RESULTS: The investigations revealed induction of plasma P-selectin and CD40 ligand (sCD40L) in moderate COVID-19 cases, which were significantly abolished with the progression of COVID-19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID-19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI-1 more prominently in moderate, and tPA particularly in severe COVID-19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease. CONCLUSIONS: In summary, induction of soluble P-selectin, sCD40L, fibrinogen, and PAI-1 suggests the activation of platelets and coagulation system at the moderate stage before COVID-19 patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID-19 severity.

Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections.

To investigate intracellular heterogeneity, cell capture of particular cell populations followed by transcriptome analysis has been highly effective in freshly isolated tissues. However, this approach has been quite challenging in immunostained formalin-fixed paraffin-embedded (FFPE) sections. This study aimed at combining the standard pathology techniques, immunostaining and laser capture microdissection, with whole RNA-sequencing and bioinformatics analysis to characterize FFPE breast cancer cell populations with heterogeneous expression of progesterone receptor (PR). Immunocytochemical analysis revealed that 60% of MCF-7 cells admixture highly express PR. Immunocytochemistry-based targeted RNA-seq (ICC-RNAseq) and in silico functional analysis revealed that the PR-high cell population is associated with upregulation in transcripts implicated in immunomodulatory and inflammatory pathways (e.g. NF-κB and interferon signaling). In contrast, the PR-low cell population is associated with upregulation of genes involved in metabolism and mitochondrial processes as well as EGFR and MAPK signaling. These findings were cross-validated and confirmed in FACS-sorted PR high and PR-low MCF-7 cells and in MDA-MB-231 cells ectopically overexpressing PR. Significantly, ICC-RNAseq could be extended to analyze samples captured at specific spatio-temporal states to investigate gene expression profiles using diverse biomarkers. This would also facilitate our understanding of cell population-specific molecular events driving cancer and potentially other diseases.