Evaluation of Mitochondrial Phagy (Mitophagy) in Human Non-small Adenocarcinoma Tumor Cells.

Non-small cell lung cancer (NSCLC) is a predominant form of lung cancer characterized by its aggressive nature and high mortality rate, primarily due to late-stage diagnosis and metastatic spread. Recent studies underscore the pivotal role of mitophagy, a selective form of autophagy targeting damaged or superfluous mitochondria, in cancer biology, including NSCLC. Mitophagy regulation may influence cancer cell survival, proliferation, and metastasis by modulating mitochondrial quality and cellular energy homeostasis. Herein, we present a comprehensive methodology developed in our laboratory for the evaluation of mitophagy in NSCLC tumor cells. Utilizing a combination of immunoblotting, immunocytochemistry, and fluorescent microscopy, we detail the steps to quantify early and late mitophagy markers and mitochondrial dynamics. Our findings highlight the potential of targeting mitophagy pathways as a novel therapeutic strategy in NSCLC, offering insights into the complex interplay between mitochondrial dysfunction and tumor progression. This study not only sheds light on the significance of mitophagy in NSCLC but also establishes a foundational approach for its investigation, paving way for future research in this critical area of cancer biology.

Prioritization of genes for translation: a computational approach.

INTRODUCTION: Gene identification for genetic diseases is critical for the development of new diagnostic approaches and personalized treatment options. Prioritization of gene translation is an important consideration in the molecular biology field, allowing researchers to focus on the most promising candidates for further investigation. AREAS COVERED: In this paper, we discussed different approaches to prioritize genes for translation, including the use of computational tools and machine learning algorithms, as well as experimental techniques such as knockdown and overexpression studies. We also explored the potential biases and limitations of these approaches and proposed strategies to improve the accuracy and reliability of gene prioritization methods. Although numerous computational methods have been developed for this purpose, there is a need for computational methods that incorporate tissue-specific information to enable more accurate prioritization of candidate genes. Such methods should provide tissue-specific predictions, insights into underlying disease mechanisms, and more accurate prioritization of genes. EXPERT OPINION: Using advanced computational tools and machine learning algorithms to prioritize genes, we can identify potential targets for therapeutic intervention of complex diseases. This represents an up-and-coming method for drug development and personalized medicine.

Transforming Growth Factor Beta and Alveolar Rhabdomyosarcoma: A Challenge of Tumor Differentiation and Chemotherapy Response.

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.

A Lipidomics Approach to Determine the Role of Lipids and Its Crosstalk with Autophagy in Lung Cancer Metastasis.

Non-small cell lung cancer (NSCLC) is among the most malignant tumors with high propensity for metastasis and is the leading cause of cancer-related death globally. Most patients present with regional and distant metastasis, associated with poor prognosis. Lipids may play an essential role in either activating or inhibiting detachment-induced apoptosis (anoikis), where the latter is a crucial mechanism to prevent metastasis, and it may have a cross-talk with autophagy. Autophagy has been shown to be induced in various human cancer metastasis, modulating tumor cell motility and invasion, cancer cell differentiation, resistance to anoikis, and epithelial to mesenchymal transition. Hence, it may play a crucial role in the transition of benign to malignant phenotypes, the core of metastasis initiation. Here, we provide a method we have established in our laboratory for detecting lipids in attached and detached non-small lung cancer cells and show how to analyze lipidomics data to find its correlation with autophagy-related pathways.

The role of BCL2L13 in glioblastoma: turning a need into a target.

Glioblastoma (GBM) is the most common aggressive central nervous system cancer. GBM has a high mortality rate, with a median survival time of 12-15 months after diagnosis. A poor prognosis and a shorter life expectancy may result from resistance to standard treatments such as radiation and chemotherapy. Temozolomide has been the mainstay treatment for GBM, but unfortunately, there are high rates of resistance with GBM bypassing apoptosis. A proposed mechanism for bypassing apoptosis is decreased ceramide levels, and previous research has shown that within GBM cells, B cell lymphoma 2-like 13 (BCL2L13) can inhibit ceramide synthase. This review aims to discuss the causes of resistance in GBM cells, followed by a brief description of BCL2L13 and an explanation of its mechanism of action. Further, lipids, specifically ceramide, will be discussed concerning cancer and GBM cells, focusing on ceramide synthase and its role in developing GBM. By gathering all current information on BCL2L13 and ceramide synthase, this review seeks to enable an understanding of these pieces of GBM in the hope of finding an effective treatment for this disease.

Ceramides and ceramide synthases in cancer: Focus on apoptosis and autophagy.

Different studies corroborate a role for ceramide synthases and their downstream products, ceramides, in modulation of apoptosis and autophagy in the context of cancer. These mechanisms of regulation, however, appear to be context dependent in terms of ceramides' fatty acid chain length, subcellular localization, and the presence or absence of their downstream targets. Our current understanding of the role of ceramide synthases and ceramides in regulation of apoptosis and autophagy could be harnessed to pioneer the development of new treatments to activate or inhibit a single type of ceramide synthase, thereby regulating the apoptosis induction or cross talk of apoptosis and autophagy in cancer cells. Moreover, the apoptotic function of ceramide suggests that ceramide analogues can pave the way for the development of novel cancer treatments. Therefore, in the current review paper we discuss the impact of ceramide synthases and ceramides in regulation of apoptosis and autophagy in context of different types of cancers. We also briefly introduce the latest information on ceramide synthase inhibitors, their application in diseases including cancer therapy, and discuss approaches for drug discovery in the field of ceramide synthase inhibitors. We finally discussed strategies for developing strategies to use lipids and ceramides analysis in biological fluids for developing early biomarkers for cancer.

Integrating Multi-Omics Analysis for Enhanced Diagnosis and Treatment of Glioblastoma: A Comprehensive Data-Driven Approach.

The most aggressive primary malignant brain tumor in adults is glioblastoma (GBM), which has poor overall survival (OS). There is a high relapse rate among patients with GBM despite maximally safe surgery, radiation therapy, temozolomide (TMZ), and aggressive treatment. Hence, there is an urgent and unmet clinical need for new approaches to managing GBM. The current study identified modules (MYC, EGFR, PIK3CA, SUZ12, and SPRK2) involved in GBM disease through the NeDRex plugin. Furthermore, hub genes were identified in a comprehensive interaction network containing 7560 proteins related to GBM disease and 3860 proteins associated with signaling pathways involved in GBM. By integrating the results of the analyses mentioned above and again performing centrality analysis, eleven key genes involved in GBM disease were identified. ProteomicsDB and Gliovis databases were used for determining the gene expression in normal and tumor brain tissue. The NetworkAnalyst and the mGWAS-Explorer tools identified miRNAs, SNPs, and metabolites associated with these 11 genes. Moreover, a literature review of recent studies revealed other lists of metabolites related to GBM disease. The enrichment analysis of identified genes, miRNAs, and metabolites associated with GBM disease was performed using ExpressAnalyst, miEAA, and MetaboAnalyst tools. Further investigation of metabolite roles in GBM was performed using pathway, joint pathway, and network analyses. The results of this study allowed us to identify 11 genes (UBC, HDAC1, CTNNB1, TRIM28, CSNK2A1, RBBP4, TP53, APP, DAB1, PINK1, and RELN), five miRNAs (hsa-mir-221-3p, hsa-mir-30a-5p, hsa-mir-15a-5p, hsa-mir-130a-3p, and hsa-let-7b-5p), six metabolites (HDL, N6-acetyl-L-lysine, cholesterol, formate, N, N-dimethylglycine/xylose, and X2. piperidinone) and 15 distinct signaling pathways that play an indispensable role in GBM disease development. The identified top genes, miRNAs, and metabolite signatures can be targeted to establish early diagnostic methods and plan personalized GBM treatment strategies.

New Visions on Natural Products and Cancer Therapy: Autophagy and Related Regulatory Pathways.

Macroautophagy (autophagy) has been a highly conserved process throughout evolution and allows cells to degrade aggregated/misfolded proteins, dysfunctional or superfluous organelles and damaged macromolecules, in order to recycle them for biosynthetic and/or energetic purposes to preserve cellular homeostasis and health. Changes in autophagy are indeed correlated with several pathological disorders such as neurodegenerative and cardiovascular diseases, infections, cancer and inflammatory diseases. Conversely, autophagy controls both apoptosis and the unfolded protein response (UPR) in the cells. Therefore, any changes in the autophagy pathway will affect both the UPR and apoptosis. Recent evidence has shown that several natural products can modulate (induce or inhibit) the autophagy pathway. Natural products may target different regulatory components of the autophagy pathway, including specific kinases or phosphatases. In this review, we evaluated ~100 natural compounds and plant species and their impact on different types of cancers via the autophagy pathway. We also discuss the impact of these compounds on the UPR and apoptosis via the autophagy pathway. A multitude of preclinical findings have shown the function of botanicals in regulating cell autophagy and its potential impact on cancer therapy; however, the number of related clinical trials to date remains low. In this regard, further pre-clinical and clinical studies are warranted to better clarify the utility of natural compounds and their modulatory effects on autophagy, as fine-tuning of autophagy could be translated into therapeutic applications for several cancers.

Potential role of TGFΒ and autophagy in early crebellum development.

During development, the interconnected generation of various neural cell types within the cerebellar primordium is essential. Over embryonic (E) days E9-E13, Purkinje cells (PCs), and cerebellar nuclei (CN) neurons are among the created primordial neurons. The molecular and cellular mechanisms fundamental for the early cerebellar neurogenesis, migration/differentiation, and connectivity are not clear yet. Autophagy has a vital role in controlling cellular phenotypes, such as epithelial-to-mesenchymal transition (EMT) and endothelial to mesenchymal transition (EndMT). Transforming growth factor-beta 1 (TGF-ß1) is the main player in pre-and postnatal development and controlling cellular morphological type via various mechanisms, such as autophagy. Thus, we hypothesized that TGF-ß1 may regulate early cerebellar development by modifying the levels of cell adhesion molecules (CAMs) and consequently autophagy pathway in the mouse cerebellar primordium. We demonstrated the stimulation of the canonical TGF-ß1 signaling pathway at the point that concurs with the generation of the nuclear transitory zone and PC plate in mice. Furthermore, our data show that the stimulated TGF-ß1 signaling pathway progressively and chronologically could upregulate the expression of ß-catenin (CTNNB1) and N-cadherin (CDH2) with the most expression at E11 and E12, leading to upregulation of chromodomain helicase DNA binding protein 8 (CDH8) and neural cell adhesion molecule 1 (NCAM1) expression, at E12 and E13. Finally, we demonstrated that the stimulated TGF-ß signaling pathway may impede the autophagic flux at E11/E12. Nevertheless, basal autophagy flux happens at earlier developmental phases from E9-E10. Our study determined potential role of the TGF-ß signaling and its regulatory impacts on autophagic flux during cerebellar development and cadherin expression, which can facilitate the proliferation, migration/differentiation, and placement of PCs and the CN neurons in their designated areas.

Adiponectin Synthesis, Secretion and Extravasation from Circulation to Interstitial Space.

Adiponectin, an adipokine that circulates as multiple multimeric complexes at high levels in serum, has antidiabetic, anti-inflammatory, antiatherogenic, and cardioprotective properties. Understanding the mechanisms regulating adiponectin's physiological effects is likely to provide critical insight into the development of adiponectin-based therapeutics to treat various metabolic-related diseases. In this review, we summarize our current understanding on adiponectin action in its various target tissues and in cellular models. We also focus on recent advances in two particular regulatory aspects; namely, the regulation of adiponectin gene expression, multimerization, and secretion, as well as extravasation of circulating adiponectin to the interstitial space and its degradation. Finally, we discuss some potential therapeutic approaches using adiponectin as a target and the current challenges facing adiponectin-based therapeutic interventions.

BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance.Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3ß: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.

Mechanisms of muscle insulin resistance and the cross-talk with liver and adipose tissue.

Insulin resistance is a metabolic disorder affecting multiple tissues and is a precursor event to type 2 diabetes (T2D). As T2D affects over 425 million people globally, there is an imperative need for research into insulin resistance to better understand the underlying mechanisms. The proposed mechanisms involved in insulin resistance include both whole body aspects, such as inflammation and metabolic inflexibility; as well as cellular phenomena, such as lipotoxicity, ER stress, and mitochondrial dysfunction. Despite numerous studies emphasizing the role of lipotoxicity in the pathogenesis of insulin resistance, an understanding of the interplay between tissues and these proposed mechanisms is still emerging. Furthermore, the tissue-specific and unique responses each of the three major insulin target tissues and how each interconnect to regulate the whole body insulin response has become a new priority in metabolic research. With an emphasis on skeletal muscle, this mini-review highlights key similarities and differences in insulin signaling and resistance between different target-tissues, and presents the latest findings related to how these tissues communicate to control whole body metabolism.

Pleiotropic effects of statins: A focus on cancer.

The statin drugs ('statins') potently inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase by competitively blocking the active site of the enzyme. Statins decrease de novo cholesterol biosynthesis and thereby reduce plasma cholesterol levels. Statins exhibit "pleiotropic" properties that are independent of their lipid-lowering effects. For example, preclinical evidence suggests that statins inhibit tumor growth and induce apoptosis in specific cancer cell types. Furthermore, statins show chemo-sensitizing effects by impairing Ras family GTPase signaling. However, whether statins have clinically meaningful anti-cancer effects remains an area of active investigation. Both preclinical and clinical studies on the potential mechanisms of action of statins in several cancers have been reviewed in the literature. Considering the contradictory data on their efficacy, we present an up-to-date summary of the pleiotropic effects of statins in cancer therapy and review their impact on different malignancies. We also discuss the synergistic anti-cancer effects of statins when combined with other more conventional anti-cancer drugs to highlight areas of potential therapeutic development.

Mechanisms of simvastatin myotoxicity: The role of autophagy flux inhibition.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.

Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells significantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co-treatment with TMZ significantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not significantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is significantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death.