Polyploidy as an Adaptation against Loss of Heterozygosity in Cancer

Polyploidy is common in cancer cells and has implications for tumor progression and resistance to therapies, but it is unclear whether it is an adaptation of the tumor or the non-adaptive effect of genomic instability. I discuss the possibility that polyploidy reduces the deleterious effects of loss of heterozygosity, which arises as a consequence of mitotic recombination, and which in diploid cells leads instead to the rapid loss of complementation of recessive deleterious mutations. I use computational predictions of loss of heterozygosity to show that a population of diploid cells dividing by mitosis with recombination can be easily invaded by mutant polyploid cells or cells that divide by endomitosis, which reduces loss of complementation, or by mutant cells that occasionally fuse, which restores heterozygosity. A similar selective advantage of polyploidy has been shown for the evolution of different types of asexual reproduction in nature. This provides an adaptive explanation for cyclical ploidy, mitotic slippage and cell fusion in cancer cells.     

AXL Knock-Out in SNU475 Hepatocellular Carcinoma Cells Provides Evidence for Lethal Effect Associated with G2 Arrest and Polyploidization

AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients.     

Cancer Development,Progression, and Therapy: An Epigenetic Overview

Carcinogenesis involves uncontrolled cell growth, which follows the activation of oncogenes and/or the deactivation of tumor suppression genes. Metastasis requires down-regulation of cell adhesion receptors necessary for tissue-specific, cell–cell attachment, as well as up-regulation of receptors that enhance cell motility. Epigenetic changes, including histone modifications, DNA methylation, and DNA hydroxymethylation, can modify these characteristics. Targets for these epigenetic changes include signaling pathways that regulate apoptosis and autophagy, as well as microRNA. We propose that predisposed normal cells convert to cancer progenitor cells that, after growing, undergo an epithelial-mesenchymal transition. This process, which is partially under epigenetic control, can create a metastatic form of both progenitor and full-fledged cancer cells, after which metastasis to a distant location may occur. Identification of epigenetic regulatory mechanisms has provided potential therapeutic avenues. In particular, epigenetic drugs appear to potentiate the action of traditional therapeutics, often by demethylating and re-expressing tumor suppressor genes to inhibit tumorigenesis. Epigenetic drugs may inhibit both the formation and growth of cancer progenitor cells, thus reducing the recurrence of cancer. Adopting epigenetic alteration as a new hallmark of cancer is a logical and necessary step that will further encourage the development of novel epigenetic biomarkers and therapeutics.     

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer.     

Implication of tumor microenvironment in chemoresistance: tumor-associated stromal cells protect tumor cells from cell death

Tumor development principally occurs following the accumulation of genetic and epigenetic alterations in tumor cells. These changes pave the way for the transformation of chemosensitive cells to chemoresistant ones by influencing the uptake, metabolism, or export of drugs at the cellular level. Numerous reports have revealed the complexity of tumors and their microenvironment with tumor cells located within a heterogeneous population of stromal cells. These stromal cells (fibroblasts, endothelial or mesothelial cells, adipocytes or adipose tissue-derived stromal cells, immune cells and bone marrow-derived stem cells) could be involved in the chemoresistance that is acquired by tumor cells via several mechanisms: (i) cell-cell and cell-matrix interactions influencing the cancer cell sensitivity to apoptosis; (ii) local release of soluble factors promoting survival and tumor growth (crosstalk between stromal and tumor cells); (iii) direct cell-cell interactions with tumor cells (crosstalk or oncologic trogocytosis); (iv) generation of specific niches within the tumor microenvironment that facilitate the acquisition of drug resistance; or (v) conversion of the cancer cells to cancer-initiating cells or cancer stem cells. This review will focus on the implication of each member of the heterogeneous population of stromal cells in conferring resistance to cytotoxins and physiological mediators of cell death.     

3D-Printed Collagen-Based Waveform Microfibrous Scaffold for Periodontal Ligament Reconstruction

Reconstruction of the periodontal ligament (PDL) to fulfill functional requirement remains a challenge. This study sought to develop a biomimetic microfibrous system capable of withstanding the functional load to assist PDL regeneration. Collagen-based straight and waveform microfibers to guide PDL cell growth were prepared using an extrusion-based bioprinter, and a laminar flow-based bioreactor was used to generate fluidic shear stress. PDL cells were seeded on the respective microfibers with 0 or 6 dynes/cm² fluidic shear stress for 1–4 h. The viability, morphology, adhesion pattern, and gene expression levels of PDL cells were assessed. The results revealed that upon bioprinting optimization, collagen-based microfibers were successfully fabricated. The straight microfibers were 189.9 ± 11.44 μm wide and the waveform microfibers were 235.9 ± 11.22 μm wide. Under 6 dynes/cm² shear stress, PDL cells were successfully seeded, and cytoskeleton expansion, adhesion, and viability were greater. Cyclin D, E-cadherin, and periostin were upregulated on the waveform microfibers. In conclusion, 3D-printed collagen-based waveform microfibers preserved PDL cell viability and exhibited an enhanced tendency to promote healing and regeneration under shear stress. This approach is promising for the development of a guiding scaffold for PDL regeneration.     

Diabetic Kinome Inhibitors—A New Opportunity for β-Cells Restoration

Diabetes, and several diseases related to diabetes, including cancer, cardiovascular diseases and neurological disorders, represent one of the major ongoing threats to human life, becoming a true pandemic of the 21st century. Current treatment strategies for diabetes mainly involve promoting β-cell differentiation, and one of the most widely studied targets for β-cell regeneration is DYRK1A kinase, a member of the DYRK family. DYRK1A has been characterized as a key regulator of cell growth, differentiation, and signal transduction in various organisms, while further roles and substrates are the subjects of extensive investigation. The targets of interest in this review are implicated in the regulation of β-cells through DYRK1A inhibition—through driving their transition from highly inefficient and death-prone populations into efficient and sufficient precursors of islet regeneration. Increasing evidence for the role of DYRK1A in diabetes progression and β-cell proliferation expands the potential for pharmaceutical applications of DYRK1A inhibitors. The variety of new compounds and binding modes, determined by crystal structure and in vitro studies, may lead to new strategies for diabetes treatment. This review provides recent insights into the initial self-activation of DYRK1A by tyrosine autophosphorylation. Moreover, the importance of developing novel DYRK1A inhibitors and their implications for the treatment of diabetes are thoroughly discussed. The evolving understanding of DYRK kinase structure and function and emerging high-throughput screening technologies have been described. As a final point of this work, we intend to promote the term “diabetic kinome” as part of scientific terminology to emphasize the role of the synergistic action of multiple kinases in governing the molecular processes that underlie this particular group of diseases.     

Adipose-Derived Stem Cells Preincubated with Green Tea EGCG Enhance Pancreatic Tissue Regeneration in Rats with Type 1 Diabetes through ROS/Sirt1 Signaling Regulation

Type 1 diabetes stem-cell-based therapy is one of the best therapeutic approaches for pancreatic damage treatment due to stem cell tissue regeneration. Epigallocatechin gallate (EGCG) is one of the active components found in green tea. Experimental results suggest that EGCG shows beneficial effects on cell protection. This study explores whether a better pancreatic regeneration therapeutic effect could be found in mesenchymal stem cells pretreated with EGCG compared to stem cells without EGCG pretreatment. A cell model confirmed that adipose-derived stem cells (ADSC) incubated with EGCG increase cell viability under high-glucose (HG) stress. This is due to survival marker p-Akt expression. In an animal model, type 1 diabetes induced the activation of several pathological signals, including islet size reduction, extracellular fibrotic collagen deposition, oxidative stress elevation, survival pathway suppression, apoptosis signaling induction, and Sirt1 antioxidant pathway downregulation. Ordinary ADSC transplantation slightly improved the above pathological signals. Further, EGCG-pretreated ADSC transplantation significantly improved the above pathological conditions. Taken together, EGCG-pretreated ADSCs show clinical potential in the treatment of patients with type 1 diabetes through the regeneration of damaged pancreatic tissues.     

High Concentrations of Genistein Decrease Cell Viability Depending on Oxidative Stress and Inflammation in Colon Cancer Cell Lines

Genistein could play a crucial role in modulating three closely linked physiological processes altered during cancer: oxidative stress, mitochondrial biogenesis, and inflammation. However, genistein’s role in colorectal cancer remains unclear. We aimed to determine genistein’s effects in two colon cancer cells: HT29 and SW620, primary and metastatic cancer cells, respectively. After genistein treatment for 48 h, cell viability and hydrogen peroxide (H₂O₂) production were studied. The cell cycle was studied by flow cytometry, mRNA and protein levels were analyzed by RT-qPCR and Western blot, respectively, and finally, cytoskeleton remodeling and NF-κB translocation were determined by confocal microscopy. Genistein 100 µM decreased cell viability and produced G₂/M arrest, increased H₂O₂, and produced filopodia in SW620 cells. In HT29 cells, genistein produced an increase of cell death, H₂O₂ production, and in the number of stress fibers. In HT29 cells, mitochondrial biogenesis was increased, however, in SW620 cells, it was decreased. Finally, the expression of inflammation-related genes increased in both cell lines, being greater in SW620 cells, where NF-κB translocation to the nucleus was higher. These results indicate that high concentrations of genistein could increase oxidative stress and inflammation in colon cancer cells and, ultimately, decrease cell viability.     

Apoptosis as Driver of Therapy-Induced Cancer Repopulation and Acquired Cell-Resistance (CRAC): A Simple In Vitro Model of Phoenix Rising in Prostate Cancer

Apoptotic cells stimulate compensatory proliferation through the caspase-3-cPLA-2-COX-2-PGE-2-STAT3 Phoenix Rising pathway as a healing process in normal tissues. Phoenix Rising is however usurped in cancer, potentially nullifying pro-apoptotic therapies. Cytotoxic therapies also promote cancer cell plasticity through epigenetic reprogramming, leading to epithelial-to-mesenchymal-transition (EMT), chemo-resistance and tumor progression. We explored the relationship between such scenarios, setting-up an innovative, straightforward one-pot in vitro model of therapy-induced prostate cancer repopulation. Cancer (castration-resistant PC3 and androgen-sensitive LNCaP), or normal (RWPE-1) prostate cells, are treated with etoposide and left recovering for 18 days. After a robust apoptotic phase, PC3 setup a coordinate tissue-like response, repopulating and acquiring EMT and chemo-resistance; repopulation occurs via Phoenix Rising, being dependent on high PGE-2 levels achieved through caspase-3-promoted signaling; epigenetic inhibitors interrupt Phoenix Rising after PGE-2, preventing repopulation. Instead, RWPE-1 repopulate via Phoenix Rising without reprogramming, EMT or chemo-resistance, indicating that only cancer cells require reprogramming to complete Phoenix Rising. Intriguingly, LNCaP stop Phoenix-Rising after PGE-2, failing repopulating, suggesting that the propensity to engage/complete Phoenix Rising may influence the outcome of pro-apoptotic therapies. Concluding, we established a reliable system where to study prostate cancer repopulation, showing that epigenetic reprogramming assists Phoenix Rising to promote post-therapy cancer repopulation and acquired cell-resistance (CRAC).     

The Dynamics and Plasticity of Epigenetics in Diabetic Kidney Disease: Therapeutic Applications Vis-à-Vis

Chronic kidney disease (CKD) refers to the phenomenon of progressive decline in the glomerular filtration rate accompanied by adverse consequences, including fluid retention, electrolyte imbalance, and an increased cardiovascular risk compared to those with normal renal function. The triggers for the irreversible renal function deterioration are multifactorial, and diabetes mellitus serves as a major contributor to the development of CKD, namely diabetic kidney disease (DKD). Recently, epigenetic dysregulation emerged as a pivotal player steering the progression of DKD, partly resulting from hyperglycemia-associated metabolic disturbances, rising oxidative stress, and/or uncontrolled inflammation. In this review, we describe the major epigenetic molecular mechanisms, followed by summarizing current understandings of the epigenetic alterations pertaining to DKD. We highlight the epigenetic regulatory processes involved in several crucial renal cell types: Mesangial cells, podocytes, tubular epithelia, and glomerular endothelial cells. Finally, we highlight epigenetic biomarkers and related therapeutic candidates that hold promising potential for the early detection of DKD and the amelioration of its progression.     

GPR55 Receptor Activation by the N-Acyl Dopamine Family Lipids Induces Apoptosis in Cancer Cells via the Nitric Oxide Synthase (nNOS) Over-Stimulation

GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca²⁺, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential.     

Inactivation of the Akt/FOXM1 Signaling Pathway by Panobinostat Suppresses the Proliferation and Metastasis of Gastric Cancer Cells

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide. Histone deacetylase (HDAC) inhibitors are a new class of cytostatic agents available for the treatment of various cancers and diseases. Although numerous clinical and pre-clinical trials on the anticancer effects of panobinostat have been conducted, only a few reports have investigated its efficacy in gastric cancer. The present study aimed to investigate the effects of panobinostat in gastric cancer cells. Panobinostat significantly inhibited the cell viability and proliferation of the gastric cancer cell lines SNU484 and SNU638 in a dose-dependent manner; it reduced the colony-forming ability of these cells. Moreover, it induced apoptosis as indicated by increased protein levels of cleaved poly ADP-ribose polymerase and cleaved caspase-3. Panobinostat induced the G2/M cell cycle arrest in SNU484 and SNU638 cells and subsequently decreased the G2/M phase regulatory-associated protein expression of p-Wee1, Myt1, and Cdc2. Furthermore, panobinostat significantly inhibited the metastasis of SNU484 and SNU638 cells by regulating the expression of MMP-9 and E-cadherin. Further, it decreased the protein levels of p-Akt and forkhead box protein M1 (FOXM1). These effects were reversed by the Akt agonist SC79 and were accelerated by the Akt inhibitor LY2940002. Moreover, tumor growth in xenograft animal experiments was suppressed by panobinostat. These results indicated that panobinostat inhibits the proliferation, metastasis, and cell cycle progression of gastric cancer cells by promoting apoptosis and inactivating Akt/FOXM1 signaling. Cumulatively, our present study suggests that panobinostat is a potential drug for the treatment of gastric cancer.     

Oxidative Stress and Epigenetic Regulation in Ageing and Age-Related Diseases

Recent statistics indicate that the human population is ageing rapidly. Healthy, but also diseased, elderly people are increasing. This trend is particularly evident in Western countries, where healthier living conditions and better cures are available. To understand the process leading to age-associated alterations is, therefore, of the highest relevance for the development of new treatments for age-associated diseases, such as cancer, diabetes, Alzheimer and cardiovascular accidents. Mechanistically, it is well accepted that the accumulation of intracellular damage determined by reactive oxygen species (ROS) might orchestrate the progressive loss of control over biological homeostasis and the functional impairment typical of aged tissues. Here, we review how epigenetics takes part in the control of stress stimuli and the mechanisms of ageing physiology and physiopathology. Alteration of epigenetic enzyme activity, histone modifications and DNA-methylation is, in fact, typically associated with the ageing process. Specifically, ageing presents peculiar epigenetic markers that, taken altogether, form the still ill-defined “ageing epigenome”. The comprehension of mechanisms and pathways leading to epigenetic modifications associated with ageing may help the development of anti-ageing therapies.     

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca²⁺ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.     

Strategies to Improve the Efficiency of Somatic Cell Nuclear Transfer

Mammalian oocytes can reprogram differentiated somatic cells into a totipotent state through somatic cell nuclear transfer (SCNT), which is known as cloning. Although many mammalian species have been successfully cloned, the majority of cloned embryos failed to develop to term, resulting in the overall cloning efficiency being still low. There are many factors contributing to the cloning success. Aberrant epigenetic reprogramming is a major cause for the developmental failure of cloned embryos and abnormalities in the cloned offspring. Numerous research groups attempted multiple strategies to technically improve each step of the SCNT procedure and rescue abnormal epigenetic reprogramming by modulating DNA methylation and histone modifications, overexpression or repression of embryonic-related genes, etc. Here, we review the recent approaches for technical SCNT improvement and ameliorating epigenetic modifications in donor cells, oocytes, and cloned embryos in order to enhance cloning efficiency.     

Using Small Molecules to Dissect Non‐apoptotic Programmed Cell Death: Necroptosis,Ferroptosis, and Pyroptosis

Genetically programmed cell death is a universal and fundamental cellular process in multicellular organisms. Apoptosis and necroptosis, two common forms of programmed cell death, play vital roles in maintenance of homeostasis in metazoans. Dysfunction of the regulatory machinery of these processes can lead to carcinogenesis or autoimmune diseases. Inappropriate death of essential cells can lead to organ dysfunction or even death; ischemia–reperfusion injury and neurodegenerative disorders are examples of this. Recently, novel forms of non‐apoptotic programmed cell death have been identified. Although these forms of cell death play significant roles in both physiological and pathological conditions, the detailed molecular mechanisms underlying them are still poorly understood. Here, we discuss progress in using small molecules to dissect three forms of non‐apoptotic programmed cell death: necroptosis, ferroptosis, and pyroptosis.