Low zinc environment induces stress signaling, senescence and mixed cell death modalities in colon cancer cells.

Currently it is not clear what type of the final cellular response (i.e. cell death modality or senescence) is induced upon chronic intracellular zinc depletion in colon cancer cells. To address this question, isogenic colon cancer lines SW480 and SW620 exposed to low zinc environment were studied over the period of 6 weeks. Low zinc environment reduced total as well as free intracellular zinc content in both cell lines. Decreased intracellular zinc content resulted in changes in cellular proliferation, cell cycle distribution and activation of stress signaling. In addition, colonocytes with low zinc content displayed increased levels of oxidative stress, changes in mitochondrial activity but in the absence of significant DNA damage. Towards the end of treatment (4th-6th week), exposed cells started to change morphologically, and typical markers of senescence as well as cell death appeared. Of two examined colon cancer cell lines, SW480 cells proved to activate predominantly senescent phenotype, with frequent form of demise being necrosis and mixed cell death modality but not apoptosis. Conversely, SW620 cells activated mostly cell death, with relatively equal distribution of apoptosis and mixed types, while senescent phenotypes and necrosis were present only in a small fraction of cell populations. Addition of zinc at the beginning of 4th week of treatment significantly suppressed cell death phenotypes in both cell lines but had no significant effect on senescence. In conclusion, presented results demonstrate variability of responses to chronic zinc depletion in colon cancer as modeled in vitro.

Chloroquine Enhances Death in Lung Adenocarcinoma A549 Cells Exposed to Cold Atmospheric Plasma Jet.

Cold atmospheric plasma (CAP) is an intensively-studied approach for the treatment of malignant neoplasms. Various active oxygen and nitrogen compounds are believed to be the main cytotoxic effectors on biotargets; however, the comprehensive mechanism of CAP interaction with living cells and tissues remains elusive. In this study, we experimentally determined the optimal discharge regime (or semi-selective regime) for the direct CAP jet treatment of cancer cells, under which lung adenocarcinoma A549, A427 and NCI-H23 cells demonstrated substantial suppression of viability, coupled with a weak viability decrease of healthy lung fibroblasts Wi-38 and MRC-5. The death of CAP-exposed cancer and healthy cells under semi-selective conditions was caspase-dependent. We showed that there was an accumulation of lysosomes in the treated cells. The increased activity of lysosomal protease Cathepsin D, the transcriptional upregulation of autophagy-related MAPLC3B gene in cancer cells and the changes in autophagy-related proteins may have indicated the activation of autophagy. The addition of the autophagy inhibitor chloroquine (CQ) after the CAP jet treatment increased the death of A549 cancer cells in a synergistic manner and showed a low effect on the viability of CAP-treated Wi-38 cells. Downregulation of Drp1 mitochondrial protein and upregulation of PINK1 protein in CAP + CQ treated cells indicated that CQ increased the CAP-dependent destabilization of mitochondria. We concluded that CAP weakly activated pro-survival autophagy in irradiated cells, and CQ promoted CAP-dependent cell death due to the destabilization of autophagosomes formation and mitochondria homeostasis. To summarize, the combination of CAP treatment with CQ could be useful for the development of cold plasma-based antitumor approaches for clinical application.

Polytonic Drug Release via Multi-Hierarchical Microstructures Enabled by Nano-Metamaterials.

″Nano-metamaterials″, rationally designed novel class metamaterials with multilevel microarchitectures and both characteristic sizes and whole sizes at the nanoscale, are introduced into the area of drug delivery system (DDS), and the relationship between release profile and treatment efficacy at the single-cell level is revealed for the first time. Fe3+ -core-shell-corona nano-metamaterials (Fe3+ -CSCs) are synthesized using a dual-kinetic control strategy. The hierarchical structure of Fe3+ -CSCs, with a homogeneous interior core, an onion-like shell, and a hierarchically porous corona. A novel polytonic drug release profile occurred, which consists of three sequential stages: burst release, metronomic release, and sustained release. The Fe3+ -CSCs results in overwhelming accumulation of lipid reactive oxygen species (ROS), cytoplasm ROS, and mitochondrial ROS in tumor cells and induces unregulated cell death. This cell death modality causes cell membranes to form blebs, seriously corrupting cell membranes to significantly overcome the drug-resistance issues. It is first demonstrated that nano-metamaterials of well-defined microstructures can modulate drug release profile at the single cell level, which in turn alters the downstream biochemical reactions and subsequent cell death modalities. This concept has significant implications in the drug delivery area and can serve to assist in designing potential intelligent nanostructures for novel molecular-based diagnostics and therapeutics.

Mitochondrial sirtuin 3 and various cell death modalities.

Sirtuin 3, a member of the mammalian sirtuin family of proteins, is involved in the regulation of multiple processes in cells. It is a major mitochondrial NAD+-dependent deacetylase with a broad range of functions, such as regulation of oxidative stress, reprogramming of tumor cell energy pathways, and metabolic homeostasis. One of the intriguing functions of sirtuin 3 is the regulation of mitochondrial outer membrane permeabilization, a key step in apoptosis initiation/progression. Moreover, sirtuin 3 is involved in the execution of various cell death modalities, which makes sirtuin 3 a possible regulator of crosstalk between them. This review is focused on the role of sirtuin 3 as a target for tumor cell elimination and how mitochondria and reactive oxygen species (ROS) are implicated in this process.

From Differential DNA Methylation in COPD to Mitochondria: Regulation of AHRR Expression Affects Airway Epithelial Response to Cigarette Smoke.

Cigarette smoking causes hypomethylation of the gene Aryl Hydrocarbon Receptor Repressor (AHRR), which regulates detoxification and oxidative stress-responses. We investigated whether AHRR DNA methylation is related to chronic obstructive pulmonary disease (COPD) and studied its function in airway epithelial cells (AECs). The association with COPD was assessed in blood from never and current smokers with/without COPD, and in AECs from ex-smoking non-COPD controls and GOLD stage II-IV COPD patients cultured with/without cigarette smoke extract (CSE). The effect of CRISPR/Cas9-induced AHRR knockout on proliferation, CSE-induced mitochondrial membrane potential and apoptosis/necrosis in human bronchial epithelial 16HBE cells was studied. In blood, DNA methylation of AHRR at cg05575921 and cg21161138 was lower in smoking COPD subjects than smoking controls. In vitro, AHRR DNA methylation at these CpG-sites was lower in COPD-derived than control-derived AECs only upon CSE exposure. Upon AHRR knockout, we found a lower proliferation rate at baseline, stronger CSE-induced decrease in mitochondrial membrane potential, and higher CSE-induced late apoptosis/necroptosis. Together, our results show lower DNA methylation of AHRR upon smoking in COPD patients compared to non-COPD controls. Our data suggest that higher airway epithelial AHRR expression may lead to impaired cigarette smoke-induced mitochondrial dysfunction and apoptosis/necroptosis, potentially promoting unprogrammed/immunogenic cell death.

Aqueous extract of Stevia rebaudiana (Bertoni) Bertoni abrogates death-related signaling pathways via boosting the expression profile of oxidative defense systems.

Indigenous inhabitants of South America and other areas have been using stevia as a traditional medicine for years, but its impact on cell signaling pathways has not been well studied yet. We evaluated the impacts of aqueous extract of Stevia rebaudiana (Bertoni) Bertoni on the expression of the selected genes involved in significant cell death modalities, including p53-DNA damage and the cellular antioxidative defense in pancreatic tissues in STZ-induced diabetic rats and murine pancreatic cell lines. The in vivo study revealed that aqueous extract of Stevia significantly upregulated the expression of GSTM1 and P1 and GPX (4.67, 12.08, and 2.81 fold, respectively; all p < .05) along with significant downregulation of the genes which were upregulated by STZ, including apoptotic genes caspase-3 and -9 (-9.80 and -4.16 fold, p < .05, respectively) and necroptotic genes, RIP1K, 2 K, and 3 K (-9.48, -2.70, and -12.9 fold, respectively, all p < .05). In vitro studies also revealed comparable results. In conclusion, the observed clinical improvements in diabetic rats are the result of overexpression of major genes of antioxidative defense systems in the course of a significant downregulation of major cell death modalities. PRACTICAL APPLICATIONS: The popularity of noncaloric sweeteners, including stevia, has rocketed in recent years, but the consumption of stevia as traditional medicine has a long history. The findings of the current study provide strong mechanistic lines of evidence supporting the beneficial biological effects of stevia as a noncaloric sweetener in diabetes.

Cell death at the cross roads of host-pathogen interaction in Mycobacterium tuberculosis infection.

Tuberculosis (TB) continues to be the leading cause of death by any single infectious agent, accounting for around 1.7 million annual deaths globally, despite several interventions and support programs by national and international agencies. With the development of drug resistance in Mycobacterium tuberculosis (M. tb), there has been a paradigm shift in TB research towards host-directed therapy. The potential targets include the interactions between host and bacterial proteins that are crucial for pathogenesis. Hence, collective efforts are being made to understand the molecular details of host-pathogen interaction for possible translation into host-directed therapy. The present review focuses on 'host cell death modalities' of host-pathogen interaction, which play a crucial role in determining the outcome of TB disease progression. Several cell death modalities that occur in response to mycobacterial infection have been identified in human macrophages either as host defences for bacterial clearance or as pathogen strategies for multiplication and dissemination. These cell death modalities include apoptosis, necrosis, pyroptosis, necroptosis, pyronecrosis, NETosis, and autophagy. These processes are highly overlapping with several mycobacterial proteins participating in more than one cell death pathway. Until now, reviews in M. tb and host cell death have discussed either focusing on host evasion strategies, apoptosis, autophagy, and necrosis or describing all these forms with limited discussions of their role in host-pathogen interactions. Here, we present a comprehensive review of various mycobacterial factors modulating host cell death pathways and the cross-talk between them. Besides this, we have discussed the networking of host cell death pathways including the interference of host miRNA during M. tb infection with their respective targets. Through this review, we present the host targets that overlap across several cell death modalities and the technical limitations of methodology in cell death research. Given the compelling need to discover alternative drug target(s), this review identifies these overlapping cell death factors as potential targets for host-directed therapy.

Autophagy and the invisible line between life and death.

For a considerable time cell death has been considered to represent mutually exclusive states with cell death modalities that are governed by their inherent and unique mode of action involving specific molecular entities and have therefore been studied primarily in isolation. It is now, however, becoming increasingly clear that these modalities are regulated by similar pathways and share a number of initiator and effector molecules that control both cell death as well as cell survival mechanisms, demanding a newly aligned and integrative approach of cell death assessment. Frequently cell death is triggered through a dual action that incorporates signaling events associated with more than one death modality. Apoptosis and necrosis regularly co-operate in a tightly balanced interplay that involves autophagy to serve context dependently either as a pro-survival or a pro-death mechanism. In this review we will assess current cell death modalities and their molecular overlap with the goal of clarifying the controversial role of autophagy in the cell death response. By dissecting the key molecular pathways and their positioning within a network of regulatory signalling hubs and checkpoints we discuss a distinct approach that integrates autophagy with a resultant cell death manifestation. In doing so, former classifications of cell death modalities fade and reveal the intricate molecular proportions and complexities of the cell death response that may contribute towards an enhanced means of cell death control.