Transcriptomics of a cytoglobin knockout mouse: Insights from hepatic stellate cells and brain.

The vertebrate respiratory protein cytoglobin (Cygb) is thought to exert multiple cellular functions. Here we studied the phenotypic effects of a Cygb knockout (KO) in mouse on the transcriptome level. RNA sequencing (RNA-Seq) was performed for the first time on sites of major endogenous Cygb expression, i.e. quiescent and activated hepatic stellate cells (HSCs) and two brain regions, hippocampus and hypothalamus. The data recapitulated the up-regulation of Cygb during HSC activation and its expression in the brain. Differential gene expression analyses suggested a role of Cygb in the response to inflammation in HSCs and its involvement in retinoid metabolism, retinoid X receptor (RXR) activation-induced xenobiotics metabolism, and RXR activation-induced lipid metabolism and signaling in activated cells. Unexpectedly, only minor effects of the Cygb KO were detected in the transcriptional profiles in hippocampus and hypothalamus, precluding any enrichment analyses. Furthermore, the transcriptome data pointed at a previously undescribed potential of the Cygb- knockout allele to produce cis-acting effects, necessitating future verification studies.

Acquired non-thermal plasma resistance mediates a shift towards aerobic glycolysis and ferroptotic cell death in melanoma.

AIMS: To gain insights into the underlying mechanisms of NTP therapy sensitivity and resistance, using the first-ever NTP-resistant cell line derived from sensitive melanoma cells (A375). METHODS: Melanoma cells were exposed to NTP and re-cultured for 12 consecutive weeks before evaluation against the parental control cells. Whole transcriptome sequencing analysis was performed to identify differentially expressed genes and enriched molecular pathways. Glucose uptake, extracellular lactate, media acidification, and mitochondrial respiration was analyzed to determine metabolic changes. Cell death inhibitors were used to assess the NTP-induced cell death mechanisms, and apoptosis and ferroptosis was further validated via Annexin V, Caspase 3/7, and lipid peroxidation analysis. RESULTS: Cells continuously exposed to NTP became 10 times more resistant to NTP compared to the parental cell line of the same passage, based on their half-maximal inhibitory concentration (IC50). Sequencing and metabolic analysis indicated that NTP-resistant cells had a preference towards aerobic glycolysis, while cell death analysis revealed that NTP-resistant cells exhibited less apoptosis but were more vulnerable to lipid peroxidation and ferroptosis. CONCLUSIONS: A preference towards aerobic glycolysis and ferroptotic cell death are key physiological changes in NTP-resistance cells, which opens new avenues for further, in-depth research into other cancer types.

The effect of local non-thermal plasma therapy on the cancer-immunity cycle in a melanoma mouse model.

Melanoma remains a deadly cancer despite significant advances in immune checkpoint blockade and targeted therapies. The incidence of melanoma is also growing worldwide, which highlights the need for novel treatment options and strategic combination of therapies. Here, we investigate non-thermal plasma (NTP), an ionized gas, as a promising, therapeutic option. In a melanoma mouse model, direct treatment of tumors with NTP results in reduced tumor burden and prolonged survival. Physical characterization of NTP treatment in situ reveals the deposited NTP energy and temperature associated with therapy response, and whole transcriptome analysis of the tumor identified several modulated pathways. NTP treatment also enhances the cancer-immunity cycle, as immune cells in both the tumor and tumor-draining lymph nodes appear more stimulated to perform their anti-cancer functions. Thus, our data suggest that local NTP therapy stimulates systemic, anti-cancer immunity. We discuss, in detail, how these fundamental insights will help direct the translation of NTP technology into the clinic and inform rational combination strategies to address the challenges in melanoma therapy.

Cytoglobin Silencing Promotes Melanoma Malignancy but Sensitizes for Ferroptosis and Pyroptosis Therapy Response.

Despite recent advances in melanoma treatment, there are still patients that either do not respond or develop resistance. This unresponsiveness and/or acquired resistance to therapy could be explained by the fact that some melanoma cells reside in a dedifferentiated state. Interestingly, this dedifferentiated state is associated with greater sensitivity to ferroptosis, a lipid peroxidation-reliant, iron-dependent form of cell death. Cytoglobin (CYGB) is an iron hexacoordinated globin that is highly enriched in melanocytes and frequently downregulated during melanomagenesis. In this study, we investigated the potential effect of CYGB on the cellular sensitivity towards (1S, 3R)-RAS-selective lethal small molecule (RSL3)-mediated ferroptosis in the G361 melanoma cells with abundant endogenous expression. Our findings show that an increased basal ROS level and higher degree of lipid peroxidation upon RSL3 treatment contribute to the increased sensitivity of CYGB knockdown G361 cells to ferroptosis. Furthermore, transcriptome analysis demonstrates the enrichment of multiple cancer malignancy pathways upon CYGB knockdown, supporting a tumor-suppressive role for CYGB. Remarkably, CYGB knockdown also triggers activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and subsequent induction of pyroptosis target genes. Altogether, we show that silencing of CYGB expression modulates cancer therapy sensitivity via regulation of ferroptosis and pyroptosis cell death signaling pathways.

Cytoglobin inhibits non-thermal plasma-induced apoptosis in melanoma cells through regulation of the NRF2-mediated antioxidant response.

Melanoma arises from pigment-producing cells called melanocytes located in the basal layers of the epidermis of the skin. Cytoglobin (CYGB) is a ubiquitously expressed hexacoordinated globin that is highly enriched in melanocytes and frequently downregulated during melanomagenesis. Previously, we showed that non-thermal plasma (NTP)-produced reactive oxygen and nitrogen species (RONS) lead to the formation of an intramolecular disulfide bridge that would allow CYGB to function as a redox-sensitive protein. Here, we investigate the cytotoxic effect of indirect NTP treatment in two melanoma cell lines with divergent endogenous CYGB expression levels, and we explore the role of CYGB in determining treatment outcome. Our findings are consistent with previous studies supporting that NTP cytotoxicity is mediated through the production of RONS and leads to apoptotic cell death in melanoma cells. Furthermore, we show that NTP-treated solutions elicit an antioxidant response through the activation of nuclear factor erythroid 2-related factor 2 (NRF2). The knockdown and overexpression of CYGB respectively sensitizes and protects melanoma cells from RONS-induced apoptotic cell death. The presence of CYGB enhances heme-oxygenase 1 (HO-1) and NRF2 protein expression levels, whereas the absence impairs their expression. Moreover, analysis of the CYGB-dependent transcriptome demonstrates the tumor suppressor long non-coding RNA maternally expressed 3 (MEG3) as a hitherto undescribed link between CYGB and NRF2. Thus, the presence of CYGB, at least in melanoma cells, seems to play a central role in determining the therapeutic outcome of RONS-inducing anticancer therapies, like NTP-treated solutions, possessing both tumor-suppressive and oncogenic features. Hence, CYGB expression could be of interest either as a biomarker or as a candidate for future targeted therapies in melanoma.

Reprogramming of glucocorticoid receptor function by hypoxia.

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance.

Plasma treatment causes structural modifications in lysozyme, and increases cytotoxicity towards cancer cells.

Bacterial and mammalian proteins, such as lysozyme, are gaining increasing interest as anticancer drugs. This study aims to modify the lysozyme structure using cold atmospheric plasma to boost its cancer cell killing effect. We investigated the structure at acidic and neutral pH using various experimental techniques (circular dichroism, fluorescence, and mass spectrometry) and molecular dynamics simulations. The controlled structural modification of lysozyme at neutral pH enhances its activity, while the activity was lost at acidic pH at the same treatment conditions. Indeed, a larger number of amino acids were oxidized at acidic pH after plasma treatment, which results in a greater distortion of the lysozyme structure, whereas only limited structural changes were observed in lysozyme after plasma treatment at neutral pH. We found that the plasma-treated lysozyme significantly induced apoptosis to the cancer cells. Our results reveal that plasma-treated lysozyme could have potential as a new cancer cell killing drug.

A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma.

Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.

Oxidation of Innate Immune Checkpoint CD47 on Cancer Cells with Non-Thermal Plasma.

Non-thermal plasma (NTP) therapy has been emerging as a promising cancer treatment strategy, and recently, its ability to locally induce immunogenic cancer cell death is being unraveled. We hypothesized that the chemical species produced by NTP reduce immunosuppressive surface proteins and checkpoints that are overexpressed on cancerous cells. Here, 3D in vitro tumor models, an in vivo mouse model, and molecular dynamics simulations are used to investigate the effect of NTP on CD47, a key innate immune checkpoint. CD47 is immediately modulated after NTP treatment and simulations reveal the potential oxidized salt-bridges responsible for conformational changes. Umbrella sampling simulations of CD47 with its receptor, signal-regulatory protein alpha (SIRPα), demonstrate that the induced-conformational changes reduce its binding affinity. Taken together, this work provides new insight into fundamental, chemical NTP-cancer cell interaction mechanisms and a previously overlooked advantage of present NTP cancer therapy: reducing immunosuppressive signals on the surface of cancer cells.

Structural modification of NADPH oxidase activator (Noxa 1) by oxidative stress: An experimental and computational study.

NADPH oxidases 1 (NOX1) derived reactive oxygen species (ROS) play an important role in the progression of cancer through signaling pathways. Therefore, in this paper, we demonstrate the effect of cold atmospheric plasma (CAP) on the structural changes of Noxa1 SH3 protein, one of the regulatory subunits of NOX1. For this purpose, firstly we purified the Noxa1 SH3 protein and analyzed the structure using X-ray crystallography, and subsequently, we treated the protein with two types of CAP reactors such as pulsed dielectric barrier discharge (DBD) and Soft Jet for different time intervals. The structural deformation of Noxa1 SH3 protein was analyzed by various experimental methods (circular dichroism, fluorescence, and NMR spectroscopy) and by MD simulations. Additionally, we demonstrate the effect of CAP (DBD and Soft Jet) on the viability and expression of NOX1 in A375 cancer cells. Our results are useful to understand the structural modification/oxidation occur in protein due to reactive oxygen and nitrogen (RONS) species generated by CAP.

Non-Thermal Plasma as a Unique Delivery System of Short-Lived Reactive Oxygen and Nitrogen Species for Immunogenic Cell Death in Melanoma Cells.

Breakthroughs in cancer immunotherapies have demonstrated considerable success, though not without limitations. Non-thermal plasma (NTP) for cancer therapy has been emerging as a potential adjuvant treatment via induction of immunogenic cell death (ICD). Cancer cells undergoing ICD stimulate a patient's immune system to mount an anticancer response. While promising, the underlying mechanisms of NTP-induced ICD must be closely examined. Here, the interaction between non-thermal plasma and cancerous cells is studied. The short-lived reactive oxygen and nitrogen species (e.g., hydroxyl radicals, atomic oxygen, nitric oxide) produced by plasma are the main effectors that elicit ICD in melanoma while, surprisingly, persistent species do not. This is demonstrated in vitro using a dielectric barrier discharge plasma system and is validated in a vaccination assay in vivo. Plasma generation of reactive species appears to be dictated by the total energy. Collectively, this work provides fundamental insight into plasma interactions with biological material. Furthermore, it lays the foundation for future development of NTP systems for clinical translation. The addition of plasma systems into the existing arsenal of cancer therapies opens the possibility for new combination strategies for safer and more robust control of cancer.

The effect of reactive oxygen and nitrogen species on the structure of cytoglobin: A potential tumor suppressor.

Many current anti-cancer therapies rely on increasing the intracellular reactive oxygen and nitrogen species (RONS) contents with the aim to induce irreparable damage, which subsequently results in tumor cell death. A novel tool in cancer therapy is the use of cold atmospheric plasma (CAP), which has been found to be very effective in the treatment of many different cancer cell types in vitro as well as in vivo, mainly through the vast generation of RONS. One of the key determinants of the cell's fate will be the interaction of RONS, generated by CAP, with important proteins, i.e. redox-regulatory proteins. One such protein is cytoglobin (CYGB), a recently discovered globin proposed to be involved in the protection of the cell against oxidative stress. In this study, the effect of plasma-produced RONS on CYGB was investigated through the treatment of CYGB with CAP for different treatment times. Spectroscopic analysis of CYGB showed that although chemical modifications occur, its secondary structure remains intact. Mass spectrometry experiments identified these modifications as oxidations of mainly sulfur-containing and aromatic amino acids. With longer treatment time, the treatment was also found to induce nitration of the heme. Furthermore, the two surface-exposed cysteine residues of CYGB were oxidized upon treatment, leading to the formation of intermolecular disulfide bridges, and potentially also intramolecular disulfide bridges. In addition, molecular dynamics and docking simulations confirmed, and further show, that the formation of an intramolecular disulfide bond, due to oxidative conditions, affects the CYGB 3D structure, thereby opening the access to the heme group, through gate functioning of His117. Altogether, the results obtained in this study (1) show that plasma-produced RONS can extensively oxidize proteins and (2) that the oxidation status of two redox-active cysteines lead to different conformations of CYGB.