Redox pathways in melanoma.

Cases of melanoma are doubling every 12 years, and in stages III and IV, the disease is associated with high mortality rates concomitant with unresectable metastases and therapeutic drug resistance. Despite some advances in treatment success, there is a marked need to understand more about the pathology of the disease. The present review provides an overview of how melanoma cells use and modulate redox pathways to facilitate thiol homeostasis and melanin biosynthesis and describes plausible redox targets that may improve therapeutic approaches in managing malignant disease and metastasis. Melanotic melanoma has some unique characteristics. Making melanin requires a considerable dedication of cellular energy resources and utilizes glutathione and glutathione transferases in certain steps in the biosynthetic pathway. Melanin is an antioxidant but is also functionally important in hematopoiesis and influential in various aspects of host immune responses, giving it unique characteristics. Together with other redox traits that are specific to melanoma, a discussion of possible therapeutic approaches is also provided.

Adaptive changes in tumor cells in response to reductive stress.

Reductive stress is characterized by an excess of cellular electron donors and can be linked with various human pathologies including cancer. We developed melanoma cell lines resistant to reductive stress agents: rotenone (ROTR), n-acetyl-L-cysteine, (NACR), or dithiothreitol (DTTR). Resistant cells divided more rapidly and had intracellular homeostatic redox-couple ratios that were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROTR cells had significant changes in mitochondrial morphology with higher numbers that were more isolated, fragmented and swollen, with greater membrane depolarization and decreased numbers of networks. These changes were accompanied by lower basal oxygen consumption and maximal respiration rates. Whole cell flux analyses and mitochondrial function assays showed that NACR and DTTR preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROTR used ketone body substrates such as D, L-ß-hydroxybutyric acid. NACR and DTTR cells had constitutively decreased levels of reactive oxygen species (ROS), although this was accompanied by activation of nuclear factor erythroid 2-related factor 2 (Nrf2), with concomitant increased expression of the downstream gene products such as glutathione S-transferase P (GSTP). Further adaptations included enhanced expression of endoplasmic reticulum proteins controlling the unfolded protein response (UPR). Although expression patterns of these UPR proteins were distinct between the resistant cells, a trend implied that resistance to reductive stress is accompanied by a constitutively increased UPR phenotype in each line. Overall, tumor cells, although tolerant of oxidative stress, can adapt their energy and survival mechanisms in lethal reductive stress conditions.

Microsomal glutathione transferase 1 in cancer and the regulation of ferroptosis.

Microsomal glutathione transferase 1 (MGST1) is a member of the MAPEG family (membrane associated proteins in eicosanoid and glutathione metabolism), defined according to enzymatic activities, sequence motifs, and structural properties. MGST1 is a homotrimer which can bind three molecules of glutathione (GSH), with one modified to a thiolate anion displaying one-third-of-sites-reactivity. MGST1 has both glutathione transferase and peroxidase activities. Each is based on stabilizing the GSH thiolate in the same active site. MGST1 is abundant in the liver and displays a broad subcellular distribution with high levels in endoplasmic reticulum and mitochondrial membranes, consistent with a physiological role in protection from reactive electrophilic intermediates and oxidative stress. In this review paper, we particularly focus on recent advances made in understanding MGST1 activation, induction, broad subcellular distribution, and the role of MGST1 in apoptosis, ferroptosis, cancer progression, and therapeutic responses.

Protein disulfide isomerase family mediated redox regulation in cancer.

Protein disulfide isomerase (PDI) and its superfamilies are mainly endoplasmic reticulum (ER) resident proteins with essential roles in maintaining cellular homeostasis, via thiol oxidation/reduction cycles, chaperoning, and isomerization of client proteins. Since PDIs play an important role in ER homeostasis, their upregulation supports cell survival and they are found in a variety of cancer types. Despite the fact that the importance of PDI to tumorigenesis remains to be understood, it is emerging as a new therapeutic target in cancer. During the past decade, several PDI inhibitors has been developed and commercialized, but none has been approved for clinical use. In this review, we discuss the properties and redox regulation of PDIs within the ER and provide an overview of the last 5 years of advances regarding PDI inhibitors.

Microsomal glutathione transferase 1 controls metastasis and therapeutic response in melanoma.

While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.

A role for microsomal glutathione transferase 1 in melanin biosynthesis and melanoma progression.

Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.

The Multifaceted Role of Glutathione S-Transferases in Health and Disease.

In humans, the cytosolic glutathione S-transferase (GST) family of proteins is encoded by 16 genes presented in seven different classes. GSTs exhibit remarkable structural similarity with some overlapping functionalities. As a primary function, GSTs play a putative role in Phase II metabolism by protecting living cells against a wide variety of toxic molecules by conjugating them with the tripeptide glutathione. This conjugation reaction is extended to forming redox sensitive post-translational modifications on proteins: S-glutathionylation. Apart from these catalytic functions, specific GSTs are involved in the regulation of stress-induced signaling pathways that govern cell proliferation and apoptosis. Recently, studies on the effects of GST genetic polymorphisms on COVID-19 disease development revealed that the individuals with higher numbers of risk-associated genotypes showed higher risk of COVID-19 prevalence and severity. Furthermore, overexpression of GSTs in many tumors is frequently associated with drug resistance phenotypes. These functional properties make these proteins promising targets for therapeutics, and a number of GST inhibitors have progressed in clinical trials for the treatment of cancer and other diseases.

Progress in biological activities and biosynthesis of edible fungi terpenoids.

The edible fungi have both edible and medicinal functions, in which terpenoids are one of the most important active ingredients. Terpenoids possess a wide range of biological activities and show great potential in the pharmaceutical and healthcare industries. In this review, the diverse biological activities of edible fungi terpenoids were summarized with emphasis on the mechanism of anti-cancer and anti-inflammation. Subsequently, this review focuses on advances in knowledge and understanding of the biosynthesis of terpenoids in edible fungi, especially in the generation of sesquiterpenes, diterpenes, and triterpenes. This paper is aim to provide an overview of biological functions and biosynthesis developed for utilizing the terpenoids in edible fungi.

A Synthetic Small RNA Homologous to the D-Loop Transcript of mtDNA Enhances Mitochondrial Bioenergetics.

Mitochondrial malfunction is a hallmark of many diseases, including neurodegenerative disorders, cardiovascular and lung diseases, and cancers. We previously found that alveolar progenitor cells, which are more resistant to cigarette smoke-induced injury than the other cells of the lung parenchyma, upregulate the mtDNA-encoded small non-coding RNA mito-ncR-805 after exposure to smoke. The mito-ncR-805 acts as a retrograde signal between the mitochondria and the nucleus. Here, we identified a region of mito-ncR-805 that is conserved in the mammalian mitochondrial genomes and generated shorter versions of mouse and human transcripts (mmu-CR805 and hsa-LDL1, respectively), which differ in a few nucleotides and which we refer to as the "functional bit". Overexpression of mouse and human functional bits in either the mouse or the human lung epithelial cells led to an increase in the activity of the Krebs cycle and oxidative phosphorylation, stabilized the mitochondrial potential, conferred faster cell division, and lowered the levels of proapoptotic pseudokinase, TRIB3. Both oligos, mmu-CR805 and hsa-LDL1 conferred cross-species beneficial effects. Our data indicate a high degree of evolutionary conservation of retrograde signaling via a functional bit of the D-loop transcript, mito-ncR-805, in the mammals. This emphasizes the importance of the pathway and suggests a potential to develop this functional bit into a therapeutic agent that enhances mitochondrial bioenergetics.

Cisplatin chemotherapy and renal function.

Cisplatin has been a mainstay of cancer chemotherapy since the 1970s. Despite its broad anticancer potential, its clinical use has regularly been constrained by kidney toxicities. This review details those biochemical pathways and metabolic conversions that underlie the kidney toxicities. A wide range of redox events contribute to the eventual physiological consequences of drug activities.

Development and validation of prognostic nomograms for single large and huge hepatocellular carcinoma after curative resection.

AIM: The prediction model of postoperative survival for single large and huge hepatocellular carcinoma (SLH-HCC, diameter > 5.0 cm) without portal vein tumour thrombus has not been well established. This study aimed to develop novel nomograms to predict postoperative recurrence and survival of these patients. METHODS: Data from 2469 patients with SLH-HCC who underwent curative resection from January 2005 to December 2015 in China were retrospectively collected. Specifically, nomograms of recurrence-free survival (RFS) and overall survival (OS) using data from a training cohort were developed with the Cox regression model (n = 1012). The modes were verified in an internal validation cohort (n = 338) and an external cohort comprising four tertiary institutions (n = 1119). RESULTS: The nomograms of RFS and OS based on tumour clinicopathologic features (diameter, differentiation, microvascular invasion, α-fetoprotein), operative factors (preoperative transcatheter arterial chemoembolisation therapy, scope of liver resection and intraoperative blood transfusion), underlying liver function (albumin-bilirubin grade) and systemic inflammatory or immune status (neutrophil-to-lymphocyte ratio) achieved high C-indexes of 0.85 (95% confidence interval [CI], 0.79-0.91) and 0.86 (95% CI, 0.79-0.93) in the training cohort, respectively, which were significantly higher than those of the five conventional HCC staging systems (0.62-0.73 for RFS, 0.63-0.75 for OS). The nomograms were validated in the internal cohort (0.83 for RFS, 0.84 for OS) and external cohort (0.87 for RFS, 0.88 for OS) and had well-fitted calibration curves. Our nomograms accurately stratified patients with SLH-HCC into low-, intermediate- and high-risk groups of postsurgical recurrence and mortality. CONCLUSIONS: The two nomograms achieved optimal prediction for postsurgical recurrence and OS for patients with SLH-HCC after curative resection.

Flavin Adenine Dinucleotide Depletion Caused by electron transfer flavoprotein subunit alpha Haploinsufficiency Leads to Hepatic Steatosis and Injury in Zebrafish.

The electron transfer flavoprotein (ETF) complex, made up of the ETF alpha subunit (ETFA), ETF beta subunit (ETFB), and ETF dehydrogenase (ETFDH), regulates fatty acid ß-oxidation activity while scavenging leaked electrons through flavin adenine dinucleotide (FAD)/reduced form FAD (FADH2) redox reactions in mitochondria. Here, we hypothesized that ETF dysfunction-mediated FAD deficiency may result in increased mitochondrial oxidative stress and steatosis and subsequent liver injury. We report that etfa haploinsufficiency caused hyperlipidemia, hypercholesterolemia, and hepatic steatosis and injury in adult zebrafish. Further, etfa+/ - mutant livers had reduced levels of FAD and glutathione and an increase in reactive oxygen species. Because FAD depletion might be critical in the pathogenesis of the liver lesion identified in etfa+/ - mutants, we used riboflavin to elevate FAD levels in the liver and found that riboflavin supplementation significantly suppressed hepatic steatosis and injury in etfa+/ - mutants through suppression of oxidative stress and de novo lipogenesis in the liver. Additionally, we found that adenosine triphosphate-linked mitochondrial oxygen consumption and mitochondrial membrane potential were reduced in etfa+/ - primary hepatocytes and that riboflavin supplementation corrected these defects. Conclusion: FAD depletion caused by etfa haploinsufficiency plays a key role in hepatic steatosis and oxidative stress-mediated hepatic injury in adult zebrafish. This raises the possibility that people with ETFA haploinsufficiency have a high risk for developing liver disease.

Development of Telintra as an Inhibitor of Glutathione S-Transferase P.

Glutathione S-transferase P (GSTP) is a component of a complex series of pathways that provide cellular redox homeostasis. It is an abundant protein in certain tumors and is over-expressed in cancer drug resistance. It has diverse cellular functions that include, thiolase activities with small electrophilic agents or susceptible cysteine residues on the protein to mediate S-glutathionylation, and chaperone binding with select protein kinases. Preclinical and clinical testing of a nanomolar inhibitor of GSTP, TLK199 (Telintra; Ezatiostat) has indicated a role for the enzyme in hematopoiesis and utility for the drug in the treatment of patients with myelodysplastic syndrome.

Altered redox regulation and S-glutathionylation of BiP contribute to bortezomib resistance in multiple myeloma.

Multiple myeloma (MM) cells have high rates of secretion of proteins rich in disulfide bonds and depend upon compartmentalized redox balance for accurate protein folding. The proteasome inhibitor bortezomib (Btz) is a successful frontline treatment for the disease, but its long-term efficacy is restricted by the acquisition of resistance. We found that MM cell lines resistant to Btz maintain high levels of oxidative stress and are cross resistant to endoplasmic reticulum (ER) stress-inducing agents thapsigargin (ThG), and tunicamycin (TuM). Moreover, cells expressing high/wild type levels of glutathione S-transferase P (GSTP) are more resistant than Gstp1/p2 knockout cells. In agreement, basal levels of S-glutathionylated proteins and redox regulation enzymes, including GSTP are elevated at mRNA and protein levels in resistant cells. GSTP mediated S-glutathionylation (SSG) regulates the activities of a number of redox active ER proteins. Here we demonstrated that the post-translational modification determines the balance between foldase and ATPase activities of the binding immunoglobulin protein (BiP), with Cys41-SSG important for ATPase, and Cys420-SSG for foldase. BiP expression and S-glutathionylation are increased in clinical specimens of bone marrow from MM patients compared to non-cancerous samples. Preventing S-glutathionylation in MM cells with a GSTP specific inhibitor restored BiP activities and reversed resistance to Btz. Therefore, S-glutathionylation of BiP confers pro-survival advantages and represents a novel mechanism of drug resistance in MM cells. We conclude that altered GSTP expression leads to S-glutathionylation of BiP, and contributes to acquired resistance to Btz in MM.

PTEN and PHLPP crosstalk in cancer cells and in TGFß-activated stem cells.

Akt kinase regulates several cellular processes, among them growth, proliferation and survival, and has been correlated to neoplastic disease. We report here crosstalk between several Akt regulatory phosphatases that controls the level of the activated form (phosphorylated) of Akt and affects tumor cell aggressiveness. In prostate cancer cell lines, we observed that transient transfection of PTEN decreased the endogenous level of PHLPPs and in contrast, the transient transfection of PHLPPs decreased the endogenous level of PTEN. Furthermore, silencing of PTEN by siRNA resulted in increased PHLPP levels. This phenomenon was not seen in non-transformed cells or in prostate stem cells. This crosstalk promoted cancer cell invasion and was controlled by epigenetically regulated processes where activation of miRs (miR-190 and miR214), the polycomb group of proteins and DNA methylation were involved. The purinergic P2X4 receptor, which has been shown to have a role in wound healing, was identified to be the mediator of this crosstalk. We also studied prostate stem cells and found this crosstalk in the TGFß1-activated epithelial-mesenchymal transition (EMT). The crosstalk seemed to be a natural part of EMT. In summary, we identify a crosstalk between Akt phosphatases which is not present in non-transformed prostate cells but occurs in cancer cells and stem cells transformed by TGFß-1. This crosstalk is important for cellular invasion. BACKGROUND: Phosphatases regulate the Akt oncogene. RESULTS: Crosstalk between Akt phosphatases in prostate cancer cells and in TGF-ß1 activated stem cells but not in non-transformed cells. CONCLUSION: This back-up mechanism facilitates invasive migration of prostate stem and cancer cells. SIGNIFICANCE: Characterization of Akt regulation may lead to a better understanding of tumor development and to novel strategies for treatment.

An Efficient Strategy for Enhancement of Bioactive Compounds in the Fruit Body of Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), by Spraying Biotic Elicitors.

Cordyceps militaris is a mushroom species with high nutritive and medicinal values based on diverse bioactive metabolites. The contents of bioactive ingredients are indicative of the quality of commercially available fruit body of this fungus. Although the application of biotic elicitors has been an efficient strategy to induce the accumulation of valuable bioactive compounds in vivo, related research in C. militaris is rarely reported. In this study, five biotic elicitors in different concentrations (0.05, 0.5, 1, and 2 mg/mL), including chitosan (CHT), 2,4-dichlorophenoxyacetic acid (2,4-D), methyl jasmonate (MeJA), gibberellic acid (GA), and triacontanol (TRIA), were first introduced to enhance the production of 10 kinds of major bioactive components in the fruit body of C. militaris. Results showed that the effect of biotic elicitors on bioactive compounds in the fruit body of C. militaris was elicitor-specific and concentration-dependent. Overall, 1 mg/L CHT was considered the most favorable for the production of 10 bioactive ingredients in C. militaris fruit body, which could increase the content of protein, polysaccharides, polyphenol, triterpenoids, flavonoids, cordyceps acid, cordycepin, and anthocyanins by 20.38-, 1.41-, 0.7-, 0.47-, 11.90-, 1.09-, 0.34-, and 2.64-fold, respectively, compared with the control. The results of this study would provide an efficient strategy for the production of a superior quality fruit body of and contribute to further elucidation of the effects of biotic elicitors on metabolite accumulation in C. militaris.

Racial disparities, cancer and response to oxidative stress.

At the intersection of genetics, biochemistry and behavioral sciences, there is a largely untapped opportunity to consider how ethnic and racial disparities contribute to individual sensitivity to reactive oxygen species and how these might influence susceptibility to various cancers and/or response to classical cancer treatment regimens that pervasively result in the formation of such chemical species. This chapter begins to explore these connections and builds a platform from which to consider how the disciplines can be strengthened further.

An evolving understanding of the S-glutathionylation cycle in pathways of redox regulation.

By nature of the reversibility of the addition of glutathione to low pKa cysteine residues, the post-translational modification of S-glutathionylation sanctions a cycle that can create a conduit for cell signaling events linked with cellular exposure to oxidative or nitrosative stress. The modification can also avert proteolysis by protection from over-oxidation of those clusters of target proteins that are substrates. Altered functions are associated with S-glutathionylation of proteins within the mitochondria and endoplasmic reticulum compartments, and these impact energy production and protein folding pathways. The existence of human polymorphisms of enzymes involved in the cycle (particularly glutathione S-transferase P) create a scenario for inter-individual variance in response to oxidative stress and a number of human diseases with associated aberrant S-glutathionylation have now been identified.

S-Glutathionylation of estrogen receptor α affects dendritic cell function.

Glutathione S-transferase Pi (GSTP) is a thiolase that catalyzes the addition of glutathione (GSH) to receptive cysteines in target proteins, producing an S-glutathionylated residue. Accordingly, previous studies have reported that S-glutathionylation is constitutively decreased in cells from mice lacking GSTP (Gstp1/p2-/-). Here, we found that bone marrow-derived dendritic cells (BMDDCs) from Gstp1/p2-/- mice have proliferation rates that are greater than those in their WT counterparts (Gstp1/p2+/+). Moreover, Gstp1/p2-/- BMDDCs had increased reactive oxygen species (ROS) levels and decreased GSH:glutathione disulfide (GSSG) ratios. Estrogen receptor α (ERα) is linked to myeloproliferation and differentiation, and we observed that its steady-state levels are elevated in Gstp1/p2-/- BMDDCs, indicating a link between GSTP and ERα activities. BMDDCs differentiated by granulocyte-macrophage colony-stimulating factor had elevated ERα levels, which were more pronounced in Gstp1/p2-/- than WT mice. When stimulated with lipopolysaccharide for maturation, Gstp1/p2-/- BMDDCs exhibited augmented endocytosis, maturation rate, cytokine secretion, and T-cell activation; heightened glucose uptake and glycolysis; increased Akt signaling (in the mTOR pathway); and decreased AMPK-mediated phosphorylation of proteins. Of note, GSTP formed a complex with ERα, stimulating ERα S-glutathionylation at cysteines 221, 245, 417, and 447; altering ERα's binding affinity for estradiol; and reducing overall binding potential (receptor density and affinity) 3-fold. Moreover, in Gstp1/p2-/- BMDDCs, ERα S-glutathionylation was constitutively decreased. Taken together, these findings suggest that GSTP-mediated S-glutathionylation of ERα controls BMDDC differentiation and affects metabolic function in dendritic cells.