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.

Nuclear factor kappa B expression in non-small cell lung cancer.

In this mini-review, we discuss the role of NF-κB, a proinflammatory transcription factor, in the expression of genes involved in inflammation, proliferation, and apoptosis pathways, and link it with prognosis of various human cancers, particularly non-small cell lung cancer (NSCLC). We and others have shown that NF-κB activity can be impacted by post-translational S-glutathionylation through reversible formation of a mixed disulfide bond between its cysteine residues and glutathione (GSH). Clinical data analysis showed that high expression of NF-κB correlated with shorter overall survival (OS) in NSCLC patients, suggesting a tumor promotion function for NF-κB. Moreover, NF-κB expression was associated with tumor stage, lymph node metastasis, and 5-year OS in these patients. NF-κB was over-expressed in the cytoplasm of tumor tissue compared to adjacent normal tissues. S-glutathionylation of NF-κB caused negative regulation by interfering with DNA binding activities of NF-κB subunits. In response to oxidants, S-glutathionylation of NF-κB also correlated with enhanced lung inflammation. Thus, S-glutathionylation is an important contributor to NF-κB regulation and clinical results highlight the importance of NF-κB in NSCLC, where NF-κB levels are associated with unfavorable prognosis.

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.

Reductive stress in cancer.

Reductive stress is defined as a condition characterized by excess accumulation of reducing equivalents (e.g., NADH, NADPH, GSH), surpassing the activity of endogenous oxidoreductases. Excessive reducing equivalents can perturb cell signaling pathways, change the formation of disulfide bonding in proteins, disturb mitochondrial homeostasis or decrease metabolism. Reductive stress is influenced by cellular antioxidant load, its flux and a subverted homeostasis that paradoxically can result in excess ROS induction. Balanced reducing equivalents and antioxidant enzymes that contribute to reductive stress can be regulated by Nrf2, typically considered as an oxidative stress induced transcription factor. Cancer cells may coordinate distinct pools of redox couples under reductive stress and these may link to biological consequences from both molecular and translational standpoints. In cancer, there is recent interest in understanding how selective induction of reductive stress may influence therapeutic management and disease progression.

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.

Nuclear PFKP promotes CXCR4-dependent infiltration by T cell acute lymphoblastic leukemia.

PFKP (phosphofructokinase, platelet), the major isoform of PFK1 expressed in T cell acute lymphoblastic leukemia (T-ALL), is predominantly expressed in the cytoplasm to carry out its glycolytic function. Our study showed that PFKP is a nucleocytoplasmic shuttling protein with functional nuclear export and nuclear localization sequences (NLSs). Cyclin D3/CDK6 facilitated PFKP nuclear translocation by dimerization and by exposing the NLS of PFKP to induce the interaction between PFKP and importin 9. Nuclear PFKP stimulated the expression of C-X-C chemokine receptor type 4 (CXCR4), a chemokine receptor regulating leukemia homing/infiltration, to promote T-ALL cell invasion, which depended on the activity of c-Myc. In vivo experiments showed that nuclear PFKP promoted leukemia homing/infiltration into the bone marrow, spleen, and liver, which could be blocked with CXCR4 antagonists. Immunohistochemical staining of tissues from a clinically well-annotated cohort of T cell lymphoma/leukemia patients showed nuclear PFKP localization in invasive cancers, but not in nonmalignant T lymph node or reactive hyperplasia. The presence of nuclear PFKP in these specimens correlated with poor survival in patients with T cell malignancy, suggesting the potential utility of nuclear PFKP as a diagnostic marker.

Nrf2 inhibition sensitizes breast cancer stem cells to ionizing radiation via suppressing DNA repair.

Radiation is widely used for cancer treatment but the radioresistance properties of cancer stem cells (CSCs) pose a significant challenge to the success of cancer therapy. Nuclear factor erythroid-2-related factor 2 (Nrf2) has emerged as a prominent regulator of cellular antioxidant responses and its over-activation is associated with drug resistant in cancer cells. However, the role of Nrf2 signaling in regulating the response of CSCs to irradiation has yet to be defined. Here, we show that exposure of triple-negative breast cancer (TNBC) cells to ionizing radiation (IR) upregulates Nrf2 expression and promotes its nuclear translocation in a reactive oxygen species (ROS)-dependent manner. Ectopic overexpression of Nrf2 attenuates, whereas knockdown of Nrf2 potentiates IR-induced killing of TNBC CSCs. Mechanistically, we found that Nrf2 knockdown increases IR-induced ROS production and impedes DNA repair at least in part via inhibition of DNA-PK. Furthermore, activation of Nrf2 by sulforaphane diminishes, whereas inhibition of Nrf2 by ML385 enhances IR-induced killing of TNBC CSCs. Collectively, these results demonstrate that IR-induced ROS production can activate Nrf2 signaling, which in turn counteracts the killing effect of irradiation. Therefore, pharmacological inhibition of IR-induced Nrf2 activation by ML385 could be a new therapeutic approach to sensitize therapy-resistant CSCs to radiotherapy.

Glutathione S-Transferase P Influences Redox Homeostasis and Response to Drugs that Induce the Unfolded Protein Response in Zebrafish.

We have created a novel glutathione S-transferase π1 (gstp1) knockout (KO) zebrafish model and used it for comparative analyses of redox homeostasis and response to drugs that cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). Under basal conditions, gstp1 KO larvae had higher expression of antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) accompanied by a more reduced larval environment and a status consistent with reductive stress. Compared with wild type, various UPR markers were decreased in KO larvae, but treatment with drugs that induce ER stress caused greater toxicities and increased expression of Nrf2 and UPR markers in KO. Tunicamycin and 02-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/nitric oxide) activated inositol-requiring protein-1/X-box binding protein 1 pathways, whereas thapsigargin caused greater activation of protein kinase-like ER kinase/activating transcription factor 4/CHOP pathways. These results suggest that this teleost model is useful for predicting how GSTP regulates organismal management of oxidative/reductive stress and is a determinant of response to drug-induced ER stress and the UPR. SIGNIFICANCE STATEMENT: A new zebrafish model has been created to study the importance of glutathione S-transferase π1 in development, redox homeostasis, and response to drugs that enact cytotoxicity through endoplasmic reticulum stress and induction of the unfolded protein response.

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.

Oxidative Stress in Cancer.

Contingent upon concentration, reactive oxygen species (ROS) influence cancer evolution in apparently contradictory ways, either initiating/stimulating tumorigenesis and supporting transformation/proliferation of cancer cells or causing cell death. To accommodate high ROS levels, tumor cells modify sulfur-based metabolism, NADPH generation, and the activity of antioxidant transcription factors. During initiation, genetic changes enable cell survival under high ROS levels by activating antioxidant transcription factors or increasing NADPH via the pentose phosphate pathway (PPP). During progression and metastasis, tumor cells adapt to oxidative stress by increasing NADPH in various ways, including activation of AMPK, the PPP, and reductive glutamine and folate metabolism.

Voltage-Dependent Anion Channels Influence Cytotoxicity of ME-344, a Therapeutic Isoflavone.

ME-344 is a second-generation cytotoxic isoflavone with anticancer activity promulgated through interference with mitochondrial functions. Using a click chemistry version of the drug together with affinity-enriched mass spectrometry, voltage-dependent anion channels (VDACs) 1 and 2 were identified as drug targets. To determine the importance of VDAC1 or 2 to cytotoxicity, we used lung cancer cells that were either sensitive (H460) or intrinsically resistant (H596) to the drug. In H460 cells, depletion of VDAC1 and VDAC2 by small interfering RNA impacted ME-344 effects by diminishing generation of reactive oxygen species (ROS), preventing mitochondrial membrane potential dissipation, and moderating ME-344-induced cytotoxicity and mitochondrial-mediated apoptosis. Mechanistically, VDAC1 and VDAC2 knockdown prevented ME-344-induced apoptosis by inhibiting Bax mitochondrial translocation and cytochrome c release as well as apoptosis in these H460 cells. We conclude that VDAC1 and 2, as mediators of the response to oxidative stress, have roles in modulating ROS generation, Bax translocation, and cytochrome c release during mitochondrial-mediated apoptosis caused by ME-344. SIGNIFICANCE STATEMENT: Dissecting preclinical drug mechanisms are of significance in development of a drug toward eventual Food and Drug Administration approval.

Nrf2-modulation by seleno-hormetic agents and its potential for radiation protection.

The trace element selenium (Se) is an essential component of selenoproteins and plays a critical role in redox signaling via regulating the activity of selenoenzymes such as thioredoxin reductase-1 and glutathione peroxidases. Se compounds and its metabolites possess a wide range of biological functions including anticancer and cytoprotection effects, modulation of hormetic genes and antioxidant enzyme activities. Radiation-induced injury of normal tissues is a significant side effect for cancer patients who receive radiotherapy in the clinic and the development of new and effective radioprotectors is an important goal of research. Others and we have shown that seleno-compounds have the potential to protect ionizing radiation-induced toxicities in various tissues and cells both in in vitro and in vivo studies. In this review, we discuss the potential utilization of Se compounds with redox-dependent hormetic activity as novel radio-protective agents to alleviate radiation toxicity. The cellular and molecular mechanisms underlying the radioprotection effects of these seleno-hormetic agents are also discussed. These include Nrf2 transcription factor modulation and the consequent upregulation of the adaptive stress response to IR in bone marrow stem cells and hematopoietic precursors.

S-Glutathionylated Serine Proteinase Inhibitors as Biomarkers for Radiation Exposure in Prostate Cancer Patients.

In biological tissues, radiation causes the formation of reactive oxygen species (ROS), some of which lead to sequential oxidation of certain protein cysteine residues. Resultant cysteinyl radicals are subject to post-translational modification through S-glutathionylation. The present clinical trial was designed to determine if S-glutathionylated serine protease inhibitors (serpins) in blood could be used as biomarkers of exposure to radiation. 56 male prostate cancer patients treated with radiotherapy were enrolled in the trial and levels of S-glutathionylated serpins A1 and A3 were assessed by immunoblotting. Patients were classified into three groups: (1) external beam radiation therapy (EBRT); (2) brachytherapy (BT); (3) both EBRT and BT. Prior to treatment, baseline plasma levels of both unmodified and S-glutathionylated serpins were similar in each group. We identified elevated plasma levels of S-glutathionylated serpin A1 monomer, trimer and serpin A3 monomer in patient blood following radiation. Maximal increased levels of these S-glutathionylated serpins were correlated with increased duration of radiotherapy treatments. We conclude that it is practical to quantify patient plasma S-glutathionylated serpins and that these post-translationally modified proteins are candidate biomarkers for measuring radiation exposure. This provides a platform for use of such biomarkers in trials with the range of drugs that, like radiation, produce ROS.

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.

Isoflavone ME-344 Disrupts Redox Homeostasis and Mitochondrial Function by Targeting Heme Oxygenase 1.

ME-344 is a second-generation isoflavone with unusual cytotoxic properties that is in clinical testing in cancer. To identify targets that contribute to its anticancer activity and therapeutic index, we used lung cancer cell lines that are naturally sensitive or resistant to ME-344. Drug-induced apoptosis was linked with enhanced levels of reactive oxygen species and this initiated a nuclear erythroid factor 2-like 2 signaling response, downstream of which, heme oxygenase 1 (HO-1) was also found to be time-dependently inhibited by ME-344. ME-344 specifically bound to, and altered, HO-1 structure and increased HO-1 translocation from the rough endoplasmic reticulum to mitochondria, but only in drug-sensitive cells. These effects did not occur in either drug-resistant or primary lung fibroblasts with lower HO-1 basal levels. HO-1 was confirmed as a drug target by using surface plasmon resonance technology and through interaction with a clickable ME-344 compound (M2F) and subsequent proteomic analyses, showing direct binding of ME-344 with HO-1. Proteomic analysis showed that clusters of mitochondrial proteins, including voltage-dependent anion-selective channels, were also impacted by ME-344. Human lung cancer biopsies expressed higher levels of Nrf2 and HO-1 compared with normal tissues. Overall, our data show that ME-344 inhibits HO-1 and impacts its mitochondrial translocation. Other mitochondrial proteins are also affected, resulting in interference in tumor cell redox homeostasis and mitochondrial function. These factors contribute to a beneficial therapeutic index and support continued clinical development of ME-344. SIGNIFICANCE: A novel cytotoxic isoflavone is shown to inhibit heme oxygenase, a desirable yet elusive target that disrupts redox homeostasis causing cell death.