Peroxiporins in Triple-Negative Breast Cancer: Biomarker Potential and Therapeutic Perspectives.

Triple-negative breast cancer (TNBC) remains one of the most challenging subtypes since it is initially characterized by the absence of specific biomarkers and corresponding targeted therapies. Advances in methodology, translational informatics, genomics, and proteomics have significantly contributed to the identification of therapeutic targets. The development of innovative treatments, such as antibody-drug conjugates and immune checkpoint inhibitors, alongside chemotherapy, has now become the standard of care. However, the quest for biomarkers defining therapy outcomes is still ongoing. Peroxiporins, which comprise a subgroup of aquaporins, which are membrane pores facilitating the transport of water, glycerol, and hydrogen peroxide, have emerged as potential biomarkers for therapy response. Research on peroxiporins reveals their involvement beyond traditional channeling activities, which is also reflected in their cellular localization and roles in cellular signaling pathways. This research on peroxiporins provides fresh insights into the mechanisms of therapy resistance in tumors, offering potential avenues for predicting treatment outcomes and tailoring successful TNBC therapies.

AQP3 and AQP5 Modulation in Response to Prolonged Oxidative Stress in Breast Cancer Cell Lines.

Aquaporins are membrane pores regulating the transport of water, glycerol, and other small molecules across membranes. Among 13 human aquaporins, six have been shown to transport H2O2 and are therefore called peroxiporins. Peroxiporins are implicated in cancer development and progression, partly due to their involvement in H2O2 transport. Oxidative stress is linked to breast cancer development but is also a mechanism of action for conventional chemotherapy. The aim of this study is to investigate the effects of prolonged oxidative stress on Aquaporin 3 (AQP3), Aquaporin 5 (AQP5), and signaling pathways in breast cancer cell lines of different malignancies alongside a non-tumorigenic breast cell line. The prolonged oxidative stress caused responses in viability only in the cancer cell lines, while it affected cell migration in the MCF7 cell line. Changes in the localization of NRF2, a transcription factor involved in oxidative stress response, were observed only in the cancer cell lines, and no effects were recorded on its downstream target proteins. Moreover, the prolonged oxidative stress caused changes in AQP3 and AQP5 expression only in the cancer cell lines, in contrast to their non-malignant counterparts. These results suggest peroxiporins are potential therapeutic targets in cancer treatment. However, further research is needed to elucidate their role in the modulation of therapy response, highlighting the importance of research on this topic.

Schiff bases and their metal complexes to target and overcome (multidrug) resistance in cancer.

Overcoming multidrug resistance (MDR) is one of the major challenges in cancer therapy. In this respect, Schiff base-related compounds (bearing a R1R2CNR3 bond) gained high interest during the past decades. Schiff bases are considered privileged ligands for various reasons, including the easiness of their preparation and the possibility to form complexes with almost all transition metal ions. Schiff bases and their metal complexes exhibit many types of biological activities and are used for the treatment and diagnosis of various diseases. Until now, 13 Schiff bases have been investigated in clinical trials for cancer treatment and hypoxia imaging. This review represents the first collection of Schiff bases and their complexes which demonstrated MDR-reversal activity. The areas of drug resistance covered in this article involve: 1) Modulation of ABC transporter function, 2) Targeting lysosomal ABCB1 overexpression, 3) Circumvention of ABC transporter-mediated drug efflux by alternative routes of drug uptake, 4) Selective activity against MDR cancer models (collateral sensitivity), 5) Targeting GSH-detoxifying systems, 6) Overcoming apoptosis resistance by inducing necrosis and paraptosis, 7) Reactivation of mutated p53, 8) Restoration of sensitivity to DNA-damaging anticancer therapy, and 9) Overcoming drug resistance through modulation of the immune system. Through this approach, we would like to draw attention to Schiff bases and their metal complexes representing highly interesting anticancer drug candidates with the ability to overcome MDR.

The Involvement of Peroxiporins and Antioxidant Transcription Factors in Breast Cancer Therapy Resistance.

Breast cancer is still the leading cause of death in women of all ages. The reason for this is therapy resistance, which leads to the progression of the disease and the formation of metastases. Multidrug resistance (MDR) is a multifactorial process that leads to therapy failure. MDR involves multiple processes and many signaling pathways that support each other, making it difficult to overcome once established. Here, we discuss cellular-oxidative-stress-modulating factors focusing on transcription factors NRF2, FOXO family, and peroxiporins, as well as their possible contribution to MDR. This is significant because oxidative stress is a consequence of radiotherapy, chemotherapy, and immunotherapy, and the activation of detoxification pathways could modulate the cellular response to therapy and could support MDR. These proteins are not directly responsible for MDR, but they support the survival of cancer cells under stress conditions.

Role of NRF2 and reactive aldehydes in acute cellular rejection in liver transplant recipients.

OBJECTIVE: To evaluate the level of oxidative stress and antioxidative response in the transplanted liver and its role in acute cellular rejection (ACR). Particular attention was paid to ACR diagnosis in patients with hepatitis C (HCV), as histopathological features of ACR and viral disease recurrence overlap. METHODS: This retrospective study included 40 liver transplant patients who underwent liver transplantation with two consecutive liver biopsies performed during one hospitalization period: 1.) initial biopsy of the donor liver (before implantation) and 2.) indication biopsy (after suspected ACR). Based on the etiology, patients were divided into two groups: 22 patients with alcoholic liver cirrhosis (EtOH group) and 18 patients with hepatitis C cirrhosis (HCV group). We analyzed the presence of acrolein, HNE (4-hydroxynonenal), and the major antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2) in both biopsies. RESULTS: The presence of acrolein and HNE in both biopsies indicates increased oxidative stress, while the decrease in these aldehydes in the indication biopsies indicates a decrease in oxidative stress over time, reflecting liver graft recovery. The absence of NRF2 in both biopsies reflects significantly reduced antioxidant protection in patients undergoing liver transplantation. CONCLUSION: The results support the role of oxidative stress in the pathogenesis of ACR. The presence of acrolein and the absence of HNE in the indication biopsy in patients with ACR could contribute to the diagnosis of ACR in clinical practice when functional antibodies are tested in the clinical setting.

Deciphering Common Traits of Breast and Ovarian Cancer Stem Cells and Possible Therapeutic Approaches.

Breast cancer (BC) and ovarian cancer (OC) are among the most common and deadly cancers affecting women worldwide. Both are complex diseases with marked heterogeneity. Despite the induction of screening programs that increase the frequency of earlier diagnosis of BC, at a stage when the cancer is more likely to respond to therapy, which does not exist for OC, more than 50% of both cancers are diagnosed at an advanced stage. Initial therapy can put the cancer into remission. However, recurrences occur frequently in both BC and OC, which are highly cancer-subtype dependent. Therapy resistance is mainly attributed to a rare subpopulation of cells, named cancer stem cells (CSC) or tumor-initiating cells, as they are capable of self-renewal, tumor initiation, and regrowth of tumor bulk. In this review, we will discuss the distinctive markers and signaling pathways that characterize CSC, their interactions with the tumor microenvironment, and the strategies they employ to evade immune surveillance. Our focus will be on identifying the common features of breast cancer stem cells (BCSC) and ovarian cancer stem cells (OCSC) and suggesting potential therapeutic approaches.

Nuclear localization of NRF2 in stroma of HER2 positive and triple-negative breast cancer.

Breast cancer is one of the leading causes of cancer-related mortality in women. During tumor growth, periods of hypoxia are followed by reoxygenation due to neovascularisation leading to disturbed redox homeostasis. ROS (Reactive Oxygen Species) produced under hypoxia activate HIF1α. ROS can also activate the major antioxidant transcription factor NRF2, but also cause damage to biomolecules. Lipids are susceptible to peroxidation, as evidenced by the formation of reactive aldehydes, among which, HNE (4-hydroxynonenal) is the most studied one. Knowing that HIF1α (Hypoxia Inducing Factor 1α) is associated with breast cancer malignancy, we aimed to investigate its correlation with HNE and NRF2 (Nuclear factor erythroid 2-related factor 2). Our results show that HIF1α is activated in breast cancer, indicating an increase in ROS but not followed by HNE production. On the other hand, NRF2 was increased in all types of breast cancer suggesting that oxidative stress is present in these pathologies, but also supporting HIF1α. Interestingly, NRF2 was activated in HER2 positive and TNBC, indicating the role of stromal NRF2 in breast cancer malignancy.

AQP3-Dependent PI3K/Akt Modulation in Breast Cancer Cells.

Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated H2O2 uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2-related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.

4-Hydroxynonenal Modulates Blood-Brain Barrier Permeability In Vitro through Changes in Lipid Composition and Oxidative Status of Endothelial Cells and Astrocytes.

Blood brain barrier (BBB) is a dynamic interface responsible for proper functioning of brain, but also a major obstacle for effective treatment of neurological diseases. Increased levels of free radicals, in high ferrous and high lipid content surrounding, induce lipid peroxidation, leading to production of 4-hydroxynonenal (HNE). HNE modifies all key proteins responsible for proper brain functioning thus playing a major role in the onset of neurological diseases. To investigate HNE effects on BBB permeability, we developed two in vitro BBB models-'physiological' and 'pathological'. The latter mimicked HNE modified extracellular matrix under oxidative stress conditions in brain pathologies. We showed that exogenous HNE induce activation of antioxidative defense systems by increasing catalase activity and glutathione content as well as reducing lipid peroxide levels in endothelial cells and astrocytes of 'physiological' model. While in 'pathological' model, exogenous HNE further increased lipid peroxidation levels of endothelial cells and astrocytes, followed by increase in Nrf2 and glutathione levels in endothelial cells. At lipid composition level, HNE caused increase in ω3 polyunsaturated fatty acid (PUFA) level in endothelial cells, followed by decrease in ω3 PUFA level and increase in monounsaturated fatty acid level in astrocytes. Using these models, we showed for the first time that HNE in 'pathological' model can reduce BBB permeability.

Oxidative Stress and Cancer Heterogeneity Orchestrate NRF2 Roles Relevant for Therapy Response.

Oxidative stress and its end-products, such as 4-hydroxynonenal (HNE), initiate activation of the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)/Kelch Like ECH Associated Protein 1 (KEAP1) signaling pathway that plays a crucial role in the maintenance of cellular redox homeostasis. However, an involvement of 4-HNE and NRF2 in processes associated with the initiation of cancer, its progression, and response to therapy includes numerous, highly complex events. They occur through interactions between cancer and stromal cells. These events are dependent on many cell-type specific features. They start with the extent of NRF2 binding to its cytoplasmic repressor, KEAP1, and extend to the permissiveness of chromatin for transcription of Antioxidant Response Element (ARE)-containing genes that are NRF2 targets. This review will explore epigenetic molecular mechanisms of NRF2 transcription through the specific molecular anatomy of its promoter. It will explain the role of NRF2 in cancer stem cells, with respect to cancer therapy resistance. Additionally, it also discusses NRF2 involvement at the cross-roads of communication between tumor associated inflammatory and stromal cells, which is also an important factor involved in the response to therapy.

Peroxiporins Are Induced upon Oxidative Stress Insult and Are Associated with Oxidative Stress Resistance in Colon Cancer Cell Lines.

Oxidative stress can induce genetic instability and change cellular processes, resulting in colorectal cancer. Additionally, adaptation of oxidative defense causes therapy resistance, a major obstacle in successful cancer treatment. Peroxiporins are aquaporin membrane channels that facilitate H2O2 membrane permeation, crucial for regulating cell proliferation and antioxidative defense. Here, we investigated four colon cancer cell lines (Caco-2, HT-29, SW620, and HCT 116) for their sensitivity to H2O2, cellular antioxidative status, and ROS intracellular accumulation after H2O2 treatment. The expression of peroxiporins AQP1, AQP3, and AQP5 and levels of NRF2, the antioxidant transcription factor, and PPARγ, a transcription factor that regulates lipid metabolism, were evaluated before and after oxidative insult. Of the four tested cell lines, HT-29 was the most resistant and showed the highest expression of all tested peroxiporins and the lowest levels of intracellular ROS, without differences in GSH levels, catalase activity, nor NF2 and PPARγ levels. Caco-2 shows high expression of AQP3 and similar resistance as HT-29. These results imply that oxidative stress resistance can be obtained by several mechanisms other than the antioxidant defense system. Regulation of intracellular ROS through modulation of peroxiporin expression may represent an additional strategy to target the therapy resistance of cancer cells.

Antimicrobial Activity of Quasi-Enantiomeric Cinchona Alkaloid Derivatives and Prediction Model Developed by Machine Learning.

Bacterial infections that do not respond to current treatments are increasing, thus there is a need for the development of new antibiotics. Series of 20 N-substituted quaternary salts of cinchonidine (CD) and their quasi-enantiomer cinchonine (CN) were prepared and their antimicrobial activity was assessed against a diverse panel of Gram-positive and Gram-negative bacteria. All tested compounds showed good antimicrobial potential (minimum inhibitory concentration (MIC) values 1.56 to 125.00 µg/mL), proved to be nontoxic to different human cell lines, and did not influence the production of reactive oxygen species (ROS). Seven compounds showed very strong bioactivity against some of the tested Gram-negative bacteria (MIC for E. coli and K. pneumoniae 6.25 µg/mL; MIC for P. aeruginosa 1.56 µg/mL). To establish a connection between antimicrobial data and potential energy surfaces (PES) of the compounds, activity/PES models using principal components of the disc diffusion assay and MIC and data towards PES data were built. An extensive machine learning procedure for the generation and cross-validation of multivariate linear regression models with a linear combination of original variables as well as their higher-order polynomial terms was performed. The best possible models with predicted R2(CD derivatives) = 0.9979 and R2(CN derivatives) = 0.9873 were established and presented. This activity/PES model can be used for accurate prediction of activities for new compounds based solely on their potential energy surfaces, which will enable wider screening and guided search for new potential leads. Based on the obtained results, N-quaternary derivatives of Cinchona alkaloids proved to be an excellent scaffold for further optimization of novel antibiotic species.

AQP3 and AQP5-Potential Regulators of Redox Status in Breast Cancer.

Breast cancer is still one of the leading causes of mortality in the female population. Despite the campaigns for early detection, the improvement in procedures and treatment, drastic improvement in survival rate is omitted. Discovery of aquaporins, at first described as cellular plumbing system, opened new insights in processes which contribute to cancer cell motility and proliferation. As we discover new pathways activated by aquaporins, the more we realize the complexity of biological processes and the necessity to fully understand the pathways affected by specific aquaporin in order to gain the desired outcome-remission of the disease. Among the 13 human aquaporins, AQP3 and AQP5 were shown to be significantly upregulated in breast cancer indicating their role in the development of this malignancy. Therefore, these two aquaporins will be discussed for their involvement in breast cancer development, regulation of oxidative stress and redox signalling pathways leading to possibly targeting them for new therapies.

Oxidative Stress Reduction by Midazolam Premedication during Oocyte Retrieval Procedure: Pilot Study.

Infertility is one of the major medical problems nowadays. Couples who opt for In Vitro Fertilization (IVF) face a great deal of stress which certainly affects the outcome of the procedure. Therefore, we aimed to reduce the stress during the oocyte retrieval procedure by applying midazolam. Total oxidant (TOC) and antioxidant (TAC) capacities of serum, as well as glutathione (GSH) content and catalase activity, were measured in both control and midazolam groups. Follicular fluid was also tested for oxidant capacity and IL1ß. Results implied that the midazolam group increased TAC at the end of the procedure. At the same time, the control group decreased GSH at the beginning of the procedure, and both groups decreased catalase activity at the end of the procedure. The results imply that stress during the procedure affects oxidative and antioxidative parameters of the patients, but did not affect the frequency of the pregnancy at the end of this pilot study. Yet, the results imply that oxidative and antioxidative mechanisms during IVF should be investigated in detail as they could affect the outcome of IVF.

Activity to Breast Cancer Cell Lines of Different Malignancy and Predicted Interaction with Protein Kinase C Isoforms of Royleanones.

Plants have been used for centuries to treat several illnesses. The Plectranthus genus has a vast variety of species that has allowed the isolation of cytotoxic compounds with notable activities. The abietane diterpenes 6,7-dehydroroyleanone (DeRoy, 1), 7α-acetoxy-6ß-hydroxyroyleanone (Roy, 2), and Parvifloron D (ParvD, 3) were obtained from Plectranthus spp. and showed promising biological activities, such as cytotoxicity. The inhibitory effects of the different natural abietanes (1-3) were compared in MFC7, SkBr3, and SUM159 cell lines, as well as SUM159 grown in cancer stem cell-inducing conditions. Based on the royleanones' bioactivity, the derivatives RoyBz (4), RoyBzCl (5), RoyPr2 (6), and DihydroxyRoy (7), previously obtained from 2, were selected for further studies. Protein kinases C (PKCs) are involved in several carcinogenic processes. Thus, PKCs are potential targets for cancer therapy. To date, the portfolio of available PKC modulators remains very limited due to the difficulty of designing isozyme-selective PKC modulators. As such, molecular docking was used to evaluate royleanones 1-6 as predicted isozyme-selective PKC binders. Subtle changes in the binding site of each PKC isoform change the predicted interaction profiles of the ligands. Subtle changes in royleanone substitution patterns, such as a double substitution only with non-substituted phenyls, or hydroxybenzoate at position four that flips the binding mode of ParvD (3), can increase the predicted interactions in certain PKC subtypes.

Free Radical Research in Cancer.

It can be challenging to find efficient therapy for cancer due to its biological diversity [...].

Chronic Oxidative Stress Promotes Molecular Changes Associated with Epithelial Mesenchymal Transition, NRF2, and Breast Cancer Stem Cell Phenotype.

Oxidative stress plays a role in carcinogenesis, but it also contributes to the modulation of tumor cells and microenvironment caused by chemotherapeutics. One of the consequences of oxidative stress is lipid peroxidation, which can, through reactive aldehydes such as 4-hydroxy-2-nonenal (HNE), affect cell signaling pathways. On the other hand, cancer stem cells (CSC) are now recognized as a major factor of malignancy by causing metastasis, relapse, and therapy resistance. Here, we evaluated whether oxidative stress and HNE modulation of the microenvironment can influence CSC growth, modifications of the epithelial to mesenchymal transition (EMT) markers, the antioxidant system, and the frequency of breast cancer stem cells (BCSC). Our results showed that oxidative changes in the microenvironment of BCSC and particularly chronic oxidative stress caused changes in the proliferation and growth of breast cancer cells. In addition, changes associated with EMT, increase in glutathione (GSH) and Nuclear factor erythroid 2-related factor 2 (NRF2) were observed in breast cancer cells grown on HNE pretreated collagen and under chronic oxidative stress. Our results suggest that chronic oxidative stress can be a bidirectional modulator of BCSC fate. Low levels of HNE can increase differentiation markers in BCSC, while higher levels increased GSH and NRF2 as well as certain EMT markers, thereby increasing therapy resistance.

Nutritional Stress in Head and Neck Cancer Originating Cell Lines: The Sensitivity of the NRF2-NQO1 Axis.

Nutritional stress disturbs the cellular redox-status, which is characterized by the increased generation of reactive oxygen species (ROS). The NRF2-NQO1 axis represents a protective mechanism against ROS. Its strength is cell type-specific. FaDu, Cal 27 and Detroit 562 cells differ with respect to basal NQO1 activity. These cells were grown for 48 hours in nutritional conditions (NC): (a) Low glucose-NC2, (b) no glucose, no glutamine-NC3, (c) no glucose with glutamine-NC4. After determining the viability, proliferation and ROS generation, NC2 and NC3 were chosen for further exploration. These conditions were also applied to IMR-90 fibroblasts. The transcripts/transcript variants of NRF2 and NQO1 were quantified and transcript variants were characterized. The proteins (NRF2, NQO1 and TP53) were analyzed by a western blot in both cellular fractions. Under NC2, the NRF2-NQO1 axis did not appear activated in the cancer cell lines. Under NC3, the NRF2-NQO1axis appeared slightly activated in Detroit 562. There are opposite trends with respect to TP53 nuclear signal when comparing Cal 27 and Detroit 562 to FaDu, under NC2 and NC3. The strong activation of the NRF2-NQO1 axis in IMR-90 resulted in an increased expression of catalytically deficient NQO1, due to NQO1*2/*2 polymorphism (rs1800566). The presented results call for a comprehensive exploration of the stress response in complex biological systems.

Short Overview of ROS as Cell Function Regulators and Their Implications in Therapy Concepts.

The importance of reactive oxygen species (ROS) has been gradually acknowledged over the last four decades. Initially perceived as unwanted products of detrimental oxidative stress, they have been upgraded since, and now ROS are also known to be essential for the regulation of physiological cellular functions through redox signaling. In the majority of cases, metabolic demands, along with other stimuli, are vital for ROS formation and their actions. In this review, we focus on the role of ROS in regulating cell functioning and communication among themselves. The relevance of ROS in therapy concepts is also addressed here.

Lipid Profile and Aquaporin Expression under Oxidative Stress in Breast Cancer Cells of Different Malignancies.

Breast cancer is the major cause of tumor-associated mortality in women worldwide, with prognosis depending on the early discovery of the disease and on the type of breast cancer diagnosed. Among many factors, lipids could contribute to breast cancer malignancy by participating in cellular processes. Also, aquaporins are membrane channels found aberrantly expressed in cancer tissues that were correlated with tumor aggressiveness, progression, and metastasis. However, the differences in lipid profile and aquaporin expression between cell types of different malignant potential have never been investigated. Here, we selected three breast cancer cell lines representing the three major breast cancer types (hormone positive, HER2NEU positive, and triple negative) and analyzed their lipid profile and steady state lipid hydroperoxide levels to correlate with cell sensitivity to H2O2. Additionally, the expression profiles of AQP1, AQP3, and AQP5 and the Nrf2 transcription factor were evaluated, before and after oxidative challenge. We found that the lipid profile was dependent on the cell type, with the HER2-positive cells having the lowest level PUFA, whereas the triple negative showed the highest. However, in triple-negative cancer cells, a lower level of the Nrf2 may be responsible for a higher sensitivity to H2O2 challenge. Interestingly, HER2-positive cells showed the highest increase in intracellular ROS after oxidative challenge, concomitant with a significantly higher level of AQP1, AQP3, and AQP5 expression compared to the other cell types, with AQP3 always being the most expressed isoform. The AQP3 gene expression was stimulated by H2O2 treatment in hormone-positive and HER2NEU cells, together with Nrf2 expression, but was downregulated in triple-negative cells that showed instead upregulation of AQP1 and AQP5. The lipid profile and AQP gene expression after oxidative challenge of these particularly aggressive cell types may represent metabolic reprogramming of cancer cells and reflect a role in adaptation to stress and therapy resistance.