Stress-mediated polysorbate 20 degradation and its potential impact on therapeutic proteins.

PURPOSE: Polysorbates are the most commonly used surfactants in formulations to stabilize therapeutic proteins against interfacial stresses. Polysorbates can undergo oxidative or enzyme-mediated hydrolytic degradation to produce free fatty acids (FFAs) and subvisible particles in formulations. To determine which product related variables contribute to PS20 degradation, we investigated the effects of storage temperature, formulation, pH, presence of hydrolytic enzymes, and specific fatty acid composition on different grades of PS20 in relation to their PS20 degradation profile and consequently the quality of protein drug products. METHODS: Bevacizumab and T-DM1 were reformulated in the freshly prepared therapeutic protein formulations containing either compendial PS20 or non-compendial PS20 with high % lauric acid and spiked with exogenous esterase or lipase. The release of FFAs and formation of particles were monitored at 4°C and 37°C. Protein quality was assessed for secondary structures, purity, and biological activity. RESULTS: Hydrolytic release of FFAs and formation of subvisible particles were found to be dependent on grades of PS20, types of enzymes used, incubation temperature, and pH. Esterase- or lipase-mediated degradation of PS20 and formation of subvisible particles in drug formulation showed no significant impact on the biological activity and stability of therapeutic proteins against degradation or aggregation. CONCLUSIONS: Our study suggests that degradation of PS20 and formation of FFA particles depend on the fatty acid composition of PS20, types of hydrolytic enzymes, pH, and temperature. The presence of FFA subvisible particles showed no significant impact on the purity and biological activity of the therapeutic proteins under the tested conditions.

Divalent ions as mediators of carbonylation in cardiac myosin binding protein C.

The dosing and efficacy of chemotherapeutic drugs can be limited by toxicity caused by off-pathway reactions. One hypothesis for how such toxicity arises is via metal-catalyzed oxidative damage of cardiac myosin binding protein C (cMyBP-C) found in cardiac tissue. Previous research indicates that metal ion mediated reactive oxygen species induce high levels of protein carbonylation, changing the structure and function of this protein. In this work, we use long timescale all-atom molecular dynamics simulations to investigate the ion environment surrounding the C0 and C1 subunits of cMyBP-C responsible for actin binding. We show that divalent cations are co-localized with protein carbonylation-prone amino acid residues and that carbonylation of these residues can lead to site-specific interruption to the actin-cMyBP-C binding.

Metal-induced oxidative stress and human plasma protein oxidation after SARS-CoV-2 infection.

Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis.

Differential Protein Citrullination in Human ER- and ER+ Tumor and Adjacent Healthy Breast Tissue.

Post-translational modification of arginine to citrulline is catalyzed by members of the peptidylarginine deiminase (PAD) family. Dysregulation of this catalysis is a significant driver of the pathogenesis of numerous inflammatory diseases, including cancer. However, dysregulation of PAD activity has not been examined in breast cancer with respect to hormone receptor status. In this study, we measured PAD enzyme levels using Western blotting and investigated protein citrullination using a mass spectrometry-based proteomics approach in primary estrogen receptor negative (ER-) or positive (ER+) breast tumor and matched adjacent normal tissue. Our findings reveal 72 and 41 citrullinated proteins in ER- tumor and adjacent healthy tissue, respectively, where 20 of these proteins are common between the two groups. We detected 64 and 49 citrullinated proteins in ER+ tumor and adjacent healthy tissue, respectively, where 32 proteins are common. Interestingly, upon comparison of ER- and ER+ tumor tissue, only 32 citrullinated proteins are shared between the two and the rest are unique to the tumor's receptor status. Using the STRING database for protein-protein interaction network analysis, these proteins are involved in protein-folding events (i.e., heat shock proteins) in ER- samples and blood-clotting events (i.e., fibulin) in ER+ samples. Constituents of the extracellular matrix structure (i.e., collagen and fibrinogen) were found in both. Herein, we establish evidence that supports the role of this unique post-translational modification in breast cancer biology. Finally, to aid drug discovery against citrullination, we developed a liquid chromatography-ultraviolet method to measure PAD enzymatic activity and optimized glucagon-like peptide II to quantitatively measure the ability of PADs to citrullinate its substrate.

Potential role of the apelin-APJ pathway in sex-related differential cardiotoxicity induced by doxorubicin in mice.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-ß2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-ß2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model.

Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species.

Biotherapeutics are exposed to common transition metal ions such as Cu(II) and Fe(II) during manufacturing processes and storage. IgG1 biotherapeutics are vulnerable to reactive oxygen species (ROS) generated via the metal-catalyzed oxidation reactions. Exposure to these metal ions can lead to potential changes to structure and function, ultimately influencing efficacy, potency, and potential immunogenicity of the molecules. Here, we stress four biotherapeutics of the IgG1 subclass (trastuzumab, trastuzumab emtansine, anti-NaPi2b, and anti-NaPi2b-vc-MMAE) with two common pharmaceutically relevant metal-induced oxidizing systems, Cu(II)/ ascorbic acid and Fe(II)/ H2O2, and evaluated oxidation, size distribution, carbonylation, Fc effector functions, antibody-dependent cellular cytotoxicity (ADCC) activity, cell anti-proliferation and autophaghic flux. Our study demonstrates that the extent of oxidation was metal ion-dependent and site-specific, leading to decreased FcγRIIIa and FcRn receptor binding and subsequently potentially reduced bioactivity, though antigen binding was not affected to a great extent. In general, the monoclonal antibody (mAb) and corresponding antibody-drug conjugate (ADC) showed similar impacts to product quality when exposed to the same metal ion, either Cu(II) or Fe(II). Our study clearly demonstrates that transition metal ion binding to therapeutic IgG1 mAbs and ADCs is not random and that oxidation products show unique structural and functional ramifications. A critical outcome from this study is our highlighting of key process parameters, route of degradation, especially oxidation (metal catalyzed or via ROS), on the CH1 and Fc region of full-length mAbs and ADCs.Abbreviations: DNPH 2,4-dinitrophenylhydrazine; ADC Antibody drug conjugate; ADCC Antibody-dependent cellular cytotoxicity; CDR Complementary determining region; DTT Dithiothreitol; HMWF high molecular weight form; LC-MS Liquid chromatography-mass spectrometry; LMWF low molecular weight forms; MOA Mechanism of action; MCO Metal-catalyzed oxidation; MetO Methionine sulfoxide; mAbs Monoclonal antibodies; MyBPC Myosin binding protein C; ROS Reactive oxygen species; SEC Size exclusion chromatography.

Effect of Fatty Acid Composition in Polysorbate 80 on the Stability of Therapeutic Protein Formulations.

PURPOSE: Polysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions. METHODS: Two polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity. RESULTS: The addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions. CONCLUSIONS: These results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.

Acute total body ionizing gamma radiation induces long-term adverse effects and immediate changes in cardiac protein oxidative carbonylation in the rat.

Radiation-induced heart disease presents a significant challenge in the event of an accidental radiation exposure as well as to cancer patients who receive acute doses of irradiation as part of radiation therapy. We utilized the spontaneously hypertensive Wistar-Kyoto rat model, previously shown to demonstrate drug-induced cardiomyopathy, to evaluate the acute and long-term effects of sub-lethal total body gamma irradiation at two, four, and fifty-two weeks. We further examined irreversible oxidative protein carbonylation in the heart immediately following irradiation in the normotensive Wistar-Kyoto rat. Both males and females sustained weight loss and anemic conditions compared to untreated controls over a one-year period as reflected by reduced body weight and low red blood cell count. Increased inflammation was detected by elevated IL-6 serum levels selectively in males at four weeks. Serum cardiac troponin T and I analyses revealed signs of cardiomyopathy at earlier timepoints, but high variability was observed, especially at one year. Echocardiography at two weeks following 5.0Gy treatment revealed a significant decrease in cardiac output in females and a significant decrease in both diastolic and systolic volumes in males. Following 10.0Gy irradiation in the normotensive Wistar-Kyoto rat, the heart tissue showed an increase in total protein oxidative carbonylation accompanied by DNA damage indicated by an increase in γ-H2AX. Using proteomic analyses, we identified several novel proteins which showed a marked difference in carbonylation including those of mitochondrial origin and most notably, cardiac troponin T, one of the key proteins involved in cardiomyocyte contractility. Overall, we present findings of acute oxidative protein damage, DNA damage, cardiac troponin T carbonylation, and long-term cardiomyopathy in the irradiated animals.

Mitochondrial dysfunction generates aggregates that resist lysosomal degradation in human breast cancer cells.

Disrupting functional protein homeostasis is an established therapeutic strategy for certain tumors. Ongoing studies are evaluating autophagy inhibition for overcoming chemotherapeutic resistance to such therapies by neutralizing lysosomal pH. New and sensitive methods to monitor autophagy in patients are needed to improve trial design and interpretation. We report that mitochondrial-damaged breast cancer cells and rat breast tumors accumulate p53-positive protein aggregates that resist lysosomal degradation. These aggregates were localized to enzymatically-active autolysosomes that were degrading autophagosomes and the autophagic receptor proteins TAX1BP1 and NDP52. NDP52 was identified to associate with aggregated proteins and knocking down NDP52 led to the accumulation of protein aggregates. TAX1BP1 was identified to partly localize with aggregates, and knocking down TAX1BP1 enhanced aggregate formation, suppressed autophagy, impaired NDP52 autophagic degradation and induced cell death. We propose that quantifying aggregates and autophagic receptors are two potential methods to evaluate autophagy and lysosomal degradation, as confirmed using primary human tumor samples. Collectively, this report establishes protein aggregates and autophagy receptors, TAX1BP1 and NDP52, as potential endpoints for monitoring autophagy during drug development and clinical studies.

FDA Approval Summary: Tagraxofusp-erzs For Treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm.

Tagraxofusp-erzs (Elzonris, Stemline) is a cytotoxin that targets CD123-expressing cells. On December 21, 2018, FDA approved tagraxofusp-erzs for the treatment of blastic plasmacytoid dendritic cell neoplasms (BPDCN) in adult and pediatric patients 2 years and older. Approval was based on the response rate in a single-arm trial, Study STML-401-0114; the pivotal cohort included 13 patients with treatment-naïve BPDCN who received tagraxofusp-erzs monotherapy. The complete response or clinical complete response (CR/CRc) rate in the pivotal cohort was 54% (95% CI: 25%-81%), and the median duration of CR/CRc was not reached with a median follow-up of 11.5 months (range: 0.2-12.7). In a separate exploratory cohort, a CR/CRc was achieved by 2 (13%) patients with R/R BPDCN. Safety was assessed in 94 patients with myeloid neoplasms treated with tagraxofusp-erzs at the approved dose and schedule. The major toxicity was capillary leak syndrome (CLS), which occurred in 55% of patients and was fatal in 2%. Hepatotoxicity and hypersensitivity reactions were reported in 88% and 46% of patients, respectively. Other common (≥30%) adverse reactions were nausea, fatigue, peripheral edema, pyrexia, and weight increase. A high proportion of patients (85%) developed neutralizing antidrug antibodies. Tagraxofusp-erzs is the first FDA-approved treatment for BPDCN.

Doxorubicin-induced cardiotoxicity is suppressed by estrous-staged treatment and exogenous 17ß-estradiol in female tumor-bearing spontaneously hypertensive rats.

BACKGROUND: Doxorubicin (DOX), an anthracycline therapeutic, is widely used to treat a variety of cancer types and known to induce cardiomyopathy in a time and dose-dependent manner. Postmenopausal and hypertensive females are two high-risk groups for developing adverse effects following DOX treatment. This may suggest that endogenous reproductive hormones can in part suppress DOX-induced cardiotoxicity. Here, we investigated if the endogenous fluctuations in 17ß-estradiol (E2) and progesterone (P4) can in part suppress DOX-induced cardiomyopathy in SST-2 tumor-bearing spontaneously hypersensitive rats (SHRs) and evaluate if exogenous administration of E2 and P4 can suppress DOX-induced cardiotoxicity in tumor-bearing ovariectomized SHRs (ovaSHRs). METHODS: Vaginal cytology was performed on all animals to identify the stage of the estrous cycle. Estrous-staged SHRs received a single injection of saline, DOX, dexrazoxane (DRZ), or DOX combined with DRZ. OvaSHRs were implanted with time-releasing pellets that contained a carrier matrix (control), E2, P4, Tamoxifen (Tam), and combinations of E2 with P4 and Tam. Hormone pellet-implanted ovaSHRs received a single injection of saline or DOX. Cardiac troponin I (cTnI), E2, and P4 serum concentrations were measured before and after treatment in all animals. Cardiac damage and function were further assessed by echocardiography and histopathology. Weight, tumor size, and uterine width were measured for all animals. RESULTS: In SHRs, estrous-staged DOX treatment altered acute estrous cycling that ultimately resulted in prolonged diestrus. Twelve days after DOX administration, all SHRs had comparable endogenous circulating E2. Thirteen days after DOX treatment, SHRs treated during proestrus had decreased cardiac output and increased cTnI as compared to animals treated during estrus and diestrus. DOX-induced tumor reduction was not affected by estrous-staged treatments. In ovaSHRs, exogenous administration of E2 suppressed DOX-induced cardiotoxicity, while P4-implanted ovaSHRs were partly resistant. However, ovaSHRs treated with E2 and P4 did not have cardioprotection against DOX-induced damage. CONCLUSIONS: This study demonstrates that estrous-staged treatments can alter the extent of cardiac damage caused by DOX in female SHRs. The study also supports that exogenous E2 can suppress DOX-induced myocardial damage in ovaSHRs.

Specific protein carbonylation in human breast cancer tissue compared to adjacent healthy epithelial tissue.

Protein carbonylation is an irreversible post-translational modification induced by severe oxidative stress. Reactive oxygen species (ROS) are constantly produced in cells and play important roles in both cancer progression and cancer suppression. ROS levels can be higher in tumor compared to surrounding healthy tissue but ROS-induced specific protein carbonylation and its unique role in cancer progression or suppression is poorly understood. In this study, we utilized previously validated ELISA and western blot methods to analyze the total and specific protein carbonylation in flash-frozen human breast cancer and matched adjacent healthy tissue to compare relative total, and specific protein carbonylation. Mass spectrometry, two-color western, and immunoprecipitation methods were used to identify and confirm the specifically carbonylated proteins in breast tumor tissue. Superoxide dismutase (SOD) activity was measured as an indicator of antioxidant activity, and LC3-II protein level was analyzed for autophagy by western blot. Findings were further confirmed using the immortalized MDA-MB-231 and MDA-MB-468 breast cancer and MCF-12A noncancerous human epithelial breast cell lines. Our results indicate that tumor tissue has greater total protein carbonylation, lower SOD1 and SOD2 protein levels, lower total SOD activity, and higher LC3-II levels compared to adjacent healthy tissue. We identified and confirmed three specific proteins of interest; filamin A, heat shock protein 90ß (HSP90ß), and bifunctional glutamate/proline-tRNA ligase (EPRS), that were selectively carbonylated in tumor tissue compared to matched adjacent healthy tissue. Correspondingly, compared to noncancerous MCF-12A epithelial cells, MDA-MB-231 cancer cells exhibited an increase in filamin A and EPRS protein carbonylation, decreased total SOD activity, and increased autophagy, but not increased HSP90ß protein carbonylation. Identification of selectively carbonylated proteins and defining their roles in cancer progression may promote the development of targeted therapeutic approaches toward mitigating oxidative damage of these proteins.

Deficiency in Cardiolipin Reduces Doxorubicin-Induced Oxidative Stress and Mitochondrial Damage in Human B-Lymphocytes.

Cardiolipin (CL) is an inner mitochondrial membrane phospholipid which plays an important role in mitochondrial function. Perturbation in CL biosynthesis alters mitochondrial bioenergetics causing a severe genetic disorder commonly known as Barth syndrome. Barth syndrome patients are known to have a reduced concentration and altered composition of CL. Cardiolipin is also known to have a high affinity for the chemotherapeutic agent doxorubicin (Dox), resulting in an extensive mitochondrial accumulation of the drug. Our results indicate that B-lymphocytes from healthy individuals are more sensitive to Dox-induced oxidative stress and cellular toxicity compared to the B-lymphocytes from Barth syndrome as indicated by greater cell death and greater level of cleaved caspase-3 following Dox treatment. Barth lymphocytes, when compared to healthy lymphocytes, showed a greater basal level of mitochondrial reactive oxygen species (mito-ROS), yet exhibited a lower level of induced mito-ROS production in response to Dox. Significantly less ATP content and slightly greater OXPHOS protein levels were detected in healthy cells compared to Barth cells after Dox treatment. Consistent with greater mitochondrial ROS, treatment with Dox induced a higher level of lipid peroxidation and protein carbonylation in healthy lymphocytes compared to Barth lymphocytes. The final remodeling of CL during CL synthesis is catalyzed by the tafazzin protein. Knockdown of tafazzin gene in H9c2 cardiomyocytes using siRNA showed decreased oxidant-induced damage, as observed in Barth lymphocytes. Our findings demonstrate that a deficiency in CL might provide a therapeutic advantage in favor of oxidant-induced anticancer activities.

Distinct oxidative cleavage and modification of bovine [Cu- Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule.

We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5Å from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction.

Reproductive hormone levels and differential mitochondria-related oxidative gene expression as potential mechanisms for gender differences in cardiosensitivity to Doxorubicin in tumor-bearing spontaneously hypertensive rats.

PURPOSE: Chemotherapy with doxorubicin (Dox) causes dose-limiting cardiotoxicity. We investigated the role that gender has on cardiosensitivity to Dox treatment by evaluating reproductive hormone levels in male, castrated male (c-male), female and ovariectomized female (o-female) adult spontaneously hypertensive rats (SHRs) and expression of mitochondria-related genes in male and female adult SHRs. METHODS: SST-2 breast tumor-bearing SHRs were treated with saline, Dox, dexrazoxane (Drz) or both Dox and Drz and monitored for 14 days. Tumor size was used to monitor anticancer activity. Heart weight, cardiac lesion score and serum levels of cardiac troponin T (cTnT) were used to determine cardiotoxicity. Serum estradiol (E2) and testosterone were evaluated using electrochemiluminescence immunoassays. Expression of mitochondria-related genes was profiled in heart by MitoChip array analyses. RESULTS: Dox significantly reduced tumor volume (±Drz) and increased heart weight in all genders (13-30% vs. control). Higher heart lesion scores were observed in reproductively normal animals (male 2.9, female 2.2) than in hormone-deficient animals (c-male 1.7, o-female 1.9). Lesion score and cTnT inversely correlated with hormone levels. Reduced levels of both sex hormones were observed after Dox treatment. Gene expression analyses of Dox-treated hearts showed significant differential expression of oxidative stress genes in male hearts and apoptotic genes in both male and female hearts. CONCLUSIONS: Our results demonstrate that adult tumor-bearing male SHRs are more cardiosensitive to Dox than female or hormone-deficient animals. We provide evidence to suggest that reproductive hormones negatively regulate or are inhibited by Dox-induced cardiotoxicity and the selective cytotoxic mechanism likely functions through the greater activation of oxidative stress and apoptosis in male SHRs.

Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress.

The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ.

Doxorubicin-induced carbonylation and degradation of cardiac myosin binding protein C promote cardiotoxicity.

Dose-dependent oxidative stress by the anthracycline doxorubicin (Dox) and other chemotherapeutic agents causes irreversible cardiac damage, restricting their clinical effectiveness. We hypothesized that the resultant protein oxidation could be monitored and correlated with physiological functional impairment. We focused on protein carbonylation as an indicator of severe oxidative damage because it is irreversible and results in proteasomal degradation. We identified and investigated a specific high-molecular weight cardiac protein that showed a significant increase in carbonylation under Dox-induced cardiotoxic conditions in a spontaneously hypertensive rat model. We confirmed carbonylation and degradation of this protein under oxidative stress and prevention of such effect in the presence of the iron chelator dexrazoxane. Using MS, the Dox-induced carbonylated protein was identified as the 140-kDa cardiac myosin binding protein C (MyBPC). We confirmed the carbonylation and degradation of MyBPC using HL-1 cardiomyocytes and a purified recombinant untagged cardiac MyBPC under metal-catalyzed oxidative stress conditions. The carbonylation and degradation of MyBPC were time- and drug concentration-dependent. We demonstrated that carbonylated MyBPC undergoes proteasome-mediated degradation under Dox-induced oxidative stress. Cosedimentation, immunoprecipitation, and actin binding assays were used to study the functional consequences of carbonylated MyBPC. Carbonylation of MyBPC showed significant functional impairment associated with its actin binding properties. The dissociation constant of carbonylated recombinant MyBPC for actin was 7.35 ± 1.9 µM compared with 2.7 ± 0.6 µM for native MyBPC. Overall, our findings indicate that MyBPC carbonylation serves as a critical determinant of cardiotoxicity and could serve as a mechanistic indicator for Dox-induced cardiotoxicity.

Mito-tempol and dexrazoxane exhibit cardioprotective and chemotherapeutic effects through specific protein oxidation and autophagy in a syngeneic breast tumor preclinical model.

Several front-line chemotherapeutics cause mitochondria-derived, oxidative stress-mediated cardiotoxicity. Iron chelators and other antioxidants have not completely succeeded in mitigating this effect. One hindrance to the development of cardioprotectants is the lack of physiologically-relevant animal models to simultaneously study antitumor activity and cardioprotection. Therefore, we optimized a syngeneic rat model and examined the mechanisms by which oxidative stress affects outcome. Immune-competent spontaneously hypertensive rats (SHRs) were implanted with passaged, SHR-derived, breast tumor cell line, SST-2. Tumor growth and cytokine responses (IL-1A, MCP-1, TNF-α) were observed for two weeks post-implantation. To demonstrate the utility of the SHR/SST-2 model for monitoring both anticancer efficacy and cardiotoxicity, we tested cardiotoxic doxorubicin alone and in combination with an established cardioprotectant, dexrazoxane, or a nitroxide conjugated to a triphenylphosphonium cation, Mito-Tempol (4) [Mito-T (4)]. As predicted, tumor reduction and cardiomyopathy were demonstrated by doxorubicin. We confirmed mitochondrial accumulation of Mito-T (4) in tumor and cardiac tissue. Dexrazoxane and Mito-T (4) ameliorated doxorubicin-induced cardiomyopathy without altering the antitumor activity. Both agents increased the pro-survival autophagy marker LC3-II and decreased the apoptosis marker caspase-3 in the heart, independently and in combination with doxorubicin. Histopathology and transmission electron microscopy demonstrated apoptosis, autophagy, and necrosis corresponding to cytotoxicity in the tumor and cardioprotection in the heart. Changes in serum levels of 8-oxo-dG-modified DNA and total protein carbonylation corresponded to cardioprotective activity. Finally, 2D-electrophoresis/mass spectrometry identified specific serum proteins oxidized under cardiotoxic conditions. Our results demonstrate the utility of the SHR/SST-2 model and the potential of mitochondrially-directed agents to mitigate oxidative stress-induced cardiotoxicity. Our findings also emphasize the novel role of specific protein oxidation markers and autophagic mechanisms for cardioprotection.

Iron homeostasis regulates the activity of the microRNA pathway through poly(C)-binding protein 2.

MicroRNAs (miRNAs) control gene expression by promoting degradation or repressing translation of target mRNAs. The components of the miRNA pathway are subject to diverse modifications that can modulate the abundance and function of miRNAs. Iron is essential for fundamental metabolic processes, and its homeostasis is tightly regulated. Here we identified iron chelators as a class of activator of the miRNA pathway that could promote the processing of miRNA precursors. We show that cytosolic iron could regulate the activity of the miRNA pathway through poly(C)-binding protein 2 (PCBP2). PCBP2 is associated with Dicer and promotes the processing of miRNA precursors. Cytosolic iron could modulate the association between PCBP2 and Dicer, as well as the multimerization of PCBP2 and its ability to bind to miRNA precursors, which can alter the processing of miRNA precursors. Our findings reveal a role of iron homeostasis in the regulation of miRNA biogenesis.

Submicron hard X-ray fluorescence imaging of synthetic elements.

Synchrotron-based X-ray fluorescence microscopy (XFM) using hard X-rays focused into sub-micron spots is a powerful technique for elemental quantification and mapping, as well as microspectroscopic measurements such as µ-XANES (X-ray absorption near edge structure). We have used XFM to image and simultaneously quantify the transuranic element plutonium at the L(3) or L(2)-edge as well as Th and lighter biologically essential elements in individual rat pheochromocytoma (PC12) cells after exposure to the long-lived plutonium isotope (242)Pu. Elemental maps demonstrate that plutonium localizes principally in the cytoplasm of the cells and avoids the cell nucleus, which is marked by the highest concentrations of phosphorus and zinc, under the conditions of our experiments. The minimum detection limit under typical acquisition conditions with an incident X-ray energy of 18 keV for an average 202 µm(2) cell is 1.4 fg Pu or 2.9×10(-20) moles Pu µm(-2), which is similar to the detection limit of K-edge XFM of transition metals at 10 keV. Copper electron microscopy grids were used to avoid interference from gold X-ray emissions, but traces of strontium present in naturally occurring calcium can still interfere with plutonium detection using its L(α) X-ray emission.