HIF-1-Dependent Heme Synthesis Promotes Gemcitabine Resistance In Human Non-Small Cell Lung Cancers Via Enhanced ABCB6 Expression

Background: Gemcitabine, a deoxycytidine analogue, is commonly used in the treatment of various types of tumors, including human non-small cell lung cancer (NSCLC). However, even the initial responders rapidly develop acquired resistance which limits our ability to effectively treat advanced NSCLC. The present study reports a widely prevalent mechanism of resistance to gemcitabine in NSCLC, whereby induced expression of ABCB6 is associated with poor prognosis and gemcitabine resistance in human NSCLCs which is in a HIF-1-dependent manner.Methods: We established gemcitabine resistant LUSC and LUAD cells and examined ABCB6 expression as well as heme synthesis in these cells. Additionally, we analyzed catalase protein expression and activity in gemcitabine resistant NSCLC cell lines. To validate ABCB6 gene as an HRE, we performed ChIP-qPCR assay. We also investigated the effect of inhibition of HIF-α/ABCB6 axis in vitro and in vivo.Results: The activation of HIF-1α/ABCB6 signaling leading to intracellular heme metabolic reprogramming and a corresponding increase in heme biosynthesis to enhance the activation and accumulation of catalase. Increased levels of catalase diminish the effective levels of reactive oxygen species (ROS) promoting gemcitabine-based resistance. Targeting HIF-1α or ABCB6, in combination with gemcitabine, strongly restrains tumor proliferation, increases tumor cell apoptosis and prolongs animal survival.Conclusion: In combination with gemcitabine-based chemotherapy, targeting HIF-1α/ABCB6 signaling could result in enhanced tumor sensitivity to chemotherapeutic agent and may improve outcome in NSCLCs.

Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment epithelia utilizes microtubule-associated protein 1 light chain 3B (LC3B)

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange, and waste removal. Each day the RPE removes the oldest ~10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation, and cell death. OSs are rich in very long chain fatty acids, which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblot analysis, and catalase activity assays, we investigated peroxisome abundance and activity at 6 AM, 7 AM (light onset), 8 AM, and 3 PM, in wild-type (WT) mice and mice lacking microtubule-associated protein 1 light chain 3B ( Lc3b), which have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 PM, in RPE of WT, but not Lc3b−/−, mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice; however, numbers were elevated overall in Lc3b−/− mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.

Evaluation of Potential Mechanisms Controlling the Catalase Expression in Breast Cancer Cells

Development of cancer cell resistance against prooxidant drugs limits its potential clinical use. MCF-7 breast cancer cells chronically exposed to ascorbate/menadione became resistant (Resox cells) by increasing mainly catalase activity. Since catalase appears as an anticancer target, the elucidation of mechanisms regulating its expression is an important issue. In MCF-7 and Resox cells, karyotype analysis showed that chromosome 11 is not altered compared to healthy mammary epithelial cells. The genomic gain ofcatalaselocus observed in MCF-7 and Resox cells cannot explain the differential catalase expression. Since ROS cause DNA lesions, the activation of DNA damage signaling pathways may influence catalase expression. However, none of the related proteins (i.e., p53, ChK) was activated in Resox cells compared to MCF-7. The c-abl kinase may lead to catalase protein degradation via posttranslational modifications, but neither ubiquitination nor phosphorylation of catalase was detected after catalase immunoprecipitation. Catalase mRNA levels did not decrease after actinomycin D treatment in both cell lines. DNMT inhibitor (5-aza-2′-deoxycytidine) increased catalase protein level in MCF-7 and its resistance to prooxidant drugs. In line with our previous report, chromatin remodeling appears as the main regulator of catalase expression in breast cancer after chronic exposure to an oxidative stress.

The levels of peroxisomal catalase protein and activity modulate the onset of cell death in tobacco BY-2 cells via reactive oxygen species levels and autophagy

In plant cells, peroxisomes participate in the metabolism of reactive oxygen species (ROS). One of the major regulators of cellular ROS levels – catalase (CAT) – occurs exclusively in peroxisomes. CAT activity is required for immunity-triggered autophagic programmed cell death (PCD). Autophagy has been recently demonstrated to represent a route for degradation of peroxisomes in plant cells. In the present study, the dynamics of the cellular peroxisome pool in tobacco BY-2 cell suspension cultures were used to analyse the effects of inhibition of basal autophagy with special attention to CAT activity. Numbers of peroxisomes per cell, levels of CAT protein and activity, cell viability, ROS levels and expression levels of genes encoding components of antioxidant system were analysed upon application of 3-methyladenine (3-MA), an inhibitor of autophagy, and/or aminotriazole (AT), an inhibitor of CAT. When applied separately, 3-MA and AT led to an increase in cell death, but this effect was attenuated by their simultaneous application. The obtained data suggest that both the levels of CAT protein in peroxisomes as well as CAT activity modulate the onset of cell death in tobacco BY-2 cells via ROS levels and autophagy.

Cardiac oxidative stress is involved in heart failure induced by thiamine deprivation in rats

Thiamine is an important cofactor of metabolic enzymes, and its deficiency leads to cardiovascular dysfunction. First, we characterized the metabolic status measuring resting oxygen consumption rate and lactate blood concentration after 35 days of thiamine deficiency (TD). The results pointed to a decrease in resting oxygen consumption and a twofold increase in blood lactate. Confocal microscopy showed that intracellular superoxide (∼40%) and H2O2 (2.5 times) contents had been increased. In addition, biochemical activities and protein expression of SOD, glutathione peroxidase, and catalase were evaluated in hearts isolated from rats submitted to thiamine deprivation. No difference in SOD activity was detected, but protein levels were found to be increased. Catalase activity increased 2.1 times in TD hearts. The observed gain in activity was attended by an increased catalase protein level. However, a marked decrease in glutathione peroxidase activity (control 435.3 ± 28.6 vs. TD 199.4 ± 30.2 nmol NADPH·min−1·ml−1) was paralleled by a diminution in the protein levels. Compared with control hearts, we did observe a greater proportion of apoptotic myocytes by TdT-mediated dUTP nick end labeling (TUNEL) and caspase-3 reactivity techniques. These results indicate that during TD, reactive oxygen species (ROS) production may be enhanced as a consequence of the installed acidosis. The perturbation in the cardiac myocytes redox balance was responsible for the increase in apoptosis.