Search

Hepatocellular carcinoma is one of the most frequent tumor types worldwide and oxidative stress represents a major risk factor in pathogenesis of liver diseases leading to HCC. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor activated by oxidative stress that governs the expression of many genes which constitute the antioxidant defenses of the cell. In addition, oxidative stress activates AMP-activated protein kinase (AMPK), which has emerged in recent years as a kinase that controls the redox-state of the cell. Since both AMPK and Nrf2 are involved in redox homeostasis, we investigated whether there was a crosstalk between the both signaling systems in hepatocarcinoma cells. Here, we demonstrated that AMPK activator AICAR, in contrary to the A769662 allosteric activator, induces Nrf2 activation and concomitantly modulates the basal redox state of the hepatocarcinoma cells. When the expression of Nrf2 is knocked down, AICAR failed to induce its effect on redox state. These data highlight a major role of Nrf2 signaling pathway in mediating the AICAR effect on basal oxidative state. Furthermore, we demonstrated that AICAR metabolization by the cell is required to induce Nrf2 activation while, the silencing of AMPK does not have any effect on Nrf2 activation. This suggests that AICAR-induced Nrf2 activation is independent of AMPK activity. In conclusion, we identified AICAR as a potent modulator of the redox state of human hepatocarcinoma cells, via the Nrf2 signaling pathway and in an AMPK-independent mechanism. © 2014 Elsevier Inc.

Catalase is an antioxidant enzyme that catalyzes mainly the transformation of hydrogen peroxide into water and oxygen. Although catalase is frequently down-regulated in tumors the underlying mechanism remains unclear. Few transcription factors have been reported to directly bind the human catalase promoter. Among them FoxO3a has been proposed as a positive regulator of catalase expression. Therefore, we decided to study the role of the transcription factor FoxO3a and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which regulates FoxO3a, in the expression of catalase. To this end, we developed an experimental model of mammary breast MCF-7 cancer cells that acquire resistance to oxidative stress, the so-called Resox cells, in which catalase is overexpressed as compared with MCF-7 parental cell line. In Resox cells, Akt expression is decreased but its phosphorylation is enhanced when compared with MCF-7 cells. A similar profile is observed for FoxO3a, with less total protein but more phosphorylated FoxO3a in Resox cells, correlating with its higher Akt activity. The modulation of FoxO3a expression by knockdown and overexpression strategies did not affect catalase expression, neither in MCF-7 nor in Resox cells. Inhibition of PI3K and mTOR by LY295002 and rapamycin, respectively, decreases the phosphorylation of downstream targets (i.e. GSK3β and p70S6K) and leads to an increase of catalase expression only in MCF-7 but not in Resox cells. In conclusion, FoxO3a does not appear to play a critical role in the regulation of catalase expression in both cancer cells. Only MCF-7 cells are sensitive and dependent on PI3K/Akt/mTOR signaling.

Cancer cells generally exhibit high levels of reactive oxygen species (ROS) that stimulate cell proliferation and promote genetic instability. Since this biochemical difference between normal and cancer cells represents a specific vulnerability that can be selectively targeted for cancer therapy, various ROS-generating agents are currently in clinical trials, either as single agents or in combination with standard therapy. However, little is known about the potential consequences of an increased oxidative stress for the efficacy of standard chemotherapeutic agents. In this context, we have assessed the influence of an oxidative stress generated by the combination of ascorbate and the redox-active quinone menadione on the capacity of melphalan, a common alkylating agent, to induce apoptosis in a chronic myelogenous leukemia cell line. Our data show that oxidative stress did not inhibit but rather promoted cancer cell killing by melphalan. Interestingly, we observed that, in the presence of oxidative stress, the type of cell death shifted from a caspase-3 dependent apoptosis to necrosis because of an ATP depletion which prevented caspase activation. Taken together, these data suggest that ROS-generating agents could be useful in combination with standard chemotherapy, even if all the molecular consequences of such an addition remain to be determined.

Because reactive oxygen species (ROS) are naturally produced as a consequence of aerobic metabolism, cells have developed a sophisticated set of antioxidant molecules to prevent the toxic accumulation of these species. However, compared with normal cells, malignant cells often exhibit increased levels of intracellular ROS and altered levels of antioxidant molecules. The resulting endogenous oxidative stress favors tumor growth by promoting genetic instability, cell proliferation and angiogenesis. In this context, we assessed the influence of catalase overexpression on the sensitivity of breast cancer cells towards various anticancer treatments. Our data show that catalase overexpression in MCF-7 cells leads to a 7-fold increase in catalase activity but provokes a 40% decrease in the expression of both glutathione peroxidase and peroxiredoxin II. Interestingly, proliferation and migration capacities of MCF-7 cells were impaired by the overexpression of catalase, as compared to parental cells. Regarding their sensitivity to anticancer treatments, we observed that cells overexpressing catalase were more sensitive to paclitaxel, etoposide and arsenic trioxide. However, no effect was observed on the cytotoxic response to ionizing radiations, 5-fluorouracil, cisplatin or doxorubicin. Finally, we observed that catalase overexpression protects cancer cells against the pro-oxidant combination of ascorbate and menadione, suggesting that changes in the expression of antioxidant enzymes could be a mechanism of resistance of cancer cells towards redox-based chemotherapeutic drugs.

Increase in cytosolic calcium concentration ([Ca(2+)](c)), release of endoplasmic reticulum (ER) calcium ([Ca(2+)](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca(2+)](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca(2+)](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca(2+)](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca(2+)](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca(2+)](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells.