Reactive oxygen species (ROS) stimulate cell proliferation and induce genetic instability, and their increase in cancer cells is often viewed as an adverse event. Here, we show that such abnormal increases in ROS can be exploited to selectively kill cancer cells using beta-phenylethyl isothiocyanate (PEITC). Oncogenic transformation of ovarian epithelial cells with H-Ras(V12) or expression of Bcr-Abl in hematopoietic cells causes elevated ROS generation and renders the malignant cells highly sensitive to PEITC, which effectively disables the glutathione antioxidant system and causes severe ROS accumulation preferentially in the transformed cells due to their active ROS output. Excessive ROS causes oxidative mitochondrial damage, inactivation of redox-sensitive molecules, and massive cell death. In vivo, PEITC exhibits therapeutic activity and prolongs animal survival.

Increased aerobic glycolysis and oxidative stress are important features of cancer cell metabolism, but the underlying biochemical and molecular mechanisms remain elusive. Using a tetracycline inducible model, we show that activation of K-rasG12V causes mitochondrial dysfunction, leading to decreased respiration, elevated glycolysis, and increased generation of reactive oxygen species. The K-RAS protein is associated with mitochondria, and induces a rapid suppression of respiratory chain complex-I and a decrease in mitochondrial transmembrane potential by affecting the cyclosporin-sensitive permeability transition pore. Furthermore, pre-induction of K-rasG12V expression in vitro to allow metabolic adaptation to high glycolytic metabolism enhances the ability of the transformed cells to form tumor in vivo. Our study suggests that induction of mitochondrial dysfunction is an important mechanism by which K-rasG12V causes metabolic changes and ROS stress in cancer cells, and promotes tumor development.

2-Methoxyestradiol (2-ME), a new anticancer agent currently in clinical trials, has been demonstrated to inhibit superoxide dismutase (SOD) and to induce apoptosis in leukemia cells through a free radicalmediated mechanism. Because the accumulation of superoxide (O 2 ؊ ) by inhibition of SOD depends on the cellular generation of O 2 ؊ , we hypothesized that the endogenous production of superoxide may be a critical factor that affects the antileukemia activity of 2-ME. In the present study, we investigated the relationship between cellular O 2 ؊ contents and the cytotoxic activity of 2-ME in primary leukemia cells from 50 patients with chronic lymphocytic leukemia (CLL). Quantitation of O 2؊ revealed that the basal cellular O 2 ؊ contents are heterogeneous among patients with CLL. The O 2 ؊ levels were significantly higher in CLL cells from patients with prior chemotherapy. CLL cells with higher basal O 2 ؊ contents were more sensitive to 2-ME in vitro than those with lower O 2 ؊ contents. There was a significant correlation between the 2-ME-induced O 2 ؊ increase and the loss of cell viability. Importantly, addition of arsenic trioxide, a compound capable of causing reactive oxygen species (ROS) generation, significantly enhanced the activity of 2-ME, even in the CLL cells that were resistant to 2-ME alone. These results suggest that the cellular generation of O 2 ؊ plays an important role in the cytotoxic action of 2-ME and that it is possible to use exogenous ROS-producing agents such as arsenic trioxide in combination with 2-ME to enhance the antileukemia activity and to overcome drug resistance. Introduction 2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17␤-estradiol that is present in human urine and blood. 2-ME is produced by sequential 2-hydroxylation and O-methylation. 1 This metabolic modification causes a loss of its ability to bind the estrogen receptor. 2-ME has been shown to have potent tumorinhibiting effects in a number of cancer cell lines in vitro and in tumor xenograft models in vivo. [2][3][4][5][6][7][8][9] This compound is currently in clinical trials, as a single agent or in combination with other anticancer agents, for treatment of several types of human cancer, including breast cancer, prostate cancer, and multiple myeloma. 2-ME also shows antileukemia activity in vitro with therapeutic selectivity. 10

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, and resistance to fludarabine-based therapies is a major challenge in CLL treatment. Because CLL cells are known to have elevated levels of reactive oxygen species (ROS), we aimed to test a novel ROS-mediated strategy to eliminate fludarabine-resistant CLL cells based on this redox alteration. Using primary CLL cells and normal lymphocytes from patients (n ‫؍‬ 58) and healthy subjects (n ‫؍‬ 12), we showed that both fludarabineresistant and -sensitive CLL cells were highly sensitive to ␤-phenylethyl isothiocyanate (PEITC) with mean IC 50 values of 5.4 M and 5.1 M, respectively. Normal lymphocytes were significantly less sensitive to PEITC (IC 50 ‫؍‬ 27 M, P < .001). CLL cells exhibited intrinsically higher ROS level and lower cellular glutathione, which were shown to be the critical determinants of CLL sensitivity to PEITC. Exposure of CLL cells to PEITC induced severe glutathione depletion, ROS accumulation, and oxidation of mitochondrial cardiolipin leading to massive cell death. Such ROS stress also caused deglutathionylation of MCL1, followed by a rapid degradation of this cell survival molecule. Our study demonstrated that the natural compound PEITC is effective in eliminating fludarabine-resistant CLL cells through a redox-mediated mechanism with low toxicity to normal lymphocytes, and warrants further clinical evaluation.

(8,17). Analysis of blood samples from 30 ovarian cancer patients and an equal number of age-matched normal subjects shows significantly increased concentrations of plasma thiobarbituric acid-reactive substances and conjugated dienes in the patient specimens, indicating increased oxidative stress in ovarian cancer (18). However, the same study also shows low levels of SOD, catalase, vitamin C, and vitamin E in the plasma of the patient blood samples, possibly due to their increased utilization in scavenging lipid peroxides as well as their sequestration by tumor cells (18). This is in contrast with the increased serum Mn-SOD observed in another study (17). Decreased Mn-SOD activity and expression have also been reported in certain colorectal carcinomas and pancreatic cancer cells (19,20). These apparent conflicting observations are likely because of the different assays and various cell types used in these studies. Thus, it is important to clarify this issue by further examining the expression levels of SOD in a large number of primary human cancer tissues in comparison with normal tissues. Tissue microarray analysis provides an effective tool for such analyses. This new technique was used in the present study to compare the expression of Mn-SOD and Cu,Zn-SOD in primary human ovarian cancer tissues, benign ovarian lesions, and normal tissues. Although the biochemical activity of Mn-SOD in catalyzing the conversion of O 2 Ϫ to H 2 O 2 in the mitochondria has been well characterized, the potential role of Mn-SOD in cancer development remains to be defined. Because the Mn-SOD level seems decreased in certain cancer cells and forced expression of Mn-SOD appears to suppress malignant phenotypes in certain experimental models, this molecule has been con-* This study was supported in part by Grants CA85563, CA100428, and CA109041 (to P. H.) from the NCI, National Institutes of Health, and RSG-04-028-1-CCE (to J. L.) from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 These authors contributed equally to this work.

Although mitochondrial dysfunction and reactive oxygen species (ROS) stress have long been observed in cancer cells, their role in promoting malignant cell behavior remains unclear. Here, we show that perturbation of the mitochondrial respiratory chain in breast cancer cells leads to a generation of subclones of cells with increased ROS, active proliferation, high cellular motility, and invasive behaviors in vitro and in vivo. Gene expression analysis using microarrays revealed that all subclones overexpressed CXCL14, a novel chemokine with undefined function. We further show that CXCL14 expression is up-regulated by ROS through the activator protein-1 signaling pathway and promotes cell motility through elevation of cytosolic Ca 2+ by binding to the inositol 1,4,5-trisphosphate receptor on the endoplasmic reticulum. Abrogation of CXCL14 expression using a decoy approach suppressed cell motility and invasion. Our data suggest that mitochondrial dysfunction and ROS stress promote cancer cell motility through a novel pathway mediated by CXCL14.

Recent studies suggest that a small subpopulation of malignant cells with stem-like properties is resistant to chemotherapy and may be responsible for the existence of residual cancer after treatment. We have isolated highly tumorigenic cancer cells with 100-fold increase in tumor initiating capacity from the tumor xenografts of human glioblastoma U87 cells in mice. These cells exhibit stem-like properties and show unique energy metabolic characteristics including low mitochondrial respiration, increased glycolysis for ATP generation, and preference for hypoxia to maintain their stemness and tumor forming capacity. Mechanistically, mitochondrial depression in the highly tumorigenic cells occurs mainly at complex II of the electron transport chain with a down-regulation of the succinate dehydrogenase subunit B, leading to deregulation of hypoxia-inducible factors. Under hypoxia, the stem-like cancer cells are resistant to conventional anticancer agents but are sensitive to glycolytic inhibition. Furthermore, combination of glycolytic inhibition with standard therapeutic agents is effective in killing the tumor-initiating cells in vitro and inhibits tumor formation in vivo. Our study suggests that stem-like cancer cells prefer a low oxygen microenvironment and actively utilize the glycolytic pathway for ATP generation. Inhibition of glycolysis may be an effective strategy to eradicate residual cancer stem cells that are otherwise resistant to chemotherapeutic agents in their hypoxic niches.

Cancer cells are under intrinsic increased oxidative stress and vulnerable to free radical-induced apoptosis. Here, we report a strategy to hinder mitochondrial electron transport and increase superoxide (O 2 . ) radical generation in human leukemia cells as a novel mechanism to enhance apoptosis induced by anticancer agents. This strategy was first tested in a proof-of-principle study using rotenone, a specific inhibitor of mitochondrial electron transport complex I.