Drug resistance to 5-FU linked to reactive oxygen species modulator 1

While acute oxidative stress triggers cell apoptosis or necrosis, persistent oxidative stress induces genomic instability and has been implicated in tumor progression and drug resistance. In a previous report, we demonstrated that reactive oxygen species modulator 1 (Romo1) expression was up-regulated in most cancer cell lines and suggested that increased Romo1 expression might confer chronic oxidative stress to tumor cells. In this study, we show that enforced Romo1 expression induces reactive oxygen species (ROS) production in the mitochondria leading to massive cell death. However, tumor cells that adapt to oxidative stress by increasing manganese superoxide dismutase (MnSOD), Prx I, and Bcl-2 showed drug resistance to 5-FU. To elucidate the relationship between 5-FU-induced ROS production and Romo1 expression, Romo1 siRNA was used to inhibit 5-FU-triggered Romo1 induction. Romo1 siRNA treatment efficiently blocked 5-FU-induced ROS generation, demonstrating that 5-FU treatment stimulated ROS production through Romo1 induction. Based on these results we suggest that cellular adaptive response to Romo1-induced ROS is another mechanism of drug resistance to 5-FU and Romo1 expression may provide a new clinical implication in drug resistance of cancer chemotherapy.     

A novel protein, Romo1, induces ROS production in the mitochondria

The majority of endogenous reactive oxygen species (ROS) are produced in the mitochondrial respiratory chain. An imbalance in ROS production alters the intracellular redox homeostasis, triggers DNA damage, and contributes to cancer development and progression. This study identified a novel protein, reactive oxygen species modulator 1 (Romo1), which is localized in the mitochondria. Romo1 was found to increase the level of ROS in the cells. Increased Romo1 expression was observed in various cancer cell lines. This suggests that the increased Romo1 expression during cancer progression may cause persistent oxidative stress to tumor cells, which can increase their malignancy.     

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H₂O₂) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H₂O₂ treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.     

Serum deprivation-induced reactive oxygen species production is mediated by Romo1

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.     

Replicative senescence induced by Romo1-derived reactive oxygen species

Persistent accumulation of DNA damage induced by reactive oxygen species (ROS) is proposed to be a major contributor toward the aging process. Furthermore, an increase in age-associated ROS is strongly correlated with aging in various species, including humans. Here we showed that the enforced expression of the ROS modulator 1 (Romo1) triggered premature senescence by ROS production, and this also contributed toward induction of DNA damage. Romo1-derived ROS was found to originate in the mitochondrial electron transport chain. Romo1 expression gradually increased in proportion to population doublings of IMR-90 human fibroblasts. An increase in ROS production in these cells with high population doubling was blocked by the Romo1 knockdown using Romo1 small interfering RNA. Romo1 knockdown also inhibited the progression of replicative senescence. Based on these results, we suggest that age-related ROS levels increase, and this contributes to replicative senescence, which is directly associated with Romo1 expression.     

Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species

Cancer cells have been found to express immunoglobulin G (IgG), but the exact functions and underlying mechanisms of cancer-derived IgG remain elusive. In this study, we first confirmed that downregulation of IgG restrained the growth and proliferation of cancer cells in vitro and in vivo. To elucidate its mechanism, we carried out a co-immunoprecipitation assay in HeLa cells and identified 27 potential IgG-interacting proteins. Among them, receptor of activated protein kinase C 1 (RACK1), ras-related nuclear protein (RAN) and peroxiredoxin 1 (PRDX1) are closely related to cell growth and oxidative stress, which prompted us to investigate the mechanism of action of IgG in the above phenomena. Upon confirmation of the interactions between IgG and the three proteins, further experiments revealed that downregulation of cancer-derived IgG lowered levels of intracellular reactive oxygen species (ROS) by enhancing cellular total antioxidant capacity. In addition, a few ROS scavengers, including catalase (CAT), dimethylsulfoxide (DMSO), n-acetylcysteine (NAC) and superoxide dismutase (SOD), further inhibited the growth of IgG-deficient cancer cells through suppressing mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) signaling pathway induced by a low level of intracellular ROS, whereas exogenous hydrogen peroxide (H₂O₂) at low concentration promoted their survival via increasing intracellular ROS levels. Similar results were obtained in an animal model and human tissues. Taken together, our results demonstrate that cancer-derived IgG can enhance the growth and proliferation of cancer cells via inducing the production of ROS at low level. These findings provide new clues for understanding tumor proliferation and designing cancer therapy.     

Establishment of lal-/- Myeloid Lineage Cell Line That Resembles Myeloid-Derived Suppressive Cells

Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4⁺ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.     

Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production

Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.     

OGDHL Is a Modifier of AKT-Dependent Signaling and NF-κB Function

Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multi-enzyme OGDH complex (OGDHC) whose malfunction is associated with neuro-degeneration. The essential role of this complex is in the degradation of glucose and glutamate and the OGDHL gene (one component of OGDHC) is down-regulated by promoter hypermethylation in many different cancer types. These properties suggest a potential growth modulating role of OGDHL in cancer; however, the molecular mechanism through which OGDHL exerts its growth modulating function has not been elucidated.Here, we report that restoration of OGDHL expression in cervical cancer cells lacking endogenous OGDHL expression suppressed cell proliferation, invasion and soft agar colony formation in vitro. Knockdown of OGDHL expression in cervical cancer cells expressing endogenous OGDHL had the opposite effect. Forced expression of OGDHL increased the production of reactive oxygen species (ROS) leading to apoptosis through caspase 3 mediated down-regulation of the AKT signaling cascade and decreased NF-κB phosphorylation. Conversely, silencing OGDHL stimulated the signaling pathway via increased AKT phosphorylation. Moreover, the addition of caspase 3 or ROS inhibitors in the presence of OGDHL increased AKT signaling and cervical cancer cell proliferation.Taken together, these data suggest that inactivation of OGDHL can contribute to cervical tumorigenesis via activation of the AKT signaling pathway and thus support it as an important anti-proliferative gene in cervical cancer.     

Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress

Superoxide dismutases (SODs) are important antioxidant enzymes responsible for the elimination of superoxide radical (O(2)(-)). The manganese-containing SOD (Mn-SOD) has been suggested to have tumor suppressor function and is located in the mitochondria where the majority of O(2)(-) is generated during respiration. Although increased reactive oxygen species (ROS) in cancer cells has long been recognized, the expression of Mn-SOD in cancer and its role in cancer development remain elusive. The present study used a human tissue microarray to analyze Mn-SOD expression in primary ovarian cancer tissues, benign ovarian lesions, and normal ovary epithelium. Significantly higher levels of Mn-SOD protein expression were detected in the malignant tissues compared with normal tissues (p < 0.05). In experimental systems, suppression of Mn-SOD expression by small interfering RNA caused a 70% increase of superoxide in ovarian cancer cells, leading to stimulation of cell proliferation in vitro and more aggressive tumor growth in vivo. Furthermore, stimulation of mitochondrial O(2)(-) production induced an increase of Mn-SOD expression. Our findings suggest that the increase in Mn-SOD expression in ovarian cancer is a cellular response to intrinsic ROS stress and that scavenging of superoxide by SOD may alleviate the ROS stress and thus reduce the simulating effect of ROS on cell growth.     

Overexpression of Lon contributes to survival and aggressive phenotype of cancer cells through mitochondrial complex I-mediated generation of reactive oxygen species

Lon protease is a multifunction protein and operates in protein quality control and stress response pathways in mitochondria. Human Lon is upregulated under oxidative and hypoxic stresses that represent the stress phenotypes of cancer. However, little literature undertakes comprehensive and detailed investigations on the tumorigenic role of Lon. Overexpression of Lon promotes cell proliferation, apoptotic resistance to stresses, and transformation. Furthermore, Lon overexpression induces the production of mitochondrial reactive oxygen species (ROS) that result from Lon-mediated upregulation of NDUFS8, a mitochondrial Fe-S protein in complex I of electron transport chain. Increased level of mitochondrial ROS promotes cell proliferation, cell survival, cell migration, and epithelial–mesenchymal transition through mitogen-activated protein kinase (MAPK) and Ras-ERK activation. Overall, the present report for the first time demonstrates the role of Lon overexpression in tumorigenesis. Lon overexpression gives an apoptotic resistance to stresses and induces mitochondrial ROS production through Complex I as signaling molecules to activate Ras and MAPK signaling, giving the survival advantages and adaptation to cancer cells. Finally, in silico and immunohistochemistry analysis showed that Lon is overexpressed specifically in various types of cancer tissue including oral cancer.     

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H₂O₂ levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.     

NADPH oxidase subunit p22phox-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22^phox expression are greatly increased in human prostate cancer tissues, and knockdown of p22^phox by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22^phox in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22^phox resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22^phox in tumor angiogenesis and tumor growth, and suggest that p22^phox is a potential novel target for prostate cancer treatment.     

Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia

Mitochondria play important roles as the powerhouse of the cell. After cerebral ischemia, mitochondria overproduce reactive oxygen species (ROS), which have been thoroughly studied with the use of superoxide dismutase transgenic or knockout animals. ROS directly damage lipids, proteins, and nucleic acids in the cell. Moreover, ROS activate various molecular signaling pathways. Apoptosis-related signals return to mitochondria, then mitochondria induce cell death through the release of pro-apoptotic proteins such as cytochrome c or apoptosis-inducing factor. Although the mechanisms of cell death after cerebral ischemia remain unclear, mitochondria obviously play a role by activating signaling pathways through ROS production and by regulating mitochondria-dependent apoptosis pathways.     

Ca2+-mediated Mitochondrial Reactive Oxygen Species Metabolism Augments Wnt/β-Catenin Pathway Activation to Facilitate Cell Differentiation

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/β-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/β-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca²⁺ from the endoplasmic reticulum stores. Ca²⁺ subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca²⁺ uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/β-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca²⁺-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/β-catenin signaling output.     

The role of compartmentalized signaling pathways in the control of mitochondrial activities in cancer cells

Mitochondria are the powerhouse organelles present in all eukaryotic cells. They play a fundamental role in cell respiration, survival and metabolism. Stimulation of G-protein coupled receptors (GPCRs) by dedicated ligands and consequent activation of the cAMP·PKA pathway finely couple energy production and metabolism to cell growth and survival. Compartmentalization of PKA signaling at mitochondria by A-Kinase Anchor Proteins (AKAPs) ensures efficient transduction of signals generated at the cell membrane to the organelles, controlling important aspects of mitochondrial biology. Emerging evidence implicates mitochondria as essential bioenergetic elements of cancer cells that promote and support tumor growth and metastasis. In this context, mitochondria provide the building blocks for cellular organelles, cytoskeleton and membranes, and supply all the metabolic needs for the expansion and dissemination of actively replicating cancer cells. Functional interference with mitochondrial activity deeply impacts on cancer cell survival and proliferation. Therefore, mitochondria represent valuable targets of novel therapeutic approaches for the treatment of cancer patients. Understanding the biology of mitochondria, uncovering the molecular mechanisms regulating mitochondrial activity andmapping the relevant metabolic and signaling networks operating in cancer cells will undoubtly contribute to create a molecular platform to be used for the treatment of proliferative disorders.Here, we will highlight the emerging roles of signaling pathways acting downstream to GPCRs and their intersection with the ubiquitin proteasome system in the control of mitochondrial activity in different aspects of cancer cell biology.     

Antiproliferative effects of goniothalamin on Ca9-22 oral cancer cells through apoptosis, DNA damage and ROS induction

Goniothalamin (GTN), a plant bioactive styryl-lactone, is a natural product with potent anti-tumorigenesis effects for several types of cancer. Nonetheless, the anticancer effect of GTN has not been examined in oral cancer. The present study was designed to evaluate its potential anticancer effects in an oral squamous cell carcinoma (OSCC) model and to determine the possible mechanisms with respect to apoptosis, DNA damage, reactive oxygen species (ROS) induction, and mitochondrial membrane potential. Our data demonstrated that cell proliferation was significantly inhibited by GTN in Ca9-22 OSCC cancer cells in concentration- and time-dependent manners (p<0.05). For cell cycle and apoptotic effects of GTN-treated Ca9-22 cancer cells, the sub-G1 population and annexin V-intensity significantly increased in a concentration-dependent manner (p<0.001). For the analysis of DNA double strand breaks, γH2AX intensity significantly increased in GTN-treated Ca9-22 cancer cells in concentration-response relationship (p<0.05). Moreover, GTN significantly induced intracellular ROS levels in Ca9-22 cancer cells in a concentration- and time-dependent manner (p<0.05). For membrane depolarization of mitochondria, the DiOC(2)(3) (3,3'-diethyloxacarbocyanine iodide) intensity of GTN-treated Ca9-22 cancer cells was significantly decreased in concentration- and time-dependent relationships (p<0.001). Taken together, these results suggest that the anticancer effect of GTN against oral cancer cells is valid and GTN-induced growth inhibition and apoptosis influence the downstream cascade including ROS induction, DNA damage, and mitochondria membrane depolarization. Therefore, GTN has potential as a chemotherapeutic agent against oral cancer.     

Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

Background

Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels.

Methods and results

In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death.

Conclusion

Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.