Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood
; however, the reason for this is unclear. Here ...we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with melanomas derived from patients, and immunocompetent mice with mouse melanomas, had more melanoma cells per microlitre in tumour-draining lymph than in tumour-draining blood. Cells that metastasized through blood, but not those that metastasized through lymph, became dependent on the ferroptosis inhibitor GPX4. Cells that were pretreated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid and less free iron in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumours. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than did melanoma cells from subcutaneous tumours. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.
•FLASH radiation dose-rates consume all the local tissue O2 to form reactive organic hydroperoxides.•Fenton type reactions will be limited in normal vs. cancer tissues due to lower levels of labile ...Fe.•Normal tissues are expected to remove organic hydroperoxides more effectively relative to tumor tissues.•Since tumor tissue cannot remove hydroperoxides as effectively, FLASH and conventional dose rate irradiation are more isoefficient at killing tumor cells compared to normal cells.
For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1–0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors.
Although breast cancer stem cells (BCSCs) display plasticity transitioning between quiescent mesenchymal-like (M) and proliferative epithelial-like (E) states, how this plasticity is regulated by ...metabolic or oxidative stress remains poorly understood. Here, we show that M- and E-BCSCs rely on distinct metabolic pathways and display markedly different sensitivities to inhibitors of glycolysis and redox metabolism. Metabolic or oxidative stress generated by 2DG, H2O2, or hypoxia promotes the transition of ROSlo M-BCSCs to a ROShi E-state. This transition is reversed by N-acetylcysteine and mediated by activation of the AMPK-HIF1α axis. Moreover, E-BCSCs exhibit robust NRF2-mediated antioxidant responses, rendering them vulnerable to ROS-induced differentiation and cytotoxicity following suppression of NRF2 or downstream thioredoxin (TXN) and glutathione (GSH) antioxidant pathways. Co-inhibition of glycolysis and TXN and GSH pathways suppresses tumor growth, tumor-initiating potential, and metastasis by eliminating both M- and E-BCSCs. Exploiting metabolic vulnerabilities of distinct BCSC states provides a novel therapeutic approach targeting this critical tumor cell population.
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•E- and M-BCSCs have divergent sensitivities to glycolysis or redox metabolism inhibition•Hypoxic or oxidant stress promotes M to E state transition by activating AMPK-HIF1α•E-BCSCs are more oxidative (OXPHOS) and reliant on NRF2 antioxidant responses•Co-inhibition of glycolysis and TXN and GSH pathways targets both M- and E-BCSCs
Luo et al. report that metabolic stressors modulate breast cancer stem cell (BCSC) state dynamics through ROS-mediated activation of the AMPK-HIF1α axis. They further describe the metabolic pathways and vulnerabilities of epithelial- and mesenchymal-like BCSCs and build a conceptual framework to effectively target both BCSC states in PDX and systemic metastasis models of TNBC.
Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose-deprivation-induced cytotoxicity. To determine whether oxidative stress mediated by O(2)(*-) and hydroperoxides ...contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, the oxidation of DHE (dihydroethidine; for O(2)(*-)) and CDCFH(2) 5- (and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate; for hydroperoxides was measured in human colon and breast cancer cells (HT29, HCT116, SW480 and MB231) and compared with that in normal human cells FHC cells, 33Co cells and HMECs (human mammary epithelial cells). Cancer cells showed significant increases in DHE (2-20-fold) and CDCFH(2) (1.8-10-fold) oxidation, relative to normal cells, that were more pronounced in the presence of the mitochondrial electron-transport-chain blocker, antimycin A. Furthermore, HCT116 and MB231 cells were more susceptible to glucose-deprivation-induced cytotoxicity and oxidative stress, relative to 33Co cells and HMECs. HT29 cells were also more susceptible to 2DG (2-deoxyglucose)-induced cytotoxicity, relative to FHC cells. Overexpression of manganese SOD (superoxide dismutase) and mitochondrially targeted catalase significantly protected HCT116 and MB231 cells from glucose-deprivation-induced cytotoxicity and oxidative stress and also protected HT29 cells from 2DG-induced cytotoxicity. These results show that cancer cells (relative to normal cells) demonstrate increased steady-state levels of ROS (reactive oxygen species; i.e. O(2)(*-) and H(2)O(2)) that contribute to differential susceptibility to glucose-deprivation-induced cytotoxicity and oxidative stress. These studies support the hypotheses that cancer cells increase glucose metabolism to compensate for excess metabolic production of ROS and that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.
Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter ...is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.
The biological effects of ionizing radiation (IR) from environmental, medical, and man-made sources, as well as from space exploration are of broad health concern. During the last 40 years it has ...become evident that, in addition to short-lived free radical-mediated events initiated within microseconds of exposure and generally thought to dissipate within milliseconds, IR-induced production of reactive oxygen and nitrogen species as well as changes in redox signaling linked to disruption of metabolic processes persist long after radiation exposure. Furthermore, persistent IR-induced increases in the metabolic production of reactive oxygen and nitrogen species appear to significantly contribute to the delayed effects of IR exposure, including induction of adaptive responses at low doses as well as carcinogenesis, fibrosis, inflammation, genomic instability, and acceleration of the onset of degenerative tissue injury processes associated with aging. The ability to identify the specific metabolic mechanisms and dose-response relationships that contribute to adaptive responses as well as persistent IR-induced injury processes holds great promise for identifying novel strategies to mitigate the deleterious effects of IR exposure as well as for gathering mechanistic information critical for risk assessment. This Forum contains original and review articles authored by experts in the field of radiobiology focusing on novel mechanisms involving redox biology and metabolism that significantly contribute to the persistent biological effects seen following IR exposure.
Precisely how silver nanoparticles (AgNPs) kill mammalian cells still is not fully understood. It is not clear if AgNP-induced damage differs from silver cation (Ag
), nor is it known how AgNP damage ...is transmitted from cell membranes, including endosomes, to other organelles. Cells can differ in relative sensitivity to AgNPs or Ag
, which adds another layer of complexity to identifying specific mechanisms of action. Therefore, we determined if there were specific effects of AgNPs that differed from Ag
in cells with high or low sensitivity to either toxicant.
Cells were exposed to intact AgNPs, Ag
, or defined mixtures of AgNPs with Ag
, and viability was assessed. The level of dissolved Ag
in AgNP suspensions was determined using inductively coupled plasma mass spectrometry. Changes in reactive oxygen species following AgNP or Ag
exposure were quantified, and treatment with catalase, an enzyme that catalyzes the decomposition of H
O
to water and oxygen, was used to determine selectively the contribution of H
O
to AgNP and Ag
induced cell death. Lipid peroxides, formation of 4-hydroxynonenol protein adducts, protein thiol oxidation, protein aggregation, and activation of the integrated stress response after AgNP or Ag
exposure were quantified. Lastly, cell membrane integrity and indications of apoptosis or necrosis in AgNP and Ag
treated cells were examined by flow cytometry.
We identified AgNPs with negligible Ag
contamination. We found that SUM159 cells, which are a triple-negative breast cancer cell line, were more sensitive to AgNP exposure less sensitive to Ag
compared to iMECs, an immortalized, breast epithelial cell line. This indicates that high sensitivity to AgNPs was not predictive of similar sensitivity to Ag
. Exposure to AgNPs increased protein thiol oxidation, misfolded proteins, and activation of the integrated stress response in AgNP sensitive SUM159 cells but not in iMEC cells. In contrast, Ag
cause similar damage in Ag
sensitive iMEC cells but not in SUM159 cells. Both Ag
and AgNP exposure increased H
O
levels; however, treatment with catalase rescued cells from Ag
cytotoxicity but not from AgNPs. Instead, our data support a mechanism by which damage from AgNP exposure propagates through cells by generation of lipid peroxides, subsequent lipid peroxide mediated oxidation of proteins, and via generation of 4-hydroxynonenal (4-HNE) protein adducts.
There are distinct differences in the responses of cells to AgNPs and Ag
. Specifically, AgNPs drive cell death through lipid peroxidation leading to proteotoxicity and necrotic cell death, whereas Ag
increases H
O
, which drives oxidative stress and apoptotic cell death. This work identifies a previously unknown mechanism by which AgNPs kill mammalian cells that is not dependent upon the contribution of Ag
released in extracellular media. Understanding precisely which factors drive the toxicity of AgNPs is essential for biomedical applications such as cancer therapy, and of importance to identifying consequences of unintended exposures.
Genetic deletion of the mitochondrial deacetylase sirtuin-3 (
Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a ...nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3
−
/− livers at 3 months of age. Livers of Sirt3
−/− mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null
Sirt3 into Sirt3
−/− MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD
K122-R), increased MnSOD activity when expressed in MnSOD
−/− MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3
−/− MEFs with lenti-MnSOD
K122-R inhibited in vitro immortalization by an oncogene (
Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3
−/− livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.
► Mitochondrial Sirt3 deacetylates MnSOD after fasting and radiation ► MnSOD contains a reversible acetyl-lysine at amino acid 122 ► MnSOD lysine 122 acetylation regulates enzymatic activity ►
Sirt3 knockout mice have a radiation damage liver phenotype
Mammalian cells use a complex network of redox-dependent processes necessary to maintain cellular integrity during oxidative metabolism, as well as to protect against and/or adapt to stress. The ...disruption of these redox-dependent processes, including those in the mitochondria, creates a cellular environment permissive for progression to a malignant phenotype and the development of resistance to commonly used anticancer agents. An extension of this paradigm is that when these mitochondrial functions are altered by the events leading to transformation and ensuing downstream metabolic processes, they can be used as molecular biomarkers or targets in the development of new therapeutic interventions to selectively kill and/or sensitize cancer versus normal cells. In this Review we propose that mitochondrial oxidative metabolism is altered in tumor cells, and the central theme of this dysregulation is electron transport chain activity, folate metabolism, NADH/NADPH metabolism, thiol-mediated detoxification pathways, and redox-active metal ion metabolism. It is proposed that specific subgroups of human malignancies display distinct mitochondrial transformative and/or tumor signatures that may benefit from agents that target these pathways.
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