Cellular response of MCF-7 and MCF-12A cell line to conventional versus ultra-high dose rate radiotherapy: analysis of proteins involved in the protection of cells against oxidative stress and the regulation of reactive oxygen species (ROS)

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

FLASH with carbon ions: Tumor control, normal tissue sparing, and distal metastasis in a mouse osteosarcoma model.

Recent reports have shown that very high dose rate radiation (35–100 Gy/second) referred to as FLASH tends to spare the normal tissues while retaining the therapeutic effect on tumor. We undertook a series of experiments to assess if ultra-high dose rate of 35 Gy/second can spare the immune system in models of radiation induced lymphopenia. We compared the tumoricidal potency of ultra-high dose rate and conventional dose rate radiation using a classical clonogenic assay in murine pancreatic cancer cell lines. We also assessed the lymphocyte sparing potential in cardiac and splenic irradiation models of lymphopenia and assessed the severity of radiation-induced gastrointestinal toxicity triggered by the two dose rate regimes in vivo. Ultra-high dose rate irradiation more potently induces clonogenic cell death than conventional dose rate irradiation with a dose enhancement factor at 10% survival (DEF10) of 1.310 and 1.365 for KPC and Panc02 cell lines, respectively. Ultra-high dose rate was equally potent in depleting CD3, CD4, CD8, and CD19 lymphocyte populations in both cardiac and splenic irradiation models of lymphopenia. Radiation-induced gastrointestinal toxicity was more pronounced and mouse survival (7 days vs. 15 days, p = 0.0001) was inferior in the ultra-high dose rate arm compared to conventional dose rate arm. These results suggest that, contrary to published data in other models of radiation-induced acute and chronic toxicity, dose rates of 35 Gy/s do not protect mice from the detrimental side effects of irradiation in our models of cardiac and splenic radiation-induced lymphopenia or gastrointestinal mucosal injury.

Is singlet oxygen involved in FLASH-RT?

Ultra-high dose rate (UHDR) radiotherapy has become a large area of research due to observed normal tissue sparing without sacrificing tumor control, termed the FLASH effect. The purpose of this study was to compare reactive oxygen species (ROS) production and DNA damage across various O2 levels at UHDR and conventional dose rates (CDR) in solutions without repair enzymes and radical scavengers. Solution assays of both ROS and DNA damage assessed dose rate and oxygen dependent (0-20% O2) changes between UHDR and CDR from an IntraOp Mobetron. For ROS reporters Amplex UltraRed (H2O2), and CellROX Deep Red (non-H2O2) were quantified via intensity per unit dose. DNA damage assayed plasmid pBR322 gel electrophoresis, to differentiate both single (SSB) and double strand breaks (DSB). For ROS assays, a significant reduction was noted from CDR to UHDR across all measured oxygen levels. The generation of H2O2 decreased when departing from physiologically relevant oxygen levels (1-5%), with generation 30-40% lower at UHDR. The DNA damage assay showed no trends in the SSB or DSB values with O2. Examination of trends between ROS and DNA damage from factors such as oxygen can help elucidate FLASH mechanisms. The H2O2 yield has maximum yield at physiological oxygenation levels (1-5%), and UHDR further diminishes yield. In DNA damage no trend was observed. It is possible that these mechanisms have underlying effects on the FLASH effect in vivo. This study is the first to directly compare radiation chemistry differences caused by UHDR to biologically relevant DNA damage in identical solutions.

Reactive oxygen species (ROS) are important biological radicals essential for determining different stages and phenotypes of cells from quiescence to proliferation, differentiation, self-renewal and even apoptosis. Low ROS favours quiescence and self-renewal in contrast to high ROS that dictates proliferation, differentiation or apoptosis. Such wide variety of cell fates depends upon specific signalling pathways that regulate the cellular ROS, thus contributing to tissue homeostasis. Imbalance of ROS causes several pathological conditions including cancer which is associated with higher level of ROS that supports tumour development and progression. However, to restrain from the excessive oxidative damage of ROS, cancer cells efficiently control the antioxidative pathways, thus favouring its own survival and maintenance at the same time. Furthermore, importance of ROS has been an active field of research in ‘cancer stem cells’ (CSCs), a subpopulation of cancer cells with stem cell-like properties and features. CSCs possess low ROS level that make them resistant to the existing chemotherapy or radiotherapy that ultimately leads to cancer recurrence. Though several evidences have proved the role of ROS in self-renewal and stemness of CSCs, there is a lot to explore about ROS-regulated signalling mechanisms in CSCs. An understanding of ROS regulation in CSCs can provide an idea about the application of oxidative stress as a therapeutic strategy in treatment of cancer. In this book chapter, we have raised the debate as to whether ROS acts as ‘friend or foe’ for cancer cells. Moreover, exploring the significance of ROS and redox regulation in lung cancer stem cells has been our major focus. Finally, it is suggested that in order to get an effective treatment and recurrence-free survival, sensitization of the cancer stem cells to high ROS environment is a must.

Mdm2 Is Required for Survival of Hematopoietic Stem Cells/Progenitors via Dampening of ROS-Induced p53 Activity

Background: The hypothalamic paraventricular nucleus (PVN) is an important nucleus in the brain that plays a key role in regulating sympathetic nerve activity (SNA) and blood pressure. Silent mating-type information regulation 2 homolog-1 (sirtuin1, SIRT1) not only protects cardiovascular function but also reduces inflammation and oxidative stress in the periphery. However, its role in the central regulation of hypertension remains unknown. It is hypothesized that SIRT1 activation by resveratrol may reduce SNA and lower blood pressure through the regulation of intracellular reactive oxygen species (ROS) and neurotransmitters in the PVN. Methods: The two-kidney one-clip (2K1C) method was used to induce renovascular hypertension in male Sprague-Dawley rats. Then, bilaterally injections of vehicle (artificial cerebrospinal fluid, aCSF, 0.4 μL) or resveratrol (a SIRT1 agonist, 160 μmol/L, 0.4 μL) into rat PVN were performed for four weeks. Results: PVN SIRT1 expression was lower in the hypertension group than the sham surgery (SHAM) group. Activated SIRT1 within the PVN lowered systolic blood pressure and plasma norepinephrine (NE) levels. It was found that PVN of 2K1C animals injected with resveratrol exhibited increased expression of SIRT1, copper-zinc superoxide dismutase (SOD1), and glutamic acid decarboxylase (GAD67), as well as decreased activity of nuclear factor-kappa B (NF-κB) p65 and NAD(P)H oxidase (NOX), particularly NOX4. Treatment with resveratrol also decreased expression of ROS and tyrosine hydroxylase (TH). Conclusion: Resveratrol within the PVN attenuates hypertension via the SIRT1/NF-κB pathway to decrease ROS and restore the balance of excitatory and inhibitory neurotransmitters.

Oxidative Stress-Mediated Activation of AKT/mTOR Signaling Pathway Leads to Myeloproliferative Syndrome in FoxO3 Null Mice: A Role for Lnk Adaptor Protein

Conventional radiotherapy based on X rays is used to treat more than 50% of cancers. Although effective, radiotherapy can damage healthy tissues around the tumor due to the X-ray dose deposition profile, as well as the safety margin needed to compensate for dose uncertainties. A notable side effect is cellular senescence, characterized by the cessation of cell division while maintaining metabolic activity and promoting the secretion of various components, called the senescence-associated secretory phenotype. To minimize toxicity in healthy tissues, proton therapy holds great promise as it enables tumors to be targeted more precisely while sparing healthy tissues beyond the tumor site. Another innovative method is ultra-high dose rate irradiation, which seems to induce less damage to healthy tissues while generating an anti-tumor response similar to standard dose rate irradiation. In this work, we aimed to compare the effects of X rays and protons at conventional dose rate (2 Gy/min) and ultra-high dose rate (454 Gy/s), on the induction of senescence in primary normal human dermal fibroblasts by analyzing several senescence biomarkers. Irradiation with ultra-high dose rate protons caused more pronounced cellular and nuclear morphological changes in normal human dermal fibroblasts than irradiation with conventional protons or X-rays. For other biomarkers, all three types of irradiations induced an increase in the proportion of senescence-associated beta-gal-positive cells, an irreversible cell cycle arrest and an accumulation of unrepaired DNA damage, but did not affect senescence-associated secretory phenotype.

SIRT3 is a key regulator of mitochondrial reactive oxygen species as well as mitochondrial function. The retina is one of the highest energy-demanding tissues, in which the regulation of reactive oxygen species is critical to prevent retinal neurodegeneration. Although previous reports have demonstrated that SIRT3 is highly expressed in the retina and important in neuroprotection, function of SIRT3 in regulating reactive oxygen species in the retina is largely unknown. In this study, we investigated the role of retinal SIRT3 in a light-induced retinal degeneration model using SIRT3 knockout mice. We demonstrate that SIRT3 deficiency causes acute reactive oxygen species accumulation and endoplasmic reticulum stress in the retina after the light exposure, which leads to increased photoreceptor death, retinal thinning, and decreased retinal function. Using a photoreceptor-derived cell line, we revealed that reactive oxygen species were the upstream initiators of endoplasmic reticulum stress. Under SIRT3 knockdown condition, we demonstrated that decreased superoxide dismutase 2 activity led to elevated intracellular reactive oxygen species. These studies have helped to elucidate the critical role of SIRT3 in photoreceptor neuronal survival, and suggest that SIRT3 might be a therapeutic target for oxidative stress-induced retinal disorders.

Purpose:Ultra-high dose rate (>40 Gy/s, FLASH) radiation therapy (RT) provides equivalent tumor control while reducing normal tissue toxicity relative to conventional dose rate (CONV) RT. However, the mechanisms underlying the observed FLASH effect are unknown. We hypothesized that the preservation of mitochondrial integrity in nontumorigenic cells by FLASH RT could be a key factor in reducing normal tissue toxicity and improving overall treatment outcomes.Methods:We examined mitochondrial health and function after CONV and FLASH in vitro, ex vivo, and in vivo through assays of metabolic flux, mitochondrial membrane potential, mitochondrial reactive oxygen species (ROS), mitochondrial DNA damage and copy number, mitochondrial morphology, and tumor growth and survival.Results:In in vitro assays, murine pancreatic cancer (PDAC) cells showed evidence of equal mitochondrial damage in response to CONV and FLASH, but nontumorigenic pancreatic cells were spared by FLASH. These results were recapitulated ex vivo, and mice treated with FLASH showed higher response rates and longer survival time than mice treated with CONV in an in vivo tumor model.Conclusions:Collectively, these results suggest that FLASH spares mitochondrial function in nontumorigenic cells, but not in PDAC cells, relative to CONV. The preservation of mitochondrial integrity in nontumorigenic cells may be a key mechanism underlying the reduced normal tissue toxicity observed with FLASH RT.

Increased production of reactive oxygen species (ROS) has been linked to the pathogenesis of congestive heart failure. However, emerging evidence suggests the involvement of ROS in the regulation of various physiological cellular processes in the myocardium. In this review, we summarize the latest findings regarding the role of ROS in the acute regulation of cardiac contractility. We discuss ROS-dependent modulation of the inotropic responses to G protein-coupled receptor agonists (e.g. β-adrenergic receptor agonists and endothelin-1), the potential cellular sources of ROS (e.g. NAD(P)H oxidases and mitochondria) and the proposed end-targets and signalling pathways by which ROS affect contractility. Accumulating new data supports the fundamental role of endogenously generated ROS to regulate cardiac function under physiological conditions.

Objectives: To advance our knowledge of disease mechanisms and therapeutic options, understanding cell cycle regulation is critical. Recent research has highlighted the importance of reactive oxygen species (ROS) in cell cycle regulation. Although excessive ROS levels can lead to age-related pathologies, ROS also play an essential role in normal cellular functions. Many cell cycle regulatory proteins are affected by their redox status, but the precise mechanisms and conditions under which ROS promote or inhibit cell proliferation are not fully understood. Methods: This review presents data from the scientific literature and publicly available databases on changes in redox state during the cell cycle and their effects on key regulatory proteins. Results: We identified redox-sensitive targets within the cell cycle machinery and analysed different effects of ROS (type, concentration, duration of exposure) on cell cycle phases. For example, moderate levels of ROS can promote cell proliferation by activating signalling pathways involved in cell cycle progression, whereas excessive ROS levels can induce DNA damage and trigger cell cycle arrest or cell death. Discussion: Our findings encourage future research focused on identifying redox-sensitive targets in the cell cycle machinery, potentially leading to new treatments for diseases with dysregulated cell proliferation.

Proton and Electron Ultrahigh-Dose-Rate Isodose Irradiations Produce Differences in Reactive Oxygen Species Yields