Radioprotection and Radiomitigation: From the Bench to Clinical Practice.
The development of protective agents against harmful radiations has been a subject of investigation for decades. However, effective (ideal) radioprotectors and radiomitigators remain an unsolved problem. Because ionizing radiation-induced cellular damage is primarily attributed to free radicals, radical scavengers are promising as potential radioprotectors. Early development of such agents focused on thiol synthetic compounds, e.g., amifostine (2-(3-aminopropylamino) ethylsulfanylphosphonic acid), approved as a radioprotector by the Food and Drug Administration (FDA, USA) but for limited clinical indications and not for nonclinical uses. To date, no new chemical entity has been approved by …
Down-regulation of Glutathione and Bcl-2 Synthesis in Mouse B16 Melanoma Cells Avoids Their Survival during Interaction with the Vascular Endothelium
B16 melanoma (B16M) cells with high GSH content show high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (approximately 5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from approximately 19% (controls) to approximately 97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucl…
Oxidative stress and antioxidants in the pathophysiology of malignant melanoma.
Abstract The high number of somatic mutations in the melanoma genome associated with cumulative ultra violet (UV) exposure has rendered it one of the most difficult of cancers to treat. With new treatment approaches based on targeted and immune therapies, drug resistance has appeared as a consistent problem. Redox biology, including reactive oxygen and nitrogen species (ROS and RNS), plays a central role in all aspects of melanoma pathophysiology, from initiation to progression and to metastatic cells. The involvement of melanin production and UV radiation in ROS/RNS generation has rendered the melanocytic lineage a unique system for studying redox biology. Overall, an elevated oxidative st…
Melanoma in the liver: Oxidative stress and the mechanisms of metastatic cell survival.
Abstract Metastatic melanoma is a fatal disease with a rapid systemic dissemination. The most frequent target sites are the liver, bone, and brain. Melanoma metastases represent a heterogeneous cell population, which associates with genomic instability and resistance to therapy. Interaction of melanoma cells with the hepatic sinusoidal endothelium initiates a signaling cascade involving cytokines, growth factors, bioactive lipids, and reactive oxygen and nitrogen species produced by the cancer cell, the endothelium, and also by different immune cells. Endothelial cell-derived NO and H2O2 and the action of immune cells cause the death of most melanoma cells that reach the hepatic microvascul…
Abstract 1390: A combination of natural polyphenols, a NAD+ booster, and a Toll-like receptor 2/6 agonist exerts high radioprotection in normal tissues without interfering with the anticancer efficacy of ionizing radiations
Abstract Ionizing radiation damages cells via direct ionization of DNA and other cellular targets as well as by indirect effects through reactive oxygen species. The response to radiation exposure depends on the cell type and dose of radiation, inherent tissue sensitivity and repair, and modulating intracellular factors that include cell cycle status, O2 pressure, and levels of thiols and other antioxidants. Potentially protective agents against exposure to harmful radiation have been investigated for decades. However, no ideal radioprotector is currently available. A wide range of phytochemicals are antioxidants and, thus, potentially radioprotective. Topical administration of Pterostilben…
Polyphenolic Phytochemicals in Cancer Prevention and Therapy: Bioavailability versus Bioefficacy
Natural polyphenols are organic chemicals which contain phenol units in their structures. They show antitumor properties. However, a key problem is their short half-life and low bioavailability under in vivo conditions. Still, definitively demonstrating the human benefits of isolated polyphenolic compounds (alone or in combination) using modern scientific methodology has proved challenging. The most common discrepancy between experimental and clinical observations is the use of nonphysiologically relevant concentrations of polyphenols in mechanistic studies. Thus, it remains highly controversial how applicable underlying mechanisms are with bioavailable concentrations and biological half-li…
Regulation of tumour cell sensitivity to TNF-induced oxidative stress and cytotoxicity: Role of glutathione
Glutathione (GSH) and the rate of cellular proliferation determine tumour cell sensitivity to tumour necrosis factor (TNF). Buthionine sulphoximine (BSO), a selective inhibitor of GSH synthesis, inhibits tumour growth and increases recombinant human TNF (rhTNF)-alpha cytoxicity in vitro. Administration of sublethal doses of rhTNF-alpha to Ehrlich ascites-tumour (EAT)-bearing mice induces oxidative stress (as measured by increases in intracellular peroxide levels, O2.- generation and mitochondrial GSSG). ATP-induced selective GSH depletion, when combined with rhTNF-alpha administration, affords a 61% inhibition of tumour growth and results in a significant extent of host survival. Administra…
Mitochondrial glutathione depletion by glutamine in growing tumor cells.
The effect of L-glutamine (Gln) on mitochondrial glutathione (mtGSH) levels in tumor cells was studied in vivo in Ehrlich ascites tumor (EAT)-bearing mice. Tumor growth was similar in mice fed a Gln-enriched diet (GED; where 30% of the total dietary nitrogen was from Gln) or a nutritionally complete elemental diet (SD). As compared with non-tumor-bearing mice, tumor growth caused a decrease of blood Gln levels in mice fed an SD but not in those fed a GED. Tumor cells in mice fed a GED showed higher glutaminase and lower Gln synthetase activities than did cells isolated from mice fed an SD. Cytosolic glutamate concentration was 2-fold higher in tumor cells from mice fed a GED ( approximately…
Acceleration of glutathione efflux and inhibition of gamma-glutamyltranspeptidase sensitize metastatic B16 melanoma cells to endothelium-induced cytotoxicity.
Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP-/-1) B16M-F10 transfected with the Bcl-2 gene or in MRP-/-1 B16M-F10 cells incubated with l-methionine, wh…
Tumoricidal activity of endothelium-derived NO and the survival of metastatic cells with high GSH and Bcl-2 levels.
Metastatic spread, not primary tumor burden, is the leading cause of cancer death. Glutathione (L-gamma-glutamyl-L-cysteinyl-glycine; GSH) is the most prevalent non-protein thiol in mammalian cells, and in cancer cells is particularly relevant in regulating mutagenic mechanisms, DNA synthesis, growth, and multidrug and radiation resistance. In malignant tumors, as compared with normal tissues, that resistance associates in most cases with higher GSH levels. Interaction of metastatic cells with the vascular endothelium activates local release of proinflammatory cytokines, which act as signals promoting cancer cell adhesion, extravasation, and proliferation. A high% of metastatic cells with h…
The Link between Oxidative Stress, Redox Status, Bioenergetics and Mitochondria in the Pathophysiology of ALS
Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disease of the motor system. It is characterized by the degeneration of both upper and lower motor neurons, which leads to muscle weakness and paralysis. ALS is incurable and has a bleak prognosis, with median survival of 3–5 years after the initial symptomatology. In ALS, motor neurons gradually degenerate and die. Many features of mitochondrial dysfunction are manifested in neurodegenerative diseases, including ALS. Mitochondria have shown to be an early target in ALS pathophysiology and contribute to disease progression. Disruption of their axonal transport, excessive generation of reactive oxygen species, disruptio…
Glucocorticoid receptor knockdown decreases the antioxidant protection of B16 melanoma cells: an endocrine system-related mechanism that compromises metastatic cell resistance to vascular endothelium-induced tumor cytotoxicity.
We previously reported an interorgan system in which stress-related hormones (corticosterone and noradrenaline), interleukin-6, and glutathione (GSH) coordinately regulate metastatic growth of highly aggressive B16-F10 melanoma cells. Corticosterone, at levels measured in tumor-bearing mice, also induces apoptotic cell death in metastatic cells with low GSH content. In the present study we explored the potential role of glucocorticoids in the regulation of metastatic cell death/survival during the early stages of organ invasion. Glucocorticoid receptor (GCR) knockdown decreased the expression and activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step in GSH synthesis, in …
Pterostilbene in Cancer Therapy.
Natural polyphenols are organic chemicals which contain phenol units in their structures and possess antitumor properties. However, a key problem is their short half-life and low bioavailability under in vivo conditions. Pterostilbene (3,5-dimethoxy-4′-hydroxystilbene; PT) is a phytoalexin originally isolated from the heartwood of red sandalwood. As recently reported by our group, PT was shown to be effective in the treatment of melanoma. Counterintuitively, PT is not effective (cytotoxic) against melanoma in vitro, and only under in vivo conditions does PT display its anticancer activity. This study elucidated that PT can be effective against melanoma through the inhibition of adrenocortic…
Tumor Cytotoxicity by Endothelial Cells
High GSH content associates with high metastatic activity in B16-F10 melanoma cells cultured to low density (LD B16M). GSH homeostasis was investigated in LD B16M cells that survive after adhesion to the hepatic sinusoidal endothelium (HSE). Invasive B16M (iB16M) cells were isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting. HSE-derived NO and H(2)O(2) caused GSH depletion and a decrease in gamma-glutamylcysteine synthetase activity in iB16M cells. Overexpression of gamma-glutamylcysteine synthetase heavy and light subunits led to a rapid recovery of cytosolic GSH, whereas mitochondrial GSH (mtGSH) further decreased during the first 18 h of culture. NO …
Glutathione in metastases: From mechanisms to clinical applications.
Metastatic spread, not primary tumors, is the leading cause of cancer death. Glutathione (γ-glutamyl-cysteinyl-glycine, GSH) is particularly relevant in cancer cells as it is involved in regulating carcinogenic mechanisms, growth and dissemination, and multidrug and radiation resistance. Upon interaction of metastatic cells with the vascular endothelium, a high percentage of metastatic cells with high GSH levels survive the combined nitrosative and oxidative stresses elicited by the vascular endothelium. GSH release from different organs, mainly the liver, and its interorgan transport through the blood circulation to metastatic foci, promote their growth. This review focuses on the relation…
Glutathione in Cancer Biology and Therapy
The glutathione (GSH) content of cancer cells is particularly relevant in regulating mutagenic mechanisms, DNA synthesis, growth, and multidrug and radiation resistance. In malignant tumors, as compared with normal tissues, that resistance associates in most cases with higher GSH levels within these cancer cells. Thus, approaches to cancer treatment based on modulation of GSH should control possible growth-associated changes in GSH content and synthesis in these cells. Despite the potential benefits for cancer therapy of a selective GSH-depleting strategy, such a methodology has remained elusive up to now. Metastatic spread, not primary tumor burden, is the leading cause of cancer death. Fo…
Bcl-2 and Mn-SOD antisense oligodeoxynucleotides and a glutamine-enriched diet facilitate elimination of highly resistant B16 melanoma cells by tumor necrosis factor-alpha and chemotherapy.
Mitochondrial glutathione (mtGSH) depletion increases sensitivity of Bcl-2-overexpressing B16 melanoma (B16M)-F10 cells (high metastatic potential) to tumor necrosis factor-alpha (TNF-alpha)-induced oxidative stress and death in vitro. In vivo, mtGSH depletion in B16M-F10 cells was achieved by feeding mice (where the B16M-F10 grew as a solid tumor in the footpad) with an L-glutamine (L-Gln)-enriched diet, which promoted in the tumor cells an increase in glutaminase activity, accumulation of cytosolic L-glutamate, and competitive inhibition of GSH transport into mitochondria. L-Gln-adapted B16M-F10 cells, isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sortin…
γ-Glutamyl transpeptidase overexpression increases metastatic growth of B16 melanoma cells in the mouse liver
B16 melanoma (B16M) cells with high glutathione (GSH) content show rapid proliferation in vitro and high metastatic activity in the liver in vivo. gamma-Glutamyl transpeptidase (GGT)-mediated extracellular GSH cleavage and intracellular GSH synthesis were studied in vitro in B16M cells with high (F10) and low (F1) metastatic potential. GGT activity was modified by transfection with the human GGT gene (B16MF1/Tet-GGT cells) or by acivicin-induced inhibition. B16MF1/Tet-GGT and B16MF10 cells exhibited higher GSH content (35 +/- 6 and 40 +/- 5 nmol/10(6) cells, respectively) and GGT activity (89 +/- 9 and 37 +/- 7 mU/10(6) cells, respectively) as compared (P <.05) with B16MF1 cells (10 +/- 3 n…
Astrocytes Protect Neurons from Aβ1-42 Peptide-Induced Neurotoxicity Increasing TFAM and PGC-1 and Decreasing PPAR-γ and SIRT-1
One of the earliest neuropathological events in Alzheimer's disease is accumulation of astrocytes at sites of Aβ1-42 depositions. Our results indicate that Aβ1-42 toxic peptide increases lipid peroxidation, apoptosis and cell death in neurons but not in astrocytes in primary culture. Aβ1-42-induced deleterious neuronal effects are not present when neurons and astrocytes are mixed cultured. Stimulation of astrocytes with toxic Aβ1-42 peptide increased p-65 and decreased IκB resulting in inflammatory process. In astrocytes Aβ1-42 decreases protein expressions of sirtuin 1 (SIRT-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) and over-expresses peroxisome proliferator-activated re…
NAD+ Precursors and Antioxidants for the Treatment of Amyotrophic Lateral Sclerosis
Charcot first described amyotrophic lateral sclerosis (ALS) between 1865 and 1874 as a sporadic adult disease resulting from the idiopathic progressive degeneration of the motor neuronal system, resulting in rapid, progressive, and generalized muscle weakness and atrophy. There is no cure for ALS and no proven therapy to prevent it or reverse its course. There are two drugs specifically approved for the treatment of ALS, riluzol and edaravone, and many others have already been tested or are following clinical trials. However, at the present moment, we still cannot glimpse a true breakthrough in the treatment of this devastating disease. Nevertheless, our understanding of the pathophysiology…
Inhibition of cancer growth by resveratrol is related to its low bioavailability.
The relationship between resveratrol (RES) bioavalability and its effect on tumor growth was investigated. Tissue levels of RES were studied after i.v. and oral administration of trans-resveratrol (t-RES) to rabbits, rats, and mice. Half-life of RES in plasma, after i.v. administration of 20 mg t-RES/kg b.wt., was very short (e.g., 14.4 min in rabbits). The highest concentration of RES in plasma, either after i.v. or oral administration (e.g., 2.6 +/- 1.0 microM in mice 2.5 min after receiving 20 mg t-RES/kg orally), was reached within the first 5 min in all animals studied. Extravascular levels (brain, lung, liver, and kidney) of RES, which paralleled those in plasma, were always1 nmol/g f…
Glutathione protects metastatic melanoma cells against oxidative stress in the murine hepatic microvasculature.
Calcein-labeled B16 melanoma (B16M) cells were injected intraportally, and in vivo video microscopy was used to study the distribution and damage of cancer cells arrested in the liver microvasculature over a period of 4 hours. The contribution of glutathione (GSH)-dependent antioxidant machinery to the possible oxidative stress-resistance mechanism of B16M cell was determined by in vitro incubation with the selective inhibitor of GSH synthesis L-buthionine (S,R)-sulphoximine (BSO) before B16M cell injection in untreated and 0.5-mg/kg lipopolysaccharide (LPS)-treated mice. In addition, untreated and LPS-treated isolated syngeneic hepatic sinusoidal endothelial cells (HSE) were used to determ…
Oxidative Stress, Neuroinflammation and Mitochondria in the Pathophysiology of Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron (MN) disease. Its primary cause remains elusive, although a combination of different causal factors cannot be ruled out. There is no cure, and prognosis is poor. Most patients with ALS die due to disease-related complications, such as respiratory failure, within three years of diagnosis. While the underlying mechanisms are unclear, different cell types (microglia, astrocytes, macrophages and T cell subsets) appear to play key roles in the pathophysiology of the disease. Neuroinflammation and oxidative stress pave the way leading to neurodegeneration and MN death. ALS-associated mitochondrial dysfunction occurs at different le…
Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism
[EN] Background: Interleukin (IL)-6 (mainly of tumor origin) activates glutathione (GSH) release from hepatocytes and its interorgan transport to B16-F10 melanoma metastatic foci. We studied if this capacity to overproduce IL-6 is regulated by cancer cell-independent mechanisms. Methods: Murine B16-F10 melanoma cells were cultured, transfected with red fluorescent protein, injected i.v. into syngenic C57BL/6J mice to generate lung and liver metastases, and isolated from metastatic foci using high-performance cell sorting. Stress hormones and IL-6 levels were measured by ELISA, and CRH expression in the brain by in situ hybridization. DNA binding activity of NF-kappa B, CREB, AP-1, and NF-IL…
Nitric Oxide Mediates Natural Polyphenol-induced Bcl-2 Down-regulation and Activation of Cell Death in Metastatic B16 Melanoma
Intravenous administration to mice of trans-pterostilbene (t-PTER; 3,5-dimethoxy-4'-hydroxystilbene) and quercetin (QUER; 3,3',4',5,6-pentahydroxyflavone), two structurally related and naturally occurring small polyphenols, inhibits metastatic growth of highly malignant B16 melanoma F10 (B16M-F10) cells. t-PTER and QUER inhibit bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. However, the molecular mechanism(s) linking polyphenol signaling and bcl-2 expression are unknown. NO is a potential bioregulator of apoptosis with controversial effects on Bcl-2 regulation. Polyphenols may affect NO generation. Short-term exposure (60 min/day) t…
Facilitation of Insulin Effects by Ranolazine in Astrocytes in Primary Culture
Ranolazine (Rn) is a drug used to treat persistent chronic coronary ischemia. It has also been shown to have therapeutic benefits on the central nervous system and an anti-diabetic effect by lowering blood glucose levels and however, no effects of Rn on cellular sensitivity to insulin (Ins) have been demonstrated yet. The present study aimed to investigate the permissive effects of Rn on the actions of Ins in astrocytes in primary culture. Ins at 10-8 M, Rn (10-6 M) and Ins+Rn (10-8 M and 10−6 M respectively) were added to astrocytes during 24 h. In comparison to control cells, Rn and/or Ins caused modifications in cell viability and proliferation. p-AKT, p-ERK, p-eNOS, Mn-SOD, COX-2, and t…
Glucocorticoid receptor antagonism overcomes resistance to BRAF inhibition in BRAF(V600E)-mutated metastatic melanoma
Clinical applications of glucocorticoids (GC) in Oncology are dependent on their pro-apoptotic action to treat lymphoproliferative cancers, and to alleviate side effects induced by chemotherapy and/or radiotherapy. However, the mechanism(s) by which GC may also promote tumor progression remains unclear. GC receptor (GR) knockdown decreases the antioxidant protection of highly metastatic B16-F10 melanoma cells. We hypothesize that a GR antagonist (RU486, mifepristone) could increase the efficacy of BRAF-related therapy in BRAF(V600E)-mutated metastatic melanoma. In vivo formed spontaneous skin tumors were reinoculated into nude mice to expand the metastases of different human BRAF(V600E) mel…
Changes in Chemokines and Chemokine Receptors Expression in a Mouse Model of Alzheimer's Disease
The amyloid precursor protein plus presenilin-1 (APP/PS1) mice are a frequently-used model for Alzheimer's disease studies (AD). However, the data relevant to which proteins are involved in inflammatory mechanism are not sufficiently well-studied using the AD mouse model. Using behavioral studies, quantitative RT-PCR and Western-blot techniques, significant findings were determined by the expression of proteins involved in inflammation comparing APP/PS1 and Wild type mice. Increased GFAP expression could be associated with the elevation in number of reactive astrocytes. IL-3 is involved in inflammation and ABDF1 intervenes normally in the transport across cell membranes and both were found …
Efficacy and tolerability of EH301 for amyotrophic lateral sclerosis: a randomized, double-blind, placebo-controlled human pilot study
Background: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by progressive loss of spinal and cortical motor neurons, leading to muscular atrophy, respiratory failure, and ultimately death. There is no known cure, and the clinical benefit of the two drugs approved to treat ALS remains unclear. Novel disease-modifying therapeutics that are able to modulate the disease course are desperately needed. Our objective was to evaluate the efficacy and tolerability of Elysium Health's candidate drug EH301 in people with ALS (PALS). Methods: This was a single-center, prospective, double-blind, randomized, placebo-controlled pilot study. Thirty-two PALS we…
An Intercellular Flow of Glutathione Regulated by Interleukin 6 Links Astrocytes and the Liver in the Pathophysiology of Amyotrophic Lateral Sclerosis
Oxidative stress has been proposed as a major mechanism of damage to motor neurons associated with the progression of amyotrophic lateral sclerosis (ALS). Astrocytes are the most numerous glial cells in the central nervous system and, under physiological conditions, protect neurons from oxidative damage. However, it is uncertain how their reactive phenotype may affect motor neurons during ALS progression. In two different ALS mouse models (SOD1G93A and FUS-R521C), we found that increased levels of proinflammatory interleukin 6 facilitate glutathione (GSH) release from the liver to blood circulation, which can reach the astrocytes and be channeled towards motor neurons as a mechanism of anti…
Glutamine potentiates TNF-α-induced tumor cytotoxicity
L-glutamine (Gln) sensitizes tumor cells to tumor necrosis factor (TNF)-alpha-induced cytotoxicity. The type and mechanism of cell death induced by TNF-alpha was studied in Ehrlich ascites tumor (EAT)-bearing mice fed a Gln-enriched diet (GED; where 30% of the total dietary nitrogen was from Gln). A high rate of Gln oxidation promotes a selective depletion of mitochondrial glutathione (mtGSH) content to approximately 58% of the level found in tumor mitochondria of mice fed a nutritionally complete elemental diet (standard diet, SD). The mechanism of mtGSH depletion involves a glutamate-induced inhibition of GSH transport from the cytosol into mitochondria. The increase in reactive oxygen in…
Blood Glutathione as an Index of Radiation-Induced Oxidative Stress in Mice and Humans
Abstract The effect of x-rays on GSH and GSSG levels in blood was studied in mice and humans. An HPLC method that we recently developed was applied to accurately determine GSSG levels in blood. The glutathione redox status (GSH/GSSG) decreases after irradiation. This effect is mainly due to an increase in GSSG levels. Mice received single fraction radiotherapy, at total doses of 1.0 to 7.0 Gy. Changes in GSSG in mouse blood can be detected 10 min after irradiation and last for 6 h within a range of 2.0–7.0 Gy. The highest levels of GSSG (20.1 ± 2.9 μ M), a 4.7-fold increase as compared with controls) in mouse blood are found 2 h after radiation exposure (5 Gy). Breast and lung cancer patien…
Changes in glutathione status and the antioxidant system in blood and in cancer cells associate with tumour growth in vivo
The relationship among cancer growth, the glutathione redox cycle and the antioxidant system was studied in blood and in tumour cells. During cancer growth, the glutathione redox status (GSH/GSSG) decreases in blood of Ehrlich ascites tumour-bearing mice. This effect is mainly due to an increase in GSSG levels. Two reasons may explain the increase in blood GSSG: (a) the increase in peroxide production by the tumour that, in addition to changes affecting the glutathione-related and the antioxidant enzyme activities, can lead to GSH oxidation within the red blood cells; and (b) an increase of GSSG release from different tissues into the blood. GSH and peroxide levels are higher in the tumour …
Glutathione and the rate of cellular proliferation determine tumour cell sensitivity to tumour necrosis factor in vivo.
Low rates of cellular proliferation are associated with low GSH content and enhanced sensitivity of Ehrlich ascites-tumour (EAT) cells to the cytotoxic effects of recombinant human tumour necrosis factor (rhTNF-alpha). Buthionine sulphoximine, a selective inhibitor of GSH synthesis, inhibited tumour growth and increased rhTNF-alpha cytoxicity in vitro. Administration of sublethal doses (10(6)units/kg per day) of rhTNF-alpha to EAT-bearing mice promoted oxidative stress (as measured by increases in intracellular peroxide levels, O2(-); generation and mitochondrial GSSG) and resulted in a slight reduction (19%) in tumour cell number when controls showed the highest rate of cellular proliferat…
Neuronal effects of Sugammadex in combination with Rocuronium or Vecuronium.
Rocuronium (ROC) and Vecuronium (VEC) are the most currently used steroidal non-depolarizing neuromuscular blocking (MNB) agents. Sugammadex (SUG) rapidly reverses steroidal NMB agents after anaesthesia. The present study was conducted in order to evaluate neuronal effects of SUG alone and in combination with both ROC and VEC. Using MTT, CASP-3 activity and Western-blot we determined the toxicity of SUG, ROC or VEC in neurons in primary culture. SUG induces apoptosis/necrosis in neurons in primary culture and increases cytochrome C (CytC), apoptosis-inducing factor (AIF), Smac/Diablo and Caspase 3 (CASP-3) protein expression. Our results also demonstrated that both ROC and VEC prevent these…
Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.
Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The …
Tumoricidal Activity of Endothelial Cells
The mechanism of NO- and H(2)O(2)-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N(G)-nitro-l-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H(2)O(2) was removed by exogenous catalase. H(2)O(2) was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H(2)O(2) due to the formation of potent oxidants, likely ( small middle dot)OH and (-)OONO radicals, via a trace metal-dependent process. B16M cells culture…
WIN 55,212-2, agonist of cannabinoid receptors, prevents amyloid β1-42 effects on astrocytes in primary culture
Alzheimer's disease (AD), a neurodegenerative illness involving synaptic dysfunction with extracellular accumulation of Aβ1-42 toxic peptide, glial activation, inflammatory response and oxidative stress, can lead to neuronal death. Endogenous cannabinoid system is implicated in physiological and physiopathological events in central nervous system (CNS), and changes in this system are related to many human diseases, including AD. However, studies on the effects of cannabinoids on astrocytes functions are scarce. In primary cultured astrocytes we studied cellular viability using MTT assay. Inflammatory and oxidative stress mediators were determined by ELISA and Western-blot techniques both in…
Bcl-2 and glutathione depletion sensitizes B16 melanoma to combination therapy and eliminates metastatic disease.
Abstract Purpose: Advanced melanoma resists all current therapies, and metastases in the liver are particularly problematic. Prevalent resistance factors include elevated glutathione (GSH) and increased expression of bcl-2 in melanoma cells. GSH has pleiotropic effects promoting cell growth and broad resistance to therapy, whereas Bcl-2 inhibits the activation of apoptosis and contributes to elevation of GSH. This study determined the in vivo efficacy of combination therapies administered while GSH and Bcl-2 were individually and simultaneously decreased in metastatic melanoma lesions. Experimental Design: Highly metastatic murine B16 melanoma (B16M-F10) cells have elevated levels of both G…
Possible Mechanisms for Tumour Cell Sensitivity to TNF-a and Potential Therapeutic Applications
TNF is a macrophage / monocyte-derived cytokine with cytostatic and cytotoxic anti-tumour activity. TNF-alpha can cause haemorrhagic necrosis and regression of experimental tumours. Nevertheless, the TNF-alpha doses required to cure tumour-bearing mice lead to injury of normal tissues and, eventually, may cause a lethal shock syndrome. This toxicity implies severe limitations for the therapeutic use of TNF-a. Reactive oxygen intermediates (ROIs) are involved in TNF-a-induced cell killing. Different studies are consistent with the hypothesis that tumour cell sensitivity to TNF-alpha is related to its capacity to buffer oxidative attack. Recently, we have demonstrated that the sensitivity of …
Sugammadex, a Neuromuscular Blockade Reversal Agent, Causes Neuronal Apoptosis in Primary Cultures
Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (< 3% in rats), and thus no relevant central nervous toxicity is expected. However the blood brain barrier permeability can be altered under different conditions (i.e. neurodegenerative diseases, trauma, ischemia, infections, or immature nervous system). Using MTT, confocal microscopy, caspase-3 activity, cholesterol quantification and Western-blot we …
Elimination of Ehrlich tumours by ATP-induced growth inhibition, glutathione depletion and X-rays
ATP-induced tumour growth inhibition is accompanied by a selective decrease in the content of the tripeptide glutathione (GSH) within the cancer cells in vivo. Depletion of cellular GSH sensitizes tumours to chemotherapy and radiation, but the usefulness of this depletion depends on whether the levels of GSH can be reduced in the tumour relative to normal tissues. We report here that administration of ATP in combination with diethylmaleate and X-rays leads to complete regression of 95% of Ehrlich ascites tumours in mice. This shows that an aggressive tumour can be eliminated by using a therapy based on modulation of GSH levels in cancer cells.
Association between Pterostilbene and Quercetin Inhibits Metastatic Activity of B16 Melanoma
AbstractInhibition of cancer growth by resveratrol (trans-3,5,4'trihydroxystilbene; RESV), a phytoalexin present in many plant species, is limited by its low bioavailability. Pterostilbene (3,5-dimethoxy-4'-hydroxystilbene; PTER) and quercetin (3,3',4',5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols, show longer half-life in vivo. In vitro growth of highly malignant B16 melanoma F10 cells (B16M-F10) is inhibited (56%) by short-time exposure (60 min/day) to PTER (40 μM) and QUER (20 μM) (approximate mean values of plasma concentrations measured within the first hour after intravenous administration of 20 mg/kg of each polyphenol). Intravenou…
Intertissue Flow of Glutathione (GSH) as a Tumor Growth-promoting Mechanism
B16 melanoma F10 (B16-F10) cells with high glutathione (GSH) content show high metastatic activity in vivo. An intertissue flow of GSH, where the liver is the main reservoir, can increase GSH content in metastatic cells and promote their growth. We have studied here possible tumor-derived molecular signals that could activate GSH release from hepatocytes. GSH efflux increases in hepatocytes isolated from mice bearing liver or lung metastases, thus suggesting a systemic mechanism. Fractionation of serum-free conditioned medium from cultured B16-F10 cells and monoclonal antibody-induced neutralization techniques facilitated identification of interleukin (IL)-6 as a tumor-derived molecule prom…
Natural polyphenols facilitate elimination of HT-29 colorectal cancer xenografts by chemoradiotherapy: a Bcl-2- and superoxide dismutase 2-dependent mechanism.
AbstractColorectal cancer is one of the most common malignancies worldwide. The treatment of advanced colorectal cancer with chemotherapy and radiation has two major problems: development of tumor resistance to therapy and nonspecific toxicity towards normal tissues. Different plant-derived polyphenols show anticancer properties and are pharmacologically safe. In vitro growth of human HT-29 colorectal cancer cells is inhibited (∼56%) by bioavailable concentrations of trans-pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene; t-PTER) and quercetin (3,3′,4′,5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols. I.v. administration of t-PTER and Q…
Efficacy and tolerability of EH301 for amyotrophic lateral sclerosis: a randomized, double-blind, placebo-controlled human pilot study
Background: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by progressive loss of spinal and cortical motor neurons, leading to muscular atrophy, respiratory failure, and ultimately death. There is no known cure, and the clinical benefit of the two drugs approved to treat ALS remains unclear. Novel disease-modifying therapeutics that are able to modulate the disease course are desperately needed. Our objective was to evaluate the efficacy and tolerability of Elysium Health’s candidate drug EH301 in people with ALS (PALS). Methods: This was a single-center, prospective, double-blind, randomized, placebo-controlled pilot study. Thirty-two PALS we…