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The cytokine storm is a consequence of mitochondrial dysfunction

The cytokine storm is a consequence of mitochondrial dysfunction

The cytokine storm is a consequence of mitochondrial dysfunction

The cytokine storm is a consequence of mitochondrial dysfunction

Lipoic acid, Methylene Blue and Chlorine dioxide relieve COVID-19 spike protein toxicity

Lipoic acid, Methylene Blue and Chlorine dioxide relieve COVID-19 spike protein toxicity

Lipoic acid, Methylene Blue and Chlorine dioxide relieve COVID-19 spike protein toxicity

Lipoic acid, Methylene Blue and Chlorine dioxide relieve COVID-19 spike protein toxicity

most diseases display a form of anabolism due to mitochondrial impairment

most diseases display a form of anabolism due to mitochondrial impairment

most diseases display a form of anabolism due to mitochondrial impairment

infection by Covid-19 follows a similar pattern

chronic inflammation

Long-term effects include redox shift and cellular anabolism as a result of the Warburg effect and mitochondrial dysfunction

Long-term effects include redox shift and cellular anabolism as a result of the Warburg effect and mitochondrial dysfunction

Long-term effects include redox shift and cellular anabolism as a result of the Warburg effect and mitochondrial dysfunction

Long-term effects include redox shift and cellular anabolism as a result of the Warburg effect and mitochondrial dysfunction

infection by Covid-19 follows a similar pattern

unrelenting anabolism leads to the cytokine storm,

unrelenting anabolism leads to the cytokine storm,

unrelenting anabolism leads to the cytokine storm,

chronic inflammation

chronic inflammation

infection by Covid-19 follows a similar pattern

Lipoic acid and Methylene Blue have been shown to enhance the mitochondrial activity, relieve the Warburg effect and increase catabolism

Lipoic acid and Methylene Blue have been shown to enhance the mitochondrial activity, relieve the Warburg effect and increase catabolism

Lipoic acid and Methylene Blue have been shown to enhance the mitochondrial activity, relieve the Warburg effect and increase catabolism

Methylene Blue, Chlorine dioxide and Lipoic acid may help reduce long-term Covid-19 effects by stimulating the catabolism

Methylene Blue, Chlorine dioxide and Lipoic acid may help reduce long-term Covid-19 effects by stimulating the catabolism

Methylene Blue, Chlorine dioxide and Lipoic acid may help reduce long-term Covid-19 effects by stimulating the catabolism

direct consequence of redox iMeBalance, itself a consequence of decreased energy yield by the mitochondria

direct consequence of redox iMeBalance, itself a consequence of decreased energy yield by the mitochondria

mitochondrial dysfunction and increased levels of lactate, which are important characteristics of metabolic shift and Warburg effect in many diseases

mitochondrial dysfunction and increased levels of lactate, which are important characteristics of metabolic shift and Warburg effect in many diseases

increased lactate dehydrogenase activity (LDH) was observed in COVID-19 patients

increased lactate dehydrogenase activity (LDH) was observed in COVID-19 patients

almost every disease presents an increased anabolism

almost every disease presents an increased anabolism

cell division is the most sophisticated way to release entropy

cell division is the most sophisticated way to release entropy

transition from catabolism to anabolism is driven by a redox shift

transition from catabolism to anabolism is driven by a redox shift

viral spike protein binds to ACE2 receptor of the host cell [22,23].

redox signaling plays an important role in regulating immune function and inflammation, and disruptions in this signaling can lead to excessive cytokine production and immune system activation

Aging is associated with a poor control of the redox balance

thiol/disulfide homeostasis

reduced extracellular environment in the elderly and the increased susceptibility to Covid-19 infection

reduced extracellular environment in the elderly and the increased susceptibility to Covid-19 infection

Redox signaling tightly modulates the inflammatory response and oxidative stress has been reported in acute Covid-19

People at high risk are the elderly, patients suffering from metabolic syndrome such as obesity, or those suffering from chronic diseases such as cancer or inflammation

COVID-19 patients with severe disease have higher levels of oxidative stress markers and lower antioxidant levels

oxidative stress can activate the NLRP3 inflammasome, which is a protein complex that plays a key role in the cytokine storm

inflammation leads to the formation of ROS and RNS, while redox iMeBalance results in cellular damage, which in turn triggers an inflammatory response

persistently elevated mtROS triggers endothelial dysfunction and inflammation, which results in a vicious loop involving ROS, inflammation, and mitochondrial dysfunction

Damaged mitochondria releasing ROS induce inflammation via the NLRP3 inflammasome

Damaged mitochondria releasing ROS induce inflammation via the NLRP3 inflammasome

reduced environment during the cytokine storm

IL-2 is highly up-regulated in Covid-19 patients [37], and IL-2 is known to significantly stimulate the generation of NO in patients

Nitric acid is also the key mediator of IL-2-induced hypotension and vascular leak syndrome

mitochondrial dysfunction has been linked to the pathogenesis of Covid-19

mitochondrial dysfunction triggered by SARS-CoV-2 leads to damage to the mitochondria

mitochondrial dysfunction triggered by SARS-CoV-2 leads to damage to the mitochondria

As catabolism is decreased, entropy is released through anabolism

Elevated levels of lactate, a characteristic of the Warburg effect, were also reported in the high-risk Covid-19

elevated levels of ventricular lactic acid consistent with oxidative stress

A decrease of ΔΨm is implicated in several inflammation-related diseases

decrease in ΔΨm in leucocytes from Covid-19 patients

vaccinated with RNA or DNA vaccines triggering the synthesis of the viral spike protein in human cells

viral reactivation in varicella-zoster virus [55] or hepatitis [56], coagulopathy and resulting stroke and myocarditis following both DNA-based vaccines [57] and RNA-based vaccines

Covid-19, mitochondrial impairment

characteristic of the Warburg effect is present in almost every disease and appears to be a central feature in most of the hallmarks of cancer

inflammation, mitochondrial dysfunction and increased lactate concentrations in the extracellular fluid

In Covid-19, like any inflammation, there is a metabolic rewiring where cells rely on glycolysis

As the mitochondria are impaired, the infected cell cannot catabolize efficiently. It will release lactic acid in the blood stream

Striking similarities are seen between cancer, Alzheimer's disease and Covid-19, all related to the Warburg effect

Cancer, inflammation, Alzheimer's, and Parkinson's diseases share a common peculiarity, the inability of the cell to export entropy outside the body in the harmless form of heat

MEB relieves the Warburg effect [87], improves memory [77], is active in the treatment of depressive episodes [79,80] and reduces the importance of ischemic strokes

MEB relieves the Warburg effect [87], improves memory [77], is active in the treatment of depressive episodes [79,80] and reduces the importance of ischemic strokes

MEB has been shown to inhibit SARS-Cov-2 replication in vitro

MEB has been shown to inhibit SARS-Cov-2 replication in vitro

It has been shown that Covid-19-patients treated with MEB, have a significant reduction in hospital stay duration and mortality

MeB is an acceptor-donor molecule

MeB + can take a pair of electrons (of H atoms) and MeBH can release this pair easily, so that MeB is partially recycled like a catalyst

MeB acts as an electron bridge between a donor (FADH2, FMNH, NADH) and an acceptor (complex IV of ETC or oxygen itself)

As a coenzyme of pyruvate dehydrogenase (PDH), alpha-lipoic acid (ALA) initiates the formation of acetyl-CoA to feed the TCA cycle

ALA enhances the catabolism of carbon. cycle and therefore may reduce the Warburg effect and consequently, lactate production

Methylene Blue plays a similar role after the TCA cycle, by carrying electrons to complex IV of the electron transport chain

Drugs such as lipoic acid and MeB, which target the metabolism, decrease the redox shift by increasing catabolism

related to intracellular hydrogen peroxide generation

only be obtained by intravenous administration of AA

Preferentially kills neoplastic cells

Is virtually non-toxic at any dosage

Does not suppress the immune system, unlike most chemotherapy agents

Increases animal and human resistance to infectious agents by enhancing lymphocyte blastogenesis, enhancing cellular immunity, strengthening the extracellular matrix, and enhancing bactericidal activity of neutrophils and modulation of complement protein

Strengthens the structural integrity of the extracellular matrix which is responsible for stromal resistance to malignant invasiveness

1969, researchers at the NCI reported AA was highly toxic to Ehrlich ascites cells in vitro

In 1977, Bram et al reported preferential AA toxicity for several malignant melanoma cell lines, including four human-derived lines

Noto et al reported that AA plus vitamin K3 had growth inhibiting action against three human tumor cell lines at non-toxic levels

Metabolites of AA have also shown antitumor activity in vitro

The AA begins to reduce cell proliferation in the tumor cell line at the lowest concentration, 1.76 mg/dl, and is completely cytotoxic to the cells at 7.04 mg/dl

the normal cells grew at an enhanced rate at the low dosages (1.76 and 3.52 mg/dl)

preferential toxicity of AA for tumor cells. >95% toxicity to human endometrial adenocarcinoma and pancreatic tumor cells (ATCC AN3-CA and MIA PaCa-2) occurred at 20 and 30 mg/dl, respectively.

No toxicity or inhibition was demonstrated in the normal, human skin fibroblasts (ATCC CCD 25SK) even at the highest concentration of 50 mg/dl.

the use of very high-dose intravenous AA for the treatment of cancer was proposed as early as 1971

Cameron and Pauling have published extensive suggestive evidence for prolonged life in terminal cancer patients orally supplemented (with and without initial intravenous AA therapy) with 10 g/day of AA

AA, plasma levels during infusion were not monitored,

the long-term, oral dosage used in those experiments (10 g/day), while substantial and capable of producing immunostimulatory and extracellular matrix modulation effects, was not high enough to achieve plasma concentrations that are generally cytotoxic to tumor cells in culture

This low cytotoxic level of AA is exceedingly rare

5 — 40 mg/dl of AA is required in vitro to kill 100% of tumor cells within 3 days. The 100% kill levels of 30 mg/dl for the endometrial carcinoma cells and 40 mg/dl for the pancreatic carcinoma cells in Figure 2 are typical

normal range (95% range) of 0.39-1.13 mg/dl

1 h after beginning his first 8-h infusion of 115 g AA (Merit Pharmaceuticals, Los Angeles, CA), the plasma AA was 3.7 mg/dl and at 5 h was 19 mg/dl. During his fourth 8-h infusion, 8 days later, the 1 h plasma level was 158 mg/dl and 5 h was 185 mg/dl

plasma levels of over 100 mg/dl have been maintained in 3 patients for more than 5 h using continuous intravenous infusion

In rare instances of patients with widely disseminated and rapidly proliferating tumors, intravenous AA administration (10 — 45 g/day) precipitated widespread tumor hemorrhage and necrosis, resulting in death

Although the outcomes were disastrous in these cases, they are similar to the description of tumor-necrosis-factor-induced hemorrhage and necrosis in mice (52) and seem to demonstrate the ability of AA to kill tumor cells in vivo.

toxic effects of AA on one normal cell line were observed at 58.36 mg/dl and the lack of side effects in patients maintaining >100 mg/dl plasma levels

Although it is very rare, tumor necrosis, hemorrhage, and subsequent death should be the highest priority concern for the safety of intravenous AA for cancer patients.

Klenner, who reported no ill effects of dosages as high as 150 g intravenously over a 24-h period

Cathcart (55) who describes no ill effects with doses of up to 200 g/d in patients with various pathological conditions

following circumstances: renal insufficiency, chronic hemodialysis patients, unusual forms of iron overload, and oxalate stone formers

Screening for red cell glucose-6-phosphate dehydrogenase deficiency, which can give rise to hemolysis of red blood cells under oxidative stress (57), should also be performed

any cancer therapy should be started at a low dosage to ensure that tumor hemorrhage does not occur.

patient is orally supplementing between infusions

a scorbutic rebound effect can be avoided with oral supplementation. Because of the possibility of a rebound effect, measurement of plasma levels during the periods between infusions should be performed to ensure that no such effect takes place

Every effort should be made to monitor plasma AA levels when a patient discontinues intravenous AA therapy.

The hydroperoxyl radical () plays an important role in the chemistry of lipid peroxidation

The is a much stronger oxidant than superoxide anion-radical

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals or nonradical species attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs) that involve hydrogen abstraction from a carbon, with oxygen insertion resulting in lipid peroxyl radicals and hydroperoxides as described previously

under medium or high lipid peroxidation rates (toxic conditions) the extent of oxidative damage overwhelms repair capacity, and the cells induce apoptosis or necrosis programmed cell death

The overall process of lipid peroxidation consists of three steps: initiation, propagation, and termination

Once lipid peroxidation is initiated, a propagation of chain reactions will take place until termination products are produced.

The main primary products of lipid peroxidation are lipid hydroperoxides (LOOH)

Among the many different aldehydes which can be formed as secondary products during lipid peroxidation, malondialdehyde (MDA), propanal, hexanal, and 4-hydroxynonenal (4-HNE) have been extensively studied

MDA has been widely used for many years as a convenient biomarker for lipid peroxidation of omega-3 and omega-6 fatty acids because of its facile reaction with thiobarbituric acid (TBA)

MDA is one of the most popular and reliable markers that determine oxidative stress in clinical situations [53], and due to MDA’s high reactivity and toxicity underlying the fact that this molecule is very relevant to biomedical research community

4-HNE is considered as “second toxic messengers of free radicals,” and also as “one of the most physiologically active lipid peroxides,” “one of major generators of oxidative stress,” “a chemotactic aldehydic end-product of lipid peroxidation,” and a “major lipid peroxidation product”

MDA is an end-product generated by decomposition of arachidonic acid and larger PUFAs

Identifying in vivo MDA production and its role in biology is important as indicated by the extensive literature on the compound (over 15 800 articles in the PubMed database using the keyword “malondialdehyde lipid peroxidation” in December 2013)

MDA reactivity is pH-dependent

When pH decreases MDA exists as beta-hydroxyacrolein and its reactivity increases

MAA adducts are shown to be highly immunogenic [177–181]. MDA adducts are biologically important because they can participate in secondary deleterious reactions (e.g., crosslinking) by promoting intramolecular or intermolecular protein/DNA crosslinking that may induce profound alteration in the biochemical properties of biomolecules and accumulate during aging and in chronic diseases

MDA is an important contributor to DNA damage and mutation

This MDA-induced DNA alteration may contribute significantly to cancer and other genetic diseases.

Dietary intake of certain antioxidants such as vitamins was associated with reduced levels of markers of DNA oxidation (M1dG and 8-oxodG) measured in peripheral white blood cells of healthy subjects, which could contribute to the protective role of vitamins on cancer risk

4-HNE is an extraordinarily reactive compound

whereas the expression of SVCT-2 is ubiquitous

differential sensitivity to VC may result from variations in VC flow into cells, which is dependent on SVCT-2 expression.

high-dose VC significantly impaired both the tumorspheres initiation (Fig. 4d, e) and the growth of established tumorspheres derived from HCC cells (Fig. 4f, g) in a time-dependent and dose-dependent manner.

Hepatocellular carcinoma (HCC)

The antioxidant, N-acetyl-L-cysteine (NAC), preventing VC-induced ROS production (a ROS scavenger), completely restored the viability and colony formation among VC-treated cells

DNA double-strand damage was found following VC treatment

DNA damage was prevented by NAC

Interestingly, the combination of VC and cisplatin was even more effective in reducing tumor growth and weight

Consistent with the in vitro results, stemness-related genes expressions in tumor xenograft were remarkably reduced after VC or VC+cisplatin treatment, whereas conventional cisplatin therapy alone led to the increase of CSCs

VC is one of the numerous common hepatoprotectants.

Interestingly, at extracellular concentrations greater than 1 mM, VC induces strong cytotoxicity to cancer cells including liver cancer cells

we hypothesized that intravenous VC might reduce the risk of recurrence in HCC patients after curative liver resection.

Intriguingly, the 5-year disease-free survival (DFS) for patients who received intravenous VC was 24%, as opposed to 15% for no intravenous VC-treated patients

Median DFS time for VC users was 25.2 vs. 18 months for VC non-users

intravenous VC use is linked to improved DFS in HCC patients.

In this study, based on the elevated expression of SVCT-2, which is responsible for VC uptake, in liver CSCs, we revealed that clinically achievable concentrations of VC preferentially eradicated liver CSCs in vitro and in vivo

Additionally, we found that intravenous VC reduced the risk of post-surgical HCC progression in a retrospective cohort study.

Three hundred thirty-nine participants (55.3%) received 2 g intravenous VC for 4 or more days after initial hepatectomy

As the key protein responsible for VC uptake in the liver, SVCT-2 played crucial roles in regulating the sensitivity to ascorbate-induced cytotoxicity

we also observed that SVCT-2 was highly expressed in human HCC samples and preferentially elevated in liver CSCs

SVCT-2 might serve as a potential CSC marker and therapeutic target in HCC

CSCs play critical roles in regulating tumor initiation, relapse, and chemoresistance

we revealed that VC treatment dramatically reduced the self-renewal ability, expression levels of CSC-associated genes, and percentages of CSCs in HCC, indicating that CSCs were more susceptible to VC-induced cell death

as a drug for eradicating CSCs, VC may represent a promising strategy for treatment of HCC, alone or particularly in combination with chemotherapeutic drugs

In HCC, we found that VC-generated ROS caused genotoxic stress (DNA damage) and metabolic stress (ATP depletion), which further activated the cyclin-dependent kinase inhibitor p21, leading to G2/M phase cell cycle arrest and caspase-dependent apoptosis in HCC cells

we demonstrated a synergistic effect of VC and chemotherapeutic drug cisplatin on killing HCC both in vitro and in vivo

Intravenous VC has also been reported to reduce chemotherapy-associated toxicity of carboplatin and paclitaxel in patients,38 but the specific mechanism needs further investigation

Our retrospective cohort study also showed that intravenous VC use (2 g) was related to the improved DFS in HCC patients after initial hepatectomy

several clinical trials of high-dose intravenous VC have been conducted in patients with advanced cancer and have revealed improved quality of life and prolonged OS

high-dose VC was not toxic to immune cells and major immune cell subpopulations in vivo

high recurrence rate and heterogeneity

tumor progression, metastasis, and chemotherapy-resistance

SVCT-2 was highly expressed in HCC samples in comparison to peri-tumor tissues

high expression (grade 2+/3+) of SVCT-2 was in agreement with poorer overall survival (OS) of HCC patients (Fig. 1c) and more aggressive tumor behavior

SVCT-2 is enriched in liver CSCs

these data suggest that SVCT-2 is preferentially expressed in liver CSCs and is required for the maintenance of liver CSCs.

pharmacologic concentrations of plasma VC higher than 0.3 mM are achievable only from i.v. administration

The viabilities of HCC cells were dramatically decreased after exposure to VC in dose-dependent manner

VC and cisplatin combination further caused cell apoptosis in tumor xenograft

These results verify that VC inhibits tumor growth in HCC PDX models and SVCT-2 expression level is associated with VC response

qPCR and IHC analysis demonstrated that expression levels of CSC-associated genes and percentages of CSCs in PDXs dramatically declined after VC treatment, confirming the inhibitory role of VC in liver CSCs

Our data show that ascorbic acid selectively killed cancer but not normal cells, using concentrations that could only be achieved by i.v. administration

Ascorbate-mediated cell death was due to protein-dependent extracellular H2O2 generation, via ascorbate radical formation from ascorbate as the electron donor. Like glucose, when ascorbate is infused i.v., the resulting pharmacologic concentrations should distribute rapidly in the extracellular water space (42). We showed that such pharmacologic ascorbate concentrations in media, as a surrogate for extracellular fluid, generated ascorbate radical and H2O2. In contrast, the same pharmacologic ascorbate concentrations in whole blood generated little detectable ascorbate radical and no detectable H2O2. These findings can be accounted for by efficient and redundant H2O2 catabolic pathways in whole blood (e.g., catalase and glutathione peroxidase) relative to those in media or extracellular fluid

ascorbic acid administered i.v. in pharmacologic concentrations may serve as a pro-drug for H2O2 delivery to the extracellular milieu

H2O2 generated in blood is normally removed by catalase and glutathione peroxidase within red blood cells, with internal glutathione providing reducing equivalents

The electron source for glutathione is NADPH from the pentose shunt, via glucose-6-phosphate dehydrogenase. If activity of this enzyme is diminished, the predicted outcome is impaired H2O2 removal causing intravascular hemolysis, the observed clinical finding.

Only recently has it been understood that the discordant clinical findings can be explained by previously unrecognized fundamental pharmacokinetics properties of ascorbate

Intracellular transport of ascorbate is tightly controlled in relation to extracellular concentration

Intravenous ascorbate infusion is expected to drastically change extracellular but not intracellular concentrations

For i.v. ascorbate to be clinically useful in killing cancer cells, pharmacologic but not physiologic extracellular concentrations should be effective, independent of intracellular ascorbate concentrations.

It is unknown why ascorbate, via H2O2, killed some cancer cells but not normal cells.

There was no correlation with ascorbate-induced cell death and glutathione, catalase activity, or glutathione peroxidase activity.

H2O2, as the product of pharmacologic ascorbate concentrations, has potential therapeutic uses in addition to cancer treatment, especially in infections

Neutrophils generate H2O2 from superoxide,

i.v. ascorbate is effective in some viral infections

H2O2 is toxic to hepatitis C

Use of ascorbate as an H2O2-delivery system against sensitive pathogens, viral or bacterial, has substantial clinical implications that deserve rapid exploration.

Recent pharmacokinetics studies in men and women show that 10 g of ascorbate given i.v. is expected to produce plasma concentrations of nearly 6 mM, which are >25-fold higher than those concentrations from the same oral dose

As much as a 70-fold difference in plasma concentrations is expected between oral and i.v. administration,

Complementary and alternative medicine practitioners worldwide currently use ascorbate i.v. in some patients, in part because there is no apparent harm

Human Burkitt's lymphoma cells

We first investigated whether ascorbate in pharmacologic concentrations selectively affected the survival of cancer cells by studying nine cancer cell lines

Clinical pharmacokinetics analyses show that pharmacologic concentrations of plasma ascorbate, from 0.3 to 15 mM, are achievable only from i.v. administration

plasma ascorbate concentrations from maximum possible oral doses cannot exceed 0.22 mM because of limited intestinal absorption

For five of the nine cancer cell lines, ascorbate concentrations causing a 50% decrease in cell survival (EC50 values) were less than 5 mM, a concentration easily achievable from i.v. infusion

All tested normal cells were insensitive to 20 mM ascorbate.

Lymphoma cells were selected because of their sensitivity to ascorbate

As ascorbate concentration increased, the pattern of death changed from apoptosis to pyknosis/necrosis, a pattern suggestive of H2O2-mediated cell death

Apoptosis occurred by 6 h after exposure, and cell death by pyknosis was ≈90% at 14 h after exposure

In contrast to lymphoma cells, there was little or no killing of normal lymphocytes and monocytes by ascorbate

Ascorbate is transported into cells as such by sodium-dependent transporters, whereas dehydroascorbic acid is transported into cells by glucose transporters and then immediately reduced internally to ascorbate

Whether or not intracellular ascorbate was preloaded, extracellular ascorbate induced the same amount and type of death.

extracellular ascorbate in pharmacologic concentrations mediates death of lymphoma cells by apoptosis and pyknosis/necrosis, independently of intracellular ascorbate.

H2O2 as the effector species mediating pharmacologic ascorbate-induced cell death

Superoxide dismutase was not protective

Because these data implicated H2O2 in cell killing, we added H2O2 to lymphoma cells and studied death patterns using nuclear staining (19, 28). The death patterns found with exogenous H2O2 exposure were similar to those found with ascorbate

For both ascorbate and H2O2, death changed from apoptosis to pyknosis/necrosis as concentrations increased

Sensitivity to direct exposure to H2O2 was greater in lymphoma cells compared with normal lymphocytes and normal monocytes

There was no association between the EC50 for ascorbate-mediated cell death and intracellular glutathione concentrations, catalase activity, or glutathione peroxidase activity

H2O2 generation was dependent on time, ascorbate concentration, and the presence of trace amounts of serum in media

ascorbate radical is a surrogate marker for H2O2 formation.

whatever H2O2 is generated should be removed by glutathione peroxidase and catalase within red blood cells, because H2O2 is membrane permeable

The data are consistent with the hypothesis that ascorbate in pharmacologic concentrations is a pro-drug for H2O2 generation in the extracellular milieu but not in blood.

The occurrence of one predicted complication, oxalate kidney stones, is controversial

In patients with glucose-6-phosphate dehydrogenase deficiency, i.v. ascorbate is contraindicated because it causes intravascular hemolysis

ascorbate at pharmacologic concentrations in blood is a pro-drug for H2O2 delivery to tissues.

ascorbate, an electron-donor in such reactions, ironically initiates pro-oxidant chemistry and H2O2 formation

data here showed that ascorbate initiated H2O2 formation extracellularly, but H2O2 targets could be either intracellular or extracellular, because H2O2 is membrane permeant

More than 100 patients have been described, presumably without glucose-6-phosphate dehydrogenase deficiency, who received 10 g or more of i.v. ascorbate with no reported adverse effects other than tumor lysis

In normal embryogenesis and homeostasis, the canonical WNT pathway is activated by secreted WNT ligands produced in highly controlled context-dependent manners and in precise amounts. WNT activity is transduced in the cytoplasm, inactivates the APC destruction complex, and results in the translocation of activate β-CATENIN to the nucleus, where it cooperates with DNA-binding TCF/LEF factors to regulate WNT-TCF targets and the ensuing genomic response

beyond the loss of activity of the APC destruction complex, for instance throughAPC mutation, phosphorylation of β-CATENIN at C-terminal sites is required for the full activation of WNT-TCF signaling and the ensuing WNT-TCF responses in cancer.

The WNT-TCF response blockade that we describe for low doses of Ivermectin suggests an action independent to the deregulation of chloride channels

involve the repression of the levels of C-terminally phosphorylated β-CATENIN forms and of CYCLIN D1, a critical target that is an oncogene and positive cell cycle regulator.

the Avermectin single-molecule derivative Selamectin, a drug widely used in veterinarian medicine (Nolan & Lok, 2012), is ten times more potent acting in the nanomolar range

Ivermectin also diminished the protein levels of CYCLIN D1, a direct TCF target and oncogene, in both HT29 and H358 tumor cells

Activated Caspase3 was used as a marker of apoptosis by immunohistochemistry 48 h after drug treatment. Selamectin and Ivermectin induced up to a sevenfold increase in the number of activated Caspase3+ cells in two primary (CC14 and CC36) and two cell line (DLD1 and Ls174T) colon cancer cell types (Fig​(Fig2C).2C). All changes were significative

The strong downregulation of the expression of the intestinal stem cell genesASCL2 andLGR5 (van der Flieret al, 2009; Scheperset al, 2012; Zhuet al, 2012b) by Ivermectin and Selamectin (Fig​(Fig2D)2D) raised the possibility that these drugs could affect WNT-TCF-dependent colon cancer stem cell behavior

Pre-established H358 tumors responded to Ivermectin showing a ˜ 50% repression of growth

Ivermectin hasin vivo efficacy against human colon cancer xenografts sensitive to TCF inhibition with no discernable side effects

Ivermectin (Campbellet al, 1983), an off-patent drug approved for human use, and related macrocyclic lactones, have WNT-TCF pathway response blocking and anti-cancer activities

these drugs block WNT-TCF pathway responses, likely acting at the level of β-CATENIN/TCF function, affecting β-CATENIN phosphorylation status.

anti-WNT-TCF activities of Ivermectin and Selamectin

Ivermectin has a well-known anti-parasitic activity mediated via the deregulation of chloride channels, leading to paralysis and death (Hibbs & Gouaux, 2011; Lynagh & Lynch, 2012). The same mode of action has been suggested to underlie the toxicity of Ivermectin for liquid tumor cells and the potentiation or sensitization effect of Avermectin B1 on classical chemotherapeutics

the specificity of the blockade of WNT-TCF responses we document, at low micromolar doses for Ivermectin and low nanomolar doses for Selamectin, indicate that the blockade of WNT-TCF responses and chloride channel deregulation are distinct modes of action

What is key then is to find a dose and a context where the use of Ivermectin has beneficial effects in patients, paralleling our results with xenografts in mice.

Cell toxicity appears at doses greater (> 10 μM for 12 h or longer or > 5 μM for 48 h or longer for Ivermectin) than those required to block TCF responses and induce apoptosis.

Our data point to a repression of WNT-β-CATENIN/TCF transcriptional responses by Ivermectin, Selamectin and related macrocylic lactones.

(i) The ability of Avermectin B1 to inhibit the activation of WNT-TCF reporter activity by N-terminal mutant (APC-insensitive) β-CATENIN as detected in our screen

(ii) The ability of Avermectin B1, Ivermectin, Doramectin, Moxidectin and Selamectin to parallel the modulation of WNT-TCF targets by dnTCF

(iii) The finding that the specific WNT-TCF response blockade by low doses of Ivermectin and Selamectin is reversed by constitutively active TCF

(iv) The repression of key C-terminal phospho-isoforms of β-CATENIN resulting in the repression of the TCF target and positive cell cycle regulator CYCLIN D1 by Ivermectin and Selamectin

(v) The specific inhibition ofin-vivo-TCF-dependent, but notin-vivo-TCF-independent cancer cells by Ivermectin in xenografts.

These results together with the reduction of the expression of the colon cancer stem cell markersASCL2 andLGR5 (e.g., Hirschet al, 2013; Ziskinet al, 2013) raise the possibility of an inhibitory effect of Ivermectin, Selamectin and related macrocyclic lactones on TCF-dependent cancer stem cells.

the capacity of cancer cells to form 3D spheroids in culture, as well as the growth of these, is also WNT-TCF-dependent (Kanwaret al, 2010) and they were also affected by Ivermectin treatment

If Ivermectin is specific, it should only block TCF-dependent tumor growth. Indeed, the sensitivity and insensitivity of DLD1 and CC14 xenografts to Ivermectin treatment, respectively, together with the desensitization to Ivermectin actionin vivo by constitutively active TCF provide evidence of the specificity of this drug to block an activated WNT-TCF pathway in human cancer.

Ivermectin has a good safety profile since onlyin-vivo-dnTCF-sensitive cancer xenografts are responsive to Ivermectin treatment, and we have not detected side effects in Ivermectin-treated mice at the doses used

previous work has shown that side effects from systemic treatments with clinically relevant doses in humans are rare (Yang, 2012), that birth defects were not observed after exposure of pregnant mothers (Pacquéet al, 1990) and that this drug does not cross the blood–brain barrier (Kokozet al, 1999). Similarly, only dogs with mutantABCB1 (MDR1) alleles leading to a broken blood–brain barrier show Ivermectin neurotoxicity (Mealeyet al, 2001; Orzechowskiet al, 2012)

Indications may include treatment for incurable β-CATENIN/TCF-dependent advanced and metastatic human tumors of the lung, colon, endometrium, and other organs.

Ivermectin, Selamectin, or related macrocyclic lactones could also serve as topical agents for WNT-TCF-dependent skin lesions and tumors such as basal cell carcinomas

they might also be useful as routine prophylactic agents, for instance against nascent TCF-dependent intestinal tumors in patients with familial polyposis and against nascent sporadic colon tumors in the general aging population

Ovarian cancer stem cells play an important role in chemoresistance and cancer recurrence

Furthermore, recent studies indicate that niclosamide exhibits anticancer effects against various human cancer cells by acting on multiple cell signaling pathways and inducing mitochondrial uncoupling [16–21]

has low systemic bioavailability (~10%) when administered orally, which is beneficial for treating local parasitic infections of the intestines while minimizing systemic exposure

The nano-NI demonstrated significantly higher inhibitory effects on sphere formation than the original niclosamide did

the nano-NI formulation decreased the metabolic activity of ovarian cancer cells and caused a metabolic shift from oxidative phosphorylation to glycolysis

This toxicity evaluation showed that oral nano-NI had no toxic effect on either group of mice in terms of weight, plasma albumin levels, and blood cell counts, and revealed no adverse effects on vital organ function in the rodents, which suggests that nano-NI is safe for animals

niclosamide inhibits tumor cell growth by interrupting multiple pathways (Wnt, Notch, STAT3, NF-κB, and mTORc1) and the generation of reactive oxygen species in several cancer cells

The current standard therapy for ovarian cancer includes taxanes and platinum-based chemotherapy after cytoreductive surgery. Among treated patients, nearly 70 to 80% will experience disease recurrence

metabolic activity, oxygen transport, and DNA synthesis

Iron is found in the human body in the form of haemoglobin in red blood cells and growing erythroid cells.

macrophages contain considerable quantities of iron

iron is taken up by the majority of cells in the form of a transferrin (Tf)-Fe(III) complex that binds to the cell surface receptor transferrin receptor 1 (TfR1)

excess iron is retained in the liver cells

the endosomal six transmembrane epithelial antigen of the prostate 3 (STEAP3) reduces Fe(III) (ferric ion) to Fe(II) (ferrous ion), which is subsequently transferred across the endosomal membrane by divalent metal transporter 1 (DMT1)

labile iron pool (LIP)

LIP is toxic to the cells owing to the production of massive amounts of ROS.

DHA is quickly converted to Vit-C within the cell, by interacting with reduced glutathione (GSH) [45,46,47]. NADPH then recycles the oxidized glutathione (glutathione disulfide (GSSG)) and converts it back into GSH

Fe(II) catalyzes the formation of OH• and OH− during the interaction between H2O2 and O2•− (Haber–Weiss reaction)

Ascorbate can efficiently reduce free iron, thus recycling the cellular Fe(II)/Fe(III) to produce more OH• from H2O2 than can be generated during the Fenton reaction, which ultimately leads to lipid, protein, and DNA oxidation

Vit-C-stimulated iron absorption

reduce cellular iron efflux

high-dose Vit-C may elevate cellular LIP concentrations

ascorbate enhanced cancer cell LIP specifically by generating H2O2

Vit-C produces H2O2 extracellularly, which in turn inhibits tumor cells immediately

tumor cells have a need for readily available Fe(II) to survive and proliferate.

Tf has been recognized to sequester most labile Fe(II) in vivo

Asc•− and H2O2 were generated in vivo upon i.v Vit-C administration of around 0.5 g/kg of body weight and that the generation was Vit-C-dose reliant

free irons, especially Fe(II), increase Vit-C autoxidation, leading to H2O2 production

iron metabolism is altered in malignancies

increase in the expression of various iron-intake pathways or the downregulation of iron exporter proteins and storage pathways

Fe(II) ion in breast cancer cells is almost double that in normal breast tissues

macrophages in the cancer microenvironment have been revealed to increase iron shedding

Advanced breast tumor patients had substantially greater Fe(II) levels in their blood than the control groups without the disease

increased the amount of LIP inside the cells through transferrin receptor (TfR)

Warburg effect, or metabolic reprogramming,

Warburg effect is aided by KRAS or BRAF mutations

Vit-C is supplied, it oxidizes to DHA, and then is readily transported by GLUT-1 in mutant cells of KRAS or BRAF competing with glucose [46]. DHA is quickly converted into ascorbate inside the cell by NADPH and GSH [46,107]. This decrease reduces the concentration of cytosolic antioxidants and raises the intracellular ROS amounts

increased ROS inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

ROS activates poly (ADP-ribose) polymerase (PARP), which depletes NAD+ (a critical co-factor of GAPDH); thus, further reducing the GAPDH associated with a multifaceted metabolic rewiring

Hindering GAPDH can result in an “energy crisis”, due to the decrease in ATP production

high-dose Vit-C recruited metabolites and increased the enzymatic activity in the pentose phosphate pathway (PPP), blocked the tri-carboxylic acid (TCA) cycle, and increased oxygen uptake, disrupting the intracellular metabolic balance and resulting in irreversible cell death, due to an energy crisis

mega-dose Vit-C influences energy metabolism by producing tremendous amounts of H2O2

Due to its great volatility at neutral pH [76], bolus therapy with mega-dose DHA has only transitory effects on tumor cells, both in vitro and in vivo.

These data suggest that PAA may show a therapeutic advantage to blood cancers vs solid tumors because of the communication between tumor cells and blood plasma

These results strongly suggest that the mechanism of PAA killing of MM cells is indeed iron-dependent

These results suggest that PAA administration in SMM may be able to prevent progression to symtomatic MM

A recent study by Yun and colleagues demonstrated that vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH, but spares normal cells

RAS family genes show the most frequent mutations in MM. KRAS, NRAS and BRAF are mutated in 22%, 20% and 7% of MM samples

the disease stage rather than the mutation of RAS and/or BRAF is the major predictive factor for PAA sensitivity in MM treatment

Other molecular mechanisms including ATP depletion and ATM-AMPK signaling have been reported to explain PAA-induced cell death

our pilot study also suggested that PAA could overcome drug resistance to bortezomib in MM cells

Our findings complement reported studies and further address the mechanism of action using clinical samples in which we observed that PAA killed tumor cells with high iron content, suggesting that iron might be the initiator of PAA cytotoxicity

combination of PAA with standard therapeutic drugs, such as melphalan, may significantly reduce the dose of melphalan needed

Combined treatment of reduced dose melphalan with PAA achieved a significantly longer progression-free survival than the same dose of melphalan alone.

These data also suggest that the bone marrow suppression induced by high-dose melphalan can be ameliorated by the combination of PAA with lower dose of melphalan because of the lack of toxicity of PAA on normal cells with low iron content.

if creatinine clearance is <30 mL/min, high dose ascorbic acid should be not administrated.

In MM preclinical and clinical studies, ascorbate was used as an adjunct drug and showed controversial results (Harvey et al., 2009, Perrone et al., 2009, Held et al., 2013, Sharma et al., 2012, Nakano et al., 2011, Takahashi, 2010, Sharma et al., 2009, Qazilbash et al., 2008). However, none of these tests used pharmacological doses of ascorbate and intravenous administration

Multiple myeloma (MM) is a plasma cell neoplasm.

Cameron and Pauling reported that high doses of vitamin C increased survival of patients with cancer

pharmacologically dosed ascorbic acid (PAA) 50–100 g (Chen et al., 2008, Padayatty et al., 2004, Hoffer et al., 2008, Padayatty et al., 2006, Welsh et al., 2013), administered intravenously, has potent anti-cancer activity and its role as anti-cancer therapy is being studied at the University of Iowa and in other centers

In the presence of catalytic metal ions like iron, PAA administered intravenously exerts pro-oxidant effects leading to the formation of highly reactive oxygen species (ROS), resulting in cell death

the labile iron pool (LIP) is significantly elevated in MM cells

The survival of CD138+ cells in vitro was significantly decreased following PAA treatment in all 9 MM

In contrast, no significant change of cell viability was observed in CD138− BM cells from the same patients

The same effect of PAA was also observed in the SMM patients

no response to PAA was detected in CD138+ cells from the 2 MGUS patients

the combination of melphalan plus PAA showed greater tumor burden reduction than each drug alone, suggesting a synergistic activity between these two drugs

Both catalase and NAC protect cells from oxidative damage

cells pretreated with NAC and catalase became resistant to PAA even at high doses

adding deferoxamine (DFO), an iron chelator, to OCI-MY5 cells before PAA treatment was also sufficient to prevent PAA-induced cellular death

iron is essential for PAA to achieve its anti-cancer activity

PAA induced early necrosis (Fig. 3Fig. 3A, 60 min) followed by late apoptosis

results further indicated that PAA induced mitochondria-mediated apoptosis

PAA by reacting with LIP and generating ROS induces mitochondria-mediated apoptosis in which AIF1 cleavage is important for cell death.

ROS and H2O2 are well known factors mediating PAA-induced cancer cell death

PAA was sensitive to all 9 MMs and 2 SMMs

Data from the American Cancer Society show that the rate of increase in cancer deaths/year (3.4%) was two-fold greater than the rate of increase in new cases/year (1.7%) from 2013 to 2017

cancer is predicted to overtake heart disease as the leading cause of death in Western societies

cancer can also be recognized as a metabolic disease.

glucose is first split into two molecules of pyruvate through the Embden–Meyerhof–Parnas glycolytic pathway in the cytosol

Aerobic fermentation, on the other hand, involves the production of lactic acid under normoxic conditions

persistent lactic acid production in the presence of adequate oxygen is indicative of abnormal respiration

Otto Warburg first proposed that all cancers arise from damage to cellular respiration

The Crabtree effect is an artifact of the in vitro environment and involves the glucose-induced suppression of respiration with a corresponding elevation of lactic acid production even under hyperoxic (pO2 = 120–160 mmHg) conditions associated with cell culture

the Warburg theory of insufficient aerobic respiration remains as the most credible explanation for the origin of tumor cells [2, 37, 51, 52, 53, 54, 55, 56, 57].

The main points of Warburg’s theory are; 1) insufficient respiration is the predisposing initiator of tumorigenesis and ultimately cancer, 2) energy through glycolysis gradually compensates for insufficient energy through respiration, 3) cancer cells continue to produce lactic acid in the presence of oxygen, and 4) respiratory insufficiency eventually becomes irreversible

Efraim Racker coined the term “Warburg effect”, which refers to the aerobic glycolysis that occurs in cancer cells

Warburg clearly demonstrated that aerobic fermentation (aerobic glycolysis) is an effect, and not the cause, of insufficient respiration

all tumor cells that have been examined to date contain abnormalities in the content or composition of cardiolipin

The evidence supporting Warburg’s original theory comes from a broad range of cancers and is now overwhelming

respiratory insufficiency, arising from any number mitochondrial defects, can contribute to the fermentation metabolism seen in tumor cells.

data from the nuclear and mitochondrial transfer experiments suggest that oncogene changes are effects, rather than causes, of tumorigenesis

Normal mitochondria can suppress tumorigenesis, whereas abnormal mitochondria can enhance tumorigenesis

In addition to glucose, cancer cells also rely heavily on glutamine for growth and survival

Glutamine is anapleurotic and can be rapidly metabolized to glutamate and then to α-ketoglutarate for entry into the TCA cycle

Glucose and glutamine act synergistically for driving rapid tumor cell growth