Group items tagged

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

Glutamine metabolism can produce ATP from the TCA cycle under aerobic conditions

Amino acid fermentation can generate energy through TCA cycle substrate level phosphorylation under hypoxic conditions

Hif-1α stabilization enhances aerobic fermentation

targeting glucose and glutamine will deprive the microenvironment of fermentable fuels

Although Warburg’s hypothesis on the origin of cancer has created confusion and controversy [37, 38, 39, 40], his hypothesis has never been disproved

Warburg referred to the phenomenon of enhanced glycolysis in cancer cells as “aerobic fermentation” to highlight the abnormal production of lactic acid in the presence of oxygen

Emerging evidence indicates that macrophages, or their fusion hybridization with neoplastic stem cells, are the origin of metastatic cancer cells

Radiation therapy can enhance fusion hybridization that could increase risk for invasive and metastatic tumor cells

Kamphorst et al. in showing that pancreatic ductal adenocarcinoma cells could obtain glutamine under nutrient poor conditions through lysosomal digestion of extracellular proteins

It will therefore become necessary to also target lysosomal digestion, under reduced glucose and glutamine conditions, to effectively manage those invasive and metastatic cancers that express cannibalism and phagocytosis.

Previous studies in yeast and mammalian cells show that disruption of aerobic respiration can cause mutations (loss of heterozygosity, chromosome instability, and epigenetic modifications etc.) in the nuclear genome

The somatic mutations and genomic instability seen in tumor cells thus arise from a protracted reliance on fermentation energy metabolism and a disruption of redox balance through excess oxidative stress.

According to the mitochondrial metabolic theory of cancer, the large genomic heterogeneity seen in tumor cells arises as a consequence, rather than as a cause, of mitochondrial dysfunction

A therapeutic strategy targeting the metabolic abnormality common to most tumor cells should therefore be more effective in managing cancer than would a strategy targeting genetic mutations that vary widely between tumors of the same histological grade and even within the same tumor

Tumor cells are more fit than normal cells to survive in the hypoxic niche of the tumor microenvironment

Hypoxic adaptation of tumor cells allows for them to avoid apoptosis due to their metabolic reprograming following a gradual loss of respiratory function

The high rates of tumor cell glycolysis and glutaminolysis will also make them resistant to apoptosis, ROS, and chemotherapy drugs

Despite having high levels of ROS, glutamate-derived from glutamine contributes to glutathione production that can protect tumor cells from ROS

It is clear that adaptability to environmental stress is greater in normal cells than in tumor cells, as normal cells can transition from the metabolism of glucose to the metabolism of ketone bodies when glucose becomes limiting

Mitochondrial respiratory chain defects will prevent tumor cells from using ketone bodies for energy

glycolysis-dependent tumor cells are less adaptable to metabolic stress than are the normal cells. This vulnerability can be exploited for targeting tumor cell energy metabolism

In contrast to dietary energy reduction, radiation and toxic drugs can damage the microenvironment and transform normal cells into tumor cells while also creating tumor cells that become highly resistant to drugs and radiation

Drug-resistant tumor cells arise in large part from the damage to respiration in bystander pre-cancerous cells

Because energy generated through substrate level phosphorylation is greater in tumor cells than in normal cells, tumor cells are more dependent than normal cells on the availability of fermentable fuels (glucose and glutamine)

Ketone bodies and fats are non-fermentable fuels

Although some tumor cells might appear to oxidize ketone bodies by the presence of ketolytic enzymes [181], it is not clear if ketone bodies and fats can provide sufficient energy for cell viability in the absence of glucose and glutamine

Apoptosis under energy stress is greater in tumor cells than in normal cells

A calorie restricted ketogenic diet or dietary energy reduction creates chronic metabolic stress in the body

. This energy stress acts as a press disturbance

Drugs that target availability of glucose and glutamine would act as pulse disturbances

Hyperbaric oxygen therapy can also be considered another pulse disturbance

The KD can more effectively reduce glucose and elevate blood ketone bodies than can CR alone making the KD potentially more therapeutic against tumors than CR

Campbell showed that tumor growth in rats is greater under high protein (>20%) than under low protein content (<10%) in the diet

Protein amino acids can be metabolized to glucose through the Cori cycle

The fats in KDs used clinically also contain more medium chain triglycerides

Calorie restriction, fasting, and restricted KDs are anti-angiogenic, anti-inflammatory, and pro-apoptotic and thus can target and eliminate tumor cells through multiple mechanisms

Ketogenic diets can also spare muscle protein, enhance immunity, and delay cancer cachexia, which is a major problem in managing metastatic cancer

GKI values of 1.0 or below are considered therapeutic

The GKI can therefore serve as a biomarker to assess the therapeutic efficacy of various diets in a broad range of cancers.

It is important to remember that insulin drives glycolysis through stimulation of the pyruvate dehydrogenase complex

The water-soluble ketone bodies (D-β-hydroxybutyrate and acetoacetate) are produced largely in the liver from adipocyte-derived fatty acids and ketogenic dietary fat. Ketone bodies bypass glycolysis and directly enter the mitochondria for metabolism to acetyl-CoA

Due to mitochondrial defects, tumor cells cannot exploit the therapeutic benefits of burning ketone bodies as normal cells would

Therapeutic ketosis with racemic ketone esters can also make it feasible to safely sustain hypoglycemia for inducing metabolic stress on cancer cells

ketone bodies can inhibit histone deacetylases (HDAC) [229]. HDAC inhibitors play a role in targeting the cancer epigenome

Therapeutic ketosis reduces circulating inflammatory markers, and ketones directly inhibit the NLRP3 inflammasome, an important pro-inflammatory pathway linked to carcinogenesis and an important target for cancer treatment response

Chronic psychological stress is known to promote tumorigenesis through elevations of blood glucose, glucocorticoids, catecholamines, and insulin-like growth factor (IGF-1)

In addition to calorie-restricted ketogenic diets, psychological stress management involving exercise, yoga, music etc. also act as press disturbances that can help reduce fatigue, depression, and anxiety in cancer patients and in animal models

Ketone supplementation has also been shown to reduce anxiety behavior in animal models

This physiological state also enhances the efficacy of chemotherapy and radiation therapy, while reducing the side effects

lower dosages of chemotherapeutic drugs can be used when administered together with calorie restriction or restricted ketogenic diets (KD-R)

Besides 2-DG, a range of other glycolysis inhibitors might also produce similar therapeutic effects when combined with the KD-R including 3-bromopyruvate, oxaloacetate, and lonidamine

A synergistic interaction of the KD diet plus radiation was seen

It is important to recognize, however, that the radiotherapy used in glioma patients can damage the respiration of normal cells and increase availability of glutamine in the microenvironment, which can increase risk of tumor recurrence especially when used together with the steroid drug dexamethasone

Poff and colleagues demonstrated that hyperbaric oxygen therapy (HBOT) enhanced the ability of the KD to reduce tumor growth and metastasis

HBOT also increases oxidative stress and membrane lipid peroxidation of GBM cells in vitro

The effects of the KD and HBOT can be enhanced with administration of exogenous ketones, which further suppressed tumor growth and metastasis

Besides HBOT, intravenous vitamin C and dichloroacetate (DCA) can also be used with the KD to selectively increase oxidative stress in tumor cells

Recent evidence also shows that ketone supplementation may enhance or preserve overall physical and mental health

Some tumors use glucose as a prime fuel for growth, whereas other tumors use glutamine as a prime fuel [102, 186, 262, 263, 264]. Glutamine-dependent tumors are generally less detectable than glucose-dependent under FDG-PET imaging, but could be detected under glutamine-based PET imaging

GBM and use glutamine as a major fuel

Many of the current treatments used for cancer management are based on the view that cancer is a genetic disease

Emerging evidence indicates that cancer is a mitochondrial metabolic disease that depends on availability of fermentable fuels for tumor cell growth and survival

Glucose and glutamine are the most abundant fermentable fuels present in the circulation and in the tumor microenvironment

Low-carbohydrate, high fat-ketogenic diets coupled with glycolysis inhibitors will reduce metabolic flux through the glycolytic and pentose phosphate pathways needed for synthesis of ATP, lipids, glutathione, and nucleotides

oxidative stress and ROS, produced in cancer-associated fibroblasts, has a “bystander effect” on adjacent cancer cells, leading to DNA damage, genomic instability and aneuploidy, which appears to be driving tumor-stroma co-evolution

tumor cells produce and secrete hydrogen peroxide, thereby “fertilizing” the tumor microenvironment and driving the “reverse Warburg effect.”

This type of stromal metabolism then produces high-energy nutrients (lactate, ketones and glutamine), as well as recycled chemical building blocks (nucleotides, amino acids, fatty acids), to literally “feed” cancer cells

loss of stromal caveolin (Cav-1) is sufficient to drive mitochondrial dysfunction with increased glucose uptake in fibroblasts, mimicking the glycolytic phenotype of cancer-associated fibroblasts.

oxidative stress initiated in tumor cells is transferred to cancer-associated fibroblasts.

Then, cancer-associated fibroblasts show quantitative reductions in mitochondrial activity and compensatory increases in glucose uptake, as well as high ROS production

These findings may explain the prognostic value of a loss of stromal Cav-1 as a marker of a “lethal” tumor microenvironment

aerobic glycolysis takes place in cancer-associated fibroblasts, rather than in tumor cells, as previously suspected.

our results may also explain the “field effect” in cancer biology,5 as hydrogen peroxide secreted by cancer cells, and the propagation of ROS production, from cancer cells to fibroblasts, would create an increasing “mutagenic field” of ROS production, due to the resulting DNA damage

Interruption of this process, by addition of catalase (an enzyme that detoxifies hydrogen peroxide) to the tissue culture media, blocks ROS activity in cancer cells and leads to apoptotic cell death in cancer cells

In this new paradigm, cancer cells induce oxidative stress in neighboring cancer-associated fibroblasts

cancer-associated fibroblasts have the largest increases in glucose uptake

cancer cells secrete hydrogen peroxide, which induces ROS production in cancer-associated fibroblasts

Then, oxidative stress in cancer-associated fibroblast leads to decreases in functional mitochondrial activity, and a corresponding increase in glucose uptake, to fuel aerobic glycolysis

cancer cells show significant increases in mitochondrial activity, and decreases in glucose uptake

fibroblasts and cancer cells in co-culture become metabolically coupled, resulting in the development of a “symbiotic” or “parasitic” relationship.

cancer-associated fibroblasts undergo aerobic glycolysis (producing lactate), while cancer cells use oxidative mitochondrial metabolism.

We have previously shown that oxidative stress in cancer-associated fibroblasts drives a loss of stromal Cav-1, due to its destruction via autophagy/lysosomal degradation

a loss of stromal Cav-1 is sufficient to induce further oxidative stress, DNA damage and autophagy, essentially mimicking pseudo-hypoxia and driving mitochondrial dysfunction

loss of stromal Cav-1 is a powerful biomarker for identifying breast cancer patients with early tumor recurrence, lymph-node metastasis, drug-resistance and poor clinical outcome

this type of metabolism (aerobic glycolysis and autophagy in the tumor stroma) is characteristic of a lethal tumor micro-environment, as it fuels anabolic growth in cancer cells, via the production of high-energy nutrients (such as lactate, ketones and glutamine) and other chemical building blocks

the upstream tumor-initiating event appears to be the secretion of hydrogen peroxide

one such enzymatically-active protein anti-oxidant that may be of therapeutic use is catalase, as it detoxifies hydrogen peroxide to water

numerous studies show that “catalase therapy” in pre-clinical animal models is indeed sufficient to almost completely block tumor recurrence and metastasis

by eliminating oxidative stress in cancer cells and the tumor microenvironment,55 we may be able to effectively cut off the tumor's fuel supply, by blocking stromal autophagy and aerobic glycolysis

breast cancer patients show systemic evidence of increased oxidative stress and a decreased anti-oxidant defense, which increases with aging and tumor progression.68–70 Chemotherapy and radiation therapy then promote further oxidative stress.69 Unfortunately, “sub-lethal” doses of oxidative stress during cancer therapy may contribute to tumor recurrence and metastasis, via the activation of myofibroblasts.

a loss of stromal Cav-1 is associated with the increased expression of gene profiles associated with normal aging, oxidative stress, DNA damage, HIF1/hypoxia, NFκB/inflammation, glycolysis and mitochondrial dysfunction

cancer-associated fibroblasts show the largest increases in glucose uptake, while cancer cells show corresponding decreases in glucose uptake, under identical co-culture conditions

Thus, increased PET glucose avidity may actually be a surrogate marker for a loss of stromal Cav-1 in human tumors, allowing the rapid detection of a lethal tumor microenvironment.

it appears that astrocytes are actually the cell type responsible for the glucose avidity.

In the brain, astrocytes are glycolytic and undergo aerobic glycolysis. Thus, astrocytes take up and metabolically process glucose to lactate.7

Then, lactate is secreted via a mono-carboxylate transporter, namely MCT4. As a consequence, neurons use lactate as their preferred energy substrate

both astrocytes and cancer-associated fibroblasts express MCT4 (which extrudes lactate) and MCT4 is upregulated by oxidative stress in stromal fibroblasts.34

In accordance with the idea that cancer-associated fibroblasts take up the bulk of glucose, PET glucose avidity is also now routinely used to measure the extent of fibrosis in a number of human diseases, including interstitial pulmonary fibrosis, postsurgical scars, keloids, arthritis and a variety of collagen-vascular diseases.

PET glucose avidity and elevated serum inflammatory markers both correlate with poor prognosis in breast cancers.

PET signal over-estimates the actual anatomical size of the tumor, consistent with the idea that PET glucose avidity is really measuring fibrosis and inflammation in the tumor microenvironment.

human breast and lung cancer patients can be positively identified by examining their exhaled breath for the presence of hydrogen peroxide.

tumor cell production of hydrogen peroxide drives NFκB-activation in adjacent normal cells in culture6 and during metastasis,103 directly implicating the use of antioxidants, NFκB-inhibitors and anti-inflammatory agents, in the treatment of aggressive human cancers.

surgery per se can promote cancer metastasis through a series of local and systemic events

surgery results in a serious wound that disrupts the structural barrier preventing the outspreading of cancer cells, change the properties of the cancer cells and stromal cells remaining in the tumor microenvironment, or impairs the host defense systems against cancers

After the primary tumor is surgically removed, the metastases can start to grow vigorously via neoangiogenesis because the circulating inhibitors disappear

infection and inflammation during the postoperative period have been reported to increase the risk of cancer recurrence in patients

Surgeons have long suspected that surgery, even if it is a necessary step in cancer treatment, facilitates cancer metastasis

Surgery-induced cancer metastasis has been well established in animal models

tumor cell dissemination, tumor-favoring immune responses, and neoangiogenesis

the surgical resection of primary tumors is beneficial is controversial

CTCs abruptly increase just after surgery

Even externally palpitating tumors for diagnosis could increase the numbers of CTCs in skin cancer and breast cancer

excessive glucocorticoids negatively modulate immune functions

immune surveillance against tumors is considered to be impaired by surgical stress

In addition to glucocorticoids, during stimulation of the HPA axis, the catecholamine hormones epinephrine and norepinephrine are released from the adrenal medulla

NK cell suppression may be attributed to increased levels of catecholamines as well as glucocorticoids

In mice bearing a primary tumor, it was observed that the removal of the primary tumor facilitated the growth of highly vascularized metastases

primary tumors may secrete angiogenic inhibitors as well as angiogenic activators

second phase of tumor recurrence and metastasis, which are newly acquired events, rather than just outcomes of incomplete treatment.

double-edged sword

HIF-1 in neutrophils plays a critical role in NETosis and bacteria-killing activity

neutrophils play various roles in the initiation and progression of cancer

NETosis

many inflammatory and neoplastic diseases

formation of neutrophil extracellular traps (NETs), which are large extracellular complexes composed of chromatin and cytoplasmic/granular proteins1

NETosis has been highlighted as an inflammatory event that promotes cancer metastasis

Once activated, neutrophils produce intracellular precursors by using DNA, histones, and granular and cytoplasmic proteins and then spread the mature form of NETs out around themselves

A series of these events is called NETosis.

neutrophil elastase, myeloperoxidase, cathepsin G, proteinase 3, lactoferrin, gelatinase, lysozyme C, calprotectin, neutrophil defensins, and cathelicidins

innate immune response against infection

Neutrophils are the most abundant type of granulocytes, comprising 40–70% of all white blood cells

two types of NEToses, suicidal (or lytic) NETosis and vital NETosis

Suicidal NETosis mainly depends on the production of reactive oxygen species (ROS)

Since neutrophils die during this process, it is called suicidal NETosis.

vital NETosis

vital NETosis occurs independently of ROS production

Vital NETosis can be induced by Gram-negative bacteria. LPS

NETs are present in a variety of cancers, such as lung cancer, colon cancer, ovarian cancer, and leukemia

neutrophils actively undergo NETosis in the tumor microenvironment

Hypoxia

NETosis plays a pivotal role in noninfectious autoimmune diseases,

cytokines

tumor-derived proteases

tumor exosomes

NETosis generally actively progresses in the tumor microenvironment.

the proliferative cytokines TGFβ and IL-10 and the angiogenic factor VEGF are representative of neutrophil-derived tissue repair proteins.

NETosis is a defense system to protect the body from invading pathogens

when neutrophils are excessively stimulated, they produce excess NETs, thereby leading to pathological consequences

plasma levels of NETosis markers are elevated after major surgeries

local invasion, intravasation into the blood or lymphatic vessels, escape from the immune system, anchoring to capillaries in target organs, extravasation into the organs, transformation from dormant cells to proliferating cells, colonization to micrometastases, and growth to macrometastases

NETs promote metastasis at multiple steps

NETs loosen the ECM and capillary wall to promote the intravasation of cancer cells

NETs and platelets wrap CTCs, which protects them from attack by immune cells and shearing force by blood flow

NETs promote the local invasion of cancer cells by degrading the extracellular matrix (ECM)

neutrophil elastase, matrix metalloproteinase 9, and cathepsin G

NETs also promote the intravasation of cancer cells

millions of tumor cells are released into the circulation every day,

NETs can wrap up CTCs with platelets

β1-integrin plays an important role in the interaction between CTCs and NETs

NET-platelet-CTC aggregates.

After metastasizing to distant tissues, tumor cells are often found to remain dormant for a period of time and unexpectedly regrow late

NETs are believed to participate in the reactivation of dormant cancer cells in metastatic regions

NET-associated proteases NE and MMP-9 were found to be responsible for the reactivation of dormant cancer cells

The association between FDG uptake and tumor burden or stage has been well documented

advanced tumors tend to have higher FDG uptake (and thus higher SUV values)

the impact of the SUV on treatment outcome has been observed even within a given tumor stage

primary tumor SUVmax >10 predicted survival, independent of the tumor stage and diameter

FDG uptake not only reflects tumor burden/stage but also expresses, at least in part, some intrinsic biologic characteristic(s) of the tumor

SUVmax reflects the highest voxel value within the ROI or VOI. It is the most widely used parameter to measure metabolic tumor activity in oncologic FDG-PET/CT imaging

Several studies suggest that primary tumor baseline SUVmax also has predictive value in assessing the tumor burden, lymph node involvement and local extension

According to EORTC, a drop (delta between baseline and post-therapy) of 15–25% in SUVmax may represent a good treatment response

PERCIST criteria was proposed by the investigators at the Johns Hopkins Medical Institutions and suggested that a decrease in SUV normalized to lean body mass of at least 30% should be achieved before considering partial tumor response

Patients with higher inflammation or tumor burdens, as measured by CRP levels or tumor antigen levels, tend to show lower peak plasma ascorbate levels after IVC.

Patients with metastatic tumors tend to achieve lower post infusion plasma ascorbate levels than those with localized tumors.

Meta-analyses of clinical studies involving cancer and vitamins also conclude that antioxidant supplementation does not interfere with the efficacy of chemotherapeutic regiments

Most of the prostate cancer patients studied, 75±19% (95% confidence), showed reductions in PSA levels during the course of their IVC therapy

Laboratory studies suggest that, at high concentrations, ascorbate does not interfere with chemotherapy or irradiation and may enhance efficacy in some situations

Cameron and Pauling observed fourfold survival times in terminal cancer patients treated with intravenous ascorbate infusions followed by oral supplementation

The inflammatory microenvironment of cancer cells leads to increasing oxidative stress, which apparently depletes vitamin C, resulting in lower plasma ascorbate concentrations in blood samples post IVC infusion. Another explanation for this finding may be that cancers are themselves more metabolically active in their uptake of vitamin C, causing subjects to absorb more of the vitamin, and as a results show lower plasma ascorbate concentrations in blood post IVC infusion.

patients with severely elevated CRP levels attain plasma ascorbate concentrations after IVC infusions that are only 65% of those attained for subjects with normal CRP levels

The finding of decreased plasma ascorbate levels in cancer patients may relate to the molecular structure of ascorbic acid; in particular, the similarity of its oxidized form, dihydroascorbic acid, to glucose

Since tumor have increased requirement for glucose [67], transport of dehydroascorbate into the cancer cells via glucose transport molecules and ascorbate through sodium-dependent transporter may be elevated

Increased accumulation of ascorbic acid in the tumor site was supported by measurements of the level of ascorbic acid in tumors in animal experiments

patients with advanced malignancies may have lower level of ascorbic acid in tissue, creating a higher demand for the vitamin C

IVC therapy appears to reduce CRP levels in cancer patients.

CRP concentrations directly correlate with disease activity in many cases and can contribute to disease progression through a range of pro-inflammatory properties.

Being an exquisitely sensitive marker of systemic inflammation and tissue damage, CRP is very useful in screening for organic disease and monitoring treatment responses

ncreases in CRP concentrations have been associated with poorer prognosis of survival in cancer patients, particularly with advance disease independent of tumor stage

Regarding inflammation, 73±13% of subjects (95% confidence) showed a reduction in CRP levels during therapy. This was an even more dramatic 86±13% (95% confidence) in subjects who started therapy with CRP levels above 10 mg/L

patients treated by IVC with follow-up several year showed that suppression of inflammation in cancer patients by high-dose IVC is feasible and potentially beneficial

Inflammation is a marker of high cancer risk, and poor treatment outcome

The subjects with highly elevated CRP concentrations have a three-fold elevation “all-cause” mortality risk and a twenty-eight fold increase in cancer mortality risk

cancer patients may need higher doses to achieve a given plasma concentration.

patients with lower vitamin C levels may see more distribution of intravenously administered ascorbate into tissues and thus attain less in plasma.

When treating patients with IVC, the first treatment likely serves to replenish depleted tissue stores, if those subjects were vitamin C deficient at the beginning of the treatment. Then, in subsequent treatments, with increasing doses, higher plasma concentrations can be attained. On-going treatments serve to progressively reduce oxidative stress in cancer patients.

large doses given intravenously may result in maximum plasma concentrations of roughly 30 mM, a level that has been shown to be sufficient for preferential cytotoxicity against cancer cells

oral intake of vitamin C exceeded 200 mg administered once daily, it was difficult to increase plasma and tissue concentrations above roughly 200 μM.

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

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

NETosis is a special form of programmed cell death in neutrophils, which is characterized by the extrusion of DNA, histones, and antimicrobial proteins in a web-like structure known as neutrophil extracellular traps (NETs)

increased generation of reactive oxygen species (ROS) is a crucial intracellular process that causes NETosis

Another indirect route of SARS-CoV-2-induced NET production is platelet activation

When NETs are activated in the circulation, they can also induce hypercoagulability and thrombosis

In COVID-19, major NET protein cargos of NETs (i.e., NE, MPO, and histones) are significantly elevated.

SARS-CoV-2 can also infect host cells through noncanonical receptors such as C-type lectin receptors

Immunopathological manifestations, including cytokine storms and impaired adaptive immunity, are the primary drivers behind COVID-19, with neutrophil infiltration being suggested as a significant cause

NETosis and NETs are increasingly recognized as causes of vascular injury

SARS-CoV-2 and its components (e.g., spike proteins and viral RNA) attach to platelets and increase their activation and aggregation in COVID-19, resulting in vascular injury and thrombosis, both of which are linked to NET formation

NET formation may be caused by activated platelets rather than SARS-CoV-2 itself

NETosis, leading to aberrant immunity such as cytokine storms, autoimmune disorders, and immunosuppression.

early bacterial coinfections were more prevalent in COVID-19 patients than those infected with other viruses

NETosis and NETs may also have a role in the development of post COVID-19 syndromes, including lung fibrosis, neurological disorders, tumor growth, and worsening of concomitant disease

NETs and other by-products of NETosis have been shown to act as direct inflammation amplifiers. Hyperinflammation

“cytokine storm”

SARS-CoV-2 drives NETosis and NET formation to allow for the release of free DNA and by-products (e.g., elastases and histones). This may trigger surrounding macrophages and endothelial cells to secrete excessive proinflammatory cytokines and chemokines, which, in turn, enhance NET formation and form a positive feedback of cytokine storms in COVID-19

NET release enables self-antigen exposure and autoantibody production, thereby increasing the autoinflammatory response

patients with COVID-19 who have higher anti-NET antibodies are more likely to be detected with positive autoantibodies [e.g., antinuclear antibodies (ANA) and anti-neutrophil cytoplasmic antibodies (ANCA)]

COVID-19 NETs may act as potential inducers for autoimmune responses

have weakened adaptive immunity as well as a high level of inflammation

tumor-associated NETosis and NETs promote an immunosuppressive environment in which anti-tumor immunity is compromised

NETs have also been shown to enhance macrophage pyroptosis in sepsis

facilitating an immunosuppressive microenvironment

persistent immunosuppression may result in bacterial co-infection or secondary infection

can enhance this process by interacting with neutrophils through toll-like receptor 4 (TLR4), platelet factor 4 (PF4), and extracellular vesicle-dependent processes

NET-induced immunosuppression in COVID-19 in the context of co-existing bacterial infection

Following initial onset of COVID-19, an estimated 50% or more of COVID-19 survivors may develop multi-organ problems (e.g., pulmonary dysfunction and neurologic impairment) or have worsening concomitant chronic illness

NETs in the bronchoalveolar lavage fluid of severe COVID-19 patients cause EMT in lung epithelial cells

decreased E-cadherin (an epithelial marker) expression

COVID-19 also has a long-term influence on tumor progression

Patients with tumors have been shown to be more vulnerable to SARS-CoV-2 infection and subsequent development of severe COVID-19

patients who have recovered from COVID-19 may have an increased risk of developing cancer or of cancer progression and metastasis

awaken cancer cells

NETs have been shown to change the tumor microenvironment

enhance tumor progression and metastasis

vitamin C has been tested in phase 2 clinical trials aimed at reducing COVID-19-associated mortality by reducing excessive activation of the inflammatory response

vitamin C is an antioxidant that significantly attenuates PMA-induced NETosis in healthy neutrophils by scavenging ROS

vitamin C may also inhibit NETosis and NET production in COVID-19

Metformin

Vitamin C

Under steady-state conditions, intracellular GSH is maintained at millimolar concentrations, which keeps cells in a reduced environment and serves as the principal intracellular redox buffer when cells are subjected to an oxidative stressor including H2O2 (26). Glutathione peroxidase (GPx) activity catalyzes the reduction of H2O2 to water with the conversion of GSH to glutathione disulfide (GSSG). Under steady-state conditions, GSSG is recycled back to GSH by glutathione disulfide reductase using reducing equivalents from NADPH. However, under conditions of increased H2O2 flux, this recycling mechanism may become overwhelmed leading to a depletion of intracellular GSH (27, 28).

ascorbate radiosensitization can create an overwhelming oxidative stress to pancreatic cancer cells resulting in oxidation/depletion of the GSH intracellular redox buffer, resulting in cell death.

Treatment with the combination of ascorbate + IR significantly delayed tumor growth compared to controls or ascorbate alone

Ascorbate + IR also significantly increased overall survival compared to controls, IR alone or ascorbate alone

54% of mice treated with the combination of IR + ascorbate had no measurable tumors

Glutathione is a measurable marker indicative of the oxidation state of the thiol redox buffer in cells. In severe systemic oxidative stress, the GSSG/2GSH couple may become oxidized, i.e. the concentration of GSH decreases and GSSG may increase because the capacity to recycle GSSG to GSH becomes rate-limiting

This suggests that the very high levels of pharmacological ascorbate in these experiments may have a pro-oxidant toward red blood cells as seen by a decrease in the capacity of the intracellular redox buffer

These data support the hypothesis that ascorbate radiosensitization does not cause an increase in oxidative damage from lipid-derived aldehydes to other organs.

Our current study demonstrates the potential for pharmacological ascorbate as a radiosensitizer in the treatment of pancreatic cancer.

pharmacological ascorbate enhances IR-induced cell killing and DNA fragmentation leading to induction of apoptosis in HL60 leukemia cells

pharmacological ascorbate significantly decreases clonogenic survival and inhibits the growth of all pancreatic cancer cell lines as a single agent, as well as sensitizes cancer cells to IR

Hurst et al. demonstrated that pharmacological ascorbate combined with IR leads to increased numbers of double-strand DNA breaks and cell cycle arrest when compared to either treatment alone

pharmacological ascorbate could serve as a “pro-drug” for the delivery of H2O2 to tumors

the double-strand breaks induced by H2O2 were more slowly repaired

The combination of ascorbate and IR provide two distinct mechanisms of action: ascorbate-induced toxicity due to extracellular production of H2O2 that then diffuses into cells and causes damage to DNA, protein, and lipids; and radiation-induced toxicity as a result of ROS-induced damage to DNA. In addition, redox metal metals like Fe2+ may play an important role in ascorbate-induced cytotoxicity. By catalyzing the oxidation of ascorbate, labile iron can enhance the rate of formation of H2O2; labile iron can also react with H2O2. Recently our group has demonstrated that pharmacological ascorbate and IR increase the labile iron in tumor homogenates from this murine model of pancreatic cancer

we demonstrated that ascorbate or IR alone decreased tumor growth, but the combination treatment further inhibited tumor growth, indicating that pharmacological ascorbate is an effective radiosensitizer in vivo

data suggest that pharmacological ascorbate may protect the gut locally by decreasing IR-induced damage to the crypt cells, and systemically, by ameliorating increases in TNF-α