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IR also indirectly induces DNA damage by stimulating reactive oxygen species (ROS) production

IR is known to induce EMT in vitro

p53 is activated in response to IR-induced DNA damage

IR paradoxically also promotes tumour recurrence and metastasis

DNA double-strand breaks (DSBs)

cancer cells undergoing EMT acquire invasive and metastatic properties

changes in the tumour microenvironment (TME)

IR seems to induce EMT and CSC phenotypes by regulating cellular metabolism

EMT, stemness, and oncogenic metabolism are known to be associated with resistance to radiotherapy and chemotherapy

Hanahan and Weinberg proposed ten hallmarks of cancer that alter cell physiology to enhance malignant growth: 1) sustained proliferation, 2) evasion of growth suppression, 3) cell death resistance, 4) replicative immortality, 5) evasion of immune destruction, 6) tumour-promoting inflammation, 7) activation of invasion and metastasis, 8) induction of angiogenesis, 9) genome instability, and 10) alteration of metabolism

EMT is a developmental process that plays critical roles in embryogenesis, wound healing, and organ fibrosis

IR is known to induce stemness and metabolic alterations in cancer cells

transforming growth factor-β [TGF-β], epidermal growth factor [EGF]) and their associated signalling proteins (Wnt, Notch, Hedgehog, nuclear-factor kappa B [NF-κB], extracellular signal-regulated kinase [ERK], and phosphatidylinositol 3-kinase [PI3K]/Akt

activate EMT-inducing transcription factors, including Snail/Slug, ZEB1/δEF1, ZEB2/SIP1, Twist1/2, and E12/E47

Loss of E-cadherin is considered a hallmark of EMT

IR has been shown to induce EMT to enhance the motility and invasiveness of several cancer cells, including those of breast, lung, and liver cancer, and glioma cells

IR may increase metastasis in both the primary tumour site and in normal tissues under some circumstance

sublethal doses of IR have been shown to enhance the migratory and invasive behaviours of glioma cells

ROS are known to play an important role in IR-induced EMT

High levels of ROS trigger cell death by causing irreversible damage to cellular components such as proteins, nucleic acids, and lipids, whereas low levels of ROS have been shown to promote tumour progression—including tumour growth, invasion, and metastasis

hypoxia-inducible factor-1 (HIF-1) is involved in IR-induced EMT

Treatment with the N-acetylcysteine (NAC), a general ROS scavenger, prevents IR-induced EMT, adhesive affinity, and invasion of breast cancer cells

Snail has been shown to play a crucial role in IR-induced EMT, migration, and invasion

IR activates the p38 MAPK pathway, which contributes to the induction of Snail expression to promote EMT and invasion

NF-κB signalling that promotes cell migration

ROS promote EMT to allow cancer cells to avoid hostile environments

HIF-1 is a heterodimer composed of an oxygen-sensitive α subunit and a constitutively expressed β subunit.

Under normoxia, HIF-1α is rapidly degraded, whereas hypoxia induces stabilisation and accumulation of HIF-1α

levels of HIF-1α mRNA are enhanced by activation of the PI3K/Akt/mammalian target of rapamycin (mTOR)

IR is known to increase stabilisation and nuclear accumulation of HIF-1α, since hypoxia is a major condition for HIF-1 activation

IR induces vascular damage that causes hypoxia

ROS is implicated in IR-induced HIF-1 activation

IR causes the reoxygenation of hypoxic cancer cells to increase ROS production, which leads to the stabilisation and nuclear accumulation of HIF-1

IR increases glucose availability under reoxygenated conditions that promote HIF-1α translation by activating the Akt/mTOR pathway

The stabilised HIF-1α then translocates to the nucleus, dimerizes with HIF-1β, and increases gene expression— including the expression of essential EMT regulators such as Snail—to induce EMT, migration, and invasion

TGF-β signalling has been shown to play a crucial role in IR-induced EMT

AP-1 transcription factor is involved in IR-induced TGF-β1 expression

Wnt/β-catenin signalling is also implicated in IR-induced EMT

Notch signalling is known to be involved in IR-induced EMT

IR also increases Notch-1 expression [99]. Notch-1 is known to induce EMT by upregulating Snail

PAI-1 signalling is also implicated in IR-induced Akt activation that increases Snail levels to induce EMT

EGFR activation is known to be associated with IR-induced EMT, cell migration, and invasion by activating two downstream pathways: PI3K/Akt and Raf/MEK/ERK

ROS and RNS are also implicated in IR-induced EGFR activation

IR has also been shown to activate Hedgehog (Hh) signalling to induce EMT

IR has been shown to induce Akt activation through several signalling pathways (EGFR, C-X-C chemokine receptor type 4 [CXCR4]/C-X-C motif chemokine 12 [CXCL12], plasminogen activator inhibitor 1 [PAI-1]) and upstream regulators (Bmi1, PTEN) that promote EMT and invasion

CSCs possess a capacity for self-renewal, and they can persistently proliferate to initiate tumours upon serial transplantation, thus enabling them to maintain the whole tumour

Conventional cancer treatments kill most cancer cells, but CSCs survive due to their resistance to therapy, eventually leading to tumour relapse and metastasis

identification of CSCs, three types of markers are utilised: cell surface molecules, transcription factors, and signalling pathway molecules

CSCs express distinct and specific surface markers; commonly used ones are CD24, CD34, CD38, CD44, CD90, CD133, and ALDH

Transcription factors, including Oct4, Sox2, Nanog, c-Myc, and Klf4,

signalling pathways, including those of TGF-β, Wnt, Hedgehog, Notch, platelet-derived growth factor receptor (PDGFR), and JAK/STAT

microRNAs (miRNAs), including let-7, miR-22, miR-34a, miR-128, the miR-200 family, and miR-451

Non-CSCs can be reprogrammed to become CSCs by epigenetic and genetic changes

EMT-inducing transcription factors, such as Snail, ZEB1, and Twist1, are known to confer CSC properties

Signalling pathways involved in EMT, including those of TGF-β, Wnt, and Notch, have been shown to play important roles in inducing the CSC phenotype

TGF-β1 not only increases EMT markers (Slug, Twist1, β-catenin, N-cadherin), but also upregulates CSC markers (Oct4, Sox2, Nanog, Klf4) in breast and lung cancer cells

some CSC subpopulations arise independently of EMT

IR has been shown to induce the CSC phenotype in many cancers, including breast, lung, and prostate cancers, as well as melanoma

Genotoxic stress due to IR or chemotherapy promotes a CSC-like phenotype by increasing ROS production

IR has been shown to induce reprogramming of differentiated cancer cells into CSCs

In prostate cancer patients, radiotherapy increases the CD44+ cell population that exhibit CSC properties

IR also induces the re-expression of stem cell regulators, such as Sox2, Oct4, Nanog, and Klf4, to promote stemness in cancer cells

EMT-inducing transcription factors and signalling pathways, including Snail, STAT3, Notch signalling, the PI3K/Akt pathway, and the MAPK cascade, have been shown to play important roles in IR-induced CSC properties

STAT3 directly binds to the Snail promoter and increases Snail transcription, which induces the EMT and CSC phenotypes, in cisplatin-selected resistant cells

Other oncogenic metabolic pathways, including glutamine metabolism, the pentose phosphate pathway (PPP), and synthesis of fatty acids and cholesterol, are also enhanced in many cancers

metabolic reprogramming

HIF-1α, p53, and c-Myc, are known to contribute to oncogenic metabolism

metabolic reprogramming

tumour cells exhibit high mitochondrial metabolism as well as aerobic glycolysis

occurring within the same tumour

CSCs can be highly glycolytic-dependent or oxidative phosphorylation (OXPHOS)-dependen

mitochondrial function is crucial for maintaining CSC functionality

cancer cells depend on mitochondrial metabolism and increase mitochondrial production of ROS that cause pseudo-hypoxia

HIF-1 then enhances glycolysis

CAFs have defective mitochondria that lead to the cells exhibiting the Warburg effect; the cells take up glucose, and then secrete lactate to 'feed' adjacent cancer cells

lactate transporter, monocarboxylate transporter (MCT)

nutrient microenvironment

Epithelial cancer cells express MCT1, while CAFs express MCT4. MCT4-positive, hypoxic CAFs secrete lactate by aerobic glycolysis, and MCT1-expressing epithelial cancer cells then uptake and use that lactate as a substrate for the tricarboxylic acid (TCA) cycle

MCT4-positive cancer cells depend on glycolysis and then efflux lactate, while MCT1-positive cells uptake lactate and rely on OXPHOS

metabolic heterogeneity induces a lactate shuttle between hypoxic/glycolytic cells and oxidative/aerobic tumour cells

bulk tumour cells exhibit a glycolytic phenotype, with increased conversion of glucose to lactate (and enhanced lactate efflux through MCT4), CSC subsets depend on oxidative phosphorylation; most of the glucose entering the cells is converted to pyruvate to fuel the TCA cycle and the electron transport chain (ETC), thereby increasing mitochondrial ROS production

the major fraction of glucose is directed into the pentose phosphate pathway, to produce redox power through the generation of NADPH and ROS scavengers

HIF-1α, p53, and c-Myc, are known to contribute to oncogenic metabolism

regulatory molecules involved in EMT and CSCs, including Snail, Dlx-2, HIF-1, STAT3, TGF-β, Wnt, and Akt, are implicated in the metabolic reprogramming of cancer cells

HIF-1 induces the expression of glycolytic enzymes, including the glucose transporter GLUT, hexokinase, lactate dehydrogenase (LDH), and MCT, resulting in the glycolytic switch

HIF-1 represses the expression of pyruvate dehydrogenase kinase (PDK), which inhibits pyruvate dehydrogenase (PDH), thereby inhibiting mitochondrial activity

STAT3 has been implicated in EMT-induced metabolic changes as well

TGF-β and Wnt play important roles in the metabolic alteration of cancer cells

Akt is also implicated in the glycolytic switch and in promoting cancer cell invasiveness

EMT, invasion, metastasis, and stemness

pyruvate kinase M2 (PKM2), LDH, and pyruvate carboxylase (PC), are implicated in the induction of the EMT and CSC phenotypes

decreased activity of PKM2 is known to promote an overall shift in metabolism to aerobic glycolysis

LDH catalyses the bidirectional conversion of lactate to pyruvate

High levels of LDHA are positively correlated with the expression of EMT and CSC markers

IR has been shown to induce metabolic changes in cancer cells

IR enhances glycolysis by upregulating GAPDH (a glycolysis enzyme), and it increases lactate production by activating LDHA, which converts pyruvate to lactate

IR enhances glycolysis by upregulating GAPDH (a glycolysis enzyme), and it increases lactate production by activating LDHA, which converts pyruvate to lactate

IR also elevates MCT1 expression that exports lactate into the extracellular environment, leading to acidification of the tumour microenvironment

IR increases intracellular glucose, glucose 6-phosphate, fructose, and products of pyruvate (lactate and alanine), suggesting a role for IR in the upregulation of cytosolic aerobic glycolysis

Lactate can activate latent TGF-

lactate stimulates cell migration and enhances secretion of hyaluronan from CAF that promote tumour metastasis

promote tumour survival, growth, invasion, and metastasis; enhance the stiffness of the ECM; contribute to angiogenesis; and induce inflammation by releasing several growth factors and cytokines (TGF-β, VEGF, hepatocyte growth factor [HGF], PDGF, and stromal cell-derived factor 1 [SDF1]), as well as MMP

tumours recruit the host tissue’s blood vessel network to perform four mechanisms: angiogenesis (formation of new vessels), vasculogenesis (de novo formation of blood vessels from endothelial precursor cells), co-option, and modification of existing vessels within tissues.

immunosuppressive cells such as tumour-associated macrophages (TAM), MDSCs, and regulatory T cells, and the immunosuppressive cytokines, TGF-β and interleukin-10 (IL-10)

immunosuppressive cells such as tumour-associated macrophages (TAM), MDSCs, and regulatory T cells, and the immunosuppressive cytokines, TGF-β and interleukin-10 (IL-10)

intrinsic immunogenicity or induce tolerance

cancer immunoediting’

three phases: 1) elimination, 2) equilibrium, and 3) escape.

The third phase, tumour escape, is mediated by antigen loss, immunosuppressive cells (TAM, MDSCs, and regulatory T cells), and immunosuppressive cytokines (TGF-β and IL-10).

IR can elicit various changes in the TME, such as CAF activity-mediated ECM remodelling and fibrosis, cycling hypoxia, and an inflammatory response

IR activates CAFs to promote the release of growth factors and ECM modulators, including TGF-β and MMP

TGF-β directly influences tumour cells and CAFs, promotes tumour immune escape, and activates HIF-1 signalling

MMPs degrade ECM that facilitates angiogenesis, tumour cell invasion, and metastasis

IR also promotes MMP-2/9 activation in cancer cells to promote EMT, invasion, and metastasis

IR-induced Snail increases MMP-2 expression to promote EMT

Radiotherapy has the paradoxical side-effect of increasing tumour aggressiveness

IR promotes ROS production in cancer cells, which may induce the activation of oncogenes and the inactivation of tumour suppressors, which further promote oncogenic metabolism

Metabolic alterations

oncogenic metabolism

elicit various changes in the TME

Although IR activates an antitumour immune response, this signalling is frequently suppressed by tumour escape mechanisms

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

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

NO also is co linked to VEGF which in turn increases the antiapoptotic gene bcl-2

The other important influence of NO is in its inhibition of the proapoptoic caspases cascade. This in turn protects the cells from intracellular preprogrammed death.

nitric oxide in immune suppression in relation to oxygen radicals is its inhibitory effect on the binding of leukocytes (PMN) at the endothelial surface

Inhibition of inducible Nitric Oxide Synthase (iNOS)

NO from the tumor cells actually suppresses the iNOS, and in addition it reduces oxygen radicals to stop the formation of peroxynitrite in these cells. But NO is not the only inhibitor of iNOS in cancer.

Spermine and spermidine, from the rate limiting enzyme for DNA synthases, ODC, also inhibit iNOS

tolerance in the immune system that decreases the immune response to antigens on the tumors

Freund’s adjuvant

increase in kinases in these cells which phosphorylate serine, and tyrosine

responsible for activation of many growth factors and enzymes

phosphorylated amino acids suppress iNOS activity

Hexokinase II

Prostaglandin E2, released from tumor cells is also an inhibitor of iNOS, as well as suppressing the immune system

Th-1 subset of T-cells. These cells are responsible for anti-viral and anti-cancer activities, via their cytokine production including Interleukin-2, (IL-2), and Interleukin-12 which stimulates T-killer cell replication and further activation and release of tumor fighting cytokines.

Th-2 subset of lymphocytes, on the other hand are activated in allergies and parasitic infections to release Interleukin-4 and Interleukin-10

These have respectively inhibitory effects on iNOS and lymphocyte Th-1 activation

Mast cells contain histamine which when released increases the T suppressor cells, to lower the immune system and also acts directly on many tumor Histamine receptors to stimulate tumor growth

Tumor cells release IL-10, and this is thought to be one of the important areas of Th-1 suppression in cancer patients

IL-10 is also increased in cancer causing viral diseases such as HIV, HBV, HCV, and EBV

IL-10 is also a central regulator of cyclooxygenase-2 expression and prostaglandin production in tumor cells stimulating their angiogenesis and NO production

nitric oxide in tumor cells even prevents the activation of caspases responsible for apoptosis

early stages of carcinogenesis, which we call tumor promotion, one needs a strong immune system, and fewer oxygen radicals to prevent mutations but still enough to destroy the tumor cells should they develop

later stages of cancer development, the oxygen radicals are decreased around the tumors and in the tumor cells themselves, and the entire cancer fighting Th-1 cell replication and movement are suppressed. The results are a decrease in direct toxicity and apoptosis, which is prevented by NO, a suppression of the macrophage and leukocyte toxicity and finally, a suppression of the T-cell induced tumor toxicity

cGMP is increased by NO

NO in cancer is its ability to increase platelet-tumor cell aggregates, which enhances metastases

the greater the malignancies and the greater the metastatic potential of these tumors

The greater the NO production in many types of tumors,

gynecological

elevated lactic acid which neutralizes the toxicity and activity of Lymphocyte immune response and mobility

The lactic acid is also feeding fungi around tumors and that leads to elevated histamine which increases T-suppressor cells.  Histamine alone stimulates many tumor cells.

T-regulatory cells (formerly,T suppressor cells) down regulate the activity of Natural killer cells

last but not least, the Lactic acid from tumor cells and acidic diets shifts the lymphocyte activity to reduce its efficacy against cancer cells and pathogens in addition to altering the bacteria of the intestinal tract.

intestinal tract bacteria in cancer cells release sterols that suppress the immune system and down regulate anticancer activity from lymphocytes.

In addition to the lactic acid, adenosine is also released from tumors. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state

Adenosine up regulates the PD1 receptor in T-1 Lymphocytes and inhibits their activity

Adenosine is a purine nucleoside found within the interstitial fluid of solid tumors at concentrations that are able to inhibit cell-mediated immune responses to tumor cells

Adenosine appears to up-regulate the PD1 receptor in T-1 Lymphocytes and inhibits the immune system further

Mast cells with their release of histamine lower the immune system and also stimulate tumor growth and activate the metalloproteinases involved in angiogenesis and metastases

COX 2 inhibitors or all trans-retinoic acid

Cimetidine, an antihistamine has been actually shown to increase in apoptosis in MDSC via a separate mechanism than the antihistamine effect

interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis

In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-alpha (TNF-alpha)

these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-alpha-induced up-regulation of IL-8 via nuclear factor-kappaB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target

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

of the approximately 108 cannabinoids produced by C. sativa, Δ9-tetrahydrocannabinol (thc) is the most relevant because of its high potency and abundance in plant preparations

Tetrahydrocannabinol exerts a wide variety of biologic effects by mimicking endogenous substances—the endocannabinoids anandamide3 and 2-arachidonoylglycerol4,5—that engage specific cell-surface cannabinoid receptors

two major cannabinoid-specific receptors—cb1 and cb2

transient receptor potential cation channel subfamily V, member 1

orphan G protein–coupled receptor 55

Most of the effects produced by cannabinoids in the nervous system and in non-neural tissues rely on cb1 receptor activation

the cb2 receptor was initially described to be present in the immune system6, but was more recently shown to also be expressed in cells from other origins

cardiovascular tone, energy metabolism, immunity, and reproduction

cannabinoids are well known to exert palliative effects in cancer patients

best-established use is the inhibition of chemotherapy-induced nausea and vomiting

thc and other cannabinoids exhibit antitumour effects in a wide array of animal models of cancer

cannabinoid receptors and their endogenous ligands are both generally upregulated in tumour tissue compared with non-tumour tissue

cb2 promotes her2 (human epidermal growth factor receptor 2) pro-oncogenic signalling in breast cancer

pharmacologic activation of cannabinoid receptors decreases tumour growth

endocannabinoid signalling can also have a tumour-suppressive role

pharmacologic stimulation of cb receptors is, in most cases, antitumourigenic. Nonetheless, a few reports have proposed a tumour-promoting effect of cannabinoids

most prevalent effect is the induction of cancer cell death by apoptosis and the inhibition of cancer cell proliferation

impair tumour angiogenesis and block invasion and metastasis

thc and other cannabinoids induce the apoptotic death of glioma cells by cb1- and cb2-dependent stimulation

Autophagy is primarily a cytoprotective mechanism, although its activation can also lead to cell death

autophagy is important for cannabinoid antineoplastic activity

autophagy is upstream of apoptosis in the mechanism of cannabinoid-induced cell death

the effect of cannabinoids in hormone- dependent tumours might rely, at least in part, on the ability to interfere with the activation of growth factor receptors

glioma cells), pharmacologic blockade of either cb1 or cb2 prevents cannabinoid-induced cell death with similar efficacy

other types of cancer cells (pancreatic48, breast24, or hepatic43 carcinoma cells, for example), antagonists of cb2 but not of cb1 inhibit cannabinoid antitumour actions

thc promotes cancer cell death in a cb1- or cb2-dependent manner (or both) at lower concentrations

cannabidiol (cbd), a phytocannabinoid with a low affinity for cannabinoid receptors15, and other marijuana-derived cannabinoids57 have also been proposed to promote the apoptotic death of cancer cells acting independently of the cb1 and cb2 receptors

In cancer cells, cannabinoids block the activation of the vascular endothelial growth factor (vegf) pathway, an inducer of angiogenesi

In vascular endothelial cells, cannabinoid receptor activation inhibits proliferation and migration, and induces apoptosis

cb1 or cb2 receptor agonists (or both) reduce the formation of distant tumour masses in animal models of both induced and spontaneous metastasis, and inhibit adhesion, migration, and invasiveness of glioma64, breast65,66, lung67,68, and cervical68 cancer cells in culture

the ceramide/p8–regulated pathway plays a general role in the antitumour activity of cannabinoids targeting cb1 and cb2

cbd, by acting independently of the cb1 and cb2 receptors, produces a remarkable anti-tumour effect—including reduction of invasiveness and metastasis

cannabinoids can also enhance immune system–mediated tumour surveillance in some contexts

ability of thc to reduce inflammation75,76, an effect that might prevent certain types of cancer

recent observations suggest that the combined administration of cannabinoids with other anticancer drugs acts synergistically to reduce tumour growth

combined administration of gemcitabine (the benchmark agent for the treatment of pancreatic cancer) and various cannabinoid agonists synergistically reduced the viability of pancreatic cancer cells

Other reports indicated that anandamide and HU-210 might also enhance the anticancer activity of paclitaxel89 and 5-fluorouracil90 respectively

Combined administration of thc and cbd enhances the anticancer activity of thc and reduces the dose of thc needed to induce its tumour growth-inhibiting activity

Preclinical animal models have yielded data indicating that systemic (oral or intraperitoneal) administration of cannabinoids effectively decreases tumour growth

Combinations of cannabinoids with classical chemotherapeutic drugs such as the alkylating agent temozolomide (the benchmark agent for the management of glioblastoma80,84) have been shown to produce a strong anticancer action in animal models

pharmacologic inhibition of egfr, erk83, or akt enhances the cell-death-promoting action of thc in glioma cultures (unpublished observations by the authors), which suggests that targeting egfr and the akt and erk pathways could enhance the antitumour effect of cannabinoids

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