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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.

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

The CHALICE cohort of 404 individuals aged 50 years had an average vitamin C intake of ~110 mg/day, which should provide adequate plasma concentrations [14]. Despite this, a significant proportion of the participants had inadequate plasma vitamin C status

inadequate plasma vitamin C concentrations (i.e., <50 µmol/L)

adequate plasma levels (i.e., >50 µmol/L)

Higher plasma vitamin C status was associated with lower weight, BMI and waist circumference

plasma vitamin C was negatively associated with blood triglycerides, HbA1c and insulin, and positively associated with HDL levels.

No correlation was found between plasma vitamin C and the two indicators of heart health; blood pressure and cardiovascular risk score.

2.4% of 50-year-olds were deficient in vitamin C (i.e., <11 µmol/L)

hypovitaminosis C (i.e., <23 µmol/L)

A high proportion (63%) of our participants had inadequate plasma vitamin C concentrations (i.e., <50 µmol/L)

The association of low vitamin C with obesity in this study replicates results in the literature [35,40,41,42,43,44], and it is apparent that individuals with higher weight require higher intakes of vitamin C to reach adequate vitamin C status

higher plasma vitamin C status is associated with lower circulating levels of blood triglycerides, insulin and HbA1c

A role for vitamin C in the prevention or management of diabetes and/or metabolic syndrome has been suggested

In this study, we also demonstrate lower levels of mild cognitive impairment in those with high vitamin C status

The odds of mild cognitive impairment were twice as high for those below 23 μmol/L plasma vitamin C concentration.

Vitamin C is present at very high concentrations in the brain

animal models have shown that the brain is the last organ to be depleted of the vitamin during prolonged deficiency

A recent animal study has shown that moderate vitamin C deficiency may play a role in accelerating amyloid plaque accumulation in Alzheimer’s disease

related to intracellular hydrogen peroxide generation

only be obtained by intravenous administration of AA

Preferentially kills neoplastic cells

Is virtually non-toxic at any dosage

Does not suppress the immune system, unlike most chemotherapy agents

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

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

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

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

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

Metabolites of AA have also shown antitumor activity in vitro

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

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

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

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

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

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

AA, plasma levels during infusion were not monitored,

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

This low cytotoxic level of AA is exceedingly rare

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

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

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

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

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

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

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

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

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

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

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

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

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

patient is orally supplementing between infusions

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

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

The rate of water loss can be quantified through measurement of sweat rate

Pre- and postexercise body weight measurements are the most common means to estimate overall water loss but are condition specific

It appears that 1% to 2% body weight loss is well tolerated by the exercising athlete

Dehydration, defined as greater than 2% loss of body weight, can negatively affect performance

In highly trained endurance athletes, plasma volume and sodium serum concentration were preserved despite a 5% body weight loss

In Ironman triathletes, dehydration to 5% body weight loss did not correlate with occurrence of medical complications

hydration should begin hours prior to exercise, especially if known deficits are present, and fluids should be consumed at a slow, steady rate, with 5 to 7 mL/kg taken 4 hours prior to exercise

Sodium concentration did not produce significant changes in the rate of absorption but was primarily dependent on carbohydrate concentration

Replacing 150% of body weight loss over 60 minutes has been tolerated without complications

IV treatment of severe dehydration (>7% body weight loss), exertional heat illness, nausea, emesis, or diarrhea, and in those who cannot ingest oral fluids for other reasons, is clinically indicated

A recent survey of the National Football League teams revealed that 75% (24 of 32) of the teams utilized IV infusion of fluids for prehydration in at least some otherwise healthy individuals

In the National Football League, an average of 1.5 L of normal saline was administered approximately 2.5 hours prior to competition

after 2 hours of exercise, the rectal temperature was 0.6° higher in the group not receiving IV infusion. Also, stroke volume and cardiac output were 11% to 16% lower in the control group versus the IV infusion group.

Recent evidence suggests the etiology of EAMC is related to muscle fatigue and neuronal excitability

no correlation between hydration status or electrolyte concentrations with EAMC

there may be a subset of muscle cramping that is associated with a loss of both body fluid and sodium

Glycerol is the primary agent for oral hyperhydration

elevation of plasma volume by 200 to 300 mL via dextran infusion resulted in 15% increase in stroke volume, 4% increase in VO2 max, and an increase in the exercise time to fatigue

Neither the tonicity nor mode of hydration resulted in improved speed of rehydration, greater fluid retention, or improved performance

There are beneficial anecdotal reports of EAMC treatment in elite and professional-level athletes with IV hydration during the course of an event

Plasma volume was better restored during rehydration with IV fluids at preexercise and 5 minutes of exercise. At 15 minutes, there was no difference between IV and oral rehydration

More rapid restoration of plasma volume was accomplished in the IV treatment group with no advantages over oral rehydration in physiological strain, heat tolerance, ratings of perceived effort, or thermal sensations

No difference was found in exercise time to exhaustion. IV and oral rehydration methods were equally effective. Heart rates were statistically higher in the oral rehydration group through 75 minutes of exercise, and there were higher increases in norepinephrine plasma concentrations

No significant differences between the groups were found for time to recovery, number of days with pain, number of days with stiffness, sleep disturbance, fatigue, rectal temperature, and loss of appetite

The current data suggest that IV rehydration is faster than oral

There may be physiological benefits of decreased heart rate and norepinephrine in athletes rehydrated via IV route

Postexercise blood 1 hour and 24 hours showed no differences in circulating myoglobin or creatine kinase

The use of IV fluid may be beneficial for a subset of fluid sensitive athletes

this should be reserved for high-level athletes with strong histories of symptoms in well-monitored settings.

Volume expanders may also be beneficial for some athletes

pharmacologic ascorbate as a prooxidant drug for therapeutic use.

Recently we reported that pharmacologic ascorbic acid concentrations produced H2O2 concentrations of ≥25 μM, causing cancer cell death in vitro

We found that H2O2 concentrations generated in vivo were those that caused cancer cell death in vitro

When ascorbate was given parenterally, Asc•−, the product of a loss of one electron from ascorbate, was detected preferentially in extracellular fluid compared with blood

Asc•− generation in extracellular fluid depended on the ascorbate dose and the resulting concentrations

With i.v. administration of ascorbate, Asc•− concentrations were as much as 12-fold greater in extracellular fluid compared to blood and approached 250 nM

In blood, such Asc•− concentrations were never produced and were always <50 nM

These data are all consistent with the hypothesis that pharmacologic ascorbate concentrations in vivo serve as a prodrug for selective delivery of H2O2 to the extracellular space

After oral ingestion, control of intracellular and extracellular ascorbate concentrations is mediated by three mechanisms: intestinal absorption, tissue transport, and renal reabsorption

intestinal absorption, or bioavailability, declines at doses >200 mg

corresponding to plasma concentrations of ≈60 μM

at approximately this concentration, the ascorbate tissue transporter SVCT2 approaches Vmax, and tissues appear to be saturated

also at ≈60 μM, renal reabsorption approaches saturation, and excess ascorbate is excreted in urine

Parenteral administration bypasses tight control

When tight control is bypassed, H2O2 forms in the extracellular space

in vivo validation of ascorbate as a prodrug for selective H2O2 formation

Temporarily bypassing tight control with parenteral administration of ascorbate allows H2O2 to form in discrete time periods only, decreasing likelihood of harm, and provides a pharmacologic basis for therapeutic use of i.v. ascorbate

H2O2 formation results in selective cytotoxicity

Tumor cells are killed with exposure to H2O2 for ≤30 min

In vitro, killing is mediated by H2O2 rather than Asc•−

In addition to cancer treatment, another potential therapeutic use is for treatment of infections. H2O2 concentrations of 25–50 μM are bacteriostatic

virally infected cells may also be candidates

Approximately 0.3% of testosterone is converted into estradiol by aromatase (CYP19A1)

the recommendation for injectable testosterone esters is to check the serum concentration midway between injections

it is recommended for serum testosterone to be evaluated 3 to 12 hours after application of the transdermal patch

testosterone concentrations should be checked 2–3 months after initiation of therapy and after adjusting the dose

a study from 1989 utilizing testosterone transdermally containing 5, 10, or 15 mg of testosterone showed that peak concentrations of testosterone were achieved 3 to 8 hours after scrotal application in hypogonadal men

It is used for many medications and has the advantage of high bioavailability, absence of hepatic first pass metabolism, increased therapeutic efficacy, and steadiness of plasma concentrations of the drug

evaluate serum testosterone at the end of the dosing interval for testosterone pellets

increased amount of fat leads to increased extragonadal aromatase activity, resulting in increased concentrations of estradiol. High circulating concentrations of estradiol down regulate the HPG axis and decrease the amount of circulating testosterone

Up to 80% of plasma estradiol originates from aromatization of testosterone and less than 20% of estradiol in the circulation is secreted by the testes

A PSA concentration, digital rectal examination, and hematocrit should be performed at baseline and at 3 months, 6 months, then yearly after TRT is initiated.

measure serum testosterone any time after the patient has been on treatment with gel for at least 1 week

If the hematocrit rises above 54%, treatment should be discontinued

elderly men having higher estradiol serum concentrations than postmenopausal women

Hypopituitary adults with GHD have been reported to have normal serum IGF-I levels in 37–70% of patients in various studies (5, 9, 18, 21, 22). This is owing in part to the fact that multiple factors regulate serum IGF-I concentrations including nutritional status; hepatic and renal function; and circulating concentrations of thyroid hormone, androgens, and estrogens

changes in concentrations of IGF-binding proteins (IGFBPs) influence the total concentration of IGF-I in plasma

Among patients with an IGF-I sd score above −1 in the present study, 46% had a peak GH less than 2.5 μg/liter and 67% had a peak GH less than 5 μg/liter.

In summary, adult GHD can be predicted with 95% accuracy by the presence of either three or four PHDs or a serum IGF-I concentration less than 84 μg/liter

We propose that adult patients with three or four PHDs (three or four of the following deficiencies: TSH, ACTH, gonadotropins [LH and/or FSH], and AVP [central diabetes insipidus]) do not require a GH stimulation test to make the diagnosis of adult GHD

A treatment dose of 4 g ascorbate/kg body weight either once or twice daily did not produce any discernible adverse effects

Xenograft experiments showed that parenteral ascorbate as the only treatment significantly decreased both tumor growth and weight by 41–53%

Peak plasma concentrations of ascorbate approached 30 mM

Pharmacologic concentrations of ascorbate decreased tumor volumes 41–53% in diverse cancer types known for both their aggressive growth and limited treatment options.

Our findings showed that pharmacologic ascorbic acid concentrations were cytotoxic to many types of cancer cells in vitro (Fig. 1A) and significantly impeded tumor progression in vivo without toxicity to normal tissues

The amelioration of ascorbate cytotoxicity in vitro by the addition of catalase was consistent among sensitive cancer cells (Fig. 1B) and points unambiguously to H2O2 generation in the extracellular medium

the current in vivo data support that pharmacologic ascorbate concentrations, which can readily be achieved in humans (Fig. 3E), diminished growth of several aggressive cancer types in mice (Fig. 2) without causing apparent adverse effects.

These intratumoral H2O2 concentrations of >125 μM persisted for >3 h after ascorbate administration

These results show that targeting the cancer DNA methylome by combining low-dose 5-aza-CdR and vitamin C stimulates the expression of ERVs, the induction of a cell-autonomous immune activation response, and increased apoptosis of cancer cells

The addition of vitamin C to treatment protocols therefore may be a straightforward way to increase the clinical efficacy of such drugs in MDS and leukemia patients

Vitamin C deficiency has been seen previously in patients with multiple types of cancer, including hematological malignancies (35⇓–37). We predict that these patients might receive the most benefits from the combination treatment.

induction of an innate immune response

We therefore measured plasma concentrations of vitamin C in a small number of patients with miscellaneous hematologic malignancies. Strikingly, 58% of patients with hematological neoplasia who were not taking vitamin C supplements had severe vitamin C deficiency (serum concentration <11.4 μM, at which clinical features of scurvy may be manifested) (34), and 33% had vitamin C levels below the normal range

it is possible that vitamin C was oxidized to DHA before it was transported into the cells

Oral administration of vitamin C should be sufficient for the therapeutic strategy, because the concentrations reported in this study would not require i.v. administration.

Vitamin C is an essential nutrient for humans and has been reported to increase IFN levels in human cells upon virus infection

daily treatment with vitamin C alone at physiological concentrations enhanced the expression of viral-defense genes relative to untreated cells

When combined with low-dose 5-aza-CdR, physiological concentrations of vitamin C synergistically inhibited cancer-cell growth and induced apoptosis. Such synergy was associated with increased ERV expression and dsRNA in treated cells. The mechanism of action differs from that of vitamin C at higher doses, which involves its pro-oxidant activity, including GSH inhibition, to generate reactive oxygen species

This activity has been shown to induce DNA damage and to enhance the sensitivities of myeloid malignancies, multiple myeloma, and cutaneous T-cell lymphoma to arsenic trioxide (41⇓⇓–44). It also can increase chemosensitivity of ovarian cancer cells (27) and selectively kill KRAS or BRAF mutant colorectal cancer cells by inhibiting GAPDH

reactive oxygen species

subnormal plasma ascorbic acid levels are a predictable feature in patients with severe sepsis

Ascorbic acid depletion in sepsis results from ascorbic acid consumption by the reduction of plasma free iron, ascorbic acid consumption by the scavenging of aqueous free radicals (peroxyl radicals), and by the destruction of the oxidized form of ascorbic acid dehydroascorbic acid

Sepsis further inhibits intracellular reduction of dehydroascorbic acid, producing acute intracellular ascorbic acid depletion

Ascorbic acid treated patients in this study exhibited rapid and sustained increases in plasma ascorbic acid levels using an intermittent every six hours administration protocol

Septic ascorbic acid-deficient neutrophils fail to undergo normal apoptosis. Rather, they undergo necrosis thereby releasing hydrolytic enzymes in tissue beds, thus contributing to organ injury

We speculate that intravenous ascorbic acid acts to restore neutrophil ascorbic acid levels

Repletion of ascorbic acid in this way allows for normal apoptosis, thus, preventing the release of organ damaging hydrolytic enzymes.

Treatment with 3αHP subsequent to 5αP-induced tumor initiation resulted in suppression of further tumorigenesis and regression of existing tumors

Tumorigenesis of ER/PR-negative breast cells is significantly enhanced by 5αP and suppressed by 3αHP, the outcome depending on the relative concentrations of these two hormones in the microenvironment in the breast regions

The findings show that the production of 5αP greatly exceeds that of 3αHP in ER/PR-negative tumors and that treatment with 3αHP can effectively block tumorigenesis and cause existing tumors to regress

hypothesis that a high 3αHP-to-5αP concentration ratio in the microenvironment may foster normalcy in noncancerous breast regions.

a large proportion (about 30% to 60%) of breast tumors are ER and/or PR negative

about 90% of normal proliferating breast epithelial cells are receptor negative

Our previous in vitro studies had shown that breast tissues and cell lines readily convert progesterone to 5α-pregnanes, such as 5αP, and delta-4-pregnenes, such as 3αHP (Figure ​(Figure1),1), and that tumorous breast tissues [15] and tumorigenic breast cell lines [16] produce higher levels of 5αP and lower levels of 3αHP than do normal breast tissues and nontumorigenic cell lines

The progesterone metabolism studies suggested that increases in 5αP and decreases in 3αHP production accompany the shift toward breast cell neoplasia and tumorigenicity

In vitro studies on five different human breast cell lines showed that cell proliferation and detachment are significantly increased by 5αP and decreased by 3αHP

the prevailing theory of hormonal regulation of breast cancer, as well as hormone-based therapies, revolves around estrogen and/or progesterone and ER/PR-positive breast cells and tumors.

Not only do these "receptor-negative" breast cancers fail to benefit from current hormonal therapies, but they also generally exhibit more-aggressive biologic behaviors and poorer prognosis than the receptor-positive ones

The results of the studies reported here show for the first time that the progesterone metabolites, 5αP and 3αHP, act as hormones that regulate ER/PR-negative breast tumor formation, growth, and regression

The onset of the ER/PR-negative human breast cell tumors in mice was considerably accelerated, and the growth significantly stimulated, by just one or two applications of 5αP

In contrast, 3αHP retarded onset of tumor formation, suppressed tumor growth, and inhibited or regressed existing 5αP-induced tumors

When both hormones were administered simultaneously, the effects of one were abrogated by the effects of the other.

The 5αPR and 3αHPR (which are associated with the plasma membranes of both ER/PR-positive [19] and ER/PR-negative [29] cells) are distinct from each other and from known ER, PR, androgen, and corticosteroid receptors, and lack affinity for other steroids, such as progesterone, estrogen, androgens, corticosteroids, and other progesterone metabolites

Levels of 5αPR are upregulated by 5αP itself and estradiol, and downregulated by 3αHP in both ER/PR-positive and -negative cells

ndications are that 5αP acts via the surface receptor-linked mitogen-activated protein kinase (MAPK; Erk1/2) pathway; 5αP significantly stimulates activation of Erk1/2 [30], increases the Bcl-2/Bax expression ratio [18] and actin depolymerization [31], and decreases expression of actin and adhesion plaque-associated vinculin [31], resulting in decreased apoptosis and increased mitosis and cell detachment

3αHP appears to suppress protein kinase C (PKC), phospholipase C (PLC), Ca2+ mobilization (unpublished observations), and the Bcl-2/Bax expression ratio [18], and increases expression of the cell-cycle inhibitor p21 [18], resulting in increased apoptosis and decreased proliferation and detachment of breast cell lines.

serum from mice with tumors had significantly more 5αP than 3αHP

the tumors, which on average had about threefold higher concentrations of 5αP than the respective sera, and >10-fold higher 5αP than 3αHP levels

Previous in vitro metabolism studies showed that human breast tumor tissues convert significantly more progesterone to 5α-pregnanes like 5αP and less to 4-pregnenes like 3αHP than do paired normal (nontumorous) tissues

Similar differences in progesterone metabolism and enzyme gene expressions were observed between tumorigenic and nontumorigenic breast cell lines

breast carcinomas are able to synthesize progesterone

The current findings, along with the previous in vitro studies, suggest that the relative concentrations of 5αP and 3αHP in the breast microenvironment constitute important autocrine/paracrine determinants not only for tumorigenesis but also for potential regression of tumors and the maintenance of normalcy of ER/PR-negative breast cells/tissues.

Evidence presented here shows that a high concentration of 5αP, relative to 3αHP in the microenvironment, promotes initiation and growth of tumors, whereas a higher concentration of 3αHP, relative to 5αP, suppresses tumorigenesis and promotes normalcy

5α-reductase and 5αPR levels are upregulated by 5αP

in the 3αHP-treated mice, the elevated 3αHP levels, relative to 5αP, in the microenvironment could have opposed progression to xenograft neoplasia by its inherent anticancer actions and the suppression of 5αP synthesis and 5αPR expression

the opposing actions of the progesterone metabolites also appear to exert some control over the estrogen-regulated effects on breast cancer by their ability to modulate ER numbers in ER-positive cells

because both ER/PR-negative and ER/PR-positive, as well as normal and tumorigenic human breast cell lines, have been shown to respond to 5αP and 3αHP in vitro, it is suggested that these endogenously produced progesterone metabolites may also play regulatory hormonal roles in ER/PR-positive breast cancers, as well as in the maintenance of normalcy in nontumorous breast tissues.

The in vivo data provide further evidence that progesterone metabolites, such as 5αP and 3αHP, deserve to be considered as active hormones in their own right, rather than inactive waste products

The starting point for innate immunity activation is the recognition of conserved structures of bacteria, viruses, and fungal components through pattern-recognition receptors

TLRs are PRRs that recognize microbe-associated molecular patterns

TLRs are transmembrane proteins containing extracellular domains rich in leucine repeat sequences and a cytosolic domain homologous to the IL1 receptor intracellular domain

The major proinflammatory mediators produced by the TLR4 activation in response to endotoxin (LPS) are TNFα, IL1β and IL6, which are also elevated in obese and insulin-resistant patients

Obesity, high-fat diet, diabetes, and NAFLD are associated with higher gut permeability leading to metabolic endotoxemia.

Probiotics, prebiotics, and antibiotic treatment can reduce LPS absorption

LPS promotes hepatic insulin resistance, hypertriglyceridemia, hepatic triglyceride accumulation, and secretion of pro-inflammatory cytokines promoting the progression of fatty liver disease.

In the endothelium, LPS induces the expression of pro-inflammatory, chemotactic, and adhesion molecules, which promotes atherosclerosis development and progression.

In the adipose tissue, LPS induces adipogenesis, insulin resistance, macrophage infiltration, oxidative stress, and release of pro-inflammatory cytokines and chemokines.

the gut microbiota has been recently proposed to be an environmental factor involved in the control of body weight and energy homeostasis by modulating plasma LPS levels

dietary fats alone might not be sufficient to cause overweight and obesity, suggesting that a bacterially related factor might be responsible for high-fat diet-induced obesity.

This was accompanied in high-fat-fed mice by a change in gut microbiota composition, with reduction in Bifidobacterium and Eubacterium spp.

n humans, it was also shown that meals with high-fat and high-carbohydrate content (fast-food style western diet) were able to decrease bifidobacteria levels and increase intestinal permeability and LPS concentrations

it was demonstrated that, more than the fat amount, its composition was a critical modulator of ME (Laugerette et al. 2012). Very recently, Mani et al. (2013) demonstrated that LPS concentration was increased by a meal rich in saturated fatty acids (SFA), while decreased after a meal rich in n-3 polyunsaturated fatty acids (n-3 PUFA).

this effect seems to be due to the fact that some SFA (e.g., lauric and mystiric acids) are part of the lipid-A component of LPS and also to n-3 PUFA's role on reducing LPS potency when substituting SFA in lipid-A

these experimental results suggest a pivotal role of CD14-mediated TLR4 activation in the development of LPS-mediated nutritional changes.

This suggests a link between gut microbiota, western diet, and obesity and indicates that gut microbiota manipulation can beneficially affect the host's weight and adiposity.

endotoxemia was independently associated with energy intake but not fat intake in a multivariate analysis

in vitro that endotoxemia activates pro-inflammatory cytokine/chemokine production via NFκB and MAPK signaling in preadipocytes and decreased peroxisome proliferator-activated receptor γ activity and insulin responsiveness in adipocytes.

T2DM patients have mean values of LPS that are 76% higher than healthy controls

LPS-induced release of glucagon, GH and cortisol, which inhibit glucose uptake, both peripheral and hepatic

LPSs also seem to induce ROS-mediated apoptosis in pancreatic cells

Recent evidence has been linking ME with dyslipidemia, increased intrahepatic triglycerides, development, and progression of alcoholic and nonalcoholic fatty liver disease

The hepatocytes, rather than hepatic macrophages, are the cells responsible for its clearance, being ultimately excreted in bile

All the subclasses of plasma lipoproteins can bind and neutralize the toxic effects of LPS, both in vitro (Eichbaum et al. 1991) and in vivo (Harris et al. 1990), and this phenomenon seems to be dependent on the number of phospholipids in the lipoprotein surface (Levels et al. 2001). LDL seems to be involved in LPS clearance, but this antiatherogenic effect is outweighed by its proatherogenic features

LPS produces hypertriglyceridemia by several mechanisms, depending on LPS concentration. In animal models, low-dose LPS increases hepatic lipoprotein (such as VLDL) synthesis, whereas high-dose LPS decreases lipoprotein catabolism

When a dose of LPS similar to that observed in ME was infused in humans, a 2.5-fold increase in endothelial lipase was observed, with consequent reduction in total and HDL. This mechanism may explain low HDL levels in ‘ME’ and other inflammatory conditions such as obesity and metabolic syndrome

It is known that the high-fat diet and the ‘ME’ increase intrahepatic triglyceride accumulation, thus synergistically contributing to the development and progression of alcoholic and NAFLD, from the initial stages characterized by intrahepatic triglyceride accumulation up to chronic inflammation (nonalcoholic steatohepatitis), fibrosis, and cirrhosis

On the other hand, LPS activates Kupffer cells leading to an increased production of ROS and pro-inflammatory cytokines like TNFα

high-fat diet mice presented with ME, which positively and significantly correlated with plasminogen activator inhibitor (PAI-1), IL1, TNFα, STAMP2, NADPHox, MCP-1, and F4/80 (a specific marker of mature macrophages) mRNAs

prebiotic administration reduces intestinal permeability to LPS in obese mice and is associated with decreased systemic inflammation when compared with controls

Cani et al. also found that high-fat diet mice presented with not only ME but also higher levels of inflammatory markers, oxidative stress, and macrophage infiltration markers

This suggests that important links between gut microbiota, ME, inflammation, and oxidative stress are implicated in a high-fat diet situation

high-fat feeding is associated with adipose tissue macrophage infiltration (F4/80-positive cells) and increased levels of chemokine MCP-1, suggesting a strong link between ME, proinflammatory status, oxidative stress, and, lately, increased CV risk

LPS has been shown to promote atherosclerosis

markers of systemic inflammation such as circulating bacterial endotoxin were elevated in patients with chronic infections and were strong predictors of increased atherosclerotic risk

As a TLR4 ligand, LPS has been suggested to induce atherosclerosis development and progression, via a TLR4-mediated inflammatory state.

In adipocytes, cortisol inhibits lipid mobilization in the presence of insulin, thus leading to triglyceride accumulation and retention.

Since the density of GC receptors is higher in intra-abdominal (visceral) fat than in other fat depots, the activity of cortisol leading to accumulation of fat is accentuated in visceral adipose tissue (24, 158), providing a mechanism by which excessive endogenous or exogenous GC lead to abdominal obesity and IR

obese patients generally have normal or subnormal plasma cortisol concentrations

This may be explained by an increased intratissular/cellular concentration of cortisol in adipose tissues

Intracellular GC may be produced from recycling of GC metabolites such as cortisone in adipose tissues

Local GC recycling metabolism is mediated by 11β-hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 11β-HSD2

Cortisol also increases 11β-HSD1 expression in human adipocytes

In humans, elevated 11β-HSD1 expression in visceral adipose tissue is also associated with obesity

even if obese patients generally have normal or subnormal plasma cortisol concentrations (131, 158), triglyceride accumulation in visceral adipose tissue may be due, at least in part, to the local production of GC in insulin- and GC-responsive organs such as adipose tissue, liver, and skeletal muscle

Vitamin C inhibits GAPDH (a glycolytic enzyme) and depletes the cellular pool of glutathione, resulting in high ROS production and oxidative stress

DoxyR CSCs are between 4- to 10-fold more susceptible to the effects of Vitamin C

Doxycycline and Vitamin C may represent a new synthetic lethal drug combination for eradicating CSCs, by ultimately targeting both mitochondrial and glycolytic metabolism

inhibiting their propagation in the range of 100 to 250 µM

metabolic flexibility in cancer cells allows them to escape therapeutic eradication, leading to chemo- and radio-resistance

used doxycycline to pharmacologically induce metabolic inflexibility in CSCs, by chronically inhibiting mitochondrial biogenesis

This treatment resulted in a purely glycolytic population of surviving cancer cells

DoxyR cells are mainly glycolytic

MCF7 cells survive and develop Doxycycline-resistance, by adopting a purely glycolytic phenotype