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inhibitory action of DHT is detectable at both the level of hormone secretion as well as PVN c-fos mRNA expression

the inhibition can be mimicked by the DHT metabolite 3β-diol and by the subtype selective ERβ agonist DPN

E2 acts to enhance HPA reactivity

the ability of the ER antagonist tamoxifen, but not the AR antagonist flutamide, to block the inhibitory actions of DHT, speaks to the intracellular mechanism by which this inhibitory signal might be transduced.

the DHT metabolite 3β-diol and the ERβ-subtype-selective agonist DPN suppressed ACTH, corticosterone, and c-fos mRNA responses to restraint stress in a manner similar to DHT

metabolism of DHT to 3β-diol and subsequent binding to ERβ can be inhibitory to HPA reactivity, and this is one possible mechanism for the action of DHT.

Our data also suggest that E2 enhances the reactivity of the HPA axis to stress by acting on or near neurons of the PVN

the actions of E2 appear to be through an ERα-dependent mechanism

these studies suggest that ERβ, within the male hypothalamus, acts to inhibit the HPA axis and that the inhibitory effects of DHT may be, at least in part, via its intracellular conversion to 3β-diol and subsequent binding to ERβ

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Cancer stem cells (CSCs) are thought to be the “root cause” of tumor recurrence, distant metastasis and therapy-resistance

the conserved evolutionary similarities between aerobic bacteria and mitochondria, certain classes of antibiotics inhibit mitochondrial protein translation, as an off-target side-effect

Vitamin C was more potent than 2-DG; it inhibited DoxyR CSC propagation by > 90% at 250 µM and 100% at 500 µM

IC-50

DoxyR CSCs are between 4- to 10-fold more sensitive to Vitamin C than control MCF7 CSCs

Berberine, which is a naturally occurring antibiotic that also behaves as an OXPHOS inhibitor

treatment with Berberine effectively inhibited the propagation of the DoxyR CSCs by > 50% at 1 µM and > 80% at 10 µM.

Doxycycline, a clinically approved antibiotic, induces metabolic stress in cancer cells. This allows the remaining cancer cells to be synchronized towards a purely glycolytic phenotype, driving a form of metabolic inflexibility

Doxycycline-driven aerobic glycolysis

new synthetic lethal strategy for eradicating CSCs, by employing i) Doxycycline (to target mitochondria) and ii) Vitamin C (to target glycolysis)

Doxycycline inhibits mitochondrial biogenesis and OXPHOS,

hibits glycolytic metabolism by targeting and inhibiting the enzyme GAPDH

CSCs act as the main promoter of tumor recurrence and patient relapse

a metabolic shift from oxidative to glycolytic metabolism represents an escape mechanism for breast cancer cells chronically-treated with a mitochondrial stressor like Doxycycline, as mitochondrial dys-function leads to a stronger dependence on glucose

Vitamin C has been demonstrated to selectively kill cancer cells in vitro and to inhibit tumor growth in experimental mouse models

many of these actions have been attributed to the ability of Vitamin C to act as a glycolysis inhibitor, by targeting GAPDH and depleting the NAD pool

here we show that DoxyR CSCs are more vulnerable to the inhibitory effects of Vitamin C, at 4- to 10-fold lower concentrations, between 100 to 250 μM

concurrent use of Vitamin C, with standard chemotherapy, reduces tumor recurrence and patient mortality

after oral administration, Vitamin C plasma levels reach concentrations of ~70-220 μM

intravenous administration results in 30- to 70- fold higher plasma concentrations of Vitamin C

pro-oxidant activity results from Vitamin C’s action on metal ions, which generates free radicals and hydrogen peroxide, and is associated with cell toxicity

it has been shown that high-dose Vitamin C is more cytotoxic to cancer cells than to normal cells

This selectivity appears to be due to the higher catalase content observed in normal cells (~10-100 fold greater), as compared to tumor cells. Hence, Vitamin C may be regarded as a safe agent that selectively targets cancer cells

the concurrent use of Doxycycline and Vitamin C, in the context of this infectious disease, appeared to be highly synergistic in patients

Goc et al., 2016, showed that Doxycycline is synergistic in vitro with certain phytochemicals and micronutrients, including Vitamin C, in the in vitro killing of the vegetative spirochete form of Borrelia spp., the causative agent underlying Lyme disease

Doxycycline, an FDA-approved antibiotic, behaves as an inhibitor of mitochondrial protein translation

CSCs successfully escape from the anti-mitochondrial effects of Doxycycline, by assuming a purely glycolytic phenotype. Therefore, DoxyR CSCs are then more susceptible to other metabolic perturbations, because of their metabolic inflexibility

Serum ferritin levels correlate with total body iron storage and systemic inflammation

The level of serum ferritin, an acute phase protein, is increased in an inflammatory environment

Previous studies have reported that elevated serum ferritin levels are associated with insulin resistance syndrome, hypertension, dyslipidemia, obesity, and metabolic syndrome as risk factors of CKD

elevated serum ferritin levels in hemodialysis patients predict higher mortality

The health benefits following exercise training are elicited by gene expression changes in skeletal muscle, which are fundamental to the remodeling process

there is increasing evidence that more short-term environmental factors can influence DNA methylation

dietary factors have the potency to alter the degree of DNA methylation in different tissues, 9,10 including skeletal muscle

In one study, a single bout of endurance-type exercise was shown to affect methylation at a few promoter CpG sites

In the context of diabetes, exercise training has been shown to affect genome-wide methylation pattern in skeletal muscle,13 as well as in adipose tissue.

physiological stressors can indeed affect DNA methylation

training intervention reshapes the epigenome and induces significant changes in DNA methylation

the findings from this tightly controlled human study strongly suggest that the regulation and maintenance of exercise training adaptation is to a large degree associated to epigenetic changes, especially in regulatory enhancer regions

Endurance training [after training (T2) vs. before training (T1)] induced significant (false discovery rate, FDR< 0.05) methylation changes at 4919 sites across the genome in the trained leg

identified 4076 differentially expressed genes

a complementary approach revealed that over 600 CpG sites correlated to the increase in citrate synthase activity, an objective measure of training response (Figure S4 and Dataset S14). This might imply that some of these sites could influence the degree of training response.

As expected by a physiological environmental trigger on adult tissue, the observed effect size on DNA methylation was small in comparison to disease states such as cancer

a preferential localization outside of CpG Islands/Shelves/Shores

endurance training especially influences enhancers

negative correlation was more prominent for probes in promoter/5′UTR/1st exon regions, while gene bodies had a stronger peak of positive correlation

The significant changes in DNA methylation, that primarily occurred in enhancer regions, were to a large extent associated with relevant changes in gene expression

The main findings of this study were that 3 months of endurance training in healthy human volunteers induced significant methylation changes at almost 5000 sites across the genome and significant differential expression of approximately 4000 genes

DMPs that increased in methylation were mainly associated to structural remodeling of the muscle and glucose metabolism, while the DMPs with decreased methylation were associated to inflammatory/immunological processes and transcriptional regulation

This suggests that the changes in methylation seen with training were not a random effect across the genome but rather a controlled process that likely contributes to skeletal muscle adaptation to endurance training

Correlation of the changes in DNA methylation to the changes in gene expression showed that the majority of significant methylation/expression pairs were found in the groups representing either increases in expression with a concomitant decrease in methylation or vice versa

The fraction of genes showing both significant decrease in methylation and upregulation was 7.5% of the DEGs or 2.3% of all genes detected in muscle tissue with at least one measured DNA methylation position. Correspondingly, 7.0% of the DEGs or 2.1% of all genes showed both significant increase in methylation and downregulation

we show that DNA methylation changes are associated to gene expression changes in roughly 20% of unique genes that significantly changed with training

Examples of structural genes include COL4A1, COL4A2 and LAMA4. These genes have also been identified as important for differences in responsiveness to endurance training

methylation status could be part of the mechanism behind variable training response

Among the metabolic genes, MDH1 catalyzes the reversible oxidation of malate to oxaloacetate, utilizing the NAD/NADH cofactor system in the citric acid cycle and NDUFA8 plays an important role in transferring electrons from NADH to the respiratory chain

PPP1R12A,

In the present study, methylation predominantly changed in enhancer regions with enrichment for binding motifs for different transcription factors suggesting that enhancer methylation may be highly relevant also in exercise biology

Of special interest in the biology of endurance training may be that MRFs, through binding to the PGC-1α core promoter, can regulate this well-studied co-factor for mitochondrial biogenesis

That endurance training led to an increased methylation in enhancer regions containing motifs for the MRFs and MEFs is somewhat counterintuitive since it should lead to the repression of the action of the above discussed transcription factors

decrease with training in this study, including CDCH15, MYH3, TNNT2, RYR1 and SH3GLB1

expression of MEF2A itself decreased with training

this study demonstrates that the transcriptional alterations in skeletal muscle in response to a long-term endurance exercise intervention are coupled to DNA methylation changes

We suggest that the training-induced coordinated epigenetic reprogramming mainly targets enhancer regions, thus contributing to differences in individual response to lifestyle interventions

a physiological health-enhancing stimulus can induce highly consistent modifications in DNA methylation that are associated to gene expression changes concordant with observed phenotypic adaptations