Stress response in a animal model is blunted at the level of pituitary through a decrease in ACTH release and through increase in peripheral metabolism
Sex steroid hormones are primarily responsible for sex difference in adult HPA function; androgens inhibit whereas estrogens
enhance HPA axis activation after a stressor
the PVN contains relatively high levels of AR (Bingaman et al., 1994; Zhou et al., 1994) and ERβ (Alves et al., 1998; Hrabovszky et al., 1998; Somponpun and Sladek, 2003) but is essentially devoid of ERα
the nonaromatizable androgen DHT and the nonselective ER ligand E2 influence HPA
reactivity by acting on neurons within or surrounding the PVN
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.
that is because the interaction with the DHT metabolite is not with the AR, but with the ER-beta.
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
Previous studies from our laboratory have demonstrated that pharmacological ascorbate is cytotoxic to pancreatic cancer cells while normal cells are resistant
Ascorbate-induced cytotoxicity is mediated by the formation of H2O2 during the oxidation of ascorbate
the combination of IR + ascorbate increased the concentration of intracellular H2O2
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
These eight distinct cancer types included: DCIS, breast (ER(+) and ER(-)), ovarian, prostate, lung, and pancreatic carcinomas, as well as melanoma and glioblastoma. Doxycycline was also effective in halting the propagation of primary cultures of CSCs from breast cancer patients, with advanced metastatic disease (isolated from ascites fluid and/or pleural effusions)
Doxycycline behaves as a strong radio-sensitizer, successfully overcoming radio-resistance in breast CSCs
cancer cells can indeed escape the effects of Doxycycline, by reverting to a purely glycolytic phenotype. Fortunately, the metabolic inflexibility conferred by this escape mechanism allows Doxycycline-resistant (DoxyR) CSCs to be more effectively targeted with many other metabolic inhibitors, including Vitamin C, which functionally blocks aerobic glycolysis
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
contribution to the training response of the epigenome as a mediator between genes and environment
Differential DNA methylation was predominantly observed in enhancers, gene bodies and intergenic regions and less in CpG islands or promoters
highly consistent and associated modifications in methylation and expression, concordant with observed health-enhancing phenotypic adaptations, are induced by a physiological stimulus
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
Exercise alters gene expression via methylation--the power of epigenetics. Interestingly, the majority of the methylation was outside the CPG island regions. This 3 month study found methylation of 5,000 sites across the genome resulting in altered expression of apps 4,000 genes. The altered muscle changes of the endurance training was linked to DNA methylation changes.