Cancer is fundamentally difficult to cure because of the diversity in tumor microenvironments. Hypoxia, or the lack of sufficient oxygen in the microenvironment, allows cancer cells to become heterogenous through epigenetic adaptation. Heterogeneity in cancer cells is probably one of the main reasons for cancer’s exceptional resilience against treatment [1].
Heterogeneity is also the reason why the same type of treatment across different patients with the same type of tumor would produce different responses [2]. Heterogeneity allows the main tumor cells to develop into a wide variety of cells with distinct molecular signatures. Together with the diverse tumor microenvironment, sensitivity to treatment becomes highly varied [1]. Heterogeneity in tumor is now viewed as an independent risk factor for poor survival in many tumor types.
Tumor heterogeneity is fueled by genomic alterations which results in genomic instability. Most common causes of genomic instability are mutations in the DNA mismatch repair system or in the proofreading polymerase enzymes. Mismatch repair (MMR) systems are critical in maintaining genetic stability because their function is to repair DNA replication errors [3].
Hypoxia is now accepted to be responsible for the induction of genetic instability in tumor microenvironments [4].
HIF – Master Regulator of Cancer Biology
Tumor microenvironments often are hypoxic. Tumor cells exhibit increased oxygen demand as a result of cell expansion in a background of decreased oxygen supply due to defective tumor vascularization [5]. In response to inadequate oxygen supply, complex mechanisms, like hypoxia-inducible factors (HIFs) and their downstream gene expression networks are activated to allow cells to adapt and survive in a hostile environment [6].
The HIF signaling pathway is regarded as the major regulator of cancer characteristics such as stemness, dormancy, invasion, metastasis, angiogenesis, immunity, metabolic reprogramming and resistance to anti-cancer therapies [7, 8]. By inducing stemness, HIF signaling enhances tumor heterogeneity, rendering the tumor resistant to treatment while developing increased capacity for metastasis [9].
Genetic instability, a hallmark of tumorigenesis, is induced by increased rate of DNA mutation brought on by hypoxia. Hypoxia can alter transcription and translation of DNA damage response and repair genes. The adaptation of tumor cells to the hypoxic microenvironment therefore drives increased genetic instability and malignant progression [10]. The ability of cancer cells to escape cell death allows them to proliferate in the presence of damaged DNA, and thus acquiring even more DNA mutations [11].
Nrf2 & Stabilization of HIF-1a – the Iron Connection
Hypoxia inducible factor 1α (HIF1α) is a transcription factor that can be activated under hypoxia. HIF-1a levels are normally kept low in the presence of oxygen. When there is adequate oxygen, HIF-1a is bound by the von Hippel-Lindau (VHL) protein, which then prepares HIF-1a for degradation [12]. If HIF-1a is not degraded properly, it will be able to bind with its heterodimeric component, HIF-1β, and becomes stabilized.
Once HIF-1a is stabilized, it can activate the expression of a large number of genes associated with survival, apoptosis, and metabolic reprogramming. HIF-1a can induce the upregulation of enzymes involved in glycolysis, and directly suppress mitochondrial metabolism by decreasing the efficiency of the electron transport chain by modulating expression of cytochrome c oxidase isoforms [13].
HIF-1a is crucial for adaptation to hypoxia. However, HIF-1a can be stabilized even under conditions of normoxia where there is sufficient oxygen. Loss of control of HIF-1a is often associated with a poor disease outcome [14].
The HIF signaling pathway is a stress response pathway. Environmental stress is not limited to low oxygen supply Mild hypothermia has been observed to remodel gene expression by activating transcription factors such as HIF1 [15]. Mild hypothermia also activates another evolutionary conserved stress response molecule, Nrf2.
The nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor was first discovered for its role in erythropoiesis. Since its discovery, Nrf2 research has been focused on its functions in detoxification and cancer prevention [16]. More recent works identified many genes involved in iron storage, iron export in addition to heme synthesis and hemoglobin catabolism are also under the control of NRF2 [17].
Since Nrf2 regulates iron homeostasis, it is not surprising that Nrf2 is deeply implicated in the activation of HIF-1a.
Activation of Nrf2 and its subsequent deregulation of iron metabolism are heavily implicated in cancer development. Nrf2 can upregulate iron storage protein ferritin, leading to enhanced tumor proliferation and therapy resistance [18]. When ferritin is upregulated by Nrf2, available iron can be lowered by about 25%. Lowering of available iron can decrease the activity of the HIF-1a degradation enzyme prolyl hydroxylase by an impressive 75%, resulting in the activation of HIF-1a [18].
Iron is actually one of the several cofactors critical for the degradation of HIF1-a. Even under normoxia, conditions like inflammation can increase ferritin, lowering free iron availability. Inadequate cytosolic free iron under normoxia has been demonstrated to activate HIF-1a. Once activated, HIF-1a will actively modulate the expression of genes that control iron homeostasis such as iron transporters (DMT1), ferroportin 1 (FPN1), duodenal cytochrome b (Dcytb), and transferrin receptor (TfR). Even central systemic mediators for iron homeostasis like hepcidin and iron regulatory proteins are regulated by HIFs once they become activated and stabilized [19].
The appropriate degradation of HIF-1a is perhaps vital in cancer pathology.
Prolyl Hydroxylase, HIF-1a & Cancer – A Tale of Oxygen, Iron and Ascorbic Acid
When there is enough oxygen (normoxia), an enzyme called prolyl hydroxylase (PHD) will add a hydroxyl group (hydroxylation) to the α-subunits of HIF. This hydroxylation allows HIF-1a to be bound to the von Hippel-Lindau protein (VHL), which then initiates the degradation process of HIF-1a.
In the HIF signaling pathway, prolyl hydroxylases (PHDs) are the true oxygen sensors. When there is enough oxygen, PHD uses oxygen as substrate for the hydroxylation of HIF where PHD inserts oxygen atoms into the prolyl residue. The hydroxylation process also requires PHD to obtain electrons from another co-substrate called α-ketoglutarate [20].
Prolyl hydroxylases (PHDs) belong to a class of non-heme iron α-ketoglutarate (αKG) dioxygenase. That means their enzymatic activities depend upon the energy derived from the conversion of iron between its oxidized and reduced states [21, 22]. PHDs also require ascorbic acid as a co-factor during the enzymatic process of hydroxylation.
In January of 2019, Kulper et al. demonstrated that intracellular ascorbate levels modulate the hypoxic HIF pathway in a dose dependent manner in human clear cell renal cell carcinoma (ccRCC). Increased intracellular ascorbate dramatically elevated activity levels of prolyl hydroxylase, resulting in lower stabilization of HIF-1a [23].
In the following chart, notice the complete inactivation of HIF-1a in 10% oxygen at higher ascorbate concentrations. The effect of ascorbate is reduced upon diminishing levels of oxygen.
HIF-1a Stabilization in Various Ascorbate and Oxygen Concentrations in human ccRCC
[Source: Wohlrab C, Kuiper C, Vissers MCM, Phillips E, Robinson BA, Dachs GU Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells Dove Press journal Hypoxia Volume 2019:7 Pages 17—31 DOI https://doi.org/10.2147/HP.S201643]
A truly astounding and groundbreaking landmark study released in April 2019 by Osipyants et al. revealed how Nature uses ascorbic acid as the master key that unlocks many doors in vital biochemical processes.
The Molecular Structure of Ascorbic Acid – MasterKey to Health & Disease
Most living organisms including plants, insects and animals produce ascorbic acid. Ascorbic acid exists naturally in the form of L-ascorbic acid [24]. In physiological pH, L-ascorbic acid exists predominantly in the ionic form of L-ascorbate.
In their 2019 landmark study, Osipyants et al. not only showed that prolyl hydroxylase depended upon ascorbic acid as substrate, but demonstrated definitively for the first time that ascorbic acid MUST BE IN THE SPECIFIC MOLECULAR STRUCTURE of L-ASCORBATE in order to suppress HIF-1a [25]!!
The discovery by Osipyants et al. brings into question the long-held understanding that the role of ascorbic acid in enzymatic reactions of non-heme iron α-ketoglutarate (αKG) dioxygenases such as prolyl hydroxylase is solely confined to its reducing capacity as an antioxidant in the conversion of ferric ions to ferrous ions.
When Osipyants et al. substituted L-ascorbate with the potent cell-permeable reducing agent, N-acetyl cysteine (NAC), they observed NO effect in the suppression of HIF-1a. These brilliant scientists then proceeded to experiment with D-ascorbate, an enantiomer of L-ascorbate. To their utmost surprise, the D-isomer, even though it possessed the same reductive potency with respect to ferric iron, was also completely ineffective when compared to L-ascorbate in the suppression of HIF-1a [25].
D-ascorbic acid is a mirror image of L-ascorbic acid. It does not exist in nature but can be synthesized artificially [26]. The binding sites in the enzyme active centers of PHDs allow for the natural docking of L-ascorbate molecules, but not its D-isomer. Look at how the insertion of D-Ascorbate into HIF prolyl hydroxylase crystal structures displayed molecular interference in diagram B, versus the perfect docking of L-ascorbate in diagram A.
L-Ascorbate Docked in Prolyl Hydroxylase
D-Ascorbate Inserted in Prolyl Hydroxylase
[Source: Andrey I. Osipyants et al. Biochimie. 2018 April ; 147: 46–54. doi:10.1016/j.biochi.2017.12.011. L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?]
Ascorbic acid is known for its ability to convert iron between ferric and ferrous forms in other important biochemical processes such as the catalytic cycle during dopamine synthesis by tyrosine hydroxylase. Might L-ascorbate assume a similar identity as master key that facilitate these biological processes?
Optimal physiological intracellular ascorbate concentrations have been found to significantly increase expression of tyrosine hydroxylase proteins [27]. Deficiency of tyrosine hydroxylase can result in impaired synthesis of dopamine, epinephrine and norepinephrine. Deficiency in any of these important catecholamines will results in a wide range of diseases. For example, epinephrine and norepinephrine are signaling molecules that can activate the conversion of ATP molecules into secondary messengers known as cyclic AMP (cAMP). cAMP regulates multiple cellular functions including cell growth and specialization; protein expression; and even gene transcriptions [28].
If the molecular structure of L-ascorbate must be maintained in order for these biochemical processes to proceed, then an open question would be how the supplementation with synthetic forms of buffered ascorbic acid impact health and disease. An examination of the molecular structures of several popular ascorbate supplements show that they do not resemble L-ascorbic acid at all.
Ascorbic Acid
Sodium Ascorbate
Magnesium Ascorbate
Calcium Ascorbate
The importance of the molecular structure of ascorbic acid in biological processes also brings into attention the emerging role of ascorbic acid as an effective epigenetic regulator. DNA methylation in cancer cells are often dysregulated. Recent advancements in the field of cancer epigenetics reveal extensive epigenetic reprogramming involving DNA methylation, histone modifications, nucleosome positioning, and non-coding mRNA expression [29]. Electromagnetic radiation has been observed to cause epigenetic changes involving chromatin accessibility [30]. The fact that ascorbic acid is used by Nature as primary quantum interface now makes even more sense than ever [31].
Have you had your AA today?
References:
[1] Tumour heterogeneity and resistance to cancer therapies | Nature Reviews Clinical Oncology https://www.nature.com/articles/nrclinonc.2017.166
[2] Tumor heterogeneity: a central foe in the war on cancer | Journal of Clinical Outcomes Management https://www.mdedge.com/jcomjournal/article/168663/immunotherapy/tumor-heterogeneity-central-foe-war-cancer
[3] DNA mismatch repair and genetic instability. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/11092832
[4] HIF-1alpha induces genetic instability by transcriptionally downregulating MutSalpha expression. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/15780936
[5] Hypoxia-inducible factors and the response to hypoxic stress. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/20965423
[6] Hypoxia-Inducible Factors: Master Regulators of Cancer Progression. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/28741521
[7] Regulation of angiogenesis by hypoxia: role of the HIF system. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/12778166?dopt=Abstract
[8] Intermittent hypoxia selects for genotypes and phenotypes that increase survival, invasion, and therapy resistance. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/25811878?dopt=Abstract
[9] Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer- and metastasis-initiating cells. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/23301832?dopt=Abstract
[10] Tumor hypoxia as a driving force in genetic instability https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016142/
[11] Effects of acute versus chronic hypoxia on DNA damage responses and genomic instability. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/20103649/
[12] Hypoxia-Inducible Factors in Physiology and Medicine https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437543/
[13]Reprogramming of the Tumor in the Hypoxic Niche: The Emerging Concept and Associated Therapeutic Strategies https://www.cell.com/trends/pharmacological-sciences/fulltext/S0165-6147%2817%2930110-4
[14] Targeting hypoxia in cancer therapy. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/21606941/
[15] Hypothermia augments stress response in mammalian cells – ScienceDirect https://www.sciencedirect.com/science/article/pii/S0891584918307482
[16] NRF2, a Key Regulator of Antioxidants with Two Faces towards Cancer https://www.hindawi.com/journals/omcl/2016/2746457/
[17] Emerging Regulatory Role of Nrf2 in Iron, Heme, and Hemoglobin Metabolism in Physiology and Disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191506/
[18] Ferritin-Mediated Iron Sequestration Stabilizes Hypoxia-Inducible Factor-1α upon LPS Activation in the Presence of Ample Oxygen. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/26628374
[19] Regulation of iron metabolism by hypoxia-inducible factors http://www.actaps.com.cn/qikan/manage/wenzhang/2017-5-14.pdf
[20] HIF-independent role of prolyl hydroxylases in the cellular response to amino acids https://www.nature.com/articles/onc2012465.pdf?origin=ppub
[21] Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV═O species https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840172/
[22] Ferrous iron and α-ketoglutarate-dependent dioxygenases in the biosynthesis of microbial natural products. – PubMed – NCBI https://www.ncbi.nlm.nih.gov/pubmed/26845569
[23] Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells https://www.dovepress.com/ascorbate-modulates-the-hypoxic-pathway-by-increasing-intracellular-ac-peer-reviewed-fulltext-article-HP
[24] Ascorbic acid | HC6H7O6 – PubChem https://pubchem.ncbi.nlm.nih.gov/compound/L-ascorbic%20acid
[25] L-ascorbic acid: A true substrate for HIF prolyl hydroxylase? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460286/
[26] D-Ascorbic acid | C6H8O6 – PubChem https://pubchem.ncbi.nlm.nih.gov/compound/D-Ascorbic-acid
[27] Mechanisms of Ascorbic Acid Stimulation of Norepinephrine Synthesis in Neuronal Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3449284/
[28] The Cyclic AMP Pathway https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504441/
[29] Epigenetics in cancer https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2802667/
[30] Specific low frequency electromagnetic fields induce epigenetic and functional changes in U937 cells https://arxiv.org/ftp/arxiv/papers/1810/1810.06255.pdf
[31] Electromagnetic Radiation and Quantum Decoherence: Is Vitamin C the Ultimate Quantum Interface? | LinkedIn https://www.linkedin.com/pulse/electromagnetic-radiation-quantum-decoherence-vitamin-doris-loh/
