September 2012

Mal's Musings

Vitamin C, Lithium, Hyperthermia and Cancer

Presented 1/9/2012


The production of hydrogen peroxide in tissues formed by intravenous Ascorbate may be utilized to augment the anticancer effects of Lithium and (possibly) radio/high frequency hyperthermia treatments. The reasons for this conclusion are presented, and the protocol outlined, whereby Lithium carbonate is administered 24 h prior to intravenous Ascorbate, which is followed forthwith by radio/high frequency hyperthermia.

Ascorbate (Vitamin C) is now recognized -

a) To require intravenous administration in order to achieve blood levels that are high enough for
potential anti-cancer effects, with subsequent effects ranging from cell cycle arrest at
o/G1,through apoptosis to pyknosis/necrosis1,2 (mainly) with increasing concentration, and

b) To give rise to hydrogen peroxide (H2O2) in the interstitial fluid3,4,5 (2 mM Ascorbate generating the
level ~150 μM of H2O2 by in vitro3 testing), the intracellular reactive oxygen species (ROS)
appearing within ~30 min., needing metal ions to induce necrosis, but without an essential need for
p53 and caspases6 (the Ascorbate EC50 for the in vitro cell lines being 2.9 - 7.1 mM), and

c) That, for its anti-cancer effects in vivo, reliance is upon this H2O2 production (>75-100 μM), as it is
with in vitro studies; the in vivo studies indicating slowing of tumour growth (to ~60%6), rather
than killing, (consistent with a failure to incapacitate cancer stem/progenitor cells), and mild
therapeutic benefit7.

The H2O2 affected the cancer cells preferentially, either directly or indirectly and, in particular, the possible suggested mechanisms may be; i) by causing DNA damage, ATP is consumed by the polyADP-ribose polymerase for active DNA repair, utilizing NAD+ and depleting ATP8 (in lymphoma cells); &/or ii) regeneration of glutathione from oxidized glutathione with NADPH from the pentose shunt, thereby depleting glucose for ATP; &/or iii) a sensitivity of some cancer cell mitochondria. The outcome was called pyknosis/necrosis cell death (not apoptosis), provided the ATP supply was unprotected (however, a cell death process termed autoschizis, characterized by cytoplasmic protrusions and extrusions, has been described for in vitro cancer cell killing by ascorbate [H2O2; Gilloteaux et al. 20069], which may be associated with ATP-depletion affecting membrane bilipid assymmetry, as studied by Manno et al. 200210and possibly appears similar to the blebbing seen by Pakhomov et al. 200911 [with nsPEF] and Vimard et al. 201112 [with Cumene hydroperoxide], being changes attributable largely to membrane damage).

Whilst numerous targets may be involved and to varying degrees, two may be of particular note :

The plasma (cell) membrane.

The possible ascorbate → H2O2 effects on the plasma membrane

Oxidized glutathione and hydroperoxides, such as tert-butylhydroperoxide induced pores (channels) in the plasma membrane. In the case of oxidized glutathione, it had to be administered intracellularly13, with the external tert-butylhydroperoxide presumably causing the internal level of oxidized glutathione to rise and effect the membrane changes. This was studied further, widening the effectors to include Ultraviolet Light and hydrogen peroxide which can cross the plasma membrane14 [H2O2(extracellular)/H2O2(cytosol) = 7], with confirmation of the tert-butylhydroperoxide15 and H2O2effects, but with discrepancies for oxidized glutathione. The channels had some characteristics of ICRAC Calcium channels, being those involved in calcium signaling. In vitro, Vitamin C (as a free radical scavenger when without serum) reduced the effects, which were considered to be due to lipid or protein peroxidation of the plasma membrane, and which were irreparable in the minutes time range. Grupe et al. (2010)16 demonstrate that H2O2 can open Calcium channels in the plasma membrane, both TRPM2 and ICRAC.

Maintenance of the plasma membrane phospholipid bilayer is ATP dependent, with the aminophospholipid translocase, MgATPase, being known as flippase (Manno et al.10 2002). The suggested depletion of ATP by the actions of H2O2 may be expected to affect the maintenance of lipid bilayer membrane integrity.

Oxidizing conditions, as with H2O2, favour electroporation of cells in suspension (Vernier et al.200917; eg 30 ns & 100 μs pulses), demonstrating membrane vulnerability to these combined assaults in laboratory practice.

The plasma membrane and electric fields

Canonical Electroporation

Electric fluxes applied to cells (usually) suspended in culture media, have been utilized as part of the standard cell culture laboratory techniques. The fluxes damage the plasma cell membranes and create temporary pores, through which selected solutions or particles can be introduced into the cells. The process has been termed electropermeabilization or (more simply) electroporation. There have been developed two main types, here referred-to as Canonical Electroporation and Non-canonical electroporation. The Canonical electroporation applies an intermittent voltage flux in the order of 1,000 Volt per cm, as 8 – 10 pulses of square waves with widths of 100 μs at a frequency of 1-5,000 Hz. There have been some attempts to apply pulses like these in vivo (Frandsen et al. 201218) but the technique is probably only applicable for small, superficial and discrete lesions. The Non-canonical electroporation applies pulses with nano-second width (ie 1 x 10-9s). These have different features :

Non-Canonical Electroporation (nanosecond pulsed electric fields; nsPEF)

The problems here are like comparing assorted apples with mixed fruits :

Non-canonical electroporation was studied by groups including Vernier et al. (200319, 200920), Deng et al. (200321; 60 & 300 ns @ 0.3 - 15 MV/m), Beebe et al. (200322; 60 ns; Jurkat & HL-60), White et al. (200423; 60 ns; HL60), Tekle et al, (200524; 10 & 50 ns; COS-7, more theoretical) and more recently by Bowman et al. (201025; 600 ns - rather long; GH3, CHO & NG108) and Romeo et al. (201126; 60 ns x1 @ 1 - 2.5 MV/m), and others. In general, the observations are that (i) the shorter the pulse width (to <10 ns), the slower the nuclear decoration by the marker propidium iodide, (indicating decreasing membrane pore size), (ii) the enigmatic effects upon the cell membrane becoming more general, without polarization to one side and with less cell swelling; the conclusion being that the effects were less at the level of the plasma membrane, but more on structures inside, such as mitochondria, and including the electrophoretic features of DNA changes. Growth and viability were not necessarily affected.

Vernier et al. (2003; 30 ns x 10 with 1 - 4 Hz @ 2.5 MV/m; Jurkat T lymphoblasts) noted a very swift (within milliseconds) global flare of cytosolic Ca2+ and also cellular enlargement, [which may cause enlargement of lipid rafts (Ayuyan & Cohen 200827)]. Under the same conditions, there was no propidium iodide nuclear decoration. EGTA, that chelates Ca2+ in the culture medium, produced no appreciable change, and prior thapsigargin (a blocker of Ca2+ binding the endoplasmic reticulum, themain Ca2+ store) seemed the only modifier that reduced the response. The conclusion then, was that the Ca2+ was being released from the endoplasmic reticulum, by an unknown mechanism. They ruminated upon various possibilities, including the role of IP3, which is normally produced by membrane receptor machinery, but considered this unlikely because the nsPEF response was so very rapid, whereas the IP3 response is reportedly longer, in seconds. The voltage across the plasma membrane necessary to create nanopores under their experimental conditions (7 ns x 50 @ 2.5 - 4.0 MV/m; Jurkat) was ~0.5 V, and they concluded that the membrane defects were not caused by an influx of Ca2+ (Vernier et al. 200428) but may provide avenues for phosphatidylserine in the inner leaflet of themembrane bilayer to reach the outer leaflet, implying a weakening of the phosphatidylserine-protein (spectrin/actin/ankyrin) bonds inside the membrane (Manno et al. 2002). White et al. (2004) found that nsPEF (60 ns x1 @ 4 – 15 kV/cm) mobilized Ca2+ from internal stores, considering the possibility of shape deformations of IP3Rs.The later study by Vernier et al. (2006) shortened the pulse widths further from 30 ns to 7 ns and 4 ns and increased the fields (4 - 30 ns @ 2.5 MV/m; Jurkat cells), finding, contrary to conclusions from earlier work, that the plasma membrane changes (revealed by decoration of phosphatidyl serine rather than molecular entry) were polar. Scarlett et al. (2009)29 indicated that nsPEF (60 ns @ 5 – 10 MV/m to 500 s) affected both the plasma membrane and intracellular membranes, in particular, the endoplasmaic reticulum, releasing Calcium. Romeo (2011; 60 ns x1 with 1.0, 1.5 & 2.5 MV/m; Jurkat T lymphoblasts) compared the effects upon the propidium iodide uptake with the uptake for the dye YO-PRO-1, which is a smaller molecule. Under these different conditions, the latter dye decorated the DNA, indicating that very small pores had been created in the plasma membrane. This difference seems important, because it indicates that the pores in the plasma membrane are of molecular-range size and fairly uniform (cf Vernier et al. 2006 findings). This conclusion was supported by the work of Bowman et al. (2010; GH3, CHO-K1 & NG108 cells, 600 ns @ >1- 2 kV/cm, Thallium indicator), who found that thallium ions could pass through the pores that the larger molecules could not, putting pore sizes as <~1.0 - 1.5 nm, and without cellular polarity. Their poration seemed unaffected by Ca2+ or other ion channels. Instead of an effect similar to grape-shot through a mainsail, it is more akin to tiles in a mosaic being dislodged; the dislodged groups may still remain (like molecular deformation), but their places may be only temporarily exposed. Ibey et al. (201030; 60 ns @ 170 - 600 kV/m, GH3 cells, Thallium indicator) affirm the creation of short-term, small, generalized membrane pores referred-to as nanopores, and demonstrate that they have the characteristics of ion channels. However, at 20 sec, their image shows no convincing polar differential. The study of channels was extended (Pakhomov et al. 200931; 600 ns @ ~160 - 408 kV/m) demonstrating that the channels seemed to change ion selectivity and characteristics as the electrostatic flux increased.

These studies of Non-canonical electroporation appear to be exclusively on cells in suspension or non-confluent growth on a surface, quite devoid from the in vivo state. Also, whilst the ns-PEF seemed to be affecting intracellular membranes (eg mitochondria, ER etc.) as far as the electrostatic fluxes were concerned, the interior or the cells tended to be regarded as homogeneous blobs of jelly, which would hardly seem likely.

In the clinical situation, there is a three dimentional mass of tumour cells with cell-to-cell contacts (cadherens) and cell-to-matrix-to-cell (integrin) contacts throughout within any patient (fewer contacts in the case of adenocarcinomas with mucus pools or similar). In the clinic, where patients are treated with radio/high frequency energy (eg for hyperthermia), the electric flux is applied to the area of tumours for (typically) one hour, considerably more than the time used for electroporation in the laboratory, and should subject the cellular points of contact with specific electrostatic stresses that do not seem to have been encountered in the laboratory. If tumour stem cells and their progenitors are anything like the human embryonal stem cells32, these points and their surrounds should be particularly vulnerable to electrostatic fluxes. If the frequency of the administered electrostatic flux is (say) ~30 MHz (or ~41 MHz), then the peak-to-peak duration (wavelength, λ) is ~33 ns (or ~24 ns) respectively. A 'peak' having one polarity would then be ~16.5 ns (or ~12 ns) in width at its base (although, if there were a rectification effect, the 'peak' may be longer or shorter). This is in the range for extrapolations from the Non-canonical Electroporation studies. Naturally, perfectly smooth sine waves or sharp square waves would not be expected (but may be approximated, unless specifically specified), so the supplied waveform for treatments would probably be unique to any installation. Whether square or sine waves in the nsPEF context differ in effects may not be known at present.

Accordingly, whilst electrostatic fluxes on cells in isolation in the laboratory can have interesting and perplexing effects, the conditions that prevail in patients under the influence of administered electrostatic fluxes that are given under quite different conditions, are essentially unknown. Naturally, there will be an assumption that the voltage fluxes across individual cells, as applied or reached in the clinic, will be much less, and therefore inadequate for any effective cellular change as examined using Non-canonical Electroporation in the laboratory. But this conclusion is based upon quite different cell-to-cell relationships, and is probably without sound foundation.

If radio/high frequency power fluxes for hyperthermia are administered immediately after the infusions of vitamin C (Ascorbate), the plasma membranes may be sensitized by the H2O2 and this may augment any effects derived from the nsPEF on the membrane.

For the reasons given above, the administration of radio/high frequency electrostatic fluxes (or very high frequency fluxes ?) to solid tumours may create plasma membrane defects (and possibly other membrane defects) and not just simply heat the tumours.

If there is any appreciable membrane poration effect and the tumour bulk is large, the serum/urine Calcium may fall quickly, because the Calcium will move into the cells of the tumour mass through the pores and/or the Calcium channels that are opened. This would be expected to precede any other simple biochemical changes, and provide an economical method of assessment.

Ascorbate and Lithium

In an earlier Chapter, the biochemical steps by which Lithium may induce apoptosis in cancer cells were outlined33. Lithium stimulated the production of TNF and Fas Ligand (Chen et al. 200734) which, 16 - 24 h later, were able (provided the co-receptor complex was functional) to initiate apoptosis pathways (Juric et al. 200935, Juric et al. 201236). Since the TNF receptor (TNFR) could be sensitized, if not triggered, by H2O237 there would seem good reason to believe that Lithium, if administered as a bolus38 some 24 h prior to the Ascorbate administrations could result in augmented cancer killing, utilizing the two synergizing approaches.

There is the matter of the H2O2 possibly interfering with cell death by apoptosis, a process in which ATP is required for chromatin condensation at the nuclear periphery and apoptotic-body formation39 (~75 min., Jurkat T-cells), and not for the 'laddering' of the DNA fragments, at least. Apoptosis is more the method of disposing of the components of the dying cell, rather than the death process itself; the main disposal options being apoptosis and autophagy40. Without an adequate ATP supply, cells still can die, but by necrosis (nuclear enlargement), and still involving Cytochrome-c release from mitochondria41, (a process that can be reversed to apoptosis if/when dATP/ATP is available42, or can reduce survival in its own right43), or pyknosis/necrosis (nuclear pyknosis) or possibly autoschizis (cytoplasmic extrusions – see later). [The nuclei of the dying prostate cancer cells after long-term bolus, intermittent Lithium would seem to be of pyknosis/necrosis type44; the reason being unclear at present]. The oxidizing influence of H2O2 may lead to the slow breakdown of lysosomes45 and augment the roles of the reactive oxygen species (ROS) generated by neutral Sphingomyelinase and 5-Lipoxygenase involved in the CCN1/246 TNF/Fas apoptosis pathways (>3-4 h), as well as the activation of p38/MAPK & JNK, and assist overcoming the anti-oxidizing influences of NF-κB and disable the NF-κB-induced MAPK phosphatases. Thus, there should be lots of ROS to drive apoptosis provided the ATP supply is adequate when appropriate, or else another form of cell death pathway, as may still involve mitochondria47 is to be involved.

The CCN1/TNF pathway is believed to involve cytosolic p53 as an intermediary following on downstream from the activated p38 MAPK and JNK. The p53 is believed to have an acute translocation to the mitochondria4849. It binds to the anti-apoptotic proteins Bcl-2 & Bcl-xL, which already bind and neutralize the pro-apoptotic Bax. Bax is displaced by the p53, and by oligomerization and pore-formation, achieves the release of cytochrome-c from mitochondria by a p53-driven process that does not involve transcription50 (and may have little requirement for ATP). The cytochrome-c binds with Apaf-1 for the latter to form the apoptosome which cleaves Procaspase-9, then Procaspase-3, as ongoing processes requiring dATP/ATP. However, too much nucleoside in the cytoplasm blocks the cytochrome-c binding to Apaf-151, so a partial fall or recovery of ATP promotes apoptosis.

These considerations may not be necessary when we consider the timing of events :

Day 0                                    Day 1                                                                 Day 2
                                                                          >4 h→ → → necrosis (?) 
+ATP → → → Apoptosis

Lithium TNF & FasL ~24 h → → → → → → → →→→36 h
                                            TNF & FasL/receptors/CCN1/2/HSPG/integrinα6β1etc.
                                                                                → p53Baxcytochrome-c etc.
                                                                                                              → → Apoptosis

(a) After the Lithium dose(s) TNF/FasL production are detectable by 16 h, rising to near peak at 24 h.

(b) If, at ~24 h, intravenous vitamin C (1-1.5 g/kg) is administered over ~90 min7, H2O2 will be generated in the interstitial fluid after about 30 min. The plasma Ascorbate level may then be above 10 mmol/L reliably until ~6 h from the infusion start. For 1-2 h additionally, the TNFR and Fas (receptors) will be sensitized for the TNF and FasL that are still being actively produced following the Lithium dose a day earlier. There should be an opportunity for ATP to regenerate in that post-Ascorbate stage.

(c) The TNFR response, when combined with the cofactors CCN1/2, LRP1, integrin α6β1 & Syndecan-4 /HSPG stimulates a slow apoptosis response (3 - 4 h).

The issue around the cell death category is somewhat academic and, given the sequence of events, all types may occur over various times. Given that Lithium, administered as a bolus dose the day before Ascorbate administration, is most unlikely to affect the latter's actions adversely, the Lithium's effects will either be zero/negligible or beneficial. Since Lithium is very easy to administer, relatively safe and far cheaper than any other form of therapy that patients may try, there seems nothing to lose by adding it into an existing Ascorbate protocol. Since it may reduce the cancer stem cell pool over weeks52, more attention should be given to its use in combined therapies, but with attention to timing.

Melding the modalities

The conclusion is that there may be benefits in combining all three treatment modalities :

On day 1, patients take 1 g of Lithium carbonate orally by two 500 mg doses 6 h apart;

On day 2, approximately 24 h after the first Lithium dose, the patient receives an intravenous
infusion of Ascorbate (Vitamin C) +/- Menadione (Vit K3)53 according to the local protocol(s) and,

Immediately following the Ascorbate (+/- Menadione), the patient has Radio/high Frequency (or Very
High Frequency ?) Hyperthermia, following the usual protocol.

By following this sequence, the maximum augmentation effect of each may be achieved.

Malcolm A Traill

Copyright © MA Traill September 2012



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