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Hepatocellular Carcinomas are Promoted by Tocopheryl Acetate but Eliminated by Tocopheryl Succinate.

 Bryant L. Adams, Ph.D.1 and Richard O. Whitten, M.D.2

 1Corresponding author

Health Environment Analysis Laboratory,

 2343 63rd CT, SW;

Tumwater, WA 98512

bladams36@gmail.com   -  phone 360-412-8008  cell 360-556-3927

 

2Cellnetix Pathology Laboratory,  Olympia, WA 98506 

 There were no grants, nor sponsoring agents for this work.  All funds were from private donations to HEAL Corp.

 Conflict of interest:  None

 

Key words: Aflatoxin, tocopheryl-acetate, tocopheryl-succinate, hepatocellular carcinomas, hepatomegaly

Contents

Graphical Abstract

 Abstract

1. Introduction

2. Methods and Materials

3. Statistical Analysis

4. Results

 4.1. Results: Identification of cellular carcinomas.

4.2. Results:  Sera vitamin E: 

4.3.1. Results: Vitamin E-Acetate effect: Figure 1.

4.3.2. Results:   Vitamin E-Succinate’s beneficial effects at 10 months: Figure 2

4.3.3. Results: Vitamin E-Succinate's beneficial effects at 13 months: Figure 3.

4.3.4. Results:   Compromised diets comparing E-Ac versus E-Su: Figure 4.

4.3.5. Results: Vitamin A and D deficiency results: Figure 5.

4.3.6. Results:  Liver enlargement from E-Ac: Figure 6.  

5.  Discussion.

5.1. E-Ac promotion of HCCs and induction of hepatomegaly.

5.2. E-Su preservation of liver size, and reduction of HCCs. 

5.3 Aflatoxin mechanism of action and relation to the tocopheryl esters.

5.4 Complexity of Proliferation and Apoptosis signals

5.5 Type I (extrinsic) and Type II (intrinsic) apoptosis with E-Su and E-Ac.

5.6 The need for E-Su allies that are cancer cell specific.

5.7. Mitochondrial targeting and other cancer cell spicific agents.

Acknowledgements.

References

Abbreviations and Definitions

Photos of visible tumors and histopathology slide results of cell structure.

Appendix A: Summary of Fish Diets used.

Appendix B: Statistical evaluation of results.

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ABSTRACT

 

BACKGROUND: The major causes of hepatocellular carcinomas are Aflatoxin, hepatitis B and hepatitis C viruses.

 Alpha tocopherol (vitamin E) and its acetate and succinate esters have each been reported as counteracting cancer development in humans and rodents.  We have investigated their salutary effect in both poor and high quality diets in rainbow trout Oncorhynchus mykiss as a model.

 METHODS:  Hepatocellular carcinomas (HCCs) were induced in rainbow trout by dietary aflatoxin B1 (AfB1).  A matrix of different levels of several vitamins and vitamin analogues were included in selected diets as possible anticancer agents.  Identification of HCCs was made by histopathology.  

 RESULTS:  1.) Elevated dietary tocopheryl acetate caused a marked increase in liver size and in AfB1-induced HCCs in rainbow trout.  2.) Poor diets increased the HCC incidence.  3.) Elevated dietary tocopheryl succinate nearly eliminated HCC development in fish fed complete diets.  Tocopheryl succinate in poor diets reduced HCCs by 77% compared to tocopheryl acetate diets.  4.) trans-retinoic acid also reduced HCC incidence.  5.) Vitamins A and D deficiency caused tumor increases but had no effect on liver size.  6.)  The use of casein and dextrin in the place of soybean textured vegetable protein, in poor diets nearly eliminated the HCC risk.  7.) Trout sera showed all three vitamin forms; free α-tocopherol, tocopheryl acetate and tocopheryl succinate, from diets containing any of these vitamin  analogues.

 CONCLUSIONS:   

Increased dietary tocopheryl acetate escalated AfB1 induced HCCs and caused hepatomegaly in rainbow trout, while tocopheryl succinate eliminated the HCC risk as shown by histopathology.

 KEYWORDS: Aflatoxin; tocopheryl-acetate; tocopheryl-succinate; hepatocellular carcinomas; hepatomegaly

 

 1. Introduction:

Liver cancer is estimated to be the  6th most common type of cancer in the world with 749,700 new cases in 2008. Between 75% and 90 % of these in humans are hepatocellular carcinomas (HCCs) [1].  It is estimated to be the 3rd leading cause of cancer deaths in the world and the 9th leading cause in the United States [2].  The American Cancer Society estimates that in 2015 in the United States there would be about 35,660 new cases and 24,550 deaths due to liver cancer. http://www.cancer.org/cancer/livercancer/ detailedguide/liver-cancer-what-is-key-statistics   (accessed 16 June 2015)  

Hepatitis B virus (HBV), hepatitis C virus (HCV) and dietary aflatoxin B1 (AfB1) are the most significant contributors to the risk for HCCs [3, 4].  Modest levels of AfB1 increased the risk of HCCs by 30 fold in HBV infected Korean men [5].   

A recent cluster involved AfB1 contaminated pet food that killed as many as 76 dogs in southeast United States [6]. (http://recalledpetfoodsettlement.com)  

Aflatoxins (several isomers) are the result of fungi (Aspergillus species) growth on feedstuffs and are found in many types of improperly stored feed including peanuts, pistachio nuts, milk, corn, soybeans and other grains.  The aflatoxin limit for pistachio nuts is 20 part per billion (ppb) [7].  The US food Safety Regulations include a limit of 20 ppb for total aflatoxins (B1, B2, G1 and G2) in all foods except milk which has a limit of 0.5 ppb for aflatoxin M1. Higher limits apply in animal feeds [8].

Enhancing resistance to tumor development as well as removing the causative agents are both desirable.  Vitamin E is one of several vitamins that are both credited and discounted as being effective in preventing various types of cancer.  Vitamin E in one study had no effect on breast cancer rate in postmenopausal women but no indication was given as to the form of the vitamin assessed [9].  Induced adenocarcinoma incidence in rats was reported to not be significantly altered by E-Ac dietary increase [10].  Dietary elevation of dl- alpha tocopheryl acetate (the synthetic racemic mixture of the vitamin) in diets (1.8 gm/ Kg diet)  in double transgenic mice genetically altered for transforming growth factor α(TGF-α)/c-myc, caused a reversal of hyperplasia, and eliminated 65% of the adenomas and 100 % of the HCCs but, in contrast, a marked reduction in apoptosis [11].   

In contrast to the variable effect of E-Ac, the results with E-Su, have been consistently beneficial.  E-Su while protecting normal cells [12] caused apoptosis in human breast cancer cells [13], human prostate cancer cells [14], murine leukemia cells [12] and rat colon pre-cancer crypts [15] and was synergistic with cellular apoptosis factors and radiation treatment [16, 17, 18, 19].

 Reactive oxygen species (ROS) such as the hydroxide radical (OH .) have been implicated as causative agents in carcinogenesis against which vitamin E acts as an ROS scavenger to protect against cancer.8  However the vitamin can only act in the scavenger role when it exists as the free alcohol but not as the acetate nor succinate ester [20] and  [21].    

We sought to test the protection given against HCC incidence by vitamins A, C, E and Vitamin A metabolites cis- and trans-retinoic acids.  Rainbow trout were used as a model for combating cancer in mammals because this species is known to be susceptible to HCC induction by dietary aflatoxin B1 [22].  Our results confirm that the intact vitamin E ester forms of both acetate and succinate are retained partially in the blood and that trans-esterification of both acetate and succinate on tocopherol occurs in rainbow trout suggesting an equilibrium exists between E-Su, E-Ac and free EOH (Results not shown).  Some studies have addressed possible differences in activity of the different analogues of the vitamin as between α-tocopherol versus β, γ or δ form and especially between the acetate and the succinate ester of α-tocopherol [11] and [23].

This study reports the in vivo prevention, in rainbow trout livers, of AfB1 induced HCCs by the use of E-Su.  In contrast, increased dietary E-Ac caused a several fold increase in trout HCC incidence and up to a three-fold increase in liver size.

 2. METHODS and MATERIALS:

 Rainbow trout were obtained both as eggs and as swim-ups from Nisqually Trout Farms Inc. in Lacey, WA.  Two weeks after swim-up they were all fed the same base diet19 for a period until they were over 1 gram in size when they were then put on the respective test diets shown in Appendix 1.  All fish tanks were 28 liter and received 1 liter per minute, 10o C Spring water to maintain 9 ppm oxygen.  The complete diet feed was made into a gel from the respective pure ingredients purchased principally from Sigma chemical Co.  The compromised diets substituted food grade textured soybean protein (TVP), wheat flour and powdered milk in the place of casein, dextrin and alpha-cell.  Aflatoxin was dissolved with propylene-glycol-methyl-ether acetate and chloroform to produce the required concentration then added to the oil mixture of each diet.  The completed diet slurries were cooled then frozen at -34o C till they were used, but fresh diets were made about every month or six weeks.

Fish serum was collected after centrifugation into vials, frozen and shipped frozen on ice for Vitamin E analysis at the Michigan State University Animal Health Diagnostic Laboratory (AHDL) in East Lansing MI.  Starting at 9 through 16 months on test diets, fish were sampled from each diet group at the same length of time on the diets.  There was more than one time length of test growth period for most of the experiments.  For sampling, the fish were killed with 3 aminobenzoic acid ethyl ester (MS222) at the rate of 200 mg/liter water.

The livers were then excised intact, weighed, photographed, the visual tumor rank recorded and  then put into 10% neutral buffered formalin for later HCC histopathology and tumor identification by RW (CellNetix Pathology and Laboratories) in Olympia, WA. The histopathology results were reported as follows 1).  Was there any carcinoma (data not shown), and 2). the number of tumor nodules in each liver slide.  Histopathological interpretation was blindsided as to treatment and gross appearance of the livers.  Gross nodule appearance was consistent with carcinomas histologically.  Histology examples of the HCCs are shown in slide photos following the text references.  Following that are photos 1 to 4 of examples of the whole livers from the respective diets.

Appendix A following the photos has the ingredients for all diets used.

  3.Statistical Analysis:

 Appendix B has a summary of the statistical probability of the variance of HCCs on test diets versus the control diet groups using the Students t test of results shown for data used in Figures 1 to 6.

 4. Results

 4.1. Results: Histopathological identification of cellular carcinomas:  Tumor characteristics include enlarged cell size, an expansile nodule, an absence of cytoplasmic lipid droplets, inflammatory cells from the tumor nodules and a loss of the 2 cell cord thickness of benign liver seen on reticulin stains shown in Photos 3 and 5B. Photo 5C shows normal liver cells.  There were slides of 997 livers examined.

 4.2.  Results:  Sera vitamin E:  Analysis showed that the trout serum level of E-OH at 50 to 270 μg/ml sera was roughly related to the dietary level of E-Ac.

 In a different test comparing dietary E-AC and E-Su, serum analysis showed that both dietary E-Ac and E-Su resulted in varying levels of all three forms of the vitamin (E-OH, E-Ac and E-Su) in the trout serum.  E-Su has been found intact in human sera (50 μg/ml) an hour after ingesting 2 grams of this ester (http://www.healcorp.net/current results.html).

 4.3.1. Results: Vitamin E-Acetate effect at 1.87 gms per KG diet, compared to the base level of 0.27 gm/Kg,  produced the opposite effect to what was expected in that the average liver HCC numbers increased by 10 fold in 10 months.  As early as 6 weeks on the diets the livers were becoming pale and enlarged.  At 6 months on the highest E-Ac diets (16.25 gm/ Kg diet) livers were twice as large as the controls.  

Figure 1: The average number of carcinoma nodules per liver slide as a function of vitamin E-acetate in each of 18 different diets. With each level of vitamin E, three other vitamins were tested, A, C and retinoic acid.

Liver enlargement occurred whether aflatoxin was in the diet or not.  The affect of dietary E-Ac on the average number of HCCs per fish in each group is shown in Figure 1.  Diets 7, 11, 17 and 18 have 7 fold the base level of E-Ac while 8 and 12 have 60 fold the base level.  Trans retinoic acid in diets 13, 14 appear to prevent HCC development.  These diets are the same as diet 11 except for t-retinoic acid but have less than a third the HCC risk as group 11 which indicates protection by t-retinoic acid.  Statistically, however, the probability of difference between group 11 and the other two groups was only 82% and 78% chance of difference respectively.  See Photos 1A and 1B at: Photos 1 through 5C

 

Figure 2: Tumor promotion by Vitamin E-acetate versus the protection shown by Vit. E-succinate.
Compare diets 7, 8, 21 and 34 with pronounced HCCs versus 29 through 33 with no HCCs.

4.3.2. Results: Vitamin E-Succinate’s beneficial effects against HCC risk was pronounced.   Figure 2 and Photos 2 show results of samples taken at 10 months on the diets.  Here E-Ac alone in diets 7, 8, & 21 had a marked promotion of HCCs.  But with diets 29 & 30 no tumors were observed.  All four diets, 8, 21, 29 and 30 had the same high level of tocopherol as either the succinate ester, or the acetate ester.  Diets 31 to 34 were used to test the effect of Vitamin E succinate on a lower concentration level.  Even though all three diets, 32, 33 and 34 had the same total tocopheryl level, only diet 34 had elevated HCCs  showing that the ratio of E-Ac to E-Su was proportional to the HCCs induced.  

 

 

Figure 3: The same tests as Figure 2, but at 13 months, shows the same effect as seen at 10 months where the high ratio of E-Ac over E-Su caused a high level of HCCS.

 4.3.3. Results: Vitamin E succinate's benefit is confirmed at 13 months shown in Figure 3.  The HCCs were beginning to develop from Diet 32, where E-Su and E-Ac were equal but when the E-Su was much greater than E-Ac (diet 33) there were still no liver tumors observed, while diet 34 induced a high level of HCCS due to the greater concentraton of E-Ac over E-Su.  See Photos 3 of the histopathology slide.  

 

 

  4.3.4.  Results:   Compromised diets (60 to 70) displayed in Figure 4, were made in order to increase the base level of HCCs in all these diets. The compromised diets had lower vitamin and mineral levels than the complete diets and they had textured soybean (TVP), supplying the protein and carbohydrates   instead of casein and dextrin that was used in the complete diets and in 64 and 67.  All diets had the same percentage of protein.  The compromised control diet 60, caused a 2.9 fold increase in average HCC numbers per liver slide above the number for the complete control, diet 2.  Diets 60 and 63 through 67 & 70 all had the low basic level of  E-Ac at 0.268 gm/kg. But diets 68 & 69 had 5.0 gm/kg of E-Ac or E-Su.  Diet 62, the test blank, alone had no aflatoxin.  

Figure 4: The average number of tumor per liver slide at 13 months on compromised diets. The comparison between diet 68 (high E-SU) and 69 (high E-Ac) again shows the benefit of E-Su in preventing tumors.

Surprisingly, diets 64 & 67 decreased the HCC numbers (to 0.08 & .25 ) while comparison diets 60, 63 65 & 66 had much higher HCC numbers.  These latter 4 diets were tests of the effects of food coloring on HCC development but no differences could be seen.  All Figure 4 diets except 64 and 67 had TVP, in the place of casein and dextrin.  All Figures 1 and 2 diets had casein and dextrin and no TVP.  Significantly Figure 4 shows that, in these compromised diets, high E-Su in diet 68 reduced the average HCC numbers to 3.2 from 13.8 that was found in the high E-Ac diet 69.  It shows that for an equally high level of tocopheryl ester in the diet, only the E-Ac increased the HCC incidence.

Comparing 64 and 67 with diets 60, 63 65 and 66, shows that casein/dextrin replacing TVP caused nearly total protection in those two diets, while the toxin was present.

Because aflatoxins found in soybean products in other countries were a major factor in promoting HCCs in humans, we tested our soybean flour for aflatoxins.

Analysis of duplicate samples of the soybean flour, both showed less than 1 part per billion of total aflatoxins (B1, B2 G1 & G2) therefore aflatoxin was not the causative factor in the difference of HCC incidence for the two types of diets (dextrin/casein versus soybean).  The amino acids in casein and the soy flour were very similar.  This suggests that an unknown factor in soybean-TVP added to the HCC incidence in AfB1 containing diets.   In diet 70 the increase of all vitamins (15 gms vitamin pack instead of 5) had only small or no effect on HCC incidence (See Photo 4).

4.3.5. Results: Vitamin A and D deficiency: Figure 5 shows that increased carcinogenesis resulted from vitamins A & D deficiency.  This is in agreement with the effects of deficiency in mammals.  But it is of note that Diets 31, 32 & 33 in figures 2 and 3 are vitamin A deficient and yet HCC, in the presence of extra E-Su, development was low or non-existent.  (Vitamin A was 12,800, 2560, 500, and 50 i.u.  in diets 2, 28, 27 and 31 to 34  respectively in Figure 5 and in Figures 2 and 3.)  (See Photo 5)

Figure 6: The dramatic affect of E-Ac on liver size at 11 months on the diets is shown in contrast to E-Su, that caused normal liver size and prevented or reduced HCC development in all tests.

4.3.6. Results:  Liver enlargement of 2 and 3 fold was caused by high dietary E-Ac in both the complete (diets 7, 8, 21 & 34) and in the compromised diet 6, in Figure 6.  Aflatoxin in the diets did not affect liver size.  And again E-Su caused no liver enlargement compared to E-Ac when both esters were given at the same high levels.  This is shown in figure 6 with diets 29 and 30 versus 8 and 21 of the complete diets and in 68 versus 69 in the compromised diets.

Although elevated E-Su prevented tumor incidence and hepatomegally, discoloration replaced the natural red (red to yellowish to grey) in proportion to the level of E-Su in the diets (photos not shown).  This is consistent with the fact that we see conversion of E-Su to E-Ac and E-OH in the serum. 

5.  Discussion:

 

This study compared E-Ac versus E-Su effect on the development of hepatomegaly and aflatoxin induced hepatocellular carcinomas (HCCs) in Rainbow trout.

 

5.1. E-Ac promotion of HCCs and induction of hepatomegaly:

The elevation of HCC incidence and hepatomegaly caused by increased E-Ac shown in Figures 1 through 3 and Figure 6, respectively in trout are parallel to the down regulation of the Hippo/Yap pathway signal shown by others that caused hepatomegaly of organs and tumor formation by inhibition of apoptosis, the controlled cell death of aberrant cells.  Restriction of the Hippo (Mst1 & Mst2 kinases) signaling path or phosphorylation of the YAP/TAZ factors has been shown to increase cell growth, organ size and tumor development [24, 25, 26, 27].

  One of the main paths of apoptosis is via mitochondrial membrane disruption called the intrinsic path, as opposed to an extrinsic path that starts the apoptotic signal outside the cell and bypasses any action on the mitochondria [28].  (See more of these two pathway types in section 5.4).

E-Ac has been shown to suppress the mitochondrial gate opening thereby reducing apoptosis and the Hippo path.  1.) It conceals the mitochondrial membrane against the Bax opening [29], 2.)  It decreases the tumor suppressor p53 protein synthesis [30], 3.)  It increases the bcl-2/Bax protein ratio [30], and 4.)  It inhibits the retinoid initiation of tumor apoptosis with mitochondrial membrane gate opening [31]. Thus dietary E-Ac, depending on its concentration is an anti-apoptotic molecule.  We have shown in this study with live animals that it causes liver hepatomegaly and promotes aflatoxin induced liver tumor formation in proportion to the dietary dose.  This study together with the above reports, indicates that inhibition of the Hippo/Yap pathway at the mitochondrial membrane gate (the intrinsic path) is one of the core sites for the adverse outcome of E-Ac in rainbow trout liver.   

 

5.2.  E-Su  preservation of liver size, and reduction of HCCs: 

E-Su on the other hand, up regulates the Bax initiated mitochondrial membrane opening  that releases cytochrome C, leading to the activation  of caspase 3, the final executor of apoptosis.  The same mitochondrial site releases Smac/DIABLO [32] which counteracts the Inhibitor of Apoptosis Protein (IAP) [33, 34], there by facilitating apoptosis.  This mitochondrial gate is one of many locations in the cell control signaling pathways that are up-regulated by E-Su [13, 35, 36].  Several of the apoptotic signals are activated by the tumor suppressor protein p53, which E-Su up-regulates [16].  Since these sites are suppressed by E-Ac, they are points of competitive inhibition against the action of  E-Su on the Hippo pathway signal.  They affect both organ size and carcinogenesis in an opposite manner as we have seen in this study.  Our results show that there is in vivo trans-esterification of the tocopherol esters tending to balance the ratio of E-OH/E-Ac/E-Su which would affect the balance of the Hippo/YAP signals.  But when either of these esters is abnormally increased they would predominate in the path signaling and either cause hepatomegaly and tumors or prevent the same respectively.

It is likely that a percentage of the dietary E-Su converted to E-Ac was responsible for some, if not all, of the HCCs shown for diet 68 in Figure 4.

Other factors that are up regulated by, but are also synergistic with E-Su in the Hippo signaling pathway [17, 36, 37] are the Tumor necrosis factor Related-Apoptosis-Inducing ligand, (TRAIL) [38] and FasL [39] both of which require activation of  cellular Death Domain receptors that are up-regulated by E-Su .  In the defense system that involves this family, the tumor necrosis factor alpha (TNFα) causes cell death, but it is not specific to cancer cells and causes inflamation and organ toxicity.  Significantly, in contrast to TNFα, TRAIL is generally nontoxic to normal cells but causes apoptosis, in tumor cells including resistant malignant mesotheliomas [16, 40],  in vitro gastric cancer cells [41], in vitrocolon cancer cells including those that are p53 negative [17] and others [42].  E-Su up-regulates all of these pathway signals.  E-Su is an activator for death receptors DR4 and DR5 that are receptors for TRAIL at the cell membrane and also, like TRAIL, these receptors exhibit apoptotic selectivity to tumor cells, and are not generally adverse in normal cells [35, 43].  In one study with pancreatic cancer cell lines the results were dubious [44].

Since TRAIL and FasL can by-pass the action on the mitochondria, and directly initiate apoptosis via caspases 8 and 3, E-Su therefore facilitates both intrinsic and extrinsic apoptosis.

The value of E-Su as a cancer cell specific agent which does not affect normal cells is shown by the following list of some of the cancerous organs or cell types that have been shown by other investigators to undergo apoptosis by E-Su treatment either alone or as a synergist with other agents:

 

 

Breast [13, 42, 46, 47, 48, 49],              Lymph glands [56, 57],

Cervical [18, 19],                                Mesothelium [16, 40, 58], 

Colon [17, 33, 50, 51],                          Ovaries [18, 19],

Gastric [41, 52, 53, 54],                        Pancreas [59, 60, 61, 62, 63],

Liver [44, 54, 55],  and                         Prostate [14, 64, 65, 66, 67, 68].

But like TRAIL and other activators, E-Su as a co-activator has been shown to be more effective  than either factor alone [40, 41].  Hence E-Su in cancer therapy is likely to be most effective when used in conjunction with a synergist.  

5.3. Aflatoxin mechanism of action and relation to the tocopheryl esters:

Aflatoxin B1, after its conversion to AfB1-8,9-exo-epoxide [69, 70] has caused: 1. A two fold increase of the apoptotic inhibitor, nuclear factor kappa beta (NF-κβ), [71, 72] in broiler livers.  2. Adverse activity of caspase-3 with necrotic degeneration in the rat hepatocytes [73].   2.  Mutation of the tumor suppressor protein p53 [74, 75] in human livers, thereby crippling regulation of  apoptosis.  4. Impairment of the immune response by destruction of thymus (type II cells) and the bursa of fabricus (types I cells) [76] in chicken broilers.  5.  Decrease in catalase and increase in “excessive” oxidative stress in chick spleen lymphocytes [77].   Catalase activity has been shown to be enhanced by tocopherol in Rainbow trout  blood where its deficiency has been implicated in disease (http://www.healcorp.net/catalase.html ) and HCC risk in humans [78, 79].  Overall, as detailed above, elevated E-Ac alone causes hepatomegaly but in the presence of AfB1 also promotes HCC incidence.  E-Su normalizes apoptosis [80, 81] and inhibits the necrosis and HCCs caused by AfB1.

It is significant that E-Su suppresses hepatomegaly and aflatoxin induced HCC induction in the light of the ubiquitous presence of aflatoxins in our society and its known contribution to HCCs in humans.

5.4. The complexity of Apoptosis

Many other proteins are involved in cell proliferation versus apoptosis such as tumor necrosis factor alpha (TNF-alpha), that activates the nuclear factor kappa beta (NF-κ β) [51, 55, 68,  82, 83], certain of the Bcl family of proteins all that are apoptosis inhibitors.  Then transforming growth factor-beta (TGF-β) [42, 46, 48], TRAIL, FasL and p53 proteins that are apoptosis activators. These are respectively inhibited or activated by E-Su and give a picture of hierarchy of highly integrated interaction and cross talk in cell regulation.  If any of the factors in one of these pathways are inhibited by mutation or toxins, apoptosis is inhibited.  In some cases where such activity is inhibited, agonists have been found that re-stabilize the activity [84].  E-Su is such an activator [16, 46].

5.5. Type I (extrinsic) apoptosis and Type II (intrinsic) apoptosis with involvment of E-Su and E-Ac:

In both of these apoptotic pathways, or cell type functions, α-E-Su acts at specific identified sites of the signaling pathway [29, 85].

 With extrinsic type I Cells the signal for apoptosis originates outside the cell and is independent of mitochondrial involvement.  In this type, E-Su up regulates the cell surface death receptors (DR4 and DR5), of both TRAIL [17, 37] and FasL [14] leading to the downstream activation of the proteases, Caspase 8, then Caspase 3, the final effector of cell apoptosis but does not directly target Caspase 9 which is a Type II Cell Caspase [16].

Intrinsic type II Cells involves a mitochondrial membrane change in permeability at the location of succinic dehydrogenase of the electron transport chain [35] where the apoptotic signal is instigated through Bid formation of Bax proteins to act upon mitochondria and cause release of  Smac/Diablo and cytochrome C.  The latter then promotes the apoptosis signal with caspase 9, p53 and Caspase 3, where Smac/Diablo intercepts the inhibitors of apoptosis proteins (IAP), and thereby is an ally to TRAIL and other apoptotic proteins as it facilitates the signal to reach Caspase 3, one of the final apoptosis actuators.  E-Su facilitates the Bax, translocation for this to happen [13] while E-Ac inhibits this mitochondrial change by the Bax/Bid factors [29, 30, 37].

5.6. The need for E-Su allies that are cancer cell specific.

Most chemotherapeutic drugs that have been successful in cancer treatment are also toxic to the extent that the value of life is often compromised and the treatment has to be discontinued.  Chemotherapy induced peripheral neurodegeneration [86, 87]  illustrates the need to find treatments that destroy cancer cells but not normal cells similar to both TRAIL and E-Su.  Another adverse example is found in the current prostate cancer treatment methods that include the use of anti-androgen drugs to block the synthesis of testosterone or 5-hydroxy testosterone, hormones which in turn promote growth of cancerous prostate cells.  Lupron is an example of this class of drugs.  Used with initial radiation treatment, it has proven effective as a periodic i.m. injection done over a period of about two years.  But it has serious side effects and like other chemotherapy drugs, it is not specific to the prostate gland, nor to cancer cells but is toxic throughout the body and after each injection causes initial sever flu like symptoms.  Over the long range it causes serious muscle debilitation and mental fatigue. http://www.lupronprostatecancer.com/sideeffects.aspx  and  http://www.rxlist.com/ lupron-side-effects-drug-center.html.

Most of the other drugs used also have toxic side effects illustrated by  prostate cancer treatment with antiandrogens [88]. 

E-Su on the other hand by a different mode is reported to repress the androgen receptor expression of the cancer cells instead of the androgen itself and but it is non toxic to normal cells [64].  Furthermore the long term ablation of androgen receptors by traditional drugs in some cases, has been found to become receptor negative, so that the cancer growth proceeds regardless of the androgen presence or absence.  Since E-Su has been found to be an agonist in several apoptosis resistant or receptor negative tumors [16, 17, 46]it could prove positive for androgen receptor treatment where traditional drugs have failed.

5.7. Mitochondrial targeting and Cancer Cell Specific Agents:

E-Ac has been shown to prevent the mitochondrial gate opening (section 5.1 and 5.4) in contrast to E-Su.  With this in mind because we find that there is in situ trans-esterification on the tocopherol moiety of the analogue, the resulting E-Ac produced, could be the cause of a slight HCC presence and enlargement  from diet 68 (Figures 4 and 6).

To prevent the trans-esterification and thereby the loss of mitochondrial gate opening, several other analogues have been produced and found more effective than E-Su, namely tocopheryl ether acetate [89] which is now undergoing a clinical test [90], tocopheramine succinate [91] and triphenylphosphonium tagged E-Su [58].  Two other synergists with E-Su are exisulind [92], and TRAIL [16, 38, 40], the latter being a native member of the tumor necrosis family that is specific to cancer cells. All of these synergists induce cancer cell apoptosis but have limited affect on normal cells and do not induce necrosis.  Localized or "point" radiation [18] and [19] used with E-Su further increases treatment success.  

By using these mitochondrial targeted tocopherol analogues in cohort with E-Su all of which up-regulate apoptosis in cancer cells but not in normal cells, future treatment methods should be able to avoid the serious side effects of current drugs.  One of the reviews of the cancer specific drugs and mitochondrial membrane permiabilization is noted [81]. 

Acknowledgements:

The authors thank Robert R Sand, DVM, for the generous use of his fish hatchery  facilities and laboratory for this study and for the technical assistance of Aaron Abbot and Dustin Tucket as well as many other volunteers.

                                   

The authors verify that there is no conflict of interest in this study.

 

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Abbreviations and Definitions 

AfB1: Aflatoxin B1 : Potent carcinogen produced in moldy food by Aspergillus species.

BAK:  BAK1 is a pro-apoptotic Bcl-2 protein having four Bcl-2 homology (BH) domains: BH 1-4.

Bax: Apoptosis regulator BAX, also known as “bcl-2-like protein 4", is

bcl-2:This name stands for B cell lymphoma. Bcl-2 proteins incite either anti- or pro-apoptosis. BID: The BH3 Interacting-domain Death agonist gene is a pro-apoptotic member of the Bcl-2 protein family and is upregulated by the tulmor suppressor p53.

Caspase: cysteine-aspartic protease, or cysteine-dependent aspartate-directed protease.

Cytokine: Cell signalling or literally Cell Movement 

DIABLO: (Direct IAP Binding protein with Low pI)  Binds caspase inhibitors and thus facilitates apoptosis

DR4, DR5 etc.: Death Domain receptors that are required for the action of TRAIL & FasL.

E-Ac:  Vitamin E acetate  (d-alpha tocopheryl-acetate)

E-Su:  Vitamin E succinate  (d-alpha tocopoheryl-succinate)

Hippo/Yap pathway: Cell proliferation path signaling that controls apoptosis, originally seen in the Drosophila melanogaster fruit fly. The Hippo proteins phosphorylate the YAP factors inhibiting them and preventing out of control cell growth and proliferation.

i.u.:  International units

IAP: Inhibitor of Apoptosis Proteins

Kinases:  Enzymes that phosphorylate a target substrate

Mst 1 and Mst2:  Serine/threonine protein kinases, the mammalian homologs of the Hippo kinase from Drosophila controlling the cell proliferation, differentiation etc.

NF-κβ: Nuclear Factor kappa beta, an apoptosis inhibitor.  In normal cells it acts as modulator of cell growth and death, but in cancer cells it prevents apoptosis and therby promotes cancer growth. 

p53: tumor suppressor protein that when activated is a transcription factor that regulates many downstream target genes, including BID.  However, p53 also has a transcription-independent role in apoptosis,   in particular, promoting Bax activation and the insertion of Bax into the mitochondrial membrane.

Smac: Second Mitochondria-derived Activator of Caspases. Also called DIABLO

TNF-α: Tumor necrosis factor alpha is a cytokine that activates NF-kβ.  It causes cell death, but it is not specific to cancer cells and causes inflamation and organ toxicity.

TRAIL: Tumor necrosis factor Related-Apoptosis-Inducing Ligand. It targets only cancer cells.

YAP: Yes Associated Protein.  Up regulation of the YAP signal causes organ hypertrophy and eventual tumor development.

 

Photo 1B: Livers of groups 7 and 8 with HCC numbers and vitamin E acetate levels shown in Figure 1:
Diet 7 with extra E-Ac at 0.16% (samples 207) shows promotion of both hepatomegaly and tumors. This group at 10 months on the diet had an average of 2.9 nodules per examined slide.
Diet 8 with 1.6% E-Ac caused an average of 6.8 HCCs per liver slide in addition to morphological damage and hepatomegaly.

Photo 2: Comparing the difference in effect of E-Ac versus E -Su (See Figures 2
and 3): Only diet 34 fish had HCCs at 10 months on the diets. As seen here diet 32
caused bleaching, but it had no tumors. Note the difference in liver size of group 34
versus groups 31-33.

Photo 3: Characteristic tumor from fish 519 Blu of Diet 34, Figure 3. Diet 34 had 1.75 grams vit. E acetate and 0.13 grams vit. E succinate per KG diet.

Photo 4: Livers of diet groups 68 with 0.5% E-Su and 69 with 0.5% E-Ac.
Group 68 had an average liver size of 1.2 % of fish body weight while group 69 had enlarged livers with an average of 3.0 % of the fish body wt. Figure 4 shows the HCC numbers and the Vitamin ester level for each diet.

Photo 5A: Vitamin A deficiency at a level of 512 i.u. per Kg of diet number 27
caused an Average of 4.3 HCCs per liver slice compared with 1.5 HCCs in diet
group 28 which had 2565 i.u. per Kg diet and diet 2 with 12,825 i.u. which
caused only 0.7 HCCs average per slice examined. (See Figure 4.)
Neither diet had elevated E-Ac. Note the difference in gross appearance of
groups 27 & 28 (without extra E-Ac) versus groups 7, 8, 34 and 69 that had
elevated E-Ac in the diets.

Photo 5B: Slides from fish 496G on diet 27 with tumor juxtaposed to benign cells and showing reticulin around the 2 cell hepatic cords in benign areas and less or no reticulin in the tumor. Diet 27 was vitamin A deficient at only 500 i.u. per Kg diet.

Photo 5C: Liver slides from fish 498Y on diet 28 in Figure 5 showing normal reticulin pattern (no tumor). Diet 28 had vitamin A at 2560 i.u. per KG diet.