oxidant

8/16/2019 Oxidant

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Oxidants, antioxidants and the current

incurability of metastatic cancers

Jim Watson

Abstract

The vast majority of all agents used to directly kill cancer cells (ionizing radiation,

most chemotherapeutic agents and some targeted therapies) work through either

directly or indirectly generating reactive oxygen species that lock key steps in the

cell cycle! "s mesenchymal cancers evolve from their epithelial cell progenitors, they

almost inevitaly possess much#heightened amounts of antioxidants that e$ectively

lock otherwise highly e$ective oxidant therapies! "lso key to etter understanding

is why and how the anti#diaetic drug metformin (the world%s most prescried

pharmaceutical product) preferentially kills oxidant#de&cient mesenchymal p'

cells! " much faster timetale should e adopted towards developing more new

drugs e$ective against p' cancers!

"lthough the mortality from many cancers, particularly those of haematopoietic

cells, has een steadily falling, the more important statistic may e that so many

epithelial cancers (carcinomas) and e$ectively all mesenchymal cancers (sarcomas)

remain largely incurale! *ven though an increasing variety of intelligently designed,

gene#targeted drugs now are in clinical use, they generally only temporarily hold

ack the fatal ravages of major cancers such as those of the lung, colon and reast

that have ecome metastatic and gone eyond the reach of the skilled surgeon or

radiotherapist! *ven though we will soon have comprehensive views of how most

cancers arise and function at the genetic and iochemical level, their +curing seemsnow to many seasoned scientists an even more daunting ojective than when the

+War on -ancer was started y .resident /ixon in 0ecemer 1231!

.ropelling me then, 45 years ago, to turn the -old 6pring 7aror 8aoratory into a

major site for unravelling the genetic underpinnings of cancer was the elief that

once the gene#induced molecular pathways to cancer ecame known, medicinal

chemists would go on to develop much more e$ective gene#targeted drugs! 9nlike

most early proponents of the +War on -ancer, who thought that 0/"#damaging

chemotherapeutic agents would ring real victories in one to two decades, : thought

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three if not four more decades of focused research would need to pass efore we

would e in a position to go all out for total victory ;1<! :n fact, only after the 12==>

?55 7uman @enome .roject provided the world with the highly accurate seAuences

for three illion human 0/" letters has it een possile to egin to approach the truegenetic complexity of cancer!

2. Molecular pathways to cancer as revealed through DNA sequencing

By now we know that mutations in at least several hundred human genes (out of a

total of ?1 555 genes) ecome serious +drivers of the anormal cell growth and

division process that generates human cancer ;2<! They do so ecause they encode

the protein components of +signal transduction pathways that enale external

signals (growth factors) to move from the cell surface receptors to key promoter>

enhancer regions along the ?4 human chromosomes! There they turn up the

expression of genes needed for cell growth and division as well as the evasion of

programmed cell death, the latter of which much underlies the ever#growing

resistance of late#stage aggressive cancer cells to radio# and chemotherapeutic

therapies! Cost importantly, there exist multiple molecular pathways that ring

aout cell growth and proliferation, each with their own speci&c surface receptors,

cytoplasmic transducers, and promoters and enhancers of gene expression ;3<!

Cuch potential cross talk exists etween these pathways, allowing new 0/"

mutations to create new pathways to cancer when pre#existing ones are locked!

"lready we know that the emergence of resistance to the gene BRAF #targeted anti#

melanoma drug Deloraf freAuently results from driver pathway cross talk, as does

resistance to the targeted drugs :ressa and Tarceva when they are deployed against

*@EF#driven lung cancers! @iven the seemingly almost intrinsic genetic instaility of

many late#stage cancers, we should not e surprised when key old timers in cancer

genetics dout eing ale to truly cure most victims of widespread metastatic

cancer!

Fesistance to gene#targeted anti#cancer drugs also comes aout as a conseAuence

of the radical changes in underlying patterns of gene expression that accompany the

epithelial#to#mesenchymal cell transitions (*CTs) that cancer cells undergo when

their surrounding environments ecome hypoxic ;4<! *CTs generate free#Goating

http://rsob.royalsocietypublishing.org/content/3/1/120144#ref-1








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mesenchymal cells whose Gexile shapes and still high "T.#generating potential give

them the capacity for amoeoid cell#like movements that let them metastasize to

other ody locations (rain, liver, lungs)! Hnly when they have so moved do most

cancers ecome truly life#threatening!

3. Epithelial-to-mesenchymal transitions are a consequence o changes in

transcriptional regulation

*CTs leave intact the pre#existing order of 0/" ases while changing the way they

are read into F/" transcripts! 9nderlying transcriptional regulation are site#speci&c

0/"#inding proteins, and sometimes regulatory F/"s, that recruit to genes the

machinery reAuired to read those genes! This includes the general transcription

machinery and also enzymes that modify the histones around which chromosomal

0/" is wound, and the 0/" itself! These enzymes mediate methylation and

acetylation of histones, as well as remodelling of the nucleosomes in various ways,

and methylation of 0/" ases, changes that can inGuence how a given gene is

expressed! Fegulation of transcription extends far eyond its role in inGuencing how

cancer cells respond to changes in their environmental surroundings! This regulation

underlies all the multiple switches that accompany the transition of fertilized eggsinto the di$erentiated cells (lung, kidney, etc!) of mature organisms!

!. "#$-li%e cyto%ines drive mesenchymal cells to commence cell

prolieration

Cuch holding ack the creation of e$ective drugs against mesenchymal cancer cells

has long een ignorance of the externally driven signalling pathways propelling them

into stem cell growth and suseAuent di$erentiation! Cost attention until now has

een focused on the Wnt signalling pathway that sends I#catenin into the cell

nucleus to activate the T-E transcription factor for essential roles in *CTs as well as

stem cell functioning ;5,6<! "n even more important villain may have een virtually

staring in our faces for almost two decadesone or more of the cytokine mediators

of inGammation and immunity, in particular, the :8K interleukin! :8K lood serum

levels, for example, steadily go up as incurale cancers ecome more life#

threatening ;7,8<! "utocrine loops proaly exist where cytokine inding to theirrespective cell surface receptors sets into motion downstream gene#activating









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pathways that not only generate more :8K molecules ut give their respective cancer

cells an aura of almost true immortality y locking the major pathway to

programmed cell death (apoptosis)! Pushing by cytokines of otherwise quiescent

mesenchymal cancer cells to grow and divide probably explains why anti-inammatory agents such as aspirin lead to much less cancer in those human beings

who regularly take them;9<!

9nfortunately, the inherently very large numer of proteins whose expression goes

either up or down as the mesenchymal cancer cells move out of Auiescent states

into the cell cycle makes it still very tricky to know, eyond the cytokines, what other

driver proteins to focus on for drug development! :deally, we should largely focus

&rst on &nding inhiitors of cancer cell proliferation as opposed to inhiitors of

cancer cell growth! :nhiiting, say, the synthesis of cellular molecular uilding locks

will slow down not only the metaolism of cancer cells ut also that of our ody%s

normally functioning cells! By contrast, locking proteins speci&cally moving through

the cell cycle should leave untouched the normal functioning of the vast majority of

our ody%s cells and so generate much less unwanted side e$ects!

&. 'he gene transcription activator Myc allows cells to move through thecell cycle

8ong thought to e a key, if not the key, protein against which to develop cell#

proliferation#inhiiting drugs is the powerful gene transcription activator Cyc! Eirst

known for its role in driving cancers of lood#forming lymphocytes (e!g! Burkitt%s

lymphoma), Cyc now also has een found to e a key driver of the rapidly fatal

+small cell lung cancers as well as the likely driver of many late#stage incurale

cancers, including receptor negative and ductal reast cancers! !ots of "ycmay turn

out to be an essential feature of much of the truly incurable cancer ! :t simultaneously

turns up the synthesis of the more than 1555 di$erent proteins reAuired to move all

cells through the cell cycle! "lthough precisely how this almost 455#amino acid long

polypeptide works at the molecular level remains to e worked out, it seems to play

a uniAue role that cannot e handled y any other class of transcription factors!

9nlike our &rst hunch that Cyc was somehow an on>o$ speci&er of gene activity, it is

a nonlinear ampli&er of expression acting universally on active genes except for the

immediate early genes that ecome expressed efore "yc ;10,11<! "lready many







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serious e$orts have een made to develop drugs that lock its cell#proliferation#

promoting activities! 9nfortunately, all such direct e$orts have so far failed!

9sing a dominant negative plasmid that locks all Cyc functions, @erard *vans

laoratory, &rst at 9-6E and now in -amridge, 9L, has used mouse xenograph

models of several major human cancers to show Cyc%s indispensale role in moving

through the cell cycle ;12<! "lthough mouse stem cells in Cyc%s asence stop

growing and dividing, they resume normal functioning when "yc is turned ack on!

By contrast, the turning o$ of "yc in human cancer cells preferentially drives them

into programmed cell death (apoptosis) with one important exceptionM pancreatic

adenocarcinoma cells do not enter into apoptosis, Auite possily explaining why

pancreatic cancer is so resistant to virtually all cell#killing reagents (@! *vans ?51?,

personal communication)!

$. (romodomain ! proteins play essential roles in maintaining the Myc

levels necessary or leu%aemic cell growth and division

"n unanticipated powerful way for lowering Cyc levels in haematopoietic cancers

has emerged from the discovery that the incurale nature of "!!-AF# acute myeloid

leukaemia ("C8) depends upon the presence of the not yet well understood protein

romodomain 4 (BF04)! When JN1, developed last year to treat the BF04#driven

rare $%& midline carcinoma, was used on human "!!-AF# "C8 cells, they rapidly

stopped multiplying and di$erentiated into macrophages ;13,14<! "t the same time,

Cyc levels rapidly plunged! Cost importantly, JN1 does not lock the normal

macrophage production, suggesting that Cyc levels in macrophage#forming stem

cells do not depend upon BF04! Their formation must depend on a di$erent

chromosomal remodeller!

). Myc is turned on through multiple molecular pathways

7ow "yc is turned on not only in other cancers ut also during normal human

development remains largely to e worked out! 8ikewise not known is how the BF04

protein at the molecular level helps turn on Cyc synthesis in C88#"E2#driven

leukaemia! 9ntil JN1 goes into the clinic against leukaemia late this year, we will not

moreover know for sure whether resistance to JN1 will compromise its clinical utility!







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9nfortunately, the answer is proaly yes ecause arti&cially turning up "yc y

means that ypass BR'( causes JN1 resistance! Coreover, there are already known

multiple ways to turn on "yc expression in normal cells, each starting y signals

inding to speci&c cell surface receptors then moving through one or more layers ofsignal transducers to the nucleus to turn up the transcription of genes needed for

cell growth and division! Cyc synthesis is not only downstream of the cytokine Jak>

6tat signal transduction pathway ut also downstream of the 7*F?>F"6>F"E>67p?>

*FL pathway that helps drive the growth of much, if not most, reast cancer ;15<!

Whether they in turn feed into BF0 protein#dependent gene#activating pathways

remains for the future to reveal! " multiplicity of Cyc#inhiiting speci&c drugs may

have to e in our arsenal efore we can routinely move eyond delaying death from

incurale cancers to true lifetime long cures!

*. Detecting %ey cancer cell vulnerabilities through +NAi screens

That the BF04 protein is among the major "chilles% heels of incurale "C8 ecame

known not ecause of a chance oservation ut y using a powerful new

methodology for detecting molecular weaknesses that are cancer cell#speci&c! "t its

heart has een the deployment over the past several years y @reg 7annon at -old

6pring 7aror 8aoratory of short hairpin F/" molecules (shF/"s) speci&cally

designed to knock ack the functioning of single human genes ;16<! " genome

shF/" lirary containing multiple proes (four to six) for each human gene

possesses some 155 555 shF/"s! Testing all of them extensively against just one

type of cancer still poses a formidale, logistical challenge likely to reAuire 1# to ?#

year long intervals for even +ig science laoratories!

Cuch smaller highly focused liraries, however, now can e deployed y high#

Auality, university#level science laoratories provided there already exist hints as to

what molecular vulnerailities might e found! Eorearmed y knowledge that

invarialy incurale forms of acute myelolastic leukaemia ("C8) originate from

rearrangements of a key gene involved in epigenetic chromosomal remodelling,

-hris Oakoc and Johannes Duer at the -old 6pring 7aror 8aoratory found the

gene#activating BF04 as the most pronounced potential molecular weakness of

an "!!-AF# human "C8! They did so y screening liraries of only some 1555 proes





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designed to knockout ?4 genes coding for the key proteins involved in epigenetic#

driven gene expression!

Cost recently, Oakoc has found three other major protein players (Cenin, *zh1P? and

*ed) that work together with BF04 to make "!!-AF# "C8 incurale y currently

deployed anti#cancer drugs ;17<! 0rugs inhiiting their respective functioning should

also provide e$ective anti#"C8 agents! )*h+, and )ed code for polycom proteins

that lock speci&c gene expression, whereas the "enin gene, like the BR'( gene,

activates gene expression! 8oss of functional *zh1P? and *ed locks the expression

of the .d/na gene#encoded p1K and p12 proteins that have widespread cell#cycle#

progression#locking roles! The Cenin protein%s molecular role proaly involves its

already known inding to C88! 8ike BF04, it may have a Cyc#level#raising role!

Einding out how such chromosome remodelling dependencies emerge and evolve

during tumour progression will directly impact the clinical implementation of

epigenetic#ased anti#cancer therapies!

,. (+D! unctioning is vital not only or ast-growing leu%aemias but also

or many i not most dangerous lymphomas and myelomas

"s soon as possile, we must &nd out in more detail how far the drug JN1%s anti#

cancer actions extend eyond "!!-AF##speci&c "C8s! "lready we know that in mice

it stops eAually well the more curale, non#"!! rearranged strains of "C8 as well as

all forms of acute lymphocytic leukaemia ("88)! BF04%s capacity to heighten Cyc

levels thus proaly extends over almost all leukaemias! Whether the polycom

proteins of "88, like those of "C8, also turn o$ the cell#cycle#inhiiting .d/na#coded

proteins p1K and p12 remains to e seen! JN1 also stops the growth in mice of many

fast#growing B#, and T#cell lymphomas, suggesting that their untreated BF04 protein

maintains their high Cyc levels necessary to make them fatal! :n JN1#resistant

lymphomas (e!g! Jurkat cell), Cyc synthesis must e turned on y a di$erent route!

-ell lines from most human multiple myeloma victims also freAuently show high

sensitivity to JN1 ;18<! There, the twosome cocktail of JN1 and the now widely

deployed proteasome inhiitor Oelcade reinforce each other%s anti#myeloma actions!

When JN1 ecomes roadly availale clinically, hopefully y mid#?51, it may

consideraly lengthen the >' more years of additional life provided to most

myeloma victims y Oelcade administration!





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JN1 also signi&cantly slows down the growth of a small ut real numer of cell lines

derived from many major solid cancers (e!g! prostate and melanoma)! BF04 may

have een only called into play late as these cancers evolve to ecome more

aggressive! Hf more importance is JN1%s failure to stop the growth of the vastmajority of solid tumour cell lines! The heightened Cyc levels needed y, say,

cancers of the prostate and reast may instead e provided y the intervention of

one or more of the some ' other BF0 proteins or other chromatin regulators!

9nfortunately, we do not yet know how the vast majority of them function eyond

the fact that their BF0 pockets, y inding to the acetyl groups, help turn on, not

turn o$, gene activation! JN1%s unanticipated locking of sperm functioning most

excitingly has led to the recent discovery of a testis#speci&c romodomain (BF0T)

essential for chromatin remodelling during spermatogenesis! Hccupancy of the BF0T

acetyl#lysine pocket y JN1 generates a complete and reversile contraceptive e$ect

;19<! *arly evidence suggests that BF0T does not promote Cyc synthesis! There may

e out there soon to e found, say, reast#speci&c or prostate#speci&c BR' gene

activators! Cost important to learn is whether they also do or do not drive Cyc

synthesis!

/. 'he circadian rhythm regulator 01E+2 by negatively regulating Myclevels unctions as an important tumour suppressor

Cyc%s paramount role in moving cancer cells through the cell cycle has recently een

reinforced y two highly independent F/"i screens to &nd genes whose loss of

function selectively kills cancer cells ;20,21<! :n sampling largely di$erent sets of

genes, they oth honed in on the gene.0$/e coding for protein kinase casein kinase

? epsilon! "mong its many multiple targets for phosphorylation and suseAuent

proteosome#mediated degradation is the transcription factorP)R gene whose

selective inding to 0/" turns o$ the function of many genes including "yc! "lready

long known has een .*F:H0 ? (.*F?) involvement as a clock protein at the heart of

the circadian rhythms of higher animal cells! 8ater, Auite unexpectedly, .*F? was

found to function as a tumour suppressor, with the asence of oth its copies

causing the rate of radiation#induced cancers to rise! :t now seems ovious that its

anti#cancer action arises from its aility to turn o$ "yc! :n .*F?%s asence, Cyc

levels greatly rise, therey explaining why tumours of many types all display higher

levels of -6/Le than found in their normal cell eAuivalents! -ommon sense suggests







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that speci&c -6/Le inhiitors should soon e roadly tested against a large variety

of human cancers!

. igh-Myc-driven ast prolierating cells possess cell cycle

vulnerabilities

7igh#Cyc#level proliferating cells less eQciently proceed through the mitotic cycle

than cells driven y lower Cyc levels! Why high Cyc leads to many more mitotic#

generated chromosome anormalities has recently een explained through a large

F/"i screen designed to reveal +synthetic lethal genes that only have vital function

under conditions of high Cyc! Cost unexpectedly, they pinpointed key roles for the

69CH#activating genes 0A)+ and 0A)involved in proteasome#speci&c protein

degradation ;22<! When they are locked from functioning, large numers of Cyc#

driven genes somehow ecome switched from on to o$! "s expected, many function

in the formation and reakdown of the mitotic spindle! " much less anticipated

second class functions in uiAuitin#ased, proteasome#mediated protein degradation!

-onceivaly, the fast growth rates of high#Cyc#level#driven proliferating cells

generate more mitosis#involved proteins than their respective proteasomes can

timely reakdown! :n any case, drugs designed to lock 0A)+ and 0A) shouldpreferentially kill fast#proliferating cancer cells!

7igh#Cyc#level vulneraility is also generated y suoptimal supplies of -0 kinase 1

(-0L1) that functions with the " type cyclins during the late 6 phase of the cell

cycle! "s long as the Cyc levels are those of normal cells, proliferating cells have

suQcient -0L1! But when more Cyc leads to faster cell cycles, much more -0L1 is

reAuired to prevent failed cell divisions! 6o, it makes a prime candidate for the

development of an e$ective drug against high#Cyc#driven cancers ;23<!

2. 4electively %illing cancer cells through e5ploiting cancer-speciic

metabolic and o5idative wea%nesses

We must focus much, much more on the wide range of metaolic and oxidative

vulnerailities that arise as conseAuences of the uncontrolled growth and

proliferation capacities of cancer cells! "s human cancers ecome driven to more

aggressive glycolytic states, their ever#increasing metaolic stress makes them





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especially vulnerale to sudden lowering of their vital "T. energy supplies! #

Bromopyruvate, the powerful dual inhiitor of hexokinase as well as oxidative

phosphorylation, kills highly dangerous hepatocellular carcinoma cells more than 15

times faster than the more resilient normal liver cells and so has the capacity to trulycure, at least in rats, an otherwise highly incurale cancer ;24,25<! The structurally

very di$erent hexokinase inhiitor ?#deoxyglucose, through its aility to lock

glycolysis, also has the potential for eing an important anti#cancer drug! /ot

surprisingly, it works even etter when comined with inhiitors of "T.#generating

oxidative phosphorylation such as the mitochondrial target drug Cito N ;26<!

" key mediator of cellular response to falling "T. levels is the "C.#dependent protein

kinase "C.L, which in times of nutritional stress phosphorylates key target proteins

to push metaolism away from anaolic growth patterns ;27<! By inhiiting mTHF it

slows protein synthesis, and y phosphorylating acetyl#-o" caroxylase it slows

down lipid synthesis! The glycolytic pathways that produce the cellular uilding

locks are indirectly controlled y "C.L through its phosphorylation of the p'

transcription factor! "ctivated p' slows down glycolysis during cell cycle arrest

through turning on its &12AR gene target! :ts respective protein reaks down the key

regulator of glycolysis fructose ?,K#isphosphate as well as locking further cell

cycles through turning on the p+ gene!

3. 1reerential cancer cell %illing by apoptosis relects high p&3 levels

The enhanced apoptosis capaility of early#stage epithelial cancer cells, in

comparison with their normal cell eAuivalents, reGects their higher content of

activated p' transcription factor! Hverexpression and ampli&cation of the p'

repressors C0C? and C0C4 are common across cancer types! :n the case of

melanomas, p' function is commonly shut down y overexpression of C0C4!

"lready a drug exists that through its inhiition of C0C4 makes melanoma much

more treatale ;28<! Lnowing more aout why p' activation sometimes leads to cell

cycle arrest (senescence) and under di$erent circumstances results in apoptosis

remains an important challenge for the immediate future!











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!. 1&3 induces apoptosis by turning on the synthesis o genes whose

primary unction is the synthesis o reactive o5ygen species

7ow p' turns on apoptosis was &rst revealed through elegant gene expression

studies carried out in Bert Oogelstein%s Johns 7opkins laoratory in 1223 ;29<!

"lthough looking for genes expressed only during apoptosis, they discovered a set of

1 p'#induced genes (P12 genes), each of which are likely key players in the cellular

synthesis of reactive oxygen species (FH6R 7?H? hydrogen peroxide, the H7 radiation

and H? superoxides)! P123, for example, codes for a Auinone oxidoreductase that is a

potent generator of FH6 ;30,31<! p' target genes also play major roles in

downstream processes through turning on synthesis of some 15 di$erent

mitochondrial functioning proteins such as B"S, .9C" and /HS", as well as death

receptors such as 0F4 and 0F', that in ways yet to e elucidated help carry out the

many successive proteolysis stages in apoptosis ;32<!

*Aually important, p' turns on the synthesis of the key proteins involved in the

apoptotic (programmed cell death) elimination of cells that have no long#term future,

say, through unsustainale metaolic stress or damage to cellular chromosomes

rought aout y exposure to ultraviolet or ionizing radiation! 6o, removing suchcells are complex sets of largely mitochondrial#sited degradation events! "s the

successive stages in apoptosis unravel, the respective dying cells lose mitochondrial

functioning and release cytochrome c, culminating in 0/"#lierating cell dissolution!

&. #ea%age rom drug-impaired mitochondrial electron transport chains

raises reactive o5ygen species levels

The mitochondrial electron transport generation of "T. and heat is oligatorily

accompanied y the production of FH6 (such as the H7 radical, 7?H? and

H? superoxides)! /ormally, preventing FH6 molecules from irreversily damaging

key nucleic acid and protein molecules are potent antioxidative molecules such as

glutathione and thioredoxin ;33<! When present in normal amounts, they cannot

handle the much larger amount of FH6 generated when oxidative phosphorylation

ecomes inhiited y mitochondrial#speci&c drugs such as rotenone that lock

feeding of /"07 into the respiratory chain or y # diindolylmethane (0:C), theactive component in the long#reputed chemo#preventative Brassica vegetales,











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which inhiits the mitochondrial E1E5 "T. synthesis complex ;34<! 6till#remaining

FH6 molecules through oxidizing intra#mitochondrial targets induce the apoptotic

elimination of cells damaged from excessive oxidative stress! "lready, 0:C is used as

an adjuvant therapy for recurrent respiratory papillomatosis in humans! Themolecular mechanism(s) through which FH6 induce apoptosis remains to e found

hopefully soon! /ow, we will e surprised if they do not somehow directly oxidize and

so activate one or more of the B"S#like proteins involved in p'#mediated apoptosis!

That FH6 y themselves can mediate apoptosis was recently convincingly shown y

the &nding that the +&rst#in#class anti#cancer mitochondrial drug elesclomol

(discovered y 6ynta .harmaceuticals through screening for anti#apoptotic agents)

kills cancer cells through promoting FH6 generation ;35<! When these resulting FH6

molecules are destroyed through the simultaneous administration of the antioxidant

molecule $#acetylcysteine, preferential killing of cancer cells stops! The failure of

elesclomol to generate apoptosis in non#cancerous cells proaly arises from the

inherently lower FH6 level generated y normal mitochondrial electron transport

machinery!

$. +eactive o5ygen species may directly induce most apoptosis

That elesclomol promotes apoptosis through FH6 generation raises the Auestion

whether much more, if not most, programmed cell death caused y anti#cancer

therapies is also FH6#induced! 8ong puzzling has een why the highly oxygen

sensitive +hypoxia#inducile transcription factor 7:E1U is inactivated y oth the,

until now thought very di$erently acting, +microtuule inding anti#cancer taxanes

such as paclitaxel and the anti#cancer 0/" intercalating topoisomerases such as

topotecan or doxoruicin, as well as y frame#shifting mutagens such as acriGavine

;36,37<! "ll these seemingly unrelated facts &nally make sense y postulating that

not only does ionizing radiation produce apoptosis through FH6 ut also today%s

most e$ective anti#cancer chemotherapeutic agents as well as the most eQcient

frame#shifting mutagens induce apoptosis through generating the synthesis of FH6

;38>40<! That the taxane paclitaxel generates FH6 through its inding to 0/"

ecame known from experiments showing that its relative e$ectiveness against

cancer cell lines of widely di$erent sensitivity is inversely correlated with their

respective antioxidant capacity ;41,42<! " common FH6#mediated way through

















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which almost all anti#cancer agents induce apoptosis explains why cancers that

ecome resistant to chemotherapeutic control ecome eAually resistant to ionizing

radiotherapy!

Fecent use of a '5 555 memer chemical lirary at C:T%s Loch -ancer -enter to

search out molecules that selectively killed /-RA0#transformed human &rolasts

revealed the piperidine derivation lanperisone ;43<! FH6 generation underlies its

cancer cell killing action! 6urprisingly, this already clinically used muscle relaxant

induced non#apoptotic cell death in a p' (VV versus) independent manner!

When lanperisone was applied in the presence of the FH6#destroying antioxidant

scavenger molecules deferoxamine, utylated hydroxylamine or the antioxidant

trolox, no activity was oserved!

). (loc%age o reactive-o5ygen-species-driven apoptosis by antio5idants

"lthough we know FH6 as a positive force for life through their apoptosis#inducing

role, for much longer we have feared them for their aility to irreversily damage key

proteins and nucleic acid molecules! 6o when not needed, they are constantly eing

neutralized y antioxidative proteins such as glutathione, superoxide dismutase,

catalase and thioredoxin! -ontrolling their synthesis as well as that of many more

minor antioxidants is the /rf? transcription factor, which proaly came into

existence soon after life as we know it started! Cost importantly, at -ancer Fesearch

9L in -amridge, 0avid Tuveson%s laoratory has recently shown that /rf? synthesis

is somehow upregulated y the cell growth and division#

promoting RA0, RAF and "4. oncogenes ;44<! Biologically, this makes sense

ecause we want antioxidants present when 0/" functions to make more of itself!

The fact that cancer cells largely driven y F"6 and Cyc are among the most diQcult

to treat may thus often e due to their high levels of FH6#destroying antioxidants!

Whether their high antioxidative level totally explains the e$ective incuraility of

pancreatic cancer remains to e shown! The fact that late#stage cancers freAuently

have multiple copies of RA0 and "4.oncogenes strongly hints that their general

incuraility more than occasionally arises from high antioxidant levels! -learly

important to learn is what other molecules exist that turn on /rf? expression! 0uring

the yeast life cycle and proaly that of most organisms, oxidative phosphorylation





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is clearly separated y time from when 0/" synthesis occurs! Whether /rf? levels

also go up and down during the cell cycle remains important to e known soon!

*. Enhancing apoptotic %illing using pre-e5isting drugs that lower

antio5idant levels

"lready there exist experiments with haematopoietic cells in which the cancer#cell#

killing capacity of the FH6 generator arsenic trioxide ("s?H) has een shown to e

inversely correlated with the content levels of the major cellular antioxidant

glutathione ;45<! "s?H also knocks down the reductive power of thioredoxin

necessary for several key steps in cellular metaolism! :ts capacity to inhiit oth

thioredoxin and glutathione widens its potential for a successful deployment against

many major cancers eyond promyelolastic leukaemia! "lso capale of enhancing

the cytotoxic e$ect of "s?H is ascoric acid, which, though known for its antioxidant

role in cells, is converted into its oxidizing form dehydroascoric acid! 9nfortunately,

up until now, we do not yet have clinically e$ective ways to lower glutathione levels!

8owering its level through deployment of the drug uthionine sulphazine that locks

its synthesis leads Auickly to upregulation of the /rf? transcription factor that in turn

upregulates glutathione synthesis ;46<! " more general way to reduce antioxidantlevels deploys motexa&n gadolinium, a memer of a class of porphyrin molecules

called texaphyrins! Through a process called futile redox recycling, it transfers

hydrogen from antioxidants to produce FH6! 9nfortunately, clinical trials designed to

show its enhancement of chemo# and radiotherapies have so far shown only modest

life extensions as opposed to cures!

Through selecting for compounds that preferentially induce apoptosis in cancer cells

as opposed to normal cells, the natural product piperlongumine from the Piper

longum plant was recently revealed as a potential anti#cancer drug ;47<! Cost

exciting, it mediates its action through its inding to the active sites of several key

cellular antioxidants (e!g! glutathione 0 transferase and caronyl reductase 1) known

to participate in cellular responses to FH6#induced oxidative stress! That

piperlongumine failed to raise FH6 levels in non#cancerous cells proaly resulted

from their inherently lower levels of these antioxidants which, in turn, result from

less activation of the /rf? transcription factor!







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,. Anti-angiogenic drugs wor% only when used in con6unction with

reactive o5ygen species generators

The non#toxic anti#angiogenesis protein endostatin (discovered and promoted in the

late 1225s in Judah Eolkman%s Boston laoratory and now resurrected y ongzhang

8uo in Beijing) shows anti#cancer activity only when it is used together with

conventional chemotherapeutic agents! This fact, long puzzling to me, may e due to

the chemotherapeutic component providing the FH6 needed for cancer cell killing

;48<! By itself, the hypoxia resulting from endostatin action may not e suQcient for

cancer cell killing! " similar explanation may explain why @enentech%s avastin also

only works when comined with chemotherapy! By contrast, the killing of

mutantBRAF melanoma cells y Deloraf works very well in the asence of any

ovious direct source of FH6! -onceivaly, the metaolic stress resulting from its

turning o$ the F"6>*FL pathways somehow shuts down the /rf? pathways, letting

FH6 rise to the level needed to kill the drug#weakened melanoma cells!

2/. #ower reactive o5ygen species levels in stem cells relect higher levels

o antio5idants

Eor more than a decade, there has existed too long ignored evidence that normal

stem cells have lower FH6 levels than their di$erentiated progeny! Just a year ago,

even more convincing experimentation showed that reast cancer stem cells also

contain lower FH6 levels than those found in their cancerous epithelial#like progeny

cells ;49<! "ll stem cells, e they normal or cancerous, proaly have lower FH6

levels as a result of their corresponding higher levels of prominent antioxidant

molecules such as glutathione and thioredoxin! Cost likely, these heightened

amounts have evolved to protect chromosomal F/" from FH6#induced damage to

the more exposed region of chromosomal 0/" as it undergoes changes in

compaction as it moves through the cell cycle! Whether all dividing cells have higher

antioxidant levels remains to e worked out! :f so, all stem cells will e inherently

much more resistant to FH6#induced apoptotic killing than more di$erentiated, much

less antioxidant#rich progeny cells!





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2. Metormin selectively targets 0%ills mesenchymal cancer stem cells

"lready we have at our disposal a relatively non#toxic, excessively well#tested drug

that preferentially kills mesenchymal stem cells! :n a still much unappreciated article

pulished three years ago in .ancer Research, Levin 6truhl%s laoratory at 7arvard

Cedical 6chool &rst showed that metformin, a locker of stage ? oxidative

phosphorylation, selectively targets stem cells! When so applied with

chemotherapeutic agents to lock xenographic tumour growth, it induces prolonged

remission if not real cures ;50,51<! But when metformin was left out of these

experiments, suseAuent multiplication of unkillale mesenchymal stem cells lets

these xenographs grow into life#threatening forms, showing that chemotherapy yitself does not kill stem cells! This most widely used anti#diaetic drug%s heightened

aility to kill late#stage mesenchymal cancer cells proaly explains why those

humans who use it regularly have reduced incidences of many cancers!

Cetformin is presently eing added to a numer of anti#cancer chemotherapeutic

regimes to see whether it magni&es their e$ectiveness in humans! The fact that

metformin works much more e$ectively against p'− − cells suggests that it may e

most active against late#stage cancers, the vast majority of whose cells have lostoth of their p53 genes! By contrast, the highly chemo#radio#sensitive early#stage

cancers against which most of anti#cancer drug development has focused might very

well show little metformin e$ectiveness! By the end of ?51, we should know

whether it radically improves any current therapies now in use! 7ighly focused new

drug development should e initiated towards &nding compounds eyond metformin

that selectively kill stem cells! "nd the reason why metformin preferentially kills p'−

−stem cells should e even more actively sought out!

22. 7ree-radical-destroying antio5idative nutritional supplements may

have caused more cancers than they have prevented

Eor as long as : have een focused on the understanding and curing of cancer (:

taught a course on -ancer at 7arvard in the autumn of 12'2), well#intentioned

individuals have een consuming antioxidative nutritional supplements as cancer

preventatives if not actual therapies! The past, most prominent scienti&c proponent

of their value was the great -altech chemist, 8inus .auling, who near the end of his






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illustrious career wrote a ook with *wan -ameron in 1232, .ancer and 6itamin .,

aout vitamin -%s great potential as an anti#cancer agent ;52<! "t the time of his

death from prostate cancer in 1224, at the age of 2, 8inus was taking 1? g of

vitamin - every day! :n light of the recent data strongly hinting that much of late#stage cancer%s untreataility may arise from its possession of too many antioxidants,

the time has come to seriously ask whether antioxidant use much more likely causes

than prevents cancer!

"ll in all, the y now vast numer of nutritional intervention trials using the

antioxidants I#carotene, vitamin ", vitamin -, vitamin * and selenium have shown

no ovious e$ectiveness in preventing gastrointestinal cancer nor in lengthening

mortality ;53<! :n fact, they seem to slightly shorten the lives of those who take

them! Euture data may, in fact, show that antioxidant use, particularly that of vitamin

*, leads to a small numer of cancers that would not have come into existence ut

for antioxidant supplementation! Blueerries est e eaten ecause they taste good,

not ecause their consumption will lead to less cancer!

23. A much aster timetable or developing anti-metastatic drugs

The world of .hysics already knew ?5 years ago that it had no choice ut to go very

ig for the 7iggs oson! To the civilized world%s great relief, they now &nally have it!

Biology and Cedicine must likewise now again aim igas when we &rst promised

the world in 12== that the still to e found human genome would later prove

indispensale for the curing of most cancers and so went for it ig! :f, however, we

continue to move forward at today%s never frantic, largely &ve#day working week, the

never receding 15>?5 year away &nal victory that our cancer world now feels safe to

project will continue to sink the stomachs of informed cancer victims and their

families! That we now have no @eneral of inGuence, much less power, say an

*isenhower or even etter a .atton, leading our country%s War on -ancer says

everything! /eeded soon is a leader that has our cancer drug development world

working every day and all through the night!

The now much#touted genome#ased personal cancer therapies may turn out to e

much less important tools for future medicine than the newspapers of today lead us

to hope ;54<! 6ending more government cancer monies towards innovative, anti#







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metastatic drug development to appropriate high#Auality academic institutions would

etter use /ational -ancer :nstitute%s (/-:) monies than the large sums spent now

testing drugs for which we have little hope of true reakthroughs! The iggest

ostacle today to moving forward e$ectively towards a true war against cancer may,in fact, come from the inherently conservative nature of today%s cancer research

estalishments! They still are too closely wedded to moving forward with cocktails of

drugs targeted against the growth promoting molecules (such as 7*F?, F"6, F"E,

C*L, *FL, .:L, "LT and mTHF) of signal transduction pathways instead of against

Cyc molecules that speci&cally promote the cell cycle!

Cost needed now are many new anti#Cyc drugs eyond the exciting new BF04

inhiitors, such as JN1, as well as multiple drugs that inhiit the antioxidative

molecules that likely make, say, pancreatic cancer so incurale! They should much

enhance the e$ectiveness of all current radio# and chemotherapeutic regimes! "s

such, they will likely cure many more now incurale cancers! 7ow they will interact

as cocktail partners with the newer targeted therapies that do not directly generate

FH6 remains to e seen! *Aually important may e an expanded search for drugs

that prevent p' reakdown!

2!. A billion dollars should suice to identiy all the remaining proteins

needed or curing most metastatic cancer

The total sum of money reAuired for F/"i methodologies to reveal the remaining

major molecular targets for future anti#cancer drug development need not e more

than '55>1555 million dollars! 9nfortunately, the /-: now is unlikely to take on still

one more ig science project when it is so hard#pressed to fund currently funded

cancer programmes! 6till dominating /-:%s ig science udget is The -ancer @enome

"tlas (T-@") project, which y its very nature &nds only cancer cell drivers as

opposed to vulnerailities (synthetic lethals)! While : initially supported T-@" getting

ig monies, : no longer do so! Eurther 155 million dollar annual injections so spent

are not likely to produce the truly reakthrough drugs that we now so desperately

need!

7appily, the &rst F/"i whole genome ig screen acked y a +ig pharma &rm has

just started with .&zer working with the -old 6pring 7aror 8aoratory! *ven,

however, if several more giants working separately join in, collectively they will

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Jim Watson's (J0W%s) interest in cancer &rst pulicly expressed itself through his

teaching on tumour viruses after he joined the 7arvard 9niversity Biology

0epartment in the fall of 12'K! 8ater, for the new :ntroductory Biology ::, his last of

15 lectures focused on how cancer might e induced y 0/" tumour viruses, the

smallest of which proaly only had 0/" suQcient to code for >' proteins! :n his

12K' textook, &he "olecular Biology of the 2ene, the last chapter (+" geneticist%s

view of cancer) raised the Auestion of how a virus might have the capacity to turn

on the cell cycle! 9pon ecoming director of the -old 6pring 7aror 8aoratory in

12K=, he changed its major research emphasis from microial genetics to cancer

(through recruiting Joe 6amrook from Fenato 0ulecco%s la at the 6alk :nstitute)!

Cajor among its early -old 6pring 7aror 8aoratory eukaryotic accomplishments

was the 1233 co#discovery of F/" splicing y Fichard Foerts and .hil 6harp (C:T)!

J0W then necessarily devoted much of his time on scienti&c politics, &rst toward

gaining /ational :nstitutes of 7ealth (/:7) acceptance of the safety of recominant

0/" procedures (123>123=), and second arguing for and then leading /:7%s role inthe 7uman @enome .roject (12=K>122?)! :n ?55=, J0W%s main interest moved to the

curing of cancer focusing on the iochemistry of cancer cells as opposed to their

genetic origins!

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