ABSTRACT A new concept of a basic mechanism involved in cell pro-
liferation is presented.  It is suggested that cells are normally restrained
from proliferating by the highly viscous nature of the intercellular glyco-
s aminogl' cans.
   J     In order to proliferate, cells must escape from this re-
straint by depolymerizing the glycosaminogl' ans in their immediate
               /II"
environment.  This process is accomplished by the release of hyaluroni-
dase and kept in check by khnx physiological hyaluronidase inhibitor.  It
is suggested that physiological hyaluronidase inhibitor isFligoglycosa-
minog `can that requires ascorbic acid for its synthesis, and perhaps in-
  f
corporates EI residues of ascorbic acid.  This hypothesis provides a com-
prehensive explanation for many disease processes involving cell pro-

liferation.  It indicates the existence of a basic underlying mechanism
and suggests a common pattern of treatment for these diseases, including


cancer.  We conclude that ascorbic acid may have much greater therapeutic
value than has been generally assigned to it.


3 .J '

    Ort homolecular medicine is the preservation of good health and
the treatment of disease by varying the concentrations in the human body
of substances that are normally present in the body and are required for

health (1, 2, 3). Of these substances, the vitamins are especially important,

and ascorbic acid, in particular, may have much greater value than has
been previously ascribed to it.  Irwin Stone has advanced arguments to
support the concept that the optimum rate of intake of ascorbic acid is
about 3 g per day under ordinary conditions, and larger, up to 40 g per
day, for a person under stress (4, 5, 6).  An argument based on the fact
that only a few Z&XXIX&$- animal species require an exogenous source

of ascorbic acid and on the amounts of ascorbic acid contained in a diet of

raw natural plant food has led to the conclusion that the optimum daily in-
take of ascorbic acid for an adult human being is about 2. 3 g or more

(7).  Ascorbic acid is a substance with extremely low toxicity; many

people have ingested ten to twenty grams per day for long periods
                  &
                                             1, 0
HIX~~ serious side effects, and ingestion of as much as   grams &-f3+~F

tLme without serious side effects has been reported (8).  In this respect

ascorbic acid ~z&cxhnlp may be considered an ideal substance for ortho-
molecul ar prophylaxis and therapy.

GROUND SUBSTANCE

    Cells in the tissues of the body are embedded in ground substance.
This ubiquitous material pervades every interspace and isolates every cell
from its neighbors.  It must be traversed by every molecule entering or
le aving the cell. ~nanrnn~~~~~~~~~~~~~~~~~x

X~~9~~~~~$~~X~~~~X~~~~~~ . 1

There is evidence that the

interface between a cell membrane and the immediate extracellular environ-

ment is the crucial factor in the whole proliferative process.  Variations in

the composition of the extracellular environment exert a profound influence
on the cell behavior, and in turn the cells possess a powerful means of

modifying their immediate environment.  This interdependence is involved

in all forms of cell           is particularly important in cancer.


-2 -

The cancer cell and its immediate environment constitute a balanced
system in which each component influe rices the other.  Understanding
of this relationship end of the means of controlling it could lead to
rational methods of treating cancer and other cell-proliferative diseases.


    Until recently cancer research has tended to concentrate almost
exclusively upon the cell, and                        rolife r at ion

equation.  The intercellular                    containing
water, electrolytes, metabolites, dissolved ~XSSXX gases, trace elements,
vitamins, & enzymes, carbohydrates, fats, and proteins. The solution is
rendered highly viscous by an abundance of certain long-chain acid muco-
polysaccharide polymers, the glycosaminoglicans nd the related proteo-
glycans, reinforced at the microscopic level by a -dimensional network
         ?k-
of collagen fibrils.  The principal glycosaminogl'cans so far identified
                    8t :
are hyaluronic acid, a long-chain polymer with high molecular weight ,b     ,
                                        Jt&&~kj#.r.r. %A$
(200,000 to 500,000) and simple chemical structure (alternating   of
                      T
N-acetylglucosamine and glucuronic acid), and varieties of chon roitin
                         d
(alternating ~$43~
      J-a#Q.iAH,&~
        of N-acetylgalactosamine and glucuronic acid) and its
sulfate esters.  Other glycosaminogl  ans may also be present. The
                                          F
chemistry of the ground subktance and the intercellular environment has
been reviewed recently by Balazs iyt7i 1
   ?
    An important property of the intercellular s        its very
                                                                 .-."
high Z&SCXE&@+C viscosity and cohesiveness.      *
          &L;&;s_P;oPertY is dependent
upon the chemical integrity of the large w.  $ The viscosity
be reduced and the structural integrity destroyed by the depolymeri
action of certain related enzymes (the endohexosaminidasels, the beta-
~&IEwc&~~~~x N-acetylglucosaminidases, the beta-   - d
acetylgalactoseaminidases, and beta-glucuronidase), known ,by the generic
name "hyaluronidase". It $s {robable that most cells in the body are able
to produce hyaluronidase (0&l).  The interlacing molecular inx network of
the intercellular ground substance is n a constant state of slow dynamic
     I
change, with synthesis of glucosaminog   (p%&eri..z-&ion) balanced by


              Tf
          -&/3


their breakdown (                f hyaluronidase, and
subsequent excret     It is within this slowly changing environment,
the "milieu interieur" of Claude Bernard, that all cellular activity
takes place. The normal cell and the cancer cell both thrive and die
within this environment.


HYALURONIDASE AND CELL PROLIFERATION

cell

Some years ago the hypothesis was advanced that all forms of
proliferation depend upon one fundamental interaction between
                  fi 2-2

the cell and its immediate environment !F .  The hypothesis may be
stated as follows: til cells in the body are embedded in a highly vise d ous

environment of ground substance which physically restrains their inherent
tendency to proliferate;   liferation is init/ate*d by release
             ,4*A~+&ka,`Bq&3&% $/     of hyaluronidase

from the cells, which          the glycosaminoglicans in the imme -
                              ? "A
diate environment and allows the cells freedom to divide and to migrate
within the limits of the alteration; proliferation continues as long as hyaluroni-
dase is being released, and stops when the production of hyaluronidase
stops or when the hyaluronidase is inhibited, and the imnrrw~ environment
is allowed to revert to its normal restraining state.


   In normal healthy tissues cell division is taking place at a constant
slow rate, corresponding to normal cell replacement.  This normal "back-
ground" rate of cell division results in a slow metabolic turnover of ground
substance, with liberation into the blood stream and then escape in the
urine of the partially depolymerized fractions of the intercellular glycosamino-
glicans, produced in the immediate vicinity of the dividing cells.  These
degradation products of ground substance depolymerization can be recog-
nized and measured by a variety of biochemical methods.  Depending upon
-
the analytical procedure employed, different fractions have been given
different names .  For &lxx+- our purpose they may be grouped
together under the general term "serum polysaccharide".   In health the
serum polysaccharide concentration-remains within a relatively narrow
         j3j 19
"normal" range (l+!++).  The process is kept in check by the presence in
the tissues and the blood of a substance called "physiological hyaluroni-
dase inhibitor" (PHI).  In health the serum PHI concentration lies within
a well-defined "normal" range .@&Z&)+ ( $5, i b) ,


  In conditions in which excess cell@ proliferation i,s occurring,
such as inflammation, tissue repair, and cancer, depolytirization of
ground substance can be demonstrated histochemically in the immediate
                                      17

vicinity of the proliferating cells (g), and there i
cant increase ir$%%e~~%%%!if polysaccharide (
PHI+ (I $> f6) r

x3x also a signifi-

the serum


NEOPLASTIC CELL     PROLIFERATION

   It follows from this hypothesis that cancer may be no more than
the permanent exhibition by some cells of a fundamental biological property
possessed by all cells.  We suggest that the characteristic feature of neo-
plastic cell   proliferation is that these cells in becoming malignant have

acquired, and are able to bequeath to their descendants in perpetuo, the
                                                              -

ability to produce hyaluronidase continuously. Wherever they travel, these
cells will always prosper, multiply, and invade within the protective in-
dependence of their own self-created depolymerized environment.  The se

renegade cells are autonomous only because they possess this specific
ability, the ability to isolate themselves permanently from "contact" and
all the usual "controls" governing tissue organization and growth restraint.
   By endowing a clone of cells with this single property of continuous
                                            +ebie.aw

hyaluronidase release ~EXXIXX~&~&K~X~~X it is possible to provide a Q'$+ e$

     xplanation for  m T y of the morphological features of malignant

                 The methods whereby cells might acquire this property
in response to a wide variety of carcinogenic stimuli have also been outlin
                   . .
                U
    rp\5-
      w together with the experimental evidence in support
of the concept (
    ?Y .


THERAPEUTIC CONTROL OF CELL PROLIFERATION

AssumJng ,that cell &k ' proliferation depends upon the interaction

between depolymerization of the ground substance and its inhibition by
cellular hyaluronidase, we see that there are two methods of exerting
therapeutic control of cancer and of other diseasewt&xxsrx states in which
excessive cellular proliferation is a harmful feature.  We may&h @*
attempt to 1~~~~&21~xt2rexgxamrm~~ increase the resistance of the
ground substance to enzymatic depolymerization, that is, to strengthen
the ground substance, or to directly m3opmxx neutralize the cellular
hyaluronidase by decreasing $' its production or inhibiting its action.


TREATMENT BY STRENGTHENING THE GROUND SUBSTANCE

    The resistance of ground substance to the action of hyaluronidase
can be increased in a number of ways, some of which are already estab-
lished as use-ful methods for retarding cell proliferation.

    ?-I
     Radio t erapy, irradiation with x-rays, is an example in which

the result of the treatment is that some of the amorphous  round sub-

stance has been replaced by a dense deposit of collagen ( R ).  The direct

cytotoxic effect of radiotherapy is less powerfully reinforced by a per-
manent reduction in the susceptibility of the ground substance in the
treated region to the action of hyaluronidase, with a consequent long-lasting
diminution in proliferative activity.

    Hormone therapy is effective because the physical-chemical state
of the ground substance is profoundly influenced by many endocrine factors;

the experimental evidence has been reviewed elsewhere w .  Resistance to

the action of hyaluronidase can be increased by the administration of cortico-
steroids, estrogens, androgens, and thyroxine, and these effects are en-
hanced after adrenalectomy and hypophysectomy. These hormones, although
differing widely in their special effects on particular target cells, all exert
to a greater or lesser extent, and roughly in the order stated, the same
effect on the intercellular field,(&kxakx namely, the absorption of amorphous
(I
ground substance and its replacement by a more resistant fibrous substance.
These hormones are the ones employed with some success in the palliation

of various forms of human cancer.

    Other agents may also be effective in altering the intercellular
environment and indirectly exerting some controlling influence on the be -

                         iv
havior of cells. It has been pointed out (4) that an explanation is provided
of the `saddow paradox", that substances which are locally carcinogenic
(by creating a local intensely impermeable carcinogenic environment) have
also some anti-proliferative value when administered systemically in experi-
mental cancer (by bringing about similar generalized changes in the resistance
of the ground substance to hyaluronidase and thus decreasing cell proliferation).


   Because of the complexity of the intercellular ground substance
and its responsiveness to external influences, many of the innumerable
"cancer treatments" that have been hopefully advocated year after year
might have some elemnt of truth behind them.  It is also true, however,

that no form of cancer treatment based on the anti  eoplastic effect of
            9 f
modification of ground substance can ever be more than palliative, be-
cause to render the ground substance totally resistant to hyaluronidase
would create a situation incompatible with life itself.


- 13 -

TREATMENT BY INHIBITION OF HYALUR.ONIDASE

   Although the ' " .xlxx indirect methods of retarding cell pro-
liferation, described above, are of great interest and in special cir-
cumstances of undoubted value, the direct inhibition of cellular hyaluroni-
dase offers more spectacular therapeutic possibilities.
   Hyaluronidase may be inhibited by drugs and by immunological
methods, but the approach most likely to succeed appears to be that of

f'   `,' utilization of the naturally occurring PHI substance.
   Spontaneous regression of advanced cancer has been well docu-
mented in a number of fortunate patients as a direct consequence of
massive intercurrent infection with hyaluronidase-producing bacteria
(' 4 ).  A possible explanation for this remarkable phenomenon is that the
depolymerizing action evoked an upsurge in the serum PHI level of
sufficient magnitude to inhibit totally the malignant capability of the
neoplastic cells &7  It is known that such infections are
ciated with an increase in the serum PHI concentration I Yt lways asso-
                              ,,A
                  l-27 ).  It has
been independently demonstrated in experimental cancer that the injection
of `hear's polysaccharide "
k         mnfrxm@ induces%%%i lysis ( ) but also a
                                   * g F
sharp and significant rise in PHI concentration ( 8 ).  The problem is how
to employ this suppressive mechanism in practical therapeutics.
                        `dmnmbnwmwxx


- 14 -

ASCORBIC ACID AND HYALURONIDASE INHIBITOR

   There is strong evidence to indicate that ascorbic acid is involved
in some way in the synthesis of physiological hyaluronidase inhibitor.
A strong xxg@~~9cxar suggestion to this effect is provided by the manifestations
of scurvy, resulting from a deficiency of ascorbic acid.  If ascorbic acid
were required for the synthesis of PHI, a deficiency of ascorbic acid would
cause the serum PHI level to decrease toward zero.  In the absence of the
control of hyaluronidase by PHI, background cellular proliferation and
release of more hyaluronidase would produce a steady and progressive
ti enzymatic depolymerization of the ground substance, with disruption
and disintegration of the collagen fibrils, intraepithelial cements, base-

e

ment membranes, p&ivascular sheaths, and all the other organized co-
hesive structures of the tissues, producing in time the general tie zed patho-
logical state of scurvy.  These generalized changes,& tissue disruption,
ulceration, and hemorrhage, are identical to the local changes that occur
in the immediate vicinity of invading neoplastic cells.  This concept of

scurvy,  as in olving uncontrolled depolymerization of ground substance,
explains why F curvy is always associated with a very high level of serum
polys accharide (27  It also explains why very small amounts of ascorbic
acid have profound effects in the treatment of scurvy.  The total body
                              x3

content of ascorbic acid is estimated to be around 5 g (R), and yet this
small amount controls the health of the whole body content of interceliluar
material, which must amount to many kilograms of substance. ~BXLVZXX~X
mrW%t$aB~~& The symptoms of scurvy are relieved by the ingestion
of a few tenths of a     ram of ascorbic acid.  It seems clear that ascorbic
          .ti

acid is not an i$portant constituent unit of the intercellular ground substance,
as has been suggested; instead, it may well be involved in the synthesis of
PHI, the circulating factor that controls intercellular homeostasis. Ascorbic
acid is, of course, required for the conversion of proline to hydroxyproline,
and is accordingly essential for the synthesis of collagen.  It may well serve


- "
4 6) \!GQ

/

15 -

f    in several ways in determining the nature of tissues and the state of health

of human beings.

.- -
  -
x          a general chemical similarity to $x the glycosamino-
                          i5 h
gl can polymers of the ground substance (l&i-S).  The PHI serum concen-
  tration rises significantly in all conditions in which excessive cell prolif-
                  15
   eration is a feature (
         4 ), but PHI is known not to be a simple breakdown
                                                      I

product of ground-substance glycosaminogl&n. Its precise cg%mical
composition is still unknown.  It has recently been suggested (
                    I4 ) that

PHI is a soluble glycosaminogl r can residue in which some or all of the
glucuronic acid units are replaced by the somewhat similar molecules of

ascorbic acid.
inhibitors ($) . The general theory i! of enzyme activity and the action of
        involves the idea that the active region of the enzyme is

complementary to the intermediate complex, with the structure corresponding

I  to the maximum of the energy curve (&.&x at the saddle-point configuration,
intermediate a in structure between the reactants and the products)

that determines the rate of reaction.  This theory requires that inhibitors
of the enzyme resemble the activated complex, rather than either the
reactant molecules or the product molecules.  Accordingly it is unlikely

that PHI would be          ~&r&M%l's",'    a fragment of hyaluronic
acid or a fragment of any other glycosaminog I.& an.  It would instead involve

at least one residue of a related but different substance.  It is possible that
a residue of ascorbic acid resembles the activated complex, and that incor-
poration of such a residue would produce an altered glycosaminog ik arl3!6adx
which could function as an inhibitor of hyaluronidase.  It is also possible,
however, that the chemical activity of ascorbic acid, such as its reducing
power..a.ad~.~~-~-in.t~ C- or its power to
cau?s$ hydroxylation reactions to take place, could function to convert an
olig oglycosaminogl
r     x can into PHI.  Whatever the mode of action of ascorbic
acid in synthesis of PHI, whether it involves incorporation of an ascorbic
acid residue or some other reaction, the therapeutic implications of the
concept that ascorbic acid is involved in the synthesis of PHI are consid-

  erable.



$6&ntrodF e arJ.&r )

   The hypothesis that ascorbic acid is required for the synthesis
of PHI/'                                                              ,
    and is itself destroyed in the course of the synthesis r"'
explai *# why in such conditions XU& as inflammation, wound repair,
and cancer the individual always appears to be deficient in ascorbic
acid, on the basis of measurement of level in the serum, measurement
of urinary excretion, and saturation tests (27, 28, 29, 30).  It is clear that
the total body requirement of ascorbic acid has become abnormally high,
s&x as would result from an increased synthesis of PHI with incorporation
or destruction of ascorbic acid.


THE SYNTHESIS OF PHI AND ITS THERAPEUTIC USE

    If the basic concept of cellular proliferation is correct, PHI
might be a valuable therapeutic agent in directly controlling all forms
of excessive pro iferation, including cancer.
i                 It is a naturally occurring
substance found in the serum of all mammals (24), and should be safe

and free from   dangerous side effects.  Determination of the chemical

structure of PHI and its synthesis should not present insuperable difficulties.
However, it may not be necessary to synthesize the substance.  It is possible

that, given enough ascorbic acid, the body could synthesize a proper quan-

tity of PHI.

    The level of ascorbic acid in blood plasma is about 15 mg per liter
when the rate of intake is about 200 mg per day.  With larger rates of in-
take the plasma level increases only slowly, because of urinary excretion,
reaching about 30 mg per liter for intake of 10 g per day.  In the therapeutic
situations here envisaged, with ascorbic acid being prescribed to control
excessive cellm proliferation, a daily intake of 10 g to 20 g or even more,
and with the bulk of that administered intravenously at first, might be neces-
sary to achieve the desired effect. ti McCormick (Q-5+61 and Klenner ('
                             2-a; ty    8 )

have been advocating and using this form of treatment for many years.
Their combined clinical experience ~z&xx&.~GIx indicates that very large
doses of ascorbic acid, in the range mentioned above, can be given intra-
venously with perfect safety and with apparent benefit in a wide variety of

disease &&XXX states.


- 18 -

THE THERAPEUTIC USES OF ASCORBIC ACID

  The hypothesis that ascorbic Fcid is required for synthesis of
PHI and can thus control harmful wlwrization of glycosaminogl cans
                             v
explains why the vitamin is effective in curing scurvyxx&&ab$l~ and.in
improving the &XXZ&~~X&XXX+@ XX& healing of wounds.  But the potential
therapeutic uses of this relatively simple substance may be infinitely
greater .

    It has been postulated for years that the administration of ascorbic
acid would increase tissue resistance to bacterial and viral infections by
\

' improving the integrity of the tissues.
T--                    We are now in a position to suggest
that, through the PHI system, the administration of ascorbic acid in suffi-
ciently high dosage may provide us with a broad-spectrum antibiotic effective
f-
against all those pathogenic bacteria, and perhaps viruses, that rely @ax

upon release of hyaluronidase to establish and spread themselves throughout

the tissues.  The dramatic clinical successes reported quite independently
      lb/(r

by McCormick (A) and by Klenner  ) in a very wide variety of infective
states support this contention.
`L4

-7-  The hypothesis also indicates a safe and elegant method of control
in many inflammatory and auto-immune diseases where, although the
individual causes are still unknown, the essential harmfu$ feature is always
excessive ccl w proliferation and ground-substance depolymerization.
A trial of orthomolecular doses of ascorbic acid seems perfectly justifiable
and $u%ax~btixx eminently preferable to the use of cortico-
steroids, irradiation, and all the other indirect methods currently employed.

   Most important of all, we are led to the conclusion that the adminis-
tration of this harmless substance, ascorbic acid, might provide us with
*x an effective means of permanently suppressing neoplastic cellular
proliferation and invasiveness, in other words, an effective means of
controlling cancer.  Ascorbic acid in adequate doses might prove to be
the ideal cytostatic agent.  Dramatic regressions might be induced in a



- 19 -

very few patients with rapidly growing tumors with precarious blood
supplies, but in the great majority the effect of the treatment is expected
to be to "disarm" rather than to "kill" the malignant cells.  "Tumors"
would remain palpable and visible on x-ray examination, but all further
progressive malignant growth might be stopped. Hopefully, malignant
ulcers would heal, and pain, hemorrhage, *and all the other
secondary distressing features of neoplasia would be brought under
cant rol.  This desirable outcome might be termed carcinostasis, with
what had been lgnx neoplastic cells now rendered harmless
intact ground substance,     I       and re-embedded
             subject again to normal tissue restraints, and

persisting in the body in heterotopic situations as@&ix@xs$irarxx "paleoplastic"
collections of essentially normal cells.  A suggestion of the possibilities
of the use of ascorbic acid in the control of cancer has been provided by
the report by Schlegel and his collaborators (31) of its effectiveness
against cancer of the bladder.  It is our hope that a thorough trial will
be given to this valuable substance, ascorbic acid, which may turn out
to be the most valuable of all substances in the armamentarium of ortho-

molecular medicine.