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There has been intense
interest in learning more about the specific nature of these
proteases, since inhibiting them would go a long way toward
preventing metastasis. Much of the focus has been on the family
of proteases called
matrix metalloproteinases
(MMPs). Members of
this family can degrade various components of the
connective-tissue matrix. The coordination and physiological
functions of the different active forms of the MMPs are poorly
understood.
[You can
hardly believe that I, with no technical training, DO understand
this function? Well, read and get your own opinion.
It is nevertheless clear that they are key to the
mechanisms by which metastatic cells migrate through tissue
compartments. In an in vitro model for malignant
progression in human squamous cell carcinoma, one of us (Meade-Tollin),
with coworkers at the University of Arizona at Tucson, has shown
that three different MMPs have quite different expression
patterns. We have observed that the expression of one of these,
matrilysin, exists in higher concentrations in cells that form
benign tumors, a presumed earlier stage in the progression to
malignancy, than in cells that form invasive tumors in vivo. It
is likely that different MMPs are involved at specific stages
during which cancer cells become metastatic. Determining the
levels of active MMPs in tissue could provide crucial
information about the mechanisms of regulation of MMP activity.
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Two other protease families are also under investigation for
activity during metastasis. The role of these families—cysteine
proteases, such as cathepsin-B, and serine proteases, such as
urokinase-type plasminogen activator—in cancer-cell invasion is
less clear.
It is possible that members of
some or all of these families of proteases interact during
metastasis. One current scenario postulates that MMPs are
secreted in their proprotein form by fibroblasts, but are
activated by either cathepsin-B or urokinase-type plasminogen
activator secreted by cancer cells.
The secretion of proteases,
such as cathepsin-B, by cancer cells demonstrates another way
these cells deviate from normal. Normally, cathepsin-B is
sequestered within the cell inside membrane-bound vesicles
called lysosomes. Lysosomes are the cellular trash cans that do
double-duty as recycling centers. These vesicles contain
enzymes—cathepsin-B among them—that can break down all
macromolecules into their constituent units so they can be
reused to make new macromolecules. Bonnie Sloane and her
colleagues at Wayne State University School of Medicine in
Detroit have discovered that cathepsin-B can be found in the
extracellular environment of invading cancer cells. Furthermore,
it appears that the invading cells not only secrete cathepsin-B,
but can subsequently bind it to their surfaces.
Recently, one of us (Van
Noorden) and colleagues were able to demonstrate that the
surface-bound form of the enzyme is active. Once the metastasis
is established at its new site, cathepsin-B continues to be
expressed on the membrane, but in an inactive form. Urokinase-type
plasminogen activator, like cathepsin-B, is present and active
on invading cells. Such observations suggest that these
proteases are at the head of a cascade of proteases that
ultimately activate MMPs, which in turn chew up proteins of the
connective matrix.
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Metastasis is therefore a complex process, requiring the cancer
cells first to release themselves of their intercellular
adhesive bonds, then leave their cellular microenvironment and
migrate through the connective tissue matrix encapsulating their
tissue compartment in order to gain access to the blood vessels
that carry them to a new organ. Once there, they must re-enact
this process in reverse. They must pass through the blood-vessel
wall into the new organ and there form new connections.
Circulation
After it has successfully
eaten through an organ's connective tissue, the cancer cell
makes its way into a nearby blood vessel, squeezing between the
endothelial cells lining the vessel lumen to enter the blood
vessel itself.
[The
word is to "anthropomorphize" means to "ascribe human
characteristics to something that is not human.
The image on
the left is a curiosity from Japan -- a set of "stickers" sold
as means of conveying an emotion for something. In other
words, these shapes are anthropomorphizes into human eyeballs,
and are further anthropomorphized so that some of the "look sad"
or "happy" or whatever. These "human characteristics" are
not inherent in abstract shapes, but man often ascribes human
characteristics to things not human.
In many cases
this is useful for some reason, but when a research scientist
refers to a cancer cell as "eating through some boundary" it
gives a false and deceptive implication as to what is actually
happening. It is not rare among scientists with an agenda.
Probably a
more accurate way to state this would be to say, "The cancer
cell has a basic motivation to survive, as do all living things.
The body, itself, as a unit, certainly has the motive to
survive. The body is the larger unit and it takes
precedence in survival decisions. When the death of one
cancer cell is compared to the death of the entire unit, it is
easy to see where the balance is.
But, to
ascribe more power or intelligence to a cancer cell than it has
is misleading. The cancer cell is normally tethered in
place, like the normal cell it replaced. When a free
radical causes, directly and indirectly, damage to the tether
material, it would be deceptive to say that the cancer cell
caused that change in the tether mechanism. Rather, it
would be more accurate to say that a free radical caused damage
to a "normal tethering" material, and that that damage released
the cancer cell from its mooring.
Is
the rowboat that drifts out to sea "escaping" the land? Or
is the rowboat simply being moved by wind and wave, and no
longer tied to the dock by a rope!
You COULD
anthropomorphize the boat and say "The
boat is escaping to the sea!"
This may be poetic, but it is not science!
Although the blood vessel
provides a conduit for the metastatic cell to reach new tissues,
the journey is fraught with peril—only 1 in 10,000 cells
survives it. First, there is a large probability the cancer cell
will be mechanically destroyed by the stresses that blood cells
alone are designed to endure. In addition, the bloodstream is
the very place where a cancer cell is likely to encounter the
types of white blood cells—such as macrophages and natural
killer cells—capable of destroying them, when the cancer cells
are recognized as foreign.
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Since cancers do take hold in new tissues, they obviously have
developed mechanisms for transiting through the bloodstream.
[Foolish!
Would you say that a screen with holes large enough to allow
some sand through, but not large enough to allow large pebbles
through -- would you say that the "Small
particles of sand have developed a way to move through the
screen?" This
"anthropomorphizing" puts your attention on the "evil cancer
cell" rather than the true cause - a free radical.
For
example, they may travel in clusters, increasing the possibility
that at least one of them will survive. Or they may surround
themselves with blood cells such as platelets, which mask cancer
cells from immune surveillance. (Platelets are blood cells
involved in clotting.)
Cancer cells that survive the
trip through the bloodstream ultimately home in on a new tissue.
The selection of the new target is often quite specific to the
type of cancer cell. For example, colon-cancer cells have a high
affinity for the liver, whereas lung-cancer cells often
metastasize to the brain, bones, adrenal glands and pancreas.
The choice of target is quite likely determined by very specific
interactions between molecules on the cancer-cell surface and
molecules on the surfaces of the endothelial cells that line the
blood vessels in the new host tissue.
The entire range of specific
interactions is not yet known, but it seems likely that
carbohydrates protruding from the cancer-cell surface become
bound to a type of carbohydrate receptor on the endothelial
cells called a selectin. Normally, the carbohydrate-selectin
interactions are used by white blood cells that need to identify
particular tissues to combat local infection. Apparently cancer
cells can exploit this system as well. Each cancer-cell type
expresses a different set of carbohydrates on its surface, which
would be attracted to different selectin molecules. The
specificity of these interactions helps account for the
differential homing specificities of different types of cancer
cells.
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Once the cancer cell contacts a surface to which it can adhere,
it rolls along the blood-vessel wall, propelled by the
bloodstream, because the carbohydrate-selectin interactions are
relatively weak. The cell comes to a complete stop as bonds,
mediated by integrins, form between the cells. At this point,
the cancer cell enacts a series of events that is almost the
reverse of the events that allowed it to leave its primary
organ. The cancer cell migrates into the host tissue by passing
through the blood-vessel wall and degrading the
connective-tissue matrix with proteases. The cancer cell is now
ready to proliferate and form a new tumor in its new host
tissue.
[Could
you not say, with equal logic, that "An
area weakened by free radical damage no longer prevents
wandering cells from entrance?"
Fighting Cancer and Its Spread
It is the fondest wish of all
scientists who study cancer that it can be defeated. Scientists
ardently hope to find some very precise way to curtail cancer's
unrestrained growth without harming healthy cells, an advance
that would represent a vast improvement over therapies currently
available.
[What trash!
Those ways have been found. They don't happen to be drugs,
so these scientists have very narrow tunnel vision -- they can
only see drug remedies, and only those where a patent is
possible.
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Right now, when cancer is diagnosed, the tumor is often removed
surgically. But there is always the possibility that some cancer
cells remain at the original site, and that others may have
already started to migrate to distant organs. So the patient is
given radiation, which can eradicate cells by apoptosis.
Radiation can be applied very specifically to sites in the body
where the primary tumor was located in order to destroy any
remaining cancer cells. But if undetected metastases are present
elsewhere in the body, they go untreated.
For this reason, radiation is
often given in conjunction with chemotherapy. The chemicals used
in chemotherapy are almost all designed to curtail division and
proliferation. The rationale is that cancer cells are dividing
more rapidly than other cells in the body
[except that the immune system cells multiply far more rapidly
than cancer cells, so the chemotherapy does kill cancer cells,
yes, but also kills the very agents in the body which should
naturally defeat the cancer] and are therefore most
vulnerable to the effects of the chemotherapeutic agents.
However, chemotherapy is quite a blunt weapon, and many normal
cells with high turnover rates, such as skin, hair and blood
cells, are affected along with the cancer cells. Sometimes
cancer cells develop resistance to chemotherapy and become
insensitive to its effects. Once that happens, the cell actually
pumps out drug molecules, leaving itself and its progeny
unharmed. Scientists therefore hope to target aspects of cancer
cells that are different from healthy cells in order to develop
drugs and therapies that attack the cancer cells only.
In the past decade several new
therapies have sought to boost the patient's immune system. The
approach grows out of the assumption that in cancer patients,
the immune system is not able to effectively eliminate all of
the cancer cells. The hope is that stimulating the immune system
will increase the patient's ability to kill off cancer cells.
[They have
thrown a "sop" to helping the immune system, but generally ALL
drugs damage the immune system! The stuff that helps the
immune system does not come from drug companies, but from
vitamin companies.
So far, this approach has been
somewhat disappointing. In the test tube, experiments that
combine immune and cancer cells are very definitely promising.
The immune cells are effective in killing the cancer cells. But
the situation is not repeated in vivo, where the
complexity of whole-animal systems confounds the interactions
between immune and cancer cells. Patrizia Griffini, in the Van
Noorden laboratory, and other investigators have reported that
in vivo, cancer cells do attract the attention of
immune cells. Once the immune cells come face to face with the
cancer cells, however, nothing seems to happen. The immune cells
fail to launch the attack.
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It seems that cancer cells have acquired the means to secrete
large amounts of immunosuppressive messenger molecules, such as
interleukin-10, transforming growth factor b and prostaglandin E2.
Cancer cells also secrete molecules such as a2-macroglobulin,
which scavenge immune-activating cytokines and
cancer-cell-destroying proteases. In short, the cancer cells are
extremely effective at manipulating their own microenvironments
to their advantage.
Cancer cells may have an
additional, and quite unexpected, effect on the immune cells
that are supposed to kill them. Recent work by Jurg Tschopp and
coworkers at the University of Lausanne in Switzerland
demonstrated that the cancer cells may turn the tables on
precisely those tumor-infiltrating immune cells deployed to
fight the cancer. This work reveals that it is the cancer cells
that in some cases are killing the immune cells.
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Immune-killing cancer cells seem to have co-opted a mechanism
usually employed by the killer immune cells. Almost all cells,
including the cells of the immune system, carry a particular
molecule on their surfaces called Fas. Fas is actually a
receptor molecule that binds to another molecule called the
Fas-ligand (FasL), which is normally expressed by immune cells.
Under normal circumstances, immune cells hook their FasL
molecule into the Fas receptor of the diseased cell. This
interaction triggers a signal that causes the diseased cell to
undergo apoptosis.
The Tschopp laboratory found
that cancerous skin cells, specifically melanoma cells, also
express FasL, whereas normal skin cells do not. Furthermore,
these melanoma cells no longer express the Fas receptor. So when
melanoma cells contact immune cells, they hook their FasL
molecule into the immune cell's Fas receptor and cause the
immune cell to undergo apoptosis. In the meantime, the melanoma
cell is unaffected.
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Current research is seeking to exploit such interactions to
develop new anticancer therapies. For example, Claudia Friesen
and her coworkers at the University of Heidelberg, Germany,
found that the common anticancer drug doxorubicin enhances
expression of both Fas and FasL on cancer cells. In effect, this
drug causes cancer cells to kill themselves by inducing
apoptosis.
Another promising class of
anticancer drugs attempts to stop angiogenesis at the site of
the tumor. Several recently characterized molecules, such as
angiostatin and endostatin, have been shown to inhibit
angiogenesis in laboratory animals. When inhibitors of
angiogenesis are given to tumor-bearing animals, the tumors stop
growing or, in some cases, even shrivel up. Several of these
inhibitors of angiogenesis are in various phases of clinical
trials and may prove to be potent against both primary cancers
and secondary metastatic tumors alike. Such drugs, however, can
potentially inhibit angiogenesis when it is wanted, for example,
in wound healing. One solution to this problem may be to
administer clotting agents selectively and directly to the
tumor's blood supply, while leaving the normal blood supply
unaffected.
Many recent experimental
strategies against cancer attempt to compensate for or correct
defective genes. The ras gene, for example, is mutated
in a large number of human cancers. The Ras protein encoded by
this gene is part of a pathway that responds to external growth
signals by telling the cell to divide. The mutated gene encodes
a protein that is permanently activated. That is, it is
continually directing the cell to divide. One of the first steps
in this pathway is catalyzed by an enzyme called farnesyl
transferase, for which effective inhibitors have now been
produced.
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Other research strategies focus on restoring the function of
growth-inhibiting genes. The favored strategy right now is gene
therapy, which seeks to replace a mutated gene with a
functioning one. Although this approach is likely to generate
important therapies in the future, results so far have been
disappointing. It is difficult to get the replacement gene to
function properly and at levels that deliver therapeutic
benefits.
In addition to efforts that
seek to fight the primary tumor, much work has been focused on
halting metastasis. Two main targets have provided the focus for
new antimetastatic strategies. One target has been the adhesion
molecules that link a cell to its original organ and that later
allow it to attach to a new organ. The second focus for drug
intervention has been the proteases that allow cells to chew
themselves out of and into tissues.
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As we have already seen, cells become unattached from their
hosts by ceasing to make the adhesion molecules—such as
E-cadherin—that connect them to surrounding cells. The obvious
antimetastatic strategy would be to restore adhesion-molecule
synthesis and prevent cancer cells from leaving their original
site. One very active avenue of research involves
gene-replacement therapy, in which functioning genes for the
appropriate adhesion molecules could be delivered to cancer
cells. This approach has worked in the test tube, but it is
still far from being applicable at the bedside.
It may also be possible to
modulate the relationship between integrins and the molecules to
which they bind in the connective-tissue matrix. Integrins may
be involved in angiogenesis as well as cell migration and the
interactions between cancer and endothelial cells. For many
integrins, the specific sequence of amino acids
arginine-glycine-aspartate seems to be important for adhesion.
Interventions have targeted this particular sequence, and have,
in fact, been shown to reduce development of metastatic tumors
in experimental situations. Still, these interventions are not
yet ready for clinical applications.
Also interesting targets for
drug intervention are the various proteases that allow
metastatic cells to enter and exit tissues. Currently, only one
inhibitor of MMPs, developed by British Biotech in Annapolis,
Maryland, is in clinical trials. Typically, MMP inhibitors are
not soluble in water, which limits their usefulness as drugs.
But the new drug, called marimastat, is water-soluble. In animal
models of cancer, marimastat strongly inhibits tumor development
and metastasis and increases the animals' survival rate.
Examination of the tissue of animals that have received this
drug reveals an increase in the amount of connective tissue in
and around the tumors as compared with untreated animals,
suggesting that the drug is effective in halting the degradation
of connective tissue by MMPs. This drug is even more potent in
combination with the drug cisplatin.
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The drug has been clinically tested on people in advanced stages
of cancer. It was found to significantly increase survival with
only the minor side effects of stiffness, local pain and
discomfort. Protease inhibitors such as marimastat seem very
promising for reducing the incidence of metastasis in patients
once a primary tumor has been diagnosed.
Members of the Van Noorden
laboratory are investigating inhibitors of other proteases
involved in metastasis. We have recently been working with a
specific small, water-soluble inhibitor of cathepsin-B, which
seems to affect normal cells very little. We have found that the
drug inhibits only the cathepsin-B on the cell membrane and not
the internal lysosomal stores of the protease. This differential
and complete inhibition of extracellular, but not intracellular,
activity indicates therapeutic promise, because it blocks only
the pathologically expressed cathepsin-B and not the
physiologically required stores. We gave the drug orally to rats
with colon cancer that had metastasized to the liver and found
that the number of their tumors was reduced by one-third, and
the size of the tumors was reduced by two-thirds. The study
strongly supports the notion that cathepsin-B is involved in
colon-cancer metastasis to the liver.
Diet and Cancer
In the future, the question of
how to treat cancer and its spread may take a back seat to
strategies that prevent its development altogether. Scientists
have increasingly discovered links between the environment, diet
and the health of the individual. The hope in all of these
studies is to eliminate the environmental dangers and educate
individuals about how they can protect themselves and their
bodies from agents that promote cancer.
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Recently, much research has focused on dietary nutrients that
might actually protect people from developing cancer. Among the
agents under exploration are vitamins E and C, selenium, wine
and substances from plants, called phytochemicals.
In the Van Noorden laboratory,
we are looking at the relation between fatty acids, cancer and
metastasis. We have found that omega-3 polyunsaturated fatty
acids (PUFAs), which are found in fish such as salmon and
mackerel, may help to prevent the development and progression of
primary cancers, whereas omega-6 PUFAs from plants actually seem
to promote tumor growth. It was recently demonstrated by us that
omega-3 PUFAs inhibit proliferation of normal cells in vivo,
whereas omega-6 PUFAs did not affect cell proliferation very
much.
These effects can be explained
by the formation of lipid peroxidation products that can damage
DNA when it is uncovered during the cell cycle. Therefore, lipid
peroxidation is kept to a minimum during the cell cycle of
normal cells. However, lipid peroxidation products are generated
in large amounts in cells that contain high levels of omega-6
and omega-3 PUFAs. Our investigations indicate that the
inhibiting effect of omega-3 PUFAs on cell proliferation is
mediated by lipid peroxidation products.
Since PUFAs seem to be
important in preventing primary cancers, members of the Van
Noorden laboratory were interested in seeing whether they had
any effect on reducing metastatic tumors. Specifically, we
explored the effect of PUFAs on colon cancers that metastasized
to the liver in rats. Quite to our surprise, we found that, in
rats, fish-oil treatment actually promoted the development of
metastatic tumors in the liver. In fact, the results were
dramatic. Fish oils caused a 10-fold increase in the number of
tumors and a 30-fold increase in their size. Plant PUFAs did not
affect the number of tumors, but their size was increased by
10-fold, as compared with animals on a low-fat diet. We believe
the fish oils had the effect they did because the liver tumors
were completely lacking the connective tissue known as stroma,
which is normally found in and around tumors. It seems that
tissues often try to encapsulate the tumor in stroma to limit
its spread into the tissue. Our results would suggest that
stroma formation is far more important in defending the tissue
from cancerous invasion, at least in the liver, than we
previously appreciated.
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Our data also show that omega-3 PUFAs have at least two
contradictory effects in relation to metastases in the liver.
First, omega-3 PUFAs restrict cellular proliferation, as was
shown in the first study. However, they also have the effect of
inhibiting stroma formation in and around the tumors. This
latter effect means that tumors can grow much faster in spite of
the fact that omega-3 PUFAs inhibit cellular proliferation. We
are now designing studies to investigate the effects of omega-3
PUFAs in the diets of colon-cancer patients at risk of
developing liver metastases.
Until such a time that
scientists become informed enough to prevent cancer, it is our
hope that as processes crucial for the development of cancer and
metastasis are ever more clearly delineated, this understanding
will lead to more rational therapies. The new therapeutic
interventions discussed hold great promise for slowing down or
preventing cancer progression. The next generations of these
drugs and strategies may even cure the disease someday. Since
metastasis is the main cause of death in cancer patients, it
will be a prime target for research to develop treatments that
are now so desperately needed.
