Proteases and their inhibitors have long been investigated in numerous
tumor systems, and at the
tumor growing front, their balance has been universally found to be shifted towards higher proteolytic activities. However, out of many promising
serine and
metalloproteinase inhibitors, none are included in
cancer treatment regimens at present. The current search for active antiproteolytic compounds is in contrast to the classical approach developed by John Beard, who suggested treating advanced
cancer by fresh
pancreatic extracts whose antitumor activity was based on their proteolytic potential. We followed John Beard's recommendations by using purified pancreatic
proenzymes/
enzymes,
trypsinogen/
trypsin (TG/TR),
chymotrypsinogen/
chymotrypsin (CG/CH) and
amylase (AM). The mixture of these enzymatic activities produces potent antimetastatic and antitumor effects in cellular, animal and human systems. The treatment of cultured tumor cells with TR and CH at nanomolar [corrected] concentrations, comparable to those achieved in the blood of the patients, causes complete arrest of the directional movement of metastatic cells. Conversely, the same treatment of normal cells results in enhanced motility and an accelerated closure of the gap created in cell monolayers. Further, treatment of cells with
serine proteases results in the formation of cellular 3-dimensional structures such as lamellae, cell streams and aggregates. In some cell types, the aggregates are compacted via
cadherin-based cell-cell communication systems and form compact spheroids. In the highly metastatic cells with lower
cadherin expression, the ability to form spheroids also diminishes.
Tumor cells unable to form spheroids when treated with
proteases are subject to elimination by apoptosis. In contrast, a large proportion of cells that form spheroids remain viable, although they are metabolically suppressed.
Protease-treated
tumor cells contain a disrupted actin cytoskeleton and exhibit a loss of front-to-back polarity. We hypothesize that the provision of
zymogens, rather than the
enzymes, was of crucial importance to the clinical effectiveness in the human trials conducted by Beard and his co-workers. The precursor nature of the active
enzymes may offer protection against numerous
serpins present in the tissues and blood. Experimental evidence supports the assertion that the conversion from
proenzyme to
enzyme occurs selectively on the surface of the
tumor cells, but not on normal cells. We believe that this selectivity of activation is responsible for the antitumor/antimetastatic effect of
proenzyme therapy and low toxicity to normal cells or
tumor host. Elevated levels of
endostatin and
angiostatin appear in the blood of TG/
CG/AM-treated
tumor-bearing mice, but not in
tumor mice treated with the vehicle alone or in
proenzyme-treated
tumor-free mice. These findings support the conclusion that proteolysis is the active mechanism of the
proenzyme treatment. Future studies will focus on the molecular mechanisms of the
proenzyme therapy including the identification of molecular target(s) on the
tumor cells. In conclusion, we have discovered that
proenzyme therapy, mandated first by John Beard nearly one hundred years ago, shows remarkable selective effects that result in growth inhibition of
tumor cells with metastatic potential.