Pancreatic (pro)enzymes treatment suppresses BXPC-3 pancreatic Cancer Stem Cell subpopulation and impairs tumour engrafting.

Cancer stem cells (CSCs) subpopulation within the tumour is responsible for metastasis and cancer relapse. Here we investigate in vitro and in vivo the effects of a pancreatic (pro)enzyme mixture composed of Chymotrypsinogen and Trypsinogen (PRP) on CSCs derived from a human pancreatic cell line, BxPC3. Exposure of pancreatic CSCs spheres to PRP resulted in a significant decrease of ALDEFLUOR and specific pancreatic CSC markers (CD 326, CD 44 and CxCR4) signal tested by flow cytometry, further CSCs markers expression was also analyzed by western and immunofluorescence assays. PRP also inhibits primary and secondary sphere formation. Three RT2 Profiler PCR Arrays were used to study gene expression regulation after PRP treatment and resulted in, (i) epithelial-mesenchymal transition (EMT) inhibition; (ii) CSCs related genes suppression; (iii) enhanced expression of tumour suppressor genes; (iv) downregulation of migration and metastasis genes and (v) regulation of MAP Kinase Signalling Pathway. Finally, in vivo anti-tumor xenograft studies demonstrated high anti-tumour efficacy of PRP against tumours induced by BxPC3 human pancreatic CSCs. PRP impaired engrafting of pancreatic CSC's tumours in nude mice and displayed an antigrowth effect toward initiated xenografts. We concluded that (pro)enzymes treatment is a valuable strategy to suppress the CSC population in solid pancreatic tumours.

In vitro treatment of carcinoma cell lines with pancreatic (pro)enzymes suppresses the EMT programme and promotes cell differentiation.

BACKGROUND: Previous research has suggested a putative utility of pancreatic (pro)enzymes in cancer treatment. The aim of the present study was to investigate the in vitro effects of a mixture of two pancreatic pro-enzymes, i.e., Chymotrypsinogen and Trypsinogen, and the enzyme Amylase on three human cancer cell lines, i.e., OE33 (derived from an oesophageal carcinoma), Panc1 (derived from a pancreatic carcinoma) and Caco-2 (derived from a colon carcinoma). RESULTS: After treatment of the three cancer cell lines with different doses of the (pro)enzymes for up to 7 days, we observed (i) growth inhibition in a dose-dependent manner, (ii) enhanced expression of ß-catenin and E-cadherin and decreased expression of several epithelial-mesenchymal transition (EMT)-associated genes, such as Vimentin, Snail and Slug, (iii) differentiation of Caco-2 cells, including the appearance of cell-specific differentiated structures such as microvilli and tight junctions, the acquisition of a more regular polygonal morphology, and an increased expression of the intestinal differentiation markers alkaline phosphatase and cytokeratin 8, and (iv) differentiation of Panc1 cells, including the formation of cell aggregates, an increment on lamellar bodies and an increased expression of the pancreatic differentiation markers glucagon and insulin. CONCLUSIONS: Our results show that the treatment of three different human cancer cell lines with pancreatic (pro)enzymes results in an enhancement of cell adhesion, an attenuation of several EMT-associated markers, and an increase in the expression of several differentiation-associated markers, suggesting the acquisition of a less malignant phenotype and a decrease in proliferative capacity due to lineage-specific cellular differentiation.

Proenzyme therapy of cancer.

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.

I-Kk and H-2Kk antigens are shed as supramolecular particles in association with membrane lipids.

The form of 125I-labeled membrane alloantigens (I-Ak and H-2Kk) and immunoglobulin M shed by murine splenic lymphocytes labeled by lactoperoxidase-catalyzed radioiodination was determined by gel chromatography and ultracentrifugation. I-Ak and H-2Kk antigens are shed as particles ranging in size from 3 X 10(5) to 2 X 10(6), whereas membrane IgM is shed as an individual unit consonant with its native size of 180,000. The effects of detergents on the size of shed I-Ak and H-2Kk and the coprecipitation of (3H) lipids with these proteins suggest that I-Ak and H-2Kk are shed in association with local membrane lipids as small membrane-derived vesicles, whereas membrane IgM is shed without detectable associated lipid.

Pancreatic enzyme stimulation of intestinal chloride secretion.

Mucosal addition of pancreatic enzymes (i.e., chymotrypsinogen and amylase) to a short-circuited Ussing chamber containing a section of stripped rabbit ileum greatly increases short-circuit current (SCC). SCC increases slowly, requiring several hours to reach peak response to enzyme addition. Serosal addition of enzyme or mucosal addition of albumin produces no such response. Chloride flux in the absence of enzyme conforms to behavior predicted for predominantly paracellular movement. Chymotrypsinogen in the mucosal bath augments serosal-to-mucosal chloride flux in a manner consistent with an intracellular pathway. The chloride secretion produced by enzyme addition is of similar magnitude to the additional increment in SCC.