Corticotropin-Releasing Factor: A Key Element in Cancer Anorexia-Cachexia Syndrome

Abstract

Myelodysplastic syndrome (MDS) is clonal disease of hematopoietic precursor cells that is characterized by pancytopenia. Patients with MDS have an increase in the risk of developing acute myeloid leukemia (AML), characterized by unlimited growth and accumulation of blast cells in hematopoietic tissues. Blast cells derived from a group of patients with AML produce and secrete tryptase. These patients exhibit increased expression of bone marrow tryptase and serum tryptase levels. There is evidence that PAR-2, cleaved and activated by tryptase, contributes to tumor progression and proliferation in various cancer cell types. However, the role of PAR-2 in the context of myeloid leukemia remains unknown. Aims. The aim of the present study was to determine the effect of PAR-2 activation on leukemic cell survival and proliferation. Methods. Protein expression of PAR-2 was examined in K562 cells, a human myeloid leukemia cell line, by RT-PCR. K562 cells were treated with the PAR-2 activating peptide SLIGRL-NH2 (1, 10 and 100 μM) for up to 72 hours in serumfree media. Media containing serum and serum free media were used as positive and negative controls, respectively. Cell proliferation in response to agonist peptide was evaluated by the MTT assay after exposure to serum-free media, SLIGRL-NH2 or serum-containing media. Caspase-3 and cleaved caspase-3 protein levels were determined by Western blots in the combined presence of PAR-2 activating peptide and cyclohexamide (50 μg/ml). Results. PAR-2 was detected in K562 cells. These cells showed a significant increase in cell growth as determined by MTT assay after treatment with serum-containing media. However, PAR2 activating peptide did not significantly alter cell growth as determined absorbance by MTT assay when compared to controls at any of the time points examined. Treatment with cyclohexamide alone significantly increased cleaved caspase-3. The PAR-2 activating peptide alone did not alter K562 cell apoptosis, as indicated by measurement of cleaved caspase-3. However, the effect of cyclohexamide on caspase-3 cleavage was significantly decreased in the present of PAR-2 activating peptide, suggesting anti-apoptotic activity. Conclusions. Our studies show that PAR-2 activation does not stimulate cellular proliferation in myeloid leukemia cells. Instead, PAR-2 activating peptide reduces leukemic cell response to cyclohexamide-induced apoptosis. The results imply that enhanced tryptase levels during MDS or AML may, via activating PAR-2, offer protection of malignant cells against chemotherapeutic agents thereby result in a survival advantage of the malignant clone.