Anti-neoplastic properties of human corticotropin releasing factor: involvement of the nitric oxide pathway

We report a series of the in vivo and in vitro studies that evaluate the anti-neoplastic potential of hCRF in W256 rat mammary carcinoma. Using magnetic resonance imaging (MRI) and direct measurements of tumor and peritumoral brain water content we found that hCRF treatment (100 micrograms/kg subcutaneously twice a day for 3 days) caused significant inhibition of growth and vascular permeability of the i.c. W256 tumors. hCRF also exhibited antiproliferative and differentiation-inducing effects in W256 cells in vitro. The calculated IC50 values were 70 nM and 100 nM of hCRF, as measured by digital videomicroscopic quantitation of tumor cell population growth rate and by [3H]-thymidine incorporation assay, respectively. The observed effects in W256 cells were CRF receptor mediated. This was shown in two ways: by the presence of relatively high levels of CRF1 receptor mRNA in W256 cells, and by the fact that the tumor growth inhibitory and differentiation inducing effects of hCRF in vitro were abolished by the CRF receptor antagonist a-helical CRF (9-41). Antiproliferative and differentiation inducing effects of hCRF in W256 cells involve activation of nitric oxide synthase (NOS) and L-arginine-NO pathway. This was shown by using the inhibitor of NOS, the L-nitro-arginine methyl esther (L-NAME), which prevented the antiproliferative and differentiation inducing effects of hCRF in vitro. The cytotoxicity of NO in W256 cells was assessed by the addition of sodium nitroprusside (SNP) to the media. SPN exhibited dose-dependent cytotoxicity in W256 cells with IC50 of 100 muM SNP as measured by [3H]-thymidine incorporation assay. We conclude, that hCRF has substantial anti-neoplastic effects which include inhibition of proliferation and induction of differentiation of the tumor cells in vitro, and a decrease in tumor vascular permeability (and possibly neo-angiogenesis) in vivo.