Unique Recombinant Human Ribonuclease and Inhibition of Kaposi's Sarcoma Cell Growth

Background: Preparations of human chorionic gonadotropin (hCG) have been shown to exhibit anti-Kaposi's sarcoma (KS) activity, but the identity of the responsible agent(s) remains controversial. One candidate agent is an eosinophilderived neurotoxin (EDN)-like polypeptide that contaminates preparations of hCG. We have genetically engineered a unique form of hEDN, which is a ribonuclease, and have evaluated the cytotoxic effects of the recombinant protein on KS Y-1 cells and on cells of other cancer types. Methods: The amino-terminus of hEDN was extended by four amino acid residues, corresponding to the proximal part of the hEDN signal peptide (serine, leucine, histidine, and valine; positions -4 to -1, respectively), by use of the polymerase chain reaction and an hEDN complementary DNA. The recombinant protein was isolated from bacterial inclusion bodies. The cytotoxic activity of this hEDN variant, (-4)rhEDN, was tested on KS Y-1 cells and human glioma, melanoma, breast carcinoma, and renal carcinoma cells. Results: Approximately half of the anti-KS activity in a crude commercial preparation of hCG was associated with a polypeptide that reacted with anti-recombinant-hEDN (rhEDN) polyclonal antibodies. Although rhEDN protein displayed little cytotoxicity against KS Y-1 cells (IC50 [50% inhibition concentration] = >100 µg/mL), (-4)rhEDN markedly inhibited cell viability (IC50 = 6 µg/mL). Neither version of rhEDN inhibited the viability of other tested human cancer cell types. Conclusions: A four amino acid extension of the amino-terminus of rhEDN confers cytotoxicity against KS Y-1 cells in vitro. Design of the (-4)rhEDN variant was based on the sequence of a natural human protein associated with hCG. Our results suggest that (-4)rhEDN is one of the agents in hCG responsible for anti-KS activity. A purified molecule is thus available for in vitro and in vivo mechanistic and, possibly, future clinical studies.