Buforin IIb, a synthetic analog of antimicrobial peptide buforin II, is a cell-penetrating peptide with potent antimicrobial activity against various microorganisms. Buforin IIb showed cytotoxicity toward 62 cancer cell lines with $IC_{50}$ values in the ranges of 6-24 ug/ml, whereas normal cells were insensitive to buforin IIb up to 200㎍/ml. Buforin IIb rapidly penetrated the cell membrane without cell lysis and accumulated in the nucleus of the cancer cells. Exogenous gangliosides decreased cytotoxicity and cellular entry of buforin IIb in cancer cells. Moreover, the depletion of cell surface gangliosides or sialic acids reduced buforin IIb-induced cytotoxicity. These results indicate cell surface gangliosides or sialic acids might be the binding target of buforin IIb. Cancer cells treated with buforin IIb manifested apoptotic changes, including DNA fragmentation, annexin V staining, and activation of caspases and PARP.
Buforin IIb induced mitochondria-dependent apoptosis, as evidenced by activation of caspase-9 and cytochrome c release into cytosol. Gel retardation and Western blot analysis showed that buforin IIb can also bind to intracellular macromolecules, such as DNA, RNA, or heat shock proteins. Two-dimensional gel electrophoresis and mass spectrometric analysis showed that chaperones and heat shock proteins were over-expressed in buforin IIb-treated cells. In vivo analysis of anticancer activity showed that buforin IIb reduced the volume of fibrosarcoma masses in p53-deficient mice and killed almost all of the tumor cells. In addition, buforin IIb showed remarkable tumor suppressing activity above the concentration of 10 mg/Kg in a mouse tumor xenograft model.
