Anti-tumor effect of recombinant plasminogen kringle 1-3 in KB cells : Histological observation and immunoprecipitation-based high performance liquid chromatography

Abstract

Angiostatin containing plasminogen kringle 1-4 domain is an endogenous angiogenesis inhibitor with anti-tumor effect. The present study applied recombinant human plasminogen kringle 1-3 (rPK1-3) in KB cell culture and BALB/c-nude mice, to elucidate the in vitro and in vivo anti-tumorigenic effect. Nude mice (n = 4) treated with rPK1-3, were compared to cisplatin treated nude mice (n = 4) and saline controls (n = 2). In vitro, the KB cells were also exposed to 10 μM of rPK1-3 for 12 and 24 hours. Cells were evaluated histologically, and their protein expression was analyzed by immunoprecipitation-based high performance liquid chromatography (IP-HPLC). Results revealed that rPK1-3 potently inhibited the growth of KB cells in cell culture, but not in animal study. The KB cells treated with rPK1- 3 showed growth arrest with decreased expression of PCNA, Ki-67, and MPM2, but enhanced apoptosis and increased expression of p53, BID, BAK, BAX, and caspase 9. Reduced cellular adaptation was also observed, with decreased expression of TGF-β1, SMAD4, pAKT, mTOR, EGFR, and PKC. Anti-angiogenesis effects with decreased expression of VEGF-A, VEGFR, angiogenin, vWF, LYVE-1, D2-40, CD31, MMP-2, and bFGF were also observed in cells treated with rPK1-3. These results suggest that in vitro, rPK1-3 has potent anti-tumorigenic effects, such as growth inhibition, increased apoptosis, reduced cellular survival, and tumor anti-angiogenesis. Therefore, rPK1-3 can be used as an anti-tumor agent for tumor cells in the absence of host systemic responses, including antibody formation.