Target lethal effect of recombinant soluble Fas coupled with protein kinase C inhibitor on colorectal carcinoma cells

Abstract

To study the target killing effect of soluble Fas(sFas) coupled with protein kinase C(PKC) inhibitor on colorectal carcinoma cells. The extracellular region of Fas protein was cloned and amplified by RT-PCR, and the expressing vector pGEX-4T-1-sFas was constructed. The sFas protein was purified by GST fusion protein purification system and coupled with Calphostin C(one kind of PKC inhibitor). The killing effect of soluble Fas coupled with PKC inhibitor on FasL-positive colorectal carcinoma cells was detected. After amplifying and cloning, the extracellular region of Fas protein, a 571 bp fragment, was proved by limited enzyme cutting and DNA sequencing. The expressed and purified protein was identified by Western Blot after transformed into E. coli BL21. The coupled sFas-Calphostin C showed suppressant activity on PKC kinase by the PKC kinase activity assay kit. The growth suppression rate of FasL-positive colorectal carcinoma HR-8348 cells treated with sFas-Calphostin C was significantly higher than that of FasL-negative cells, but the killing effect of sFas-Calphostin C on normal human monocyte was not obvious. Compared with 5-Fu alone, the growth suppression rate of FasL-positive colorectal carcinoma HR-8348 cells was significantly raised by sFas-Calphostin C combined with 5-Fu. The recombinant of soluble Fas and PKC inhibitor shows target killing effect on colorectal carcinoma cells.