Objective: To investigate the effects of transfection of human plasminogen Kringle 5 gene on pancreatic cancer.
Methods: Human pancreatic cancer cells of the line PC-3 were cultured. Recombinant plasmid RINGLE 5 containing human plasminogen Kringle 5 cDNA was transfected into the PC-3 cells mediated by lipodectAMINE. RT-PCR was used to detect the expression of Kringle 5. Another 2 groups of PC3 cells: PC3 cells cultured in pure culture medium and those transfected with blank plasmids were used as controls. MTT method was used to draw the growth curves of cells. Immunohistochemistry was used to detect the expression of Kringle 5. Flow cytometry was used to observe the cell cycle and apoptosis. The morphology of cells was observed by transmission electron microscopy. Human umbilical vein endothelial cells ECV304 at logarithmic growth phase were cultured and the supernatants of the 3 groups of PC3 cells were added into the culture fluid respectively. MTT method was used to detect the absorbance and draw the growth curves. Twenty-one BALB/c nude mice were randomly divided into 3 groups of 7 mice to be inoculated with these 3 different PC3 cells. The appearance and size of tumor were observed continuously.
Results: Kringle 5 was expressed in the PC3 cells transfected with Kringle 5. The proliferation in the PC3 cells transfected with Kringle 5 became significantly lower compared with the other groups of PC3 cells 3 or 7 days respectively after transfection (P = 0.045 or P = 0.038). Four days after the addition of the different kinds of supernatant the ECV304 cells grew significantly slower in the Kringle 5 group than in the other 2 groups (P = 0.041). Immunohistochemistry showed high expression of Kringle 5 protein in the PC3 cells transfected with Kringle 5 and very weak expression in other 2 groups. The apoptotic indices of the PC3 cells, PC3/PUCKRINGLE 5 cells, and PC3/Kringle 5 cells were 6.6% +/- 1.3%, 7.3% +/- 0.9%, and 12.1% +/- 2.3% respectively (P = 0.045). The S stage change rates of the PC3 cells, PC3/PUCKRINGLE 5cells, and PC3/Kringle 5 cells were 33.3% +/- 3.7%, 19.4% +/- 2.4%, and 7.4% +/- 3.3% respectively. Transmission electron microscopy showed significant apoptosis in the PC3 cells transfected with Kringle 5 gene. Since the 5th week after inoculation the tumor grew significantly slower in the Kringle 5 group than in the other 2 groups (P = 0.032). Since the 7th week the tumor growth remarkably slowed down (P = 0.05).
Conclusion: PC3 cell successfully transfected with K5 gene can potently inhibits endothelial cell proliferation and the growth of pancreatic cancer in nude mouse. The study provides a promise for the anti-angiogenesis gene therapy in vivo.