The Influence of Angiogeneic Gene Expression Following Management with Antineoplastic Agents to the Apoptosis of Malignant Tumor Cell

Kim, Young Jae; Lee, Yoon Young; Lee, Young Me; Chang, Sung Yeoul; Kim, Seung Ryong; Kim, Kyung Tai; Hwang, Youn Yeung; Cho, Sam Hyun; Ryu, Ki Young

Abstract

OBJECTIVE
We evaluated the alteration in the expression of the genes which are related to the angiogenesis in tumor bed following the management with antineoplstic agents to the various gynecologic cancer cell lines.
METHODS
To evaluate the alteration in the expression of the genes which are related to the angiogensis in tumor bed, VEGF, bFGF, and TSP-1, in parallel with the tumor cell proliferation inhibition, apoptosis, following the management with cisplatin(0, 30, 50, and 100 uM), paclitaxel(0.00, 0.02, 0.05, and 0.1uM), and thalidomide(0, 50, 100, 200ug/ml) in a dose-dependent fashion to the cell lines, Ovarian (A2780), cisplatin resistens ovarian(A2780 CDDP), breast (MCF-7), and squamous cell carcinom of uterus(SiHa,) cancer cell lines were investigated by means of MTT staining for formazen formation, FACS analysis for DNA damage, and isolation of total RNA and RT-PCR.
RESULTS
The percentage of the cell proliferation inhibition and apoptosis after management of cisplatin were 0.00, 21.07, 39.19, and 62.45%: 9.5, 13.65, 13.25, and 25.8% in A2780, 0.00, 14.23, 24.85, and 43.48%: 14.25, 17.22, 22.76 and 39.91% in A2780 CDDP, 0.00, 3.26, 8.54, and 29.11%: 24.84, 27.56, 29.47, and 56.07% in MCF-7, but the proliferation inhibition was not heralded under the concentration of 30 uM although increased percentage of apoptosis under the serial concentrations of cisplatin in SiHa. In the cases with paclitaxel in accordance with the concentration, the degree of proliferation inhibition revealed as 0.00, 16, 92, 27.68, and 51.75% in A2780, 0.00, 28.84, 41.24, and 46.6% in A2780CDDP, 0.00, 7.92, 29.23, 41.58% in MCF-7, and 0,00, 37.24, 40.23, and 42.57% in SiHa. but the majority of apoptosis had been developed under the 1st infusion dose with 0.02 uM. Because of this phenomenon, too narrow cytotoxic effect of paclitaxel, it is difficult to define relation between apoptosis and alteration of gene expression. The proliferation inhibiton and apoptotic effect of thalidomide were 0.00, 15.39, 19.06, 59.06% : 5.99, 8.49, 11.23, and 13.04% in A2780, 0.00, 1.97, 32.25, 50.42% : 12.16, 12.62, 12.77, and 13.48% in A2780CDDP, 0.00, 9.78, 30.65, and 50.27% : 27.00, 29.41, 30.37, and 36.11% in MCF-7, and 0.00, 9.87, 26.46, and 53.45% : 6.00, 6.96, 6.34, 7.09% in SiHa. Among the targeted genes which were related angiogenesis in tumor bed, VEGF, and bFGF were amplified as size as 212 bp sized VEGF, and 238 bp sized bFGF. However, 492 bp sized TSP-1 was not amplified in MGF-7 and SiHa under the treatment with cisplatin and paclitaxel, and A2780 CDDP, MCF-7, and SiHa which was managed with thalidomide. TSP-2 was not amplified at all in each targeted cell lines. The dose-dependent effect of targeted gene expression was not shown even in the cases with paclitaxel infusion, which was belong to the type 1 antangiogenesis agent which can be a direct effector to the VEGF, or bFGF in tumor vascular beds. Furthermore, cisplatin induced cytotoxicity was not developed in parallel with the alteration of expression of bFGF.
CONCLUSION
These result suggest that ongoing research for the evaluation of antiangiogenetic effect with various cytotoxic agents, the objective tool should be proposed for the fine differentiation of the real antiangiogentic effect of cytotoxic and antiproliferating agent to endothelial cell., especially in the experiments with type 1 antiangiogenetic agent. In the case with type 2 antiangiogentic agents, iIt will be helpful to construct the condition with vector related transfection to the targeted cell lines, and to include various cytokines as a target to know the paracrine effects of the tumor cells in normal environment of the animal or other host.