[Inhibition of bladder cancer cell growth and angiogenesis by co-blockage of vascular endothelial growth factor and its receptor KDR].
OBJECTIVE
To study the effect of co-blockage of vascular endothelial growth factor (VEGF) and its receptor (KDR) on growth of bladder carcinoma T24 cells and nude mice xenograft.


METHODS
T24 cell line co-transfected with VEGF siRNA and sKDR expression plasmids was developed and its proliferation was assayed by MTT and apoptosis by FCM. The nude mice model bearing bladder carcinoma xenograft was established. The tumor cell VEGF expression, stroma microvessel density (MVD) and tumor cell topoisomerase II alpha (Topo II alpha) expression were detected by immunohistochemistry. Cell apoptosis was estimated by TUNEL assay.


RESULTS
MTT assay showed that cell proliferation in VEGF siRNA, sKDR and combination groups was 56.3% +/- 8.3%, 42.6% +/- 13.8% and 32.5% +/- 4.3%, respectively, significantly lower than that in the scramble control (97.3% +/- 11.6%, P < 0.0001). FCM showed there were sub-diploid apoptotic peaks before G1 phase in VEGF siRNA, sKDR and combination groups, and apoptosis ratio was 5.1% +/- 0.9%, 4.2% +/- 0.5% and 8.8% +/- 0.7%, respectively, all of which were higher than that in the scramble control (0.9% +/- 0.4%, P < 0.05), and the combination group had even more higher apoptosis than the two singlely treated groups (P < 0.01). In vivo test showed that tumor growth was inhibited in VEGF siRNA, sKDR and combination groups, and from day 16 the tumor volume in combination group was significantly smaller than that in scramble control (P < 0.05), and from day 28 the tumor almost lost the ability to further growth. Immunohistochemistry revealed VEGF expression in combination group was 54.37 +/- 5.28, significantly lower than that in the scramble control (141.66 +/- 8.59, P < 0.0001). MVD number was only 8.22 +/- 3.79, much less compared with that in the scramble control (61.76 +/- 5.28, P < 0.0001) or sKDR group (19.46 +/- 4.16, P = 0.0089). Tumor cell proliferation index in the combination group (1.5% +/- 0.7%) was significantly decreased compared with that in the scramble control (11.8% +/- 5.2%, P < 0.0001), and apoptosis index (67.2% +/- 8.5%) was much higher than that in the scramble control (8.7% +/- 2.7%, P < 0.0001), VEGF siRNA group (54.3% +/- 4.8%, P = 0.0492) or sKDR group (52.3% +/- 6.4%, P = 0.0293).


CONCLUSIONS
VEGF siRNA or sKDR alone can inhibit tumor cell proliferation and induce cell apoptosis, but co-blockage of VEGF and KDR by their combination shows more significant therapeutic efficacy.
Chen
Xiu-ling
XL
Department of Urology, The Second Hospital of Jilin University, Changchun 130041, China.
Liu
Lu-cheng
LC
Xu
Zong-ge
ZG
Li
Zhe
Z
Li
Ran-wei
RW
Gao
Rui-juan
RJ
Wang
Song
S
Zhang
Ming
M
Guo
Hang
H
D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D047368:Tumor Burden; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2