Reduction of hypoxia-induced angiogenesis in ovarian cancer cells by inhibition of HIF-1 alpha gene expression.
OBJECTIVE
The goal of this study was to investigate the effects of silencing HIF-1 alpha gene expression with specific small interfering RNA (siRNA) on VEGF production and angiogenesis in epithelial ovarian cancer (EOC) cells.


METHODS
Two EOC cell lines, MDAH-2774 and SKOV-3, were cultured under normoxic (20% O(2)) and hypoxic (2% O(2)) conditions using standard techniques. After EOC cells were transfected with siRNA, HIF-1 alpha and VEGF mRNA levels were measured by real-time RT-PCR. Angiogenesis was evaluated utilizing an in vitro assay model consisting of human umbilical vein endothelial cells (HUVEC) and polymerized ECM Matrix.


RESULTS
Both EOC cell lines evaluated constitutively expressed HIF-1 alpha and VEGF mRNA. HIF-1 alpha and VEGF mRNA levels were significantly increased in response to hypoxia (P < 0.05). Under hypoxic conditions, inhibition of HIF-1 alpha gene expression by a specific siRNA resulted in a significant reduction in HIF-1 alpha and VEGF mRNA levels (P < 0.05). In the in vitro angiogenesis model, supernatant from the hypoxic EOC cells induced the HUVEC to form a complex tubular network, a hallmark of angiogenesis. Semi-quantitative analysis of the angiogenesis assay revealed a significant reduction in tube formation when supernatant from HIF-1 alpha siRNA-treated hypoxic EOC cell was used (P < 0.05).


CONCLUSIONS
Inhibition of HIF-1 alpha expression by specific siRNA resulted in a significant decrease in VEGF production and angiogenesis. Further investigation of HIF-1 alpha inhibition for anti-tumor activity is warranted and may potentially prove HIF-1 alpha as a therapeutic target in the management ovarian cancer.
Division of Gynecologic Oncology, Barbara Ann Karmanos Cancer Institute, Wayne State University, Harper Professional Building, 4160 John R Street, Detroit, MI 48201, USA. csbryant@med.wayne.edu
Christopher S
CS
Bryant
Adnan R
AR
Munkarah
Sanjeev
S
Kumar
Ramesh B
RB
Batchu
Jay P
JP
Shah
Jeremy
J
Berman
Robert T
RT
Morris
Zhong L
ZL
Jiang
Ghassan M
GM
Saed
D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000188:drug therapy*; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology / Q000627:therapeutic use*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A