The suppression of hypoxia-inducible factor and vascular endothelial growth factor by siRNA does not affect the radiation sensitivity of multicellular tumor spheroids.
BACKGROUND
The hypoxic microenvironment is closely associated with the radiation resistance of tumor cells. Hypoxia induces several genes such as hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF) to promote tumor cell growth and survival. The up-regulated expression levels of HIF-1 and VEGF in tumor cells also correlate with their resistance to radiation, suggesting that these genes are potential therapeutic targets for strategies designed to enhance radiation effects. To further investigate this possibility, we investigated the effects of suppressing these genes upon the radiation sensitivity of cancer cells. We conducted these experiments using multicellular spheroids as a three-dimensional in vitro tumor model and RNA interference as the method of gene suppression.


METHODS
SQ5 human lung carcinoma cells were treated with HIF-1/VEGF siRNA and/or radiation. Reversed transfection methods were employed for the spheroids. Gene expression was analyzed using quantitative RT-PCR and western blotting. Cell toxicity was qualified by colony formation assay.


RESULTS
Compared with monolayer cells, spheroids showed up-regulated expression of HIF-1 and increased radiation resistance. Hypoxic conditions elevated the expression of HIF-1 and VEGF and enhanced the surviving fraction of spheroids after exposure to radiation. However, when the expression of HIF-1 and VEGF was down-regulated by transfection of targeting siRNA, this did not influence the cytotoxic effects of the radiation under either normoxic or hypoxic conditions.


CONCLUSIONS
We have established a method to transfect siRNA into spheroid cells. Our current data indicate that the functions of HIF-1 or VEGF are independent of radiation sensitivity in spheroids under either normoxic or hypoxic conditions.
Zou
Yuefen
Y
Department of Radiology, Jiangsu Province Hospital, First Affiliated Hospital with Nanjing Medical University, 300 Guangzhou Road, Nanjing, China.
Cheng
Chao
C
Omura-Minamisawa
Motoko
M
Kang
Yun
Y
Hara
Takamitsu
T
Guan
Xiaohong
X
Inoue
Tomio
T
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000532:radiotherapy; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D011836:Radiation Tolerance; D018874:Spheroids, Cellular / Q000528:radiation effects*; D013489:Suppression, Genetic / Q000528:radiation effects; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A