Abstract
Lysophosphatidic acid (LPA) plays an important role in mediating cell proliferation, survival, and tumor invasion and angiogenesis. This bioactive phospholipid at the concentration in ascitic fluid stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF). In the present study, we investigated whether LPA activates hypoxia inducible factor-1 (HIF-1), a key transcriptional complex in tumor progression and metastasis, thereby increasing the expression of VEGF. Immunoblotting, reverse transcription-PCR, ELISA, immunofluorescence, and chromatin immunoprecipitation assay were used to examine the expression of VEGF and HIF-1alpha in various cancer cells. Specific HIF-1alpha small interfering RNA was transfected to various cancer cells to determine the role of HIF-1alpha in LPA-induced VEGF expression. LPA induced expressions of VEGF and HIF-1alpha in OVCAR-3, CAOV-3, PC-3, and SK-Hep1 cells but not in SKOV-3 and Hep-3B cells. In OVCAR-3 and PC-3 cells, the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin/p70S6K and p42/p44 mitogen-activated protein kinase pathways were required for LPA-induced HIF-1alpha and VEGF expressions, whereas only the phosphoinositide 3-kinase/mammalian target of rapamycin/p70S6K pathway was important in SK-Hep1 cells. Immunofluorescence microscopy assay showed translocation of HIF-1alpha to nucleus by LPA, and chromatin immunoprecipitation assay revealed the binding of HIF-1alpha to the promoter of VEGF by LPA. Importantly, we found that small interfering RNA-induced reduction of HIF-1alpha expression significantly attenuated VEGF expression by LPA. Our results show for the first time that LPA induces VEGF via HIF-1alpha activation and reveal a critical role of HIF-1alpha in LPA-induced cancer cell proliferation and angiogenesis.
Highlights
Lysophosphatidic acid (LPA) plays an important role in mediating cell proliferation, survival, and tumor invasion and angiogenesis
To test whether LPA modulates the levels of vascular endothelial growth factor (VEGF) in our system, ovarian cancer (SKOV-3, OVCAR-3, and CAOV-3), prostate cancer (PC-3), and hepatoma (SK-Hep1 and Hep-3B) cells were incubated with LPA or 250 Amol/L CoCl2 for indicated period of time, and the conditioned media of each cancer cells were tested for the VEGF production through ELISA
CoCl2 did not give any influence to the expression of HIF-1a in SKOV-3 cells. These results are consistent with the data of LPA-induced VEGF expression (Fig. 1A), suggesting that there is a close relationship between the expressions of VEGF and HIF-1a by LPA in cancer cells tested
Summary
Lysophosphatidic acid (LPA) plays an important role in mediating cell proliferation, survival, and tumor invasion and angiogenesis. This bioactive phospholipid at the concentration in ascitic fluid stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF). We investigated whether LPA activates hypoxia inducible factor-1 (HIF-1), a key transcriptional complex in tumor progression and metastasis, thereby increasing the expression of VEGF. The hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression and angiogenesis This protein is a heterodimeric transcription factor, which regulates hypoxia-activated genes, and consist of a constitutively expressed HIF-1h subunit and a highly regulated HIF-1a (12 – 14). Oncogenic activation (such as Ha-ras, myc, or src) or loss of tumor suppressor function (such as p53, PTEN, or VHL) is associated with HIF-1-mediated tumor progression [23]
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