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In vitro tube formation, an assay for the capillary formation of HUVECs, is a simple but powerful tool for examining angiogenetic associations. HUVECs were grown to form capillary-like structures on the Matrigel? surface, as shown in Fig. 2C. Suramin, an angiogenesis inhibitor [32] and [33], destroyed the capillary-like structures on the Matrigel?, as expected. When HUVECs were incubated in the presence of AC1-004 at 10 and 30 μM, respectively, capillary-like structure was not formed indicating that SM 224 AC1-004 inhibited in vitro tube formation of HUVEC on matrigel by inhibiting VEGF function via blocking accumulation of HIF-1α. When cytotoxic effect of AC1-004 on HUVECs was also examined using the MTT assay, the growth of HUVECs was not affected by AC1-004, up to 30 μM (Supplementary material). This result implies that AC1-004 may have an inhibitory effect on angiogenesis.
AC1-004 regulates stability of HIF-1α through Hsp90 and Akt signaling
To address the mechanism of AC1-004 in inhibiting HIF-1α activity, changes in HIF-1α mRNA levels were investigated during AC1-004 treatment. The HIF-1α mRNA level was steady in the presence of AC1-004 (Fig. 2B), indicating that the inhibition of HIF-1α accumulation was not regulated at the transcription level. In order to further evaluate the inhibitory activity of AC1-004 on HIF-1α accumulation, its effect on the stability of HIF-1α protein was examined in HCT-116 cells pretreated with proteasomal inhibitor MG132 (Z-Leu-Leu-Leu-al) as shown in Fig. 3A. Pretreatment of cells with MG132 resulted in an accumulation of HIF-1α in normoxia, as expected. Furthermore, the inhibition of HIF-1α accumulation by AC1-004 was reversed in the presence of MG132, indicating that AC1-004 may affect HIF-1α protein stability via proteasomal-dependent degradation.