Prostate cancer classified as an adenocarcinoma is the second most common malignant tumors in American men, with estimates of 192,280 new cases and approximately 27,360 deaths in 2009 
. Betulinic acid (BA), a plant-derived pentacyclic lupane-type triterpenoid, can be extracted from various plants such as Sarracenia flava 
spp., Inga punctata 
spp., and Vauquelinia corymbosa 
. Several groups reported anti-cancer activity of BA in various cancers including lung, colorectal, breast, prostate and cervical cancer 
, but not normal cells 
. Also, BA completely inhibited tumour growth without toxicity in athymic mice bearing human melanomas 
. Moreover, anti-cancer activity of BA was exerted by inducing apoptosis in the cancer cells. For example, BA-induced apoptosis was independent of p53 in neurorectodermal tumor 
and melanoma cells 
. In neuroblastoma cells, BA induced apoptosis through loss of the mitochondrial membrane potential, reactive oxygen species (ROS) production and caspase activation 
Interestingly, Karna and colleagues recently reported that BA inhibited the expression of HIF-1α and vascular endothelial growth factor (VEGF) in human endometrial cancer cells 
. However, the regulatory mechanisms whereby BA inhibits angiogenesis are not fully understood. In the present study, we found that BA suppressed hypoxia-mediated protein accumulation, transcriptional activation and nuclear localization of HIF-1α in PC-3 cells. Consistent with the results of Karna's paper, our data also showed that BA significantly inhibited VEGF secretion and protein expression in hypoxic PC-3 cells. Additionally, in vitro
tube formation assay further confirmed anti-angiogenenic effect of BA in hypoxic PC-3 cells.
Recently, Niu and colleagues suggested that constitutively activated STAT3 up-regulated VEGF and induced tumor angiogenesis 
. Also, Wei and colleagues reported that STAT3 activation regulates the expression of VEGF and human pancreatic cancer angiogenesis Furthermore, several papers described the role of STAT3 as a potential modulator of HIF-1α-induced VEGF signaling in cancer cells 
. In this regard, the effect of BA on STAT3 and HIF-1α activation was examined in hypoxic PC-3 cells in our study. Consistent with the evidences by Pandey and colleagues that BA suppressed STAT3 activation in multiple myeloma cells 
, BA prevented hypoxia-induced tyrosine phosphorylation, DNA binding activity and nuclear translocalization of STAT3, suggesting the inhibitory effect of BA on STAT3 activation.
VEGF promoter contains various transcription factor binding sites including STAT3 
as well as HIF-1 
. Physical interaction of STAT3 with HIF-1 controls VEGF transcriptional activation by their binding to the VEGF promoter 
. In our study, hypoxia promoted the binding of STAT3 and HIF-1α to the VEGF promoter in PC-3 cells. In contrast, BA remarkably inhibited the binding of STAT3 and HIF-1α to the VEGF promoter site under hypoxic condition. Additionally, silencing STAT3 using its specific siRNA significantly enhanced BA-mediated inhibition of VEGF production, implying the involvement of STAT3 in anti-angiogenic regulation of BA in hypoxic PC-3 cells. Similar to our study, Gariboldi and colleagues reported that NVP-AEW541, a IGFR1 inhibitor, disrupted IGF/STAT3/HIF1 pathway in human glioblastoma cells 
. Leeman-Neill and colleagues also reported that Guggulsterone inhibited STAT3 and HIF-1α and suggested a biologic rationale for further clinical investigation BA for human head and neck squamous cell carcinoma (HNSCC) therapy 
Collectively, our data demonstrate that BA suppressed expression and transactivation of hypoxia-induced HIF-1α, STAT3, VEGF as well as capillary tube formation in PC-3 cells. It is noteworthy that anti-cancer activity of BA is exerted by inhibiting angiogenesis via inhibition of binding of STAT3 and HIF-1α to the VEGF promoter in PC-3 cells. Thus, our findings suggest that BA can be a potent anti-angiogenic agent by targeting STAT3/HIF-1α/VEGF signaling for prostate cancer therapy.