Abstract 4114: Silibinin inhibits hypoxia-induced proliferation, angiogenesis and lipogenesis in prostate cancer cells both in vitro and in vivo
Authors: Deep Gagan , Ramteke Anand M. , Nambiar Dhanya K. , Jain Anil K. , Serkova Natalie J. , Agarwal Chapla , Agarwal Rajesh 
Published: May 2015

Abstract
Hypoxia (low oxygen conditions) in prostate tumors is associated with aggressive phenotype and poor prognosis. Also, hypoxia is considered the first major challenge faced by growing mass of neoplastic cells, as cells in hypoxic areas are surrounded by metabolic waste, acidic pH and necrotic cells. To overcome these hostile conditions, tumor cells activate transcriptional machinery leading to neo-angiogenesis, altered metabolism and selection of resistant clones. This suggests that prostate cancer (PCa) growth and progression could be prevented via targeting hypoxia-induced signaling. Here, we analyzed silibinin effect on hypoxia-induced proliferation, angiogenesis and metabolic changes in human PCa cells both in vitro and in vivo. Silibinin inhibited LNCaP cells clone formation (exposed to 1% O2 for 48 hrs and then returned to normoxic culture conditions) by 90-100% at 25-50 µM doses. Under similar hypoxic conditions in 22Rv1 cells, silibinin inhibited the clone formation by 63-77% at 25-50 µM doses. Importantly, conditioned media collected from PCa cells under hypoxic conditions (1% O2 for 24 hrs) induced tube formation by human umbilical vein endothelial cells (HUVEC) on matrigel, whereas conditioned media from silibinin-treated hypoxic PCa cells or silibinin addition in hypoxic-conditioned media abrogated HUVEC tube formation. Interestingly, we observed higher lipid accumulation in LNCaP cells under hypoxia, which was decreased (∼70%) by silibinin. Molecular analyses showed that silibinin decreases hypoxia-induced HIF-1α expression without affecting HIF-1β in PCa cells. Furthermore, silibinin increased the level of FIH (factor inhibiting HIF) and prolyl-hydroxylase 2, involved in HIF-1α degradation. Also, silibinin decreased the level of phosphorylated mTOR and ERK1/2 in PCa cells under hypoxia, and strongly inhibited lipid synthesis via activating AMP-activated protein kinase and decreasing acetyl Co A carboxylase, fatty acid synthase, acetyl Co A synthetase, fatty-acyl Co A synthetase and lipin expression. In vivo, silibinin effect on hypoxia-induced proliferation, angiogenesis and metabolism was analyzed in 22Rv1 xenograft model. Mice with established 22Rv1 tumors were imaged at base-line as well as 6 and 15 days following silibinin administration (200 mg/kg body weight) by diffusion weighted (DWI), dynamic contrast enhanced (DCE)-MRI and 18FDG-PET. At the study end, endogenous tumor metabolites were assed by NMR and various biomarkers by IHC. In vivo results showed that silibinin feeding reduced the tumor volume (by 47%), inhibited HIF-1α signaling, decreased proliferation and angiogenesis (DCE-MRI), induced apoptotic death and modulated tumor metabolism (PET and NMR). Together, these findings further support silibinin usefulness in PCa prevention through inhibiting hypoxia-induced proliferation, angiogenesis and lipogenesis.
Citation Format: Gagan Deep, Anand M. Ramteke, Dhanya K. Nambiar, Anil K. Jain, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. Silibinin inhibits hypoxia-induced proliferation, angiogenesis and lipogenesis in prostate cancer cells both in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2014-4114