Jack A. Schalken

Significance The human prostate accumulates high luminal citrate levels to serve sperm viability. There is only indirect qualitative evidence about metabolic pathways and carbon sources maintaining these levels. Human citrate-secreting prostate cancer cells were supplied with 13 C-labeled substrates, and NMR spectra of extracellular fluid were recorded. We report absolute citrate production rates of prostate cells and direct evidence that glucose is the main carbon source for secreted citrate. Pyruvate carboxylase provides sufficient anaplerotic carbons to support citrate secretion. Glutamine carbons exchange with carbons for secreted citrate but are likely not involved in its net synthesis. Moreover, we developed metabolic models employing the 13 C distribution in extracellular citrate as input to assess intracellular pathways followed by carbons toward citrate.

Background Cell‐penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor ( AR ) pre‐mRNA, thereby preventing the generation of the splice variant AR‐V7 mRNA. AON‐mediated knockdown of AR‐V7 resulted in inhibition of androgen‐independent cell proliferation. In this study, we evaluated the CPP‐mediated delivery of this AON into castration‐resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). Methods Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter‐Glo cell viability assay. The effectivity of CPP‐mediated delivery of the splice‐correcting AON‐intronic splicing enhancer (ISE) targeting the ISE in the castration‐resistant prostate cancer (CRPC)‐derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR‐V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 ( HP1BP3 ). Western blot analysis was used to confirm AR‐V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. Results The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine‐rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier‐associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 μM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON‐ISE directed against AR pre‐mRNA achieved significant downregulation of AR‐V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen‐independent cell proliferation of DuCaP and VCaP cells. Conclusions This proof‐of‐principle study constitutes the basis for further development of CPP‐mediated delivery of AONs for targeted therapy in prostate cancer.