Knockdown of HIF-1α by siRNA-expressing plasmid delivered by attenuated Salmonella enhances the antitumor effects of cisplatin on prostate cancer

Resistance to cisplatin (DDP) and dose-related toxicity remain two important obstacles in the treatment of prostate cancer (PCa) patients with DDP-based chemotherapy. We have investigated whether the knockdown of hypoxia-inducible factor-1 alpha (HIF-1α) by siRNA could enhance the antitumor activity of DDP, and aimed to determine the underlying mechanisms. Intravenous injection of attenuated Salmonella carrying a HIF-1α siRNA-expressing plasmid was used to knockdown HIF-1α in a PC-3 xenograft model. The in vitro and in vivo effects of HIF-1α siRNA treatment and/or DPP on PCa cell proliferation, apoptosis, glycolysis, and production of reactive oxygen species (ROS) were assessed by examining molecular markers specific to each process. The results demonstrated that the treatment of tumor-bearing mice with attenuated Salmonella carrying the HIF-1α siRNA plasmid greatly enhanced the antitumor effects of low-dose DDP. Further mechanistic studies demonstrated that knockdown of HIF-1α improved the response of PCa cells to DDP by redirecting aerobic glycolysis toward mitochondrial oxidative phosphorylation, leading to cell death through overproduction of ROS. Our findings indicate that DDP-based chemotherapy combined with targeting the HIF-1α-regulated cancer metabolism pathway might be an ideal strategy to treat PCa.


Supplemental Methods and Materials
Construction of vector expressing hypoxia-inducible factor-1 alpha siRNA According to the method used in our previous publications (Ji et al., 2011;Zhang et al., 2007), an siRNA with the sequence 5'-CUGAUGACCAGCAACUUGA-3' (Genbank accession number NM_001530.3) was selected to specifically target hypoxia-inducible factor-1 alpha (HIF-1α) mRNA. The oligonucleotide contains a sense strand of 19 nucleotides followed by a short spacer (loop sequence 5-TTCAAGAGA-3), an antisense strand, and a terminator containing five Ts. Double-stranded DNA oligonucleotides were cloned into pGCsilencerU6/Neo/GFP (Jikai Chemical, Inc.) to generate the si-HIF-1α plasmid (Supplementary Figure 1).

Immunohistochemistry
The paraffin-embedded cancer tissue sections (4 µm) were deparaffinized, rehydrated, and incubated in 3% H 2 O 2 to quench an endogenous peroxidase effect. Next, tissue sections were incubated with a serum-blocking solution for 30 min, then incubated with primary antibodies against HIF-1 (Novus) and PCNA (Calbiochem) overnight, washed with phosphate-buffered saline (PBS), and incubated with biotinylated secondary antibody for 60 min. Finally, nuclei were counterstained with hematoxylin and images were captured on a light microscope. Positive and negative cells were counted in 10 random high-power fields for each sample.
Glucose-uptake and lactate assays Glucose uptake was assayed using a Glucose Uptake Assay Kit (Abcam, Cambridge, MA). Briefly, PC-3 cells (1.0 x 10 4 cells/well) were cultured on 96-well plates and treated as indicated. The PC-3 cells were washed twice with PBS and starved in 100 μL of serum-free F-12K medium (ATCC, lot: 60971339) overnight to promote increased glucose uptake, then preincubated with 100 μL Krebs-Ringer-Phosphate-Hepes buffer containing 2% bovine serum albumin for 40 min. The cells were then incubated with 10 μL of 10 mM 2-deoxyglucose (2-DG) for 20 min. The uptake reaction was performed at 37°C for 1 h and terminated by adding 80 μL extraction buffer, following the manufacturer's instructions. The amount of 2-DG in the test samples, which was proportional to the accumulated 2-DG-6-phosphate (2-DG6P), was calculated using the plotted 2-DG6P standard curve.
Lactate levels in the culture medium of PC-3 cells were measured using the EnzymChrom L-Lactate assay kit (BioAssay Systems) according to the manufacturer's instructions. The data were normalized against the total number of cells.

Flow cytometry (FCM) analysis
PC-3 xenografts that received the various treatments were ground and filtered through a cell-filter membrane, and resuspended in 100 µL PBS. Next, the cells were incubated with DNA staining solution containing 5 µL propidium iodide (Beckman Coulter, Fullerton, CA) for 30 min at room temperature in the dark. The cell-cycle distribution of the PC-3 cells was evaluated by FCM using an Epics-XL-MCL flow cytometer (Beckman Coulter).

Luciferase reporter assay
The HIF-1α reporter gene, constructed in the pGL3 vector, was obtained from Jiran Laboratories (Jiran Bioengineering, Shanghai, China). Cell lysates from PC-3, DU145, LNCaP, 22RV1, REPE-1, or BPH1 cells were collected 48 h after the transfection, and the luciferase assay was conducted with a Dual-Luciferase Reporter Assay Kit following the manufacturer's instructions (Promega).

Toxicity assessment
Possible adverse side effects were assessed based on animal body weight, appearance, behavior, appetite, diarrhea, and survival until they were sacrificed. Organs such as the heart, liver, spleen, lung, and kidney were collected and fixed in a 4% paraformaldehyde solution. Paraffin-embedded sections (4 m) were obtained for general morphological observation by hematoxylin and eosin (H&E) staining in a blinded manner under a microscope. Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were detected using an enzyme-linked immunosorbent assay (ELISA) kit from BioVision according to the manufacturer's instructions. (a) DNA fragmentation was detected with an apoptosis ELISA assay in PC-3 cells. The cells were transiently transfected with HIF-1α-specific siRNA using Lipofectamine 2000 for 48 h; 24 h after siRNA treatment, the cells were exposed to DDP or vehicle control. After treatment, cell lysates were prepared and used for analysis with the apoptosis ELISA. (b) Protein expression of Bax/Bcl-2 ratio, cleaved caspase-3, and cleaved PARP was examined in PC-3 cells subjected to various treatments, using western blot analysis. Data shown are mean ± SD of three separate experiments. * p < 0.05 versus control group; # p < 0.05 versus the si-HIF-1α group or DDP group. The original blots are presented in Supplementary Figure 10.