Article Figures & Data
Figures
- Figure 1.
Nelfinavir and its metabolite M8 induce caspase-dependent apoptosis and dose-dependent cell death in liposarcoma. A, caspase 9/6 activation was analyzed in LiSa-2 (left) and SW872 (right) cells treated with 0, 2.5, 10, or 20 μmol/L nelfinavir (NFV). After 24 hours, cells were harvested for caspase analysis. B, clonogenic assay in pretreated LiSa-2 cells with NFV. C, DIMSCAN assay was performed in NFV- or M8-treated LiSa-2 cells. A total of 1,000 cells seeded in a 96-well plate for overnight incubation, followed by treatment with NFV or M8 (0, 2.5, 10, or 20 μmol/L) for 2 or 6 days. Each experiment was done in triplicate. Results are presented as mean ± SD. *, P < 0.05; **, P < 0.01 compared with DMSO control (0 μmol/L).
- Figure 2.
Nelfinavir increases SREBP-1 protein half-life (T1/2) and inhibits intracellular trafficking of SREBP-1 and ATF6. A, SREBP-1 T1/2 determination. Lysate from SW872 cells pretreated with 10 mmol/L NFV or DMSO for 4 hours followed by cycloheximide (100 mmol/L) for the indicated time points was analyzed by Western blot. Relative SREBP-1 expression was quantitated by Image Quant (Molecular Dynamics). B, dose-dependent induction of precursor ATF6 and FASN reduction by NFV. LiSa-2 cell was treated with NFV at the indicated dose for 24 hours and lysate was harvested for Western blot analysis. C, visualization of SREBP-EGFP transport from cytoplasm to nucleus in NFV-treated cells. A total of 3 × 105 LiSa-2 cells/well were seeded in glass-bottomed 2-well chamber slide for overnight incubation and transfected with pSREBP-1-EGFP, cells were then cultured 24 hours to allow transgene expression before staining with Hoechst 33322 1 μg/ml for 5 minutes. The live cell image was captured and recorded at indicated time points using a confocal microscopy (t = 0 hour, 5 minutes after addition of 10 μmol/L nelfinavir or DMSO). D, visualization of transport of ATF6-EGFP from cytoplasm to nucleus in NFV-treated LiSa-2 cells. Cells were transfected with pATF6-EGFP for time-lapse imaging. Bar, 20 μm.
- Figure 3.
siRNA-mediated inhibition of S1P or S2P blocks intracellular trafficking of SREBP-1. A, siRNA knockdown of S1P (top) and S2P (bottom). LiSa-2 cells were transfected with S1P, S2P or scrambled siRNA by Lipofectamine RNAiMAX. The transfected cells were cultured for 48 hours and collected for S1P or S2P qRT-PCR and Western blot for SREBP-1 detection. **, P < 0.01 compared to control siRNA. B, visualization of transport of SREBP-EGFP in S1P siRNA treated LiSa-2 cells. A total of 3 × 105 cells were seeded on day 1 and S1P siRNA were transfected on day 2. On day 3, cells were transfected with pSREBP-1-EGFP. On day 4, the nucleus was Hoechst-stained for time-lapse imaging. t = 0 hour, 24 hours after pSREBP-1-EGFP transfection. C, visualization of transport of SREBP-EGFP in S2P siRNA treated LiSa-2 cells. D, visualization of transport of SREBP-EGFP in S1P and S2P siRNA treated LiSa-2 cells. Equal amounts of S1P and S2P siRNA were transfected and follow the treatment as (B). Bar, 20 μm.
- Figure 4.
A, siRNA-mediated inhibition of S1P or S2P reduces liposarcoma proliferation. A total of 2,000 LiSa-2 cells were seeded in a 96-well plate, followed by transfection with S1P, S2P, or S1P and S2P, or scrambled siRNA. Twenty-four hours later, the cells were transfected with pSREBP-1-EGFP. At 48 and 72 hours after siRNA transfection, cells proliferation was determined by DIMSCAN assay. *, P < 0.05, **, P < 0.01 compared with control siRNA. B, S1P inhibitor does not alter the expression of SREBP-1 and ATF6. LiSa-2 cells were treated at the indicated DCI dose for 4 hours then lysed for Western blot analysis of SREBP-1. pATF6-EGFP (or mock) was transfected 24 hours prior to the treatment of DCI for 4 hours for analysis of ATF6. C, S2P inhibitor alters the expression of SREBP-1 and ATF6. Western blot analysis of SREBP-1 and ATF6 as (B) for LiSa-2 cells treated with 1,10-phenanthroline for 4 hours. D, S2P inhibitor induces a dose-dependent apoptosis. LiSa-2 cells were treated with indicated dose of DCI, 1,10-phenanthroline, or NFV for 4 hours and collected for Annexin V detection by flow cytometry.
- Figure 5.
A, nelfinavir inhibits liposarcoma growth in a murine model. LiSa-2 liposarcoma xenografts were established in female SCID mice and the mice were treated by daily oral feeding of NFV at 500 mg/kg/d. Size of tumors was measured and presented as mean ± SE (n = 7 mice/group). *, P < 0.05 compared with the control animals. B, no significant toxicity associated with NFV treatment. The mice were weighed 3 times weekly. Weight was measured and presented as mean ± SE. #, P < 0.05 compared with the control animals. C, pharmacokinetics determination of NFV in SCID mice. A single oral dose of NFV at 500 mg/kg was given to the SCID mice. Blood samples of the treated mice at 0, 1, 2, 4, 8, and 24 hours after oral gavage were analyzed to determine the plasma concentration of NFV using an UPLC-tandem mass spectrometric assay.
- Figure 6.
A, nelfinavir induces ER stress. siRNA transfected (48 hours) or NFV treated (24 hours) LiSa-2 cells were lysed for Western blot detection of GRP78. TG (0.5 μmol/L) was served as a positive control. B, current model of nelfinavir in liposarcoma. SREBP and ATF6 are synthesized as ER transmembrane proteins and transported to the Golgi upon appropriate stimulus. Luminal S1P cleaves first, followed by intramembrane S2P to liberate the transcriptionally active amino-terminal segments of nSREBP and nATF6 from the Golgi membrane to migrate to the nucleus to activate transcription of lipogenic target genes such as FASN or UPR targets genes (solid arrow); NFV inhibits liposarcoma proliferation, and promotes apoptosis through inhibition of RIP processing of SREBP and ATF6. Inhibition of S2P cleavage by NFV leads to retention of precursor SREBP and ATF6 proteins in the ER or Golgi and block of transcriptional activation of target genes (FASN). Accumulation of unprocessed SREBP and ATF6 induces ER stress, which leads to caspase-dependent apoptosis (dotted arrow). NFV, nelfinavir; ER, endoplasmic reticulum; nSREBP-1, nuclear active SREBP; nATF6, nuclear active ATF6; FASN, fatty acid synthase; UPR, unfolded protein response; S1P, site-1 protease; S2P, site-2 protease; RIP, regulated intramembrane proteolysis.
Additional Files
Supplementary Data
Supplementary Figures S1-S4.
Files in this Data Supplement:
- Supplementary Data - Supplementary Figures S1-S4.
Volume 17, Issue 7
