Induction of cellular prion protein (PrPc) under hypoxia inhibits apoptosis caused by TRAIL treatment

Abstract

Hypoxia decreases cytotoxic responses to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. Cellular prion protein (PrPc) is regulated by HIF-1α in neurons. We hypothesized that PrPc is involved in hypoxia-mediated resistance to TRAIL-induced apoptosis. We found that hypoxia induced PrPc protein and inhibited TRAIL-induced apoptosis. Thus silencing of PrPc increased TRAIL-induced apoptosis under hypoxia. Overexpression of PrPc protein using an adenoviral vector inhibited TRAIL-induced apoptosis. In xenograft model in vivo, shPrPc transfected cells were more sensitive to TRAIL-induced apoptosis than in shMock transfected cells. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia.

Keywords: HIF-1α; PrPc; TRAIL; colon cancer; hypoxia.

Figure 1. Hypoxia regulates HIF-1α and PrPc

(A) Western blot analysis of HIF-1α, PrPc, Bcl-2, p-Akt, DR4 and DR5 from HCT116 cells pre-exposed to normoxia or hypoxia for 24 h and treated with or 10 μM DEF under normoxia for 24 h. β-actin was used as a loading control.

Figure 2. Hypoxia inhibits TRAIL-induced apoptosis human colon carcinoma cells

HCT116 cells were pre-exposed to normoxia or hypoxia for 24 h and further incubated with recombinant TRAIL (0–200 ng/ml) for an additional 4 h under the same conditions. (A) Treated cells were photographed with a light microscope (× 200). (B, C) Viable cells were stained with crystal violet. Viability of control cells was set at 100%, and viability relative to the control was estimated. These results are representative of three independent experiments. (D) HCT8 cells were photographed with a light microscope (× 200). (G, H) Viable cells were stained with crystal violet. Viability of control cells was set at 100%, and viability relative to the control was estimated. These results are representative of two independent experiments. Bar graph indicates the number of total cells and percent of apoptotic cells. *p < 0.05 or **p < 0.01 significant differences between control and each treatment group. In (I and J) HCT-116 and HeLa cells were exposed to hypoxia 12 hr after then treated with 100 ng/ml TRAIL for indicated time periods, respectively. Cell viability was measured by the Annexin V assay.

Figure 3. Deferoxamine inhibits TRAIL-induced apoptosis

(A) HCT116 cells were pre-treated with 10 μM deferoxamine (DEF) under normoxia for 24 h before treatment with 0–200 ng/ml of TRAIL, for 4 h. Treated cells were photographed with a light microscope (× 200). (B) Cell viability was measured by the crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control is presented. The bar graph indicates the mean ± standard error of the mean (SEM) (n = 3). *p < 0.05 or **p < 0.01 significant differences between control and each treatment group.

Figure 4. Knockdown of PrPc inhibited the HIF-1α-mediated PrPc expression

(A, B) HCT116 cells were pre-treated 20 nM PrPc RNAi for 24 h and pre-exposed to DEF or hypoxia for 24 h and then detected the protein levels of HIF-1α, PrPc. β-actin was used as a loading control. (C, D) The mRNA levels were determined by quantitative real-time PCR for HIF-1α and PrPc. This treatment is as described in C. These results are representative of three independent experiments. **p < 0.01 compared to basal conditions.

Figure 5. Knockdown of PrPc blocks protection of hypoxia in TRAIL-treated cells

HCT116 cells were pre-treated 20 nM PrPc RNAi for 24 h and pre-exposed to DEF or hypoxia for 24 h and further incubated with recombinant TRAIL (200 ng/ml) for an additional 3 h under the same conditions. (A, D) Treated cells were photographed with a light microscope (× 200). (B, C, E, F) Viable cells were stained with crystal violet. Viability of control cells was set at 100%, and viability relative to the control was estimated. These results are representative of two independent experiments. (G) Prnp gene in HCT116 cells was knocked down (PrPc siRNA) using the PrPc siRNA oligomer and then exposed to hypoxic conditions with or without TRAIL 200 ng/ml for 24 h. Cell viability was measured by PI staining assay, (H) Bar graph indicates the number of total cells. HCT116 cells viability was measured by PI staining assay. Percent of numerical value represents the population of apoptotic cells. (E, F) Bar graph indicates the number of total cells and percent of apoptotic cells. *p < 0.05, **p < 0.01: compared to basal conditions.

Figure 6. Lentiviral shRNA knock-down of PrPc sensitizes HCT116 cancer cells for TRAIL-induced colon cancer cell death

(A, B) PrPc-shRNA or mock transfected HCT116 cells were cultured in 21% or 1% oxygen tension for 24 h or pre-treated with 10 μM of deferoxamine, and then treated with the indicated dose (200 ng/ml) of TRAIL for 3 h. Cell viability was measured by the crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control is presented. The bar graph indicates the mean ± S.E.M. (n = 3). **P < 0.01, significant differences between control and each treatment group. (C) The treated cells were photographed with a light microscope (× 200). (D) Western blot analysis of HIF-1α and PrPc from PrPc-shRNA HCT116 cells pre-exposed to normoxia or hypoxia for 24 h and treated with or 10 μM DEF under normoxia for 24 h. β-actin was used as a loading control.

Figure 7. PrPc-shRNA HCT116 tumors increased TRAIL-mediated apoptosis in mouse xenograft

(A, B) shPrPc HCT116 tumors growth in mouse thigh is suppressed by intra-tumoral injection of TRAIL in BALB/c nude mice when TRAIL treatment was started at tumors size were ≥ 150 mm³ after tumor implant. (C) Western blot analysis of HIF-1α, active-caspase-3 and PrPc from shPrPc HCT116 tumors in xenograft mice. Lane 1 : shMock1, Lane 2 : shMock2, Lane 3 : shPrPc1 Lane 4 : shPrPc2. (D) Representative images of PrPc and HIF-1α expression in shPrPc HCT116 tumors described in (C)

Figure 8. PrPc overexpression inhibits TRAIL-induced apoptosis

Transfection of Ad-Prnp at multiplicity of infection (MOI) 0, 10, 20, 80with Ad-empty at multiplicity of infection 80 for 24 hand incubated with 200 ng/ml TRAIL for 3 h. (A) Treated cells were photographed with a light microscope (× 200). (B, C) Viable cells were stained with crystal violet. Viability of control cells was set at 100%, and viability relative to the control was estimated. These results are representative of two independent experiments. (D) Western blot analysis indicated that overexpression of PrPc was investigated in transfected HCT116 cells after 24 h using the specific anti-PrPc antibody 3F4. *p < 0.05, **p < 0.01 : compared to basal conditions.