Inhibition of PCSK9 protects against radiation-induced damage of prostate cancer cells

Figures & data

Figure 1 PCSK9 siRNA treatment promotes cellular viability in cells exposed to high doses of IR.

Notes: (A) LnCap cells (left) and PC-3 cells (right) were, respectively, transfected with PCSK9 siRNAs (PCSK9 siRNA-1 and siRNA-2) and control siRNA. After 48 h, the efficiency of PCSK9 knockdown was determined by real-time PCR. (B) PCSK9 protein expression in the siRNA-treated cells was analyzed by Western blot. β-Actin was included as a loading control. (C) After 24 h of PCSK9 siRNA pretreatment, relative cellular viability of the indicated cell lines after treatment with increasing doses of IR was determined by CCK-8 assay. Data are expressed as the mean ± SD (*P<0.05).
Abbreviations: CCK-8, cell counting kit-8; IR, ionizing radiation; PCSK9, proprotein convertase subtilisin/kexin type 9; siRNAs, small interfering RNAs; SD, standard deviation.

Figure 2 Inhibition of PCSK9 increases radioresistance in PCa cells exposed to IR.

Notes: After PCSK9 was inhibited by 24 h of siRNA pretreatment, LnCap cells (A) and PC-3 cells (B) were exposed to 0 or 8 Gy of IR. Cellular apoptosis was revealed by Hoechest 33342/PI staining. Apoptotic cells exhibited blue-positive staining, whereas dead cells exhibited red-positive staining. PCSK9 siRNA treatment alone did not cause cell apoptosis. IR-induced apoptosis was attenuated by PCSK9 siRNA treatment. Magnification ×200.
Abbreviations: IR, ionizing radiation; PCSK9, proprotein convertase subtilisin/kexin type 9; PI, propidium iodide; siRNA, small interfering RNA.

Figure 3 Inhibition of PCSK9 decreases MMP in LnCap cells exposed to IR.

Notes: LnCap cells transfected with control or PCSK9 siRNA were treated with 0 or 8 Gy of IR, respectively. Magnification ×400. (A) MMPs were visualized by Rho-123 staining, and representative images were given. (B) Flow cytometric analysis of MMP alteration in indicated cells and representative results are shown. (C) Mean Rho-123 signal assessed by flow cytometry in response to IR exposure. Mean ± SD of three independent experiments were performed (*P<0.05 vs control siRNA).
Abbreviations: IR, ionizing radiation; MMP, mitochondrial membrane potential; PCSK9, proprotein convertase subtilisin/kexin type 9; Rho-123, Rhodamine-123; SD, standard deviation; siRNA, small interfering RNA.

Figure 4 Radioresistance mechanisms of PCSK9 siRNA in LnCap cells.

Notes: (A) LnCap cells were treated with 8 Gy of IR. At different time points post-IR, the expression of PCSK9, cyto C, caspase-3, Bax, and Bcl-2 was measured by Western blot, with β-actin as the loading control. (B–E) PCSK9, cyto C, Bax, and Bcl-2 protein levels were quantified from three independent experiments as in (A) by measuring the intensity of the indicated protein band relative to the β-actin (loading) control (*P<0.05). The bars indicate mean ± SD (n=3). (F) LnCap cells were pretreated with control or PCSK9 siRNA prior to IR (0 or 8 Gy), and proteins were lysed 24 h after IR. The expression of cyto C, caspase-3, Bax, and Bcl-2 was measured by Western blot, with β-actin as the loading control. (G) Cyto C, caspase-3, Bax, and Bcl-2 protein levels were quantified from three independent experiments as in (F). Mean ± SD of three independent experiments were performed (*P<0.05 vs 0 Gy groups, ^#P<0.05 vs control siRNA groups).
Abbreviations: PCSK9, proprotein convertase subtilisin/kexin type 9; siRNA, small interfering RNA; IR, ionizing radiation; cyto C, cytochrome C; Bcl-2, B-cell leukemia/lymphoma 2; Bax, Bcl-2-associated X protein; SD, standard deviation.