Activating stress-activated protein kinase-mediated cell death and inhibiting epidermal growth factor receptor signaling: a promising therapeutic strategy for prostate cancer

Abstract

Epidermal growth factor receptor (EGFR) activation is an important event that regulates mitogenic signaling, such as the Raf, mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 cascades. EGFR activation has been implicated in the transition of prostate cancer from androgen dependence to independence. Therefore, inhibition of EGFR may effectively suppress prostate cancer growth and progression. The goal of this study was to determine whether the natural compound psoralidin alters EGFR-mediated signaling resulting in the inhibition of prostate cancer growth. Results suggest that inhibition of EGFR alone (by serum deprivation) fails to induce stress-mediated protein kinases (SAPK), namely, Jun NH(2)-terminal kinase/c-Jun signaling, in androgen-independent prostate cancer (AIPC) cells. Treatment with psoralidin, however, inhibited both constitutive and EGF-induced EGFR activation and simultaneously triggered SAPK signaling, resulting in the induction of apoptosis in AIPC cells. In addition, psoralidin downregulated EGFR-regulated MAPK signaling and inhibited cell proliferation in AIPC cells. Oral administration of psoralidin effectively suppressed PC-3 xenograft tumors in nude mice. Compared with control tumors, inhibition of pEGFR expression and an increase in the phosphorylation, activation, and nuclear translocation of c-Jun were observed in psoralidin-treated tumor sections. Our studies suggest that psoralidin may be a potent therapeutic agent that modulates EGFR-mediated key epigenetic events in AIPC.

Figure 1. Basal expression of EGFR in androgen-dependent and -independent PCa cells

Androgen-dependent (LNCaP) and –independent (PC-3, DU-145 and C4-2B) were grown either in complete medium or serum-free medium. Whole cell lysates were obtained and Western blot analyses were performed using (A) pEGFR (Tyr 1173) and total EGFR antibodies or (B) pJNK1/2, JNK1/2, pc-Jun and c-Jun antibodies. (C) PC-3 and DU-145 cells were serum deprived for 48 h and stimulated with 100 ng/ml of EGF for up to 120 minutes. Whole cell lysates were subjected to Western blot analysis using pEGFR and EGFR antibodies. β-actin was used as the internal loading control.

Figure 2. Psoralidin inhibits EGFR-mediated MAPK signaling in AIPC cells

Serum-deprived PC-3 and DU-145 cells were stimulated with 100 ng/ml of EGF and treated concurrently with psoralidin for 30 or 60 minutes. Whole cell lysates were subjected to Western blot analysis using (A) pEGFR, and EGFR antibodies, (B) Raf-1, MEK-4, MEKK-1 and pMEK1/2 antibodies or (C) pERK1/2, ERK1/2, pELK-1 and Elk-1 antibodies. β-actin was used as the internal loading control.

Figure 3. Psoralidin overcomes EGF-mediated inhibition of SAPK signaling and modulates pro-apoptotic machinery in AIPC cells

(A) Serum-deprived PC-3 and DU-145 cells were stimulated with 100 ng/ml of EGF and treated concurrently with psoralidin for 30 or 60 minutes. Whole cell lysates were subjected to Western blot analysis using pJNK1/2, JNK1/2, pc-Jun and c-Jun antibodies. (B) PC-3 and DU-145 cells were serum deprived for 48 h, stimulated with 100 ng/ml of EGF and treated concurrently with EGF and psoralidin for up to 12h. Whole cell lysates were subjected to Western blot analysis using survivin, Bcl-2, Bax and cleaved caspase-3 antibodies. β-actin was used as the internal loading control.

Figure 4. Psoralidin increases JNK kinase activity and potentiates JNK-mediated apoptosis in AIPC cells

(A) PC-3 and DU145 cells were treated with 60 and 45 μM (IC₅₀) psoralidin respectively for 24h. Whole cell lysates were subjected to immunoprecipitation using c-Jun fusion beads. JNK kinase activity was assayed by Western blot analysis using a pc-Jun antibody (upper panel). Bars represent a fold increase in JNK kinase activity following the treatment with psoralidin (lower panel). (B) PC-3 and DU-145 cells were treated with SP600125 alone (5 μM), psoralidin alone (60 or 45 μM, respectively) or a combination of SP600125 and psoralidin for 24 h and cells were subjected to apoptosis assay using Annexin V-FITC staining. Bars represent the percentage of apoptosis ± standard deviation (SD). (C) PC-3 cells were transfected with the c-Jun promoter-luciferase reporter construct and rennilla control vector (pGL3.4) was used to normalize transfection efficiency followed by treatment with psoralidin or vehicle. Luciferase reporter assay was performed to determine c-Jun promoter activity. Bars represent fold change in c-Jun promoter activity ± SD.

Figure 5. Psoralidin inhibits EGFR-mediated MAPK and induced SAPK signaling in AIPC cells grown in complete medium

PC-3 and DU145 cells were grown in complete medium containing 10% fetal bovine serum and treated with 60 and 45 μM (IC₅₀) psoralidin for up to 12 h. Whole cell lysates were subjected to Western blot analysis using (A) pEGFR and EGFR antibodies, (B) MEK-1, MEK-4, pMEK3/6, pMEK1/2 and MEKK-1 antibodies and (C) pERK and ERK antibodies (upper panel) and (C) PC-3 and DU145 cells were treated with 60 and 45 μM psoralidin for 24 h. Whole cell lysates were subjected to immunoprecipitation using ERK antibody. ERK kinase activity was assayed by Western blot analysis using a pElk-1 antibody. Total ELK-1 antibody was used as control (lower panel). (D) PC-3 and DU145 cells were grown in complete medium containing 10% fetal bovine serum and treated with 60 and 45 μM psoralidin for up to 12 h. Whole cell lysates were subjected to Western blot analysis using pJNK1/2, JNK1/2, pc-Jun and c-Jun antibodies. β-actin was used as the internal loading control.

Figure 6. Psoralidin inhibits tumor growth in xenografts models

(A) PC-3 cells were implanted subcutaneously in nude mice. When the tumor reached a volume of 50 mm³, the animals were randomized into two groups (control and psoralidin-treated). Control animals were orally fed sunflower oil, while psoralidin-treated animals received oral administration of psoralidin (50 mg/kg) for a four week period. Tumor growth was monitored and tumor volumes were measured and graphed. Each data point represents mean ± SD. (B) Control and psoralidin treated tumors were subjected to immunohistochemical analysis using (i) pEGFR and (ii) pc-Jun antibodies and (iii) number of apoptotic cells using TUNEL assay.