Targeting gastrin-releasing peptide as a new approach to treat aggressive refractory neuroblastomas

Abstract

Background: The overall survival for neuroblastoma remains dismal, in part due to the emergence of resistance to chemotherapeutic drugs. We have demonstrated that gastrin-releasing peptide (GRP), a gut peptide secreted by neuroblastoma, acts as an autocrine growth factor. We hypothesized that knockdown of GRP will induce apoptosis in neuroblastoma cells and potentiate the cytotoxic effects of chemotherapeutic agents.

Methods: The human neuroblastoma cell lines (JF, SK-N-SH) were transfected with small interfering (si) RNA targeted at GRP. Apoptosis was assessed by DNA fragmentation assay. Immunoblotting was used to confirm molecular markers of apoptosis, and flow cytometry was performed to determine cell cycle arrest after GRP knockdown.

Results: siGRP resulted in an increase in apoptosis in the absence of chemotherapeutic interventions. A combination of GRP silencing and chemotherapeutic drugs resulted in enhanced apoptosis when compared to either of the treatments alone. GRP silencing led to increased expression of proapoptotic proteins, p53 and p21.

Conclusion: Silencing of GRP induces apoptosis in neuroblastoma cells; it acts synergistically with chemotherapeutic effects of etoposide and vincristine. GRP knockdown-mediated apoptosis appears to be associated with upregulation of p53 in neuroblastoma cells. Targeting GRP may be postulated as a potential novel agent for combinational treatment to treat aggressive neuroblastomas.

Figure 1. Knockdown of GRP expression using siRNA in GRP-amplified neuroblastoma cell lines

JF and SK-N-SH cells were transfected with siGRP or siNTC, and gene expression was analyzed using RT-PCR. GRP expression was effectively silenced with siGRP when compared to controls (siNTC) in both cell lines examined (mean ± SEM; * = p < 0.05 vs. siNTC).

Figure 2. GRP silencing-induced apoptosis

(A) Cells treated with siGRP exhibited an increase in apoptosis in comparison to control cells (siNTC). (B) GRP silencing (siGRP) resulted in a significant decrease in cell proliferation when compared to control cells. Apoptosis and cell proliferation were analyzed using Cell Death ELISA and CCK-8, respectively (mean ± SEM; * = p < 0.05 vs. siNTC).

Figure 3. Chemotherapy-induced apoptosis

A dose-response curve for the effects of chemotherapeutic agents on apoptosis was assessed using Cell Death ELISA for JF and SK-N-SH cells at 48 h time point. (A) The lowest effective dosage of vincristine was determined as 25 nM for JF cells and 1 nM for SK-N-SH cells. (B) The lowest effective dosage of etoposide was determined as 2 μM for JF cells and 0.1 μM for SK-N-SH cells (mean ± SEM; * = p < 0.05 vs. without treatment).

Figure 4. GRP knockdown enhances chemotherapy-induced apoptosis

(A) Combination treatment with vincristine and siGRP resulted in augmentation of apoptosis in JF and SK-N-SH cells when compared to vincristine alone. (B) GRP silencing, in addition to treatment with etoposide, resulted in an increase in apoptosis in comparison to drug treatment alone in both JF and SK-N-SH cells (mean ± SEM; * = p < 0.05 vs. siNTC alone, †= p < 0.05 vs. siNTC plus drug).

Figure 5. Activation of apoptotic pathway after siGRP treatment

Combination treatment of SK-N-SH cells with chemotherapeutic drugs and siGRP resulted in increased cleavage of PARP and caspase-3 suggesting activation of apoptosis as the mechanism of cell death by GRP silencing. β-actin levels indicate equal sample loading.

Figure 6. GRP silencing-induced apoptosis is mediated by p53 and G0-G1 cell cycle arrest

(A) Treatment of SK-N-SH cells with siGRP over a time course (24-96 h) resulted in an increase in phosphorylation of p53 and its downstream target p21. A delayed decrease in phospho-ERK was observed at 72 and 96 h time points (B) siGRP or siNTC transfected SK-N-SH cells (1 × 10⁶ cells/well) were plated and analyzed for cells in different phases of the cell cycle using flow cytometry. The percentage of cells in the apoptotic sub G0/G1 phase showed a significant increase after siGRP treatment.