Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model

Abstract

Nucleolin (NCL) is highly expressed in several types of cancer and represents an interesting therapeutic target. It is expressed at the plasma membrane of tumor cells, a property which is being used as a marker for several human cancer including glioblastoma. In this study we investigated targeting NCL as a new therapeutic strategy for the treatment of this pathology. To explore this possibility, we studied the effect of an antagonist of NCL, the multivalent pseudopeptide N6L using primary culture of human glioblastoma cells. In this system, N6L inhibits cell growth with different sensitivity depending to NCL localization. Cell cycle analysis indicated that N6L-induced growth reduction was due to a block of the G1/S transition with down-regulation of the expression of cyclin D1 and B2. By monitoring autophagy markers such as p62 and LC3II, we demonstrate that autophagy is enhanced after N6L treatment. In addition, N6L-treatment of mice bearing tumor decreased in vivo tumor growth in orthotopic brain tumor model and increase mice survival. The results obtained indicated an anti-proliferative and pro-autophagic effect of N6L and point towards its possible use as adjuvant agent to the standard therapeutic protocols presently utilized for glioblastoma.

Keywords: autophagy; glioblastoma; targeted therapy.

Figure 1. Viability assay on glioblastoma primary cultures, more sensitive (panel A) and less sensitive cells (panel B) upon treatment with different N6L concentrations for different timepoints

Data are reported with respect to control untreated cells. The experiment reported is representative of 4 experiments performed in quadruplicate. Data are mean ± SE; **,p < 0.005; ***p < 0.0005. In C and D nucleolin immunolocalization in more sensitive and less sensitive cells, respectively.

Figure 2

N6L internalization by Alexafluor 488-N6L (fN6L) in the more responsive cultures A. and B. and in the less responsive ones C. and D.

Figure 3

Panel A: Proliferation assay in control and N6L-treated cells, evaluated by BrdU incorporation at different timepointsThe data are expressed as percentage of the relative control. The experiment reported is representative of 4 experiments for each culture performed in quadruplicate. Data are mean ± SE. **, p < 0.005; ***p < 0.0005. In panel B: western blotting analysis for cyclin D1 in control and N6L-treated cultures for 24 h and 48 h. A representative blotting is shown; the densitometric analysis is the mean ± SE of 4 different experiments for each culture. ***, p < 0.0005; Panel C: western blotting analysis for cyclin B2 in control and N6L-treated cultures for 24 h and 48 h. A representative blotting is shown, the densitometric analysis is the mean ± SE of 4 different experiments for each culture. **, p < 0.005; ***, p < 0.0005.

Figure 4. Cell cycle analysis measured by cytofluorimetry on glioblastoma primary cultures upon treatment with N6L for 24 and 48 h

The experiment reported is representative of quadruplicate experiments. The panel A: shows the original cytofluorimetric profiles, the panels B: and C: show the analysis of the cell population distribution along the cell cycle phases in control and treated conditions. Data are mean ± SE. *, p < 0.05.

Figure 5

Panel A: cell migration in control and N6L-treated cellsThe experiment reported is representative of 4 experiments for each culture performed in quadruplicate. Data are mean ± SE. ***, p < 0.0005. Panel B: western blotting analysis for p-EGFR in control and N6L-treated cultures for 24 h and 48 h. A representative blotting is shown; the densitometric analysis is the mean ± SE of 4 different experiments for each culture. ***, p < 0.0005; Panel C: western blotting analysis for p-ERK1,2 in control and N6L-treated cultures for 24 h and 48 h. A representative blotting is shown; the densitometric analysis is the mean ± SE of 4 different experiments for each culture. *, p < 0.05; **, p < 0.005.

Figure 6

A: western blotting analysis for p-62 in control and N6L-treated cultures for 24 h and 48 h. A representative blotting is shown; the densitometric analysis is the mean ± SE of 4 different experiments for each culture. **, p < 0.005. B: autophagic flux evaluation by CQ treatment in the presence/absence of N6L by western blotting analysis for LC3II and LC3I. A representative blotting is shown; the densitometric analysis, representing the ratio LC3II/LC3I is the mean ± SE of 4 different experiments for each culture. **p < 0.005; ***, p < 0.0005; ++, p < 0.005, N6L/CQ versus CQ C: LC3 immunolocalization in control and N6L-treated cells.

Figure 7

A: western blotting analysis for nuclear p-53 in control and N6L-treated cultures at 24 h and 48 h. A representative blotting is shown; the densitometric analysis is the mean ± SE of 4 different experiments for each culture. B: western blotting analysis for cytosolic p-53 in control and N6L-treated cultures for 24 h and 48 h. A representative blotting is shown; the densitometric analysis is the mean ± SE of 4 different experiments for each culture. **, p < 0.005. C: p-53 immunolocalization in control and N6L-treated cells.

Figure 8. in vivo studies performed on BALB/c-nu/nu athymic mice injected intracranially with U87 LUC cells and treated with 10 mg/kg bw of N6L for 5 days/week from day 1 post-cell inoculation for 6 weeks

In Panel A: representative images of the bioluminescence in control and N6L-treated mice is reported; In Panel B: quantification of the bioluminescence signal recorded at the1st, 2nd, 3rd, and 4th week from cell injection. The BP values shown are the results of the ANOVA for each group, while AP values shown are the results of the ANOVA for control group versus treated group. In Panel C: the survival function obtained by Kaplan-Meier survival plot of N6L treated and sham-treated control animals. The mean survival times were 33.3+/–1.5 and 21.6+/–0.6 days, respectively, for the treated and control groups (mean+/–SE, n = 12). There was a statistically significant increase life span in the treated animals (P = 0.0001 Mantel-Cox log rank test).