Nerve Growth Factor, Antimicrobial Peptides and Chemotherapy: Glioblastoma Combination Therapy to Improve Their Efficacy

Abstract

Glioblastoma (GBM) is an aggressive and lethal malignancy of the central nervous system with a median survival rate of 15 months. We investigated the combined anticancer effects of nerve growth factor (NGF), cathelicidin (LL-37), and protegrin-1 (PG-1) with chemotherapy (temozolomide, doxorubicin, carboplatin, cisplatin, and etoposide) in the glioblastoma U251 cell line to overcome the limitations of conventional chemotherapy and to guarantee specific treatments to succeed. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cell viability and to determine the cytotoxic effects of NGF, LL-37, and PG-1 and their combination with chemotherapy in U251 cells. Synergism or antagonism was determined using the combination index (CI) method. Caspase-3 activity was evaluated spectrophotometrically using a caspase-3 activity assay kit. Apoptosis was analyzed with flow cytometry using propidium iodide (PI) and YO-PRO-1. NGF and the peptides showed a strong cytotoxic effect on U251 glioma cells in the MTT test (IC₅₀ 0.0214, 3.1, and 26.1 μM, respectively) compared to chemotherapy. The combination of PG-1 + etoposide had a synergistic effect on apoptosis of U251 glioma cells. It should be noted that the cells were in the early and late stages of apoptosis, respectively, compared with the control cells. The caspase-3 activation analysis revealed that the caspase-3 level was not significantly (p > 0.05) increased in U251 cells following PG-1 with etoposide treatment compared with that in the untreated cells, suggesting that the combination of PG-1 and etoposide may induce caspase-independent apoptosis in U251 cells. NGF, LL-37, and PG-1 represent promising drug candidates as the treatment regimen for GBM. Furthermore, the synergistic efficacy of the combined protocol using PG-1 and etoposide may overcome some of the typical limitations of the conventional therapeutic protocols, thus representing a promising approach for GBM therapy.

Keywords: antimicrobial peptides; chemotherapy; glioblastoma; nerve growth factor; synergistic effect.

Figure 1

Graph of viability versus drug concentration. Cell viability was assessed with the MTT assay. Dose-dependent effect of NGF (A), LL-37 (B), and PG-1 (C) and their combination with chemotherapy on cell viability at 24 h time-course. Data shown are representative of three separate experiments, and values are given as mean ± SD. Statistically significant difference * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 combination effects from drug alone; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001—statistically significant difference drug or combination from control.

Figure 1

Graph of viability versus drug concentration. Cell viability was assessed with the MTT assay. Dose-dependent effect of NGF (A), LL-37 (B), and PG-1 (C) and their combination with chemotherapy on cell viability at 24 h time-course. Data shown are representative of three separate experiments, and values are given as mean ± SD. Statistically significant difference * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 combination effects from drug alone; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001—statistically significant difference drug or combination from control.

Figure 2

Computed CI values for the combination of NGF on U251 human glioma cells according to the results of the MTT test (A); LL-37 with NGF and PG-1 on U251 human glioma cells according to the results of the MTT test (B); PG-1 with LL-37 and NGF on U251 human glioma cells according to the results of the MTT test (C). The results are presented as the means (columns) ± S.D. (bars) (n = 3, in triplicate). * p < 0.05, ** p < 0.01, **** p < 0.0001 statistically significant differences between CI value of drug or its combinations and control.

Figure 2

Computed CI values for the combination of NGF on U251 human glioma cells according to the results of the MTT test (A); LL-37 with NGF and PG-1 on U251 human glioma cells according to the results of the MTT test (B); PG-1 with LL-37 and NGF on U251 human glioma cells according to the results of the MTT test (C). The results are presented as the means (columns) ± S.D. (bars) (n = 3, in triplicate). * p < 0.05, ** p < 0.01, **** p < 0.0001 statistically significant differences between CI value of drug or its combinations and control.

Figure 3

Apoptosis, necrosis, and necroptosis analysis with flow cytometry using PI and YO-PRO-1. U251 cells were treated with etoposide (B) and PG-1 (C) individually and in combination (D) (etoposide + PG-1) for 24 h compared with that in the normal controls (A). C3 represents the rate of viable cells; C1 and C2 represent the rate of late apoptosis or necrosis; C4 represents the rate of early apoptosis.

Figure 4

Caspase-3 activity of U251 cells; Data shown are representative of three separate experiments, and values are given as mean ± SD. * p < 0.05, statistically significant difference between control and PG-1.