Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 µM), TMZ (155 µM), and NGF (7.55 × 10-3 µM) inhibited 43.9%-60.3%, 73.5%-81.3%, 66.2% the clonogenicity of glioma U251 cells for 1-2 days, respectively. LL-37 (4 µM), and NGF (7.55 × 10-3 µM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1-3 days. In contrast, PG-1 (16 µM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

Expression of molecular markers and synergistic anticancer effects of chemotherapy with antimicrobial peptides on glioblastoma cells.

OBJECTIVE: Glioblastoma multiforme (GBM) is the most aggressive and fatal malignant primary brain tumor. The enhancement of the survival rate for glioma patients remains limited, even with the utilization of a combined treatment approach involving surgery, radiotherapy, and chemotherapy. This study was designed to assess the expression of IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX in glioblastoma tissue from 11 patients. We investigated the anticancer impact and combined effects of cathelicidin (LL-37), protegrin-1 (PG-1), with chemotherapy-temozolomide (TMZ), doxorubicin (DOX), carboplatin (CB), cisplatin (CPL), and etoposide (ETO) in primary GBM cells. In addition, we examined the effect of LL-37, PG-1 on normal human fibroblasts and in the C6/Wistar rat intracerebral glioma model. METHODS: For this study, 11 cases of glioblastoma were evaluated immunohistochemically for IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cells viability and to determine cytotoxic effects of LL-37, PG-1 and their combination with chemotherapy in primary GBM cells. Synergism or antagonism was determined using combination index (CI) method. Finally, we established C6 glioblastoma model in Wistar rats to investigate the antitumor activity. RESULTS: Peptides showed a strong cytotoxic effect on primary GBM cells in the MTT test (IC50 2-16 and 1-32 µM) compared to chemotherapy. The dual-drug combinations of LL-37 + DOX, LL-37 + CB (CI 0.46-0.75) and PG-1 + DOX, PG-1 + CB, PG-1 + TMZ (CI 0.11-0.77), demonstrated a synergism in primary GBM cells. In rat C6 intracerebral GBM model, survival of rats in experimental group (66.75 ± 12.6 days) was prolonged compared with that in control cohort (26.2 ± 2.66 days, p = 0.0008). After LL-37 treatment, experimental group rats showed significantly lower tumor volumes (31.00 ± 8.8 mm3) and weight (49.4 ± 13.3 mg) compared with control group rats (153.8 ± 43.53 mg, p = 0.038; 82.50 ± 7.60 mm3, respectively). CONCLUSIONS: The combination of antimicrobial peptides and chemical drugs enhances the cytotoxicity of chemotherapy and exerts synergistic antitumor effects in primary GBM cells. Moreover, in vivo study provided the first evidence that LL-37 could effectively inhibit brain tumor growth in rat C6 intracerebral GBM model. These results suggested a significant strategy for proposing a promising therapy for the treatment of GBM.