Host-defence caerin 1.1 and 1.9 peptides suppress glioblastoma U87 and U118 cell proliferation through the modulation of mitochondrial respiration and induce the downregulation of CHI3L1

Abstract

Glioblastoma, the most aggressive form of brain cancer, poses a significant global health challenge with a considerable mortality rate. With the predicted increase in glioblastoma incidence, there is an urgent need for more effective treatment strategies. In this study, we explore the potential of caerin 1.1 and 1.9, host defence peptides derived from an Australian tree frog, in inhibiting glioblastoma U87 and U118 cell growth. Our findings demonstrate the inhibitory impact of caerin 1.1 and 1.9 on cell growth through CCK8 assays. Additionally, these peptides effectively curtail the migration of glioblastoma cells in a cell scratch assay, exhibiting varying inhibitory effects among different cell lines. Notably, the peptides hinder the G0/S phase replication in both U87 and U118 cells, pointing to their impact on the cell cycle. Furthermore, caerin 1.1 and 1.9 show the ability to enter the cytoplasm of glioblastoma cells, influencing the morphology of mitochondria. Proteomics experiments reveal intriguing insights, with a decrease in CHI3L1 expression and an increase in PZP and JUNB expression after peptide treatment. These proteins play roles in cell energy metabolism and inflammatory response, suggesting a multifaceted impact on glioblastoma cells. In conclusion, our study underscores the substantial anticancer potential of caerin 1.1 and 1.9 against glioblastoma cells. These findings propose the peptides as promising candidates for further exploration in the realm of glioblastoma management, offering new avenues for developing effective treatment strategies.

Fig 1. CCK8 assay of the effects of F1/F3 against the proliferation of U87 and U118 cells.

(A) F1/F3 exerted a substantial inhibitory effect on the growth of U87 cells, with an IC50 value of 5.018 μg/mL. (B) F1/F3 significantly suppressed the proliferation of U118 cells, yielding an IC50 value of 11.18 μg/mL. Microscopic photos of the scratches in U87 (left) and U118 (right) cells recorded at 0, 6, 12, and 24 h in the control and treatment groups (F1/F3 at a concentration of 5 μg/mL, with a molar ratio 1:1), respectively (C). Comparison of the healing degree of U87 (D) and U118 (E) cells between the control and the treatment groups in the scratch experiment at different time. Data are representative of at least 3 independent experiments and are presented as the mean ± SD. ns: non-significant, *: P-value < 0.05, **: P-value < 0.01, ***: P-value < 0.001 and ****: P-value < 0.0001.

Fig 2. F1/F3 induced the apoptosis of U87 and U118 cells.

Flow cytometry images compares the apoptosis degree of U87 (A) and U118 (B) cells in the untreated, F1/F3 and P3 groups. (C) Percentage of apoptotic cells in response to different treatments in U87 and U118 cells relative to the untreated groups, respectively. Data are representative of at least 3 independent experiments and are presented as the mean ± SD. ns: non-significant, ***: P-value < 0.001, ****: P-value < 0.0001.

Fig 3. Fluorescence microscopy images of glioma cells treated with PBS (untreated), FITC-labelled F1/F3 and FITC-labelled P3 (control) after 6 hours of treatment.

The concentration of F1/F3 is 5 μg/mL, and the molar ratio of F1 to F3 is 1:1. Fluorescence microscopic images of U87 (A) and U118 (B) cells in the untreated, F1/F3 and control groups with FITC and DAPI labels, as well as the merged images. (C) The distribution of FITC-labelled F1/F3 in the cytoplasm of U87 and U118 cells, respectively. In these images, blue corresponds to the nucleus stained with the DAPI probe, while green corresponds to FITC.

Fig 4. Electron microscopy images of F1/F3 treating glioblastoma cells.

The magnification of the electron microscope is 40,000x, and the concentration of F1/F3 is 5 μg/mL with a molar ratio 1:1. Selected images of U87 (A) and U118 (B) cells treated by F1/F3 for 0, 1, 6, and 12 h. ER: endoplasmic reticulum; G: Golgi apparatus; L: lysosome; M: mitochondrion; N: nucleus; R: ribosome. The scale bar is 500 nm.

Fig 5. The comparison of mitochondrial membrane potential in glioblastoma cells with and without F1/F3 treatment.

(A) Fluorescence microscopy images show the changes in mitochondrial membrane potential in untreated (left two images) and treated (right two images) U87 cells. (B) Fluorescence microscopy images show the changes in mitochondrial membrane potential in untreated (left two images) and treated (right two images) U87 cells. In the images, red indicates that JC-1 is in a polymer state, signifying healthy mitochondria, while green indicates that JC-1 is in a monomeric state, indicating a decrease in mitochondrial transmembrane potential.

Fig 6. Quantitative proteomic analysis of glioblastoma cells with F1/F3 treatment.

(A) The UpSet graph provides a comparison of the DEPs identified between different groups: U87, U118, U87F, and U118F. The Venn graph in (A) compares the DEPs up- and downregulated in the U87 relative to U118 group (UN), as well as the U87F relative to U118F group (F1/F3). (B) The Volcano graph represents the DEPs that are upregulated and downregulated in U87 cells due to the treatment, as compared to the untreated U87 group. (C) Protein-protein interaction between DEPs in the U87F relative to the U87 groups. (D) Enrichment analysis of biological processes in the U87F with respect to the U87 groups. GSEA reveals the hallmark pathways activated in the U87F (E) and U118F (F) concerning U87 and U118 groups, respectively. The data are representative of three independent experiments. (See S1 and S2 Tables for detailed proteomic analysis results).

Fig 7. F1/F3 treatment inhibited the expression of CHI3L1 and shifted the energy metabolism of U87 cells.

At 5 μg/mL, F1/F3 downregulated the content of CHI3L1 significantly (A) and induced a concentration-dependent response (B) in U87 cells. At 5 μg/mL, F1/F3 significantly downregulated the content of CHI3L1 (C) in U118 cells, and the concentration dependence displayed in (D). (E) The TCGA glioblastoma multiforme database revealed a notable upregulation of CHI3L1 in tumours compared to normal tissues. (F) Low CHI3L1 expression was positively correlated with the survival time of glioblastoma multiforme patients, showing statistical significance. (G) Evaluation of glycolysis/OXPHOS levels in the U87 (top) and U87F (bottom) groups demonstrated that F1/F3 treatment led to a shift from glycolysis to OXPHOS in U87 cells, as indicated by changes in ATP levels and lactate production. The inhibition of glycolysis or OXPHOS pathways was facilitated using 2-DG or oligomycin, respectively. Data are representative of at least 3 independent experiments and are presented as the mean ± SD. *: P-value < 0.05, **: P-value < 0.01, ***: P-value < 0.001, and ****: P-value < 0.0001.(see S3 Table for analysis results of western-blotting and TCGA data, S1 Raw images for original western-blotting images, and S4 Table for glycolysis/OXPHOS test data).