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.

Graphical Abstract

中文翻译：

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胶质母细胞瘤细胞分子标志物的表达及抗菌肽化疗的协同抗癌作用

客观的

多形性胶质母细胞瘤（GBM）是最具侵袭性和致命性的恶性原发性脑肿瘤。即使采用手术、放疗和化疗等联合治疗方法，神经胶质瘤患者生存率的提高仍然有限。本研究旨在评估 11 名患者胶质母细胞瘤组织中 IDH1、TP53、EGFR、Ki-67、GFAP、H3K27M、MGMT、VEGF、NOS、CD99 和 ATRX 的表达。我们研究了导管素 (LL-37)、protegrin-1 (PG-1) 与化疗药物（替莫唑胺 (TMZ)、阿霉素 (DOX)、卡铂 (CB)、顺铂 (CPL) 和依托泊苷）的抗癌作用和联合作用(ETO) 在原代 GBM 细胞中。此外，我们还检测了 LL-37、PG-1 对正常人成纤维细胞和 C6/Wistar 大鼠脑胶质瘤模型的影响。

方法

在本研究中，对 11 例胶质母细胞瘤病例进​​行了 IDH1、TP53、EGFR、Ki-67、GFAP、H3K27M、MGMT、VEGF、NOS、CD99 和 ATRX 的免疫组织化学评估。MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑）测定用于研究细胞活力并确定 LL-37、PG-1 及其与化疗联合用药的细胞毒性作用。原代GBM细胞。使用组合指数（CI）方法确定协同或拮抗作用。最后，我们在Wistar大鼠中建立了C6胶质母细胞瘤模型来研究其抗肿瘤活性。

结果

与化疗相比，肽在 MTT 测试中显示出对原代 GBM 细胞的强烈细胞毒性作用（IC _{50 2-16 和 1-32 μM）。}LL-37 + DOX、LL-37 + CB (CI 0.46–0.75) 和 PG-1 + DOX、PG-1 + CB、PG-1 + TMZ (CI 0.11–0.77) 的双药组合显示出原代 GBM 细胞中的协同作用。在大鼠C6脑内GBM模型中，实验组大鼠的存活时间（66.75±12.6天）比对照组大鼠的存活时间（26.2±2.66天，p  = 0.0008）延长。^{LL-37治疗后，实验组大鼠的肿瘤体积（31.00±8.8mm 3} ）和重量（49.4±13.3mg）显着低于对照组大鼠（153.8±43.53mg，p  = 0.038；82.50±7.60mm ³，分别）。

结论

抗菌肽与化学药物联合增强化疗的细胞毒性，对原代GBM细胞发挥协同抗肿瘤作用。此外，体内研究首次证明LL-37可以有效抑制大鼠C6脑内GBM模型中的脑肿瘤生长。这些结果表明了一种重要的策略，可以提出一种有前景的 GBM 治疗方法。

图形概要