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Page 2 of 25 Han et al. Cancer Drug Resist 2024;7:16 https://dx.doi.org/10.20517/cdr.2024.01

extracted from melon pedicels, on glioma has not been investigated. This study aims to investigate the inhibitory
effect of isocuB on glioma and elucidate its underlying mechanisms, with the objective of developing it as a
potential therapeutic agent for glioma.

Methods: We used network pharmacology and bioinformatics analysis to predict potential targets and associated
pathways of isocuB in glioma. Subsequently, the inhibitory effect of isocuB on glioma and its related mechanisms
were assessed through Counting Kit-8 (CCK-8), wound healing, transwell, Western blot (WB), reverse
transcription-quantitative polymerase chain reaction (RT-qPCR), and other in vitro experiments, alongside tumor
formation experiments in nude mice.

Results: Based on this investigation, it suggested that isocuB might inhibit the growth of gliomas through the PI3K-
AKT and MAPK pathways. Additionally, we proposed that isocuB may enhance glioma drug sensitivity to
temozolomide (TMZ) via modulation of hsa-mir-1286a. The CCK-8 assay revealed that isocuB exhibited inhibitory
effects on U251 and U87 proliferation and outperformed TMZ. Wound healing and transwell experiments showed
that isocuB inhibited the invasion and migration of U251 cells by suppressing the activity of MMP-2/9, N-cadherin,
and Vimentin. The TdT-mediated dUTP-biotin nick end labeling (TUNEL) and flow cytometry (FCM) assays
revealed that isocuB induced cell apoptosis through inhibition of BCL-2. Subsequently, we conducted RT-qPCR
and WB experiments, which revealed that PI3K/AKT and MAPK pathways might be involved in the
mechanism of the inhibition isocuB on glioma. Additionally, isocuB promoted the sensitivity of glioma U251
to TMZ by inhibiting hsa-mir-1286a. Furthermore, we constructed TMZ-resistant U251 strains and demonstrated
effective inhibition by isocuB against these resistant strains. Finally, we confirmed that isocuB can inhibit tumor
growth in vivo through experiments on tumors in nude mice.

Conclusion: IsocuB may protect against glioma by acting on the PI3K/AKT and MAPK pathways and promote the
sensitivity of glioma U251 to TMZ by inhibiting hsa-mir-1286a.

Keywords: Isocucurbitacin B, glioma, network pharmacology, hsa-mir-1286a, drug sensitivity

INTRODUCTION
The most challenging malignancies worldwide encompass stomach, lung, liver, colorectal, esophageal,
breast cancer, and glioma. Gliomas account for 81% of central nervous system (CNS) malignancies and pose
significant therapeutic challenges. They are classified into four grades (1-4) ranging from low to high
[1,2]
grade . According to the fifth edition of the WHO classification of tumors of the central nervous system,
adult-type diffuse gliomas, pediatric-type diffuse low-grade gliomas, pediatric-type diffuse high-grade
[3]
gliomas, and circumscribed astrocytic gliomas were featured . There are numerous therapeutic methods for
gliomas, which are mainly categorized into surgery, immunotherapy, targeted therapy, electric field therapy,
[4,5]
and chemophototherapy . At present, the predominant approach globally involves maximal surgical
resection supplemented with temozolomide (TMZ) and radiotherapy. However, this regimen generally
[6]
yields a dismal .

Modern pharmaceutical systems commonly use natural remedies to extract active ingredients with
pharmacological properties. 60,000 years ago, plants were used as medicines, reflecting a wide range of
[7]
pharmacological properties, including anticancer activity. Numerous natural compounds , such as
quercetin , isoliquiritigenin , and lycopene , have apparent efficacy in inhibiting tumorigenesis,
[10]
[9]
[8]
inhibiting tumor metastasis, and controlling tumor growth. Isocucurbitacin B (IsocuB) is isolated from the
stalked tips of melons in the angiosperm family Cucurbitaceae. Natural cucurbitines exhibit various
biological and pharmacological activities. Almost all the Cucurbitaceae families consist of approximately
229 species. Cucurbitacin is a class of highly oxidized tetracyclic triterpenoids with potent anticancer