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Page 22 of 25 Han et al. Cancer Drug Resist 2024;7:16 https://dx.doi.org/10.20517/cdr.2024.01

Secondly, exploring the mechanism of isocuB in reversing drug resistance in glioma is necessary. We
established the U251/TMZ-resistant strain, and found hsa-mir-1286a might play an important role in
sensitizing drug-resistant cells to chemotherapy. Anyway, the combination effect of isocuB and TMZ, and
the regulatory effect of hsa-mir-1286a on TMZ-resistant xenograft, as well as PI3K-AKT and MAPK
pathways, are still unclear. In this situation, more experiments (e.g., calculating combined index) could be
conducted with the combination of isocuB and TMZ in U251/TMZ-resistant strain. Adopting more
resistant strains is beneficial for the study. For the animal study, the effect of isocuB on U251/TMZ-resistant
model could be conducted in established TMZ-resistant xenograft. Transfection of hsa-mir-1286a mimic or
inhibitor and addition of activator of PI3K-AKT and MAPK pathways benefit the investigation of the
underlying mechanism of isocuB in TMZ-resistance. Thirdly, more animal models could be used in the
study to give a more comprehensive evaluation of the effect of isocuB on glioma. We mainly utilized nude
mice to establish the xenograft with a single dose administration. Actually, administration with different
dosages in groups, at least dividing into high-dose group, medium-dose group, and low-dose group, could
assess the dose-dependent effect of isocuB in vivo. Immunohistochemistry could be utilized to detect the
protein expression in vivo and in situ hybridization could exhibit RNA expression, especially for hsa-mir-
1286a, in vivo. Establishing a glioma-bearing mouse model via orthotopic transplantation of GL261 cells in
C57BL/6 mice is also interesting for further study. Finally, according to our previous experiments, we found
that isocuB was slightly toxic to mice. Ensuring the safety of innovative drugs is necessary for preclinical
research. Thus, it is notable to conduct in vitro and in vivo experiments for evaluation and investigation of
the toxicology of isocuB, and search for novel approaches for detoxification of isocuB, such as structural
modification and addition of detoxicant.

Altogether, the underlying mechanism of anti-glioma activity of isocuB and detoxification of isocuB deserve
further investigation, providing novel insights into the drug candidate in glioma treatment.

Conclusions
The potential top five genes of the anti-glioma effect of isocuB were identified by network pharmacology as
RXRα, AKT1, ESR1, MAPK1, and HSP90AA1. Molecular docking data further indicated that isocuB could
bind to the corresponding protein of these genes with a good affinity. Moreover, AKT1 and MAPK1 mRNA
were expressed at a high level in glioma, according to the clinical database analysis results. Furthermore, GO
and KEGG analyses revealed that the PI3K/Akt and MAPK pathways were the top two connected pathways,
as AKT1 and MAPK1 are the key genes of the PI3K/AKT and MAPK pathways. IsocuB exhibited superior
efficacy over TMZ in inhibiting glioma proliferation, as evidenced by the CCK8 assay. IsocuB suppressed
the invasion and migration of U251 cells by blocking the activity of MMP2 and MMP9 in wound healing
and transwell assays. The TUNEL and FCM assays demonstrated the inhibitory effect of isocuB on BCL-2,
thereby inducing cell death. Subsequently, we conducted RT-qPCR and WB assays to determine that isocuB
could improve the sensitivity of U251 glioma to TMZ by inhibiting hsa-mir-1286a and reduce glioma
growth with the modulation of PI3K/AKT and MAPK pathways. Ultimately, our findings illustrate that
isocuB exerts an inhibitory effect on glioma growth and EMT process. The mechanism is associated with
the regulation of PI3K/AKT and MAPK pathway. Additionally, it is interesting to find that isocuB enhances
the drug sensitivity to TMZ via the decrease of hsa-mir-1286a RNA. The comprehensive evidence from
experimental demonstration, database analysis, and network pharmacology supports the therapeutic
potential of isocuB for glioma in the future.

DECLARATIONS
Authors’ contributions
Completed the network pharmacology and molecular docking experiments and prepared the original draft:
Han M