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Han et al. Cancer Drug Resist 2024;7:16 https://dx.doi.org/10.20517/cdr.2024.01 Page 11 of 25

AKT1 contains mutations, deep deletions, and amplifications in gliomas. AKT1 exhibited the highest
mutation rate of 3.81%, amplification rate of 2.02%, and deep deletion rate of 0.34% [Figure 2C]. A total of
189 samples from two studies were included in the methylation analysis. The results showed a negative
correlation between AKT1 methylation and AKT1 gene expression in glioma tissues (P < 0.001), indicating
that AKT1 gene expression may be epigenetically regulated. Copy number amplification of AKT1 was
detected in three cases, while mild AKT1 copy number loss was detected in 29 cases. According to the
pathological type, all 189 cases of GBM (100%) had low AKT1 methylation. Mutations in MAPK1 were
identified in gliomas. MAPK1 exhibited the highest mutation rate (1.9%) without any apparent significant
deletions or amplifications [Figure 2D]. Different mutations and different types of gliomas associated with
MAPK1 are shown in Figure 2D.

To investigate the relationship between AKT1 and MAPK1 mRNA expression and clinicopathologic
parameters, we used the CGGA database, which comprises 325 cases from the glioma mRNA microarray
database, for correlation analysis. The cutoff values for AKT1 and MAPK1 mRNA expression were 70.48
and 44.84, respectively. Values higher than the cutoff indicated high expression, and vice versa. The results
indicated that AKT1 and MAPK1 were present in different tumor stages. There were statistically significant
differences in the expression of glioma grades (WHO II, WHO III, and WHO IV) (P < 0.001) [Figure 3A
and B], as well as statistically significant differences among different states of isocitrate dehydrogenase
(IDH) (wild type, mutant type) (P < 0.001). There was a statistically significant difference among the various
age groups (P < 0.05) [Figure 3A and B]. Expression of AKT1 and MAPK1 was correlated with WHO
classification, IDH status, and age. The TCGA glioma data were retrieved from the GEPIA database. The
group cutoff was selected as the median to divide the high expression group and the low expression group
(cutoff-high = 50%; cutoff-low = 50%) for analyzing the mRNA expression of AKT1 and MAPK1 in glioma
to verify their prognostic relationship. To validate the analysis results from the CGGA database and assess
the prognostic significance of AKT1 and MAPK1 in glioma, survival analyses were performed using the
GEPIA database. The results, using the Kaplan-Meier method, showed that there were 676 cases of TCGA
glioma. The prognosis of the low AKT1 and MAPK1 expression groups was better than that of the high
expression group, and the differences in overall survival (OS) between the two groups were statistically
significant (P < 0.01, P < 0.0001). Disease-free survival of the low AKT1 and MAPK1 expression groups was
better than that of the high expression group. The differences were significant (P < 0.05) [Figure 3C and D].

Experimental verification results
IsocuB inhibits proliferation, migration and invasion and increases apoptosis
Our experimental results demonstrated that isocuB had a concentration- and time-dependent effect on
glioma cell proliferation in the CCK8 assay [Figure 4A]. The IC of isocuB-inhibited U251 cells is
50
0.79 µmol/L at 24 h and 10.54 µmol/L at 12 h. The IC of isocuB-inhibited U87 cells is 2.12 µmol/L at 24 h
50
[Figure 4B]. Thus, the inhibitory effect of isocuB on U251 was much greater than that on U87; therefore,
U251 was primarily used in our subsequent experiments. Our results demonstrate that isocuB inhibited
glioma migration in a time- and dose-dependent manner, as illustrated in the wound healing assay
[Figure 4C and D].

In addition, isocuB inhibited the invasion of glioma cells in the transwell invasion assay [Figure 5A and B].
The MMP family is associated with cancer cell survival, proliferation, apoptosis, invasion, and metastasis .
[25]
The mRNA and protein expression levels were significantly decreased after interference with isocuB in
U251 cells [Figure 5C-E]. Epithelial-mesenchymal transition (EMT) is a process in which cells acquire
invasive mesenchymal competence through a series of events that include loss of cellular connections,
[26]
cytoskeletal reorganization, and remodeling of the extracellular matrix . The results of the WB experiment
results showed a significant decrease in the protein levels of N-cadherin and Vimentin. This indicated that