AKR1B1 Represses Glioma Cell Proliferation through p38 MAPK-Mediated Bcl-2/BAX/Caspase-3 Apoptotic Signaling Pathways

This study aimed to investigate the regulatory role of Aldo-keto reductase family 1 member B1 (AKR1B1) in glioma cell proliferation through p38 MAPK activation to control Bcl-2/BAX/caspase-3 apoptosis signaling. AKR1B1 expression was quantified in normal human astrocytes, glioblastoma multiforme (GBM) cell lines, and normal tissues by using quantitative real-time polymerase chain reaction. The effects of AKR1B1 overexpression or knockdown and those of AKR1B1-induced p38 MAPK phosphorylation and a p38 MAPK inhibitor (SB203580) on glioma cell proliferation were determined using an MTT assay and Western blot, respectively. Furthermore, the AKR1B1 effect on BAX and Bcl-2 expression was examined in real-time by Western blot. A luminescence detection reagent was also utilized to identify the effect of AKR1B1 on caspase-3/7 activity. The early and late stages of AKR1B1-induced apoptosis were assessed by performing Annexin V-FITC/PI double-staining assays. AKR1B1 expression was significantly downregulated in glioma tissues and GBM cell lines (T98G and 8401). Glioma cell proliferation was inhibited by AKR1B1 overexpression but was slightly increased by AKR1B1 knockdown. Additionally, AKR1B1-induced p38 MAPK phosphorylation and SB203580 reversed AKR1B1′s inhibitory effect on glioma cell proliferation. AKR1B1 overexpression also inhibited Bcl-2 expression but increased BAX expression, whereas treatment with SB203580 reversed this phenomenon. Furthermore, AKR1B1 induced caspase-3/7 activity. The induction of early and late apoptosis by AKR1B1 was confirmed using an Annexin V-FITC/PI double-staining assay. In conclusion, AKR1B1 regulated glioma cell proliferation through the involvement of p38 MAPK-induced BAX/Bcl-2/caspase-3 apoptosis signaling. Therefore, AKR1B1 may serve as a new therapeutic target for glioma therapy development.


Introduction
Gliomas are the most common brain tumors among adults [1]. Its most aggressive subtype is glioblastoma multiforme (GBM), which accounts for approximately 80% of malignant gliomas, and the median survival time is only 12-14 months [2]. Considering its diffuse and infiltrative characteristics, GBM exhibits outstanding abilities in cell proliferation, invasion, and migration, thereby limiting the scope for surgical removal and the effects of clinical treatments [3,4]. Elucidating the cellular and molecular mechanisms leading to GBM proliferation and invasion would shed light on new strategies for therapeutic intervention. Gliomas are a collection of brain tumors that arise from glial cells in the pathway. Upon activation by internal or external stimuli, caspase-3 cleaves and activates downstream apoptotic proteins, culminating in the disassembly of cellular structures and functions. Therefore, the quantification of caspase-3 activity is a crucial means of evaluating the extent of apoptosis [44,45].
In this study, we aimed to investigate the roles of AKR1B1 in glioma cell proliferation and related mechanisms. We hypothesized that the AKR1B1 reduces glioma cell proliferation and activates p38 MAPK phosphorylation, thereby mediating the Bcl-2/BAX/caspase-3 pathway.

Patient Samples
All tissue samples used in this study were collected from patients undergoing surgical resection at Kaohsiung Medical University Hospital (Kaohsiung, Taiwan). Prior to the procedure, written informed consent was obtained from each participant. This study was approved by the Clinical Research Ethics Committee of Kaohsiung Medical University Hospital (KMUHIRB-G(II)-20170010). During glioma resection surgery, normal brain tissue was collected. The initial step of the procedure involved corticotomy, during which some normal brain tissue was removed. Consequently, normal brain tissue could be collected during the necessary steps of tumor resection. The specific location of the normal brain tissue was dependent on the tumor location, such as the frontal, parietal, or occipital lobe.

DNA Transfection and RNA Interference
A pCMV6-Entry vector, pCMV6-Myc-DDK-tagged-AKR1B1, or si-RNA was added into a jetPRIME buffer and then mixed gently with a jetPRIME transfection reagent according to the manufacturer's instructions. We incubated the transfection mixture for 10 min at room temperature and added it to cells in a growth medium. These cells were then incubated at 37 • C, and the transfection medium was replaced with a cell growth medium after 24 h transfection.

Caspase3/7 Activity Assay
After seeding the cells in 96-well plates at a density of 1 × 10 4 cells/well, we added 20 µL of Caspase-Glo 3/7 reagent to individual wells and control wells (growth media only). Subsequently, the plates were shaken at 300 rpm for 30 s and further incubated in the dark at 37 • C for 30 min. Luminescence was then measured using a microplate reader (Promega Corporation, Madison, WI, USA).

MTT Assay
For this assay, cells were also seeded in 96-well plates at a density of 1 × 10 4 cells/well. The viability of cells transfected with plasmids was measured at 0, 24, 48, 72, and 96 h, and that of cells treated with si-RNA was measured at 0, 1, 2, 3, 4, and 5 d. We added the MTT reagent (100 µL) to each well and incubated the cells for 4 h at 37 • C. After incubation, we carefully removed the MTT reagent and added 100 µL of dimethyl sulfoxide to solubilize the violet crystals. Next, the plates were shaken for 20 min in the dark to allow for complete solubilization. The optical density was measured at 490 nm using a microplate (ELISA) reader (Thermo Fischer, Waltham, MA, USA).

Western Blot Analysis
Cells were harvested and lysed using RIPA buffer supplemented with protease inhibitors. The protein concentration in the resulting lysate was measured using a Bradford assay (Bio-Rad, Hercules, CA, USA). Total proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. For 1 h at room temperature, the membranes were blocked in phosphate-buffered saline with 0.1% Tween 20 containing 5% nonfat milk powder. Next, we incubated these membranes with primary antibodies overnight at 4 • C, followed by incubation with appropriate secondary antibodies for 1 h at room temperature. The chemiluminescence signal was visualized using an enhanced chemiluminescence detection kit system (PerkinElmer, Shelton, CT, USA).

Annexin V-FITC/PI Double-Staining Assay
A total of 8 × 10 4 cells were plated in a 12-well culture plate, and after 24 h, they were transfected with either 0.2 µg of a control plasmid or a plasmid expressing AKR1B1. Following 24 h of transfection, cells were gently washed with 1× PBS to remove the excess medium. In accordance with the manufacturer's recommendations, an appropriate volume of Annexin V-FITC staining solution was added, mixed gently to prevent foaming and incubated at room temperature for 15-20 min in the dark. Subsequently, an appropriate volume of propidium iodide (PI) staining solution was added and mixed gently. Cells were then washed gently with 1× PBS to remove un-bound Annexin V-FITC and PI dyes. After staining, the cells were examined under a fluorescent microscope (Nikon Eclipse 800), and the corresponding fluorescent images were captured at a 20× magnification.
To quantify the percentage of Annexin V-positive and PI-positive cells, multiple fields were randomly chosen. The numbers of Annexin V-positive (green fluorescence) and PI-positive (red fluorescence) cells were enumerated for each field, along with the total number of cells using brightfield microscopy. The percentage of Annexin V-positive and PI-positive cells was calculated as the number of positive cells divided by the total number of cells and then multiplied by 100. The results were then averaged across all the fields to determine the overall percentage of Annexin V-positive and PI-positive cells for each experimental condition.

Statistical Analysis
All experimental results were analyzed using Student's t-tests. A p-value less than 0.05 was considered statistically significant.

AKR1B1 Expression in Human Glioma Tissue and Various Glioma Cell Lines
To explore whether AKR1B1 expression was inhibited in human glioma cells, we examined AKR1B1 mRNA levels in normal human brain tissues and glioma tissues. According to the analysis by quantitative reverse-transcription polymerase chain reaction, the AKR1B1 mRNA level significantly decreased by 50% in human glioma tissues ( Figure 1A), suggesting that low AKR1B1 levels participated in tumorigenesis.
ume of Annexin V-FITC staining solution was added, mixed gently to prevent foaming and incubated at room temperature for 15-20 min in the dark. Subsequently, an appropriate volume of propidium iodide (PI) staining solution was added and mixed gently. Cells were then washed gently with 1× PBS to remove un-bound Annexin V-FITC and PI dyes. After staining, the cells were examined under a fluorescent microscope (Nikon Eclipse 800), and the corresponding fluorescent images were captured at a 20× magnification. To quantify the percentage of Annexin V-positive and PI-positive cells, multiple fields were randomly chosen. The numbers of Annexin V-positive (green fluorescence) and PI-positive (red fluorescence) cells were enumerated for each field, along with the total number of cells using brightfield microscopy. The percentage of Annexin V-positive and PI-positive cells was calculated as the number of positive cells divided by the total number of cells and then multiplied by 100. The results were then averaged across all the fields to determine the overall percentage of Annexin V-positive and PI-positive cells for each experimental condition.

Statistical Analysis
All experimental results were analyzed using Student's t-tests. A p-value less than 0.05 was considered statistically significant.

AKR1B1 Expression in Human Glioma Tissue and Various Glioma Cell Lines
To explore whether AKR1B1 expression was inhibited in human glioma cells, we examined AKR1B1 mRNA levels in normal human brain tissues and glioma tissues. According to the analysis by quantitative reverse-transcription polymerase chain reaction, the AKR1B1 mRNA level significantly decreased by 50% in human glioma tissues ( Figure 1A), suggesting that low AKR1B1 levels participated in tumorigenesis.  According to our findings in human tissues, human glioma cell lines with low AKR1B1 expression should be selected as the appropriate cell model. Among normal human astrocytes (SV-1) and human glioma cell lines (U87, 8401, G5T, 05MG, and T98G), 8401 and T98G cell lines presented the lowest AKR1B1 mRNA levels ( Figure 1B). Therefore, 8401 and T98G cells were the most appropriate cell models to investigate the role of AKR1B1 in tumorigenesis.

AKR1B1 Caused a Cytotoxic Effect on Human Glioblastoma Cell Lines
To examine whether AKR1B1 could inhibit tumor growth, we overexpressed AKR1B1 levels in human glioma cells by transfecting plasmid expression into the cells (denoted as AKR1B1-expressing glioma cells). The transfection efficiency of the AKR1B1-expressing plasmid was determined by increased mRNA and protein levels in human T98G cells (Figure 2A). After plasmid transfection, we examined the cytotoxic effect every 24 h by using the MTT assay. AKR1B1 showed potently antiproliferative effects on T98G and 8401 time-dependently ( Figure 3A,B). We further examined whether inhibiting AKR1B1 could promote glioma cell proliferation. To test our hypothesis, we knocked down AKR1B1 in glioma cells through si-RNA and measured cell viability every 24 h. Diminished mRNA and protein levels in 8401 cells determined the transfection efficiency of AKR1B1 si-RNA ( Figure 2B). According to the cell viability analysis, AKR1B1 knockdown exacerbated cancer cell growth time-dependently ( Figure 3C,D). Therefore, low AKR1B1 levels directly caused tumor progression.

AKR1B1 Caused a Cytotoxic Effect on Human Glioblastoma Cell Lines
To examine whether AKR1B1 could inhibit tumor growth, we overexpress AKR1B1 levels in human glioma cells by transfecting plasmid expression into the ce (denoted as AKR1B1-expressing glioma cells). The transfection efficiency of the AKR1B expressing plasmid was determined by increased mRNA and protein levels in hum T98G cells (Figure 2A). After plasmid transfection, we examined the cytotoxic effect eve 24 h by using the MTT assay. AKR1B1 showed potently antiproliferative effects on T98 and 8401 time-dependently ( Figure 3A,B). We further examined whether inhibitin AKR1B1 could promote glioma cell proliferation. To test our hypothesis, we knocke down AKR1B1 in glioma cells through si-RNA and measured cell viability every 24 Diminished mRNA and protein levels in 8401 cells determined the transfection efficien of AKR1B1 si-RNA ( Figure 2B). According to the cell viability analysis, AKR1B1 knoc down exacerbated cancer cell growth time-dependently ( Figure 3C,D). Therefore, lo AKR1B1 levels directly caused tumor progression.  (right panel) analysis results show increased AKR1B1 protein and mRNA levels at 24 h after transfection with the AKR1B1 plasmid in T98G cells, respectively, (n = 3). (B) Western blot (left panel) and q-RT-PCR (right panel) analysis results reveal that AKR1B1 protein and mRNA levels significantly declined at 72 h after siRNA transfection in 8401 cells (n = 3). Data are presented as mean ± SEM comparing 8401 glioma cells treated with the control plasmid (CN plasmid) or the control si-RNA (si-CN); * p < 0.05 and ** p < 0.01.

AKR1B1 Activated p38 Signaling in Glioma Cells
In the mechanism of AKR1B1-induced cancer cell death, AKR1B1 might activate the p38 MAPK signaling pathway in glioma cells. To test this hypothesis, we assessed phosphorylated p38 MAPK (the active form of p38 MAPK) and total p38 MAPK expression levels in glioma cells by Western blot analysis. We observed increased phosphorylated p38 MAPK levels and no significant alteration of total p38 MAPK protein in AKR1B1-expressing T98G cells ( Figure 4A,B). Next, we assessed whether the antitumor effect of AKR1B1 could be reversed by SB203580, a p38 MAPK inhibitor. Indeed, the p38 MAPK inhibitor rescued the viability of T98G cells time-dependently ( Figure 4B). Thus, AKR1B1 directly activated p38 MAPK signaling, which played a critical role in the antitumor effect of AKR1B1.

AKR1B1 Activated p38 Signaling in Glioma Cells
In the mechanism of AKR1B1-induced cancer cell death, AKR1B1 might activate the p38 MAPK signaling pathway in glioma cells. To test this hypothesis, we assessed phosphorylated p38 MAPK (the active form of p38 MAPK) and total p38 MAPK expression levels in glioma cells by Western blot analysis. We observed increased phosphorylated p38 MAPK levels and no significant alteration of total p38 MAPK protein in AKR1B1-expressing T98G cells ( Figure 4A,B). Next, we assessed whether the antitumor effect of AKR1B1 could be reversed by SB203580, a p38 MAPK inhibitor. Indeed, the p38 MAPK inhibitor rescued the viability of T98G cells time-dependently ( Figure 4B). Thus, AKR1B1 directly activated p38 MAPK signaling, which played a critical role in the antitumor effect of AKR1B1.

AKR1B1 Induced Apoptosis in Glioma Cells
Given that AKR1B1-expressing glioma cells inhibited cell growth ( Figure 3A,B) and induced activation of p38 MAPK signaling ( Figure 4A,B), the p38 MAPK pathway has been shown to play a role in inducing apoptosis or programmed cell death in cancer cells [35,36]. AKR1B1 might exert a cytotoxic effect through an apoptotic signal pathway. Therefore, we examined the expression of apoptosis-related proteins such as BAX and Bcl-2 in AKR1B1-expressing glioma cells.
Both the mRNA and protein levels of apoptosis-associated BAX and Bcl-2 signals were examined in AKR1B1-expressing T98G cells. After AKR1B1 plasmid transfection, the mRNA level of the pro-apoptotic BAX signal increased time-dependently ( Figure 5A), whereas that of the anti-apoptotic Bcl-2 signal significantly decreased ( Figure 5B). In addition, the ratio of BAX/Bcl-2 increased time-dependently in the AKR1B1 group but did not significantly change in the control group ( Figure 5C). Regarding the protein expression of BAX and Bcl-2 in AKR1B1-expressing T98G cells, the same effect was observed ( Figure 5D).

AKR1B1 Induced Apoptosis in Glioma Cells
Given that AKR1B1-expressing glioma cells inhibited cell growth ( Figure 3A,B) and induced activation of p38 MAPK signaling ( Figure 4A,B), the p38 MAPK pathway has been shown to play a role in inducing apoptosis or programmed cell death in cancer cells [35,36]. AKR1B1 might exert a cytotoxic effect through an apoptotic signal pathway. Therefore, we examined the expression of apoptosis-related proteins such as BAX and Bcl-2 in AKR1B1-expressing glioma cells.
Both the mRNA and protein levels of apoptosis-associated BAX and Bcl-2 signals were examined in AKR1B1-expressing T98G cells. After AKR1B1 plasmid transfection, the mRNA level of the pro-apoptotic BAX signal increased time-dependently ( Figure 5A), whereas that of the anti-apoptotic Bcl-2 signal significantly decreased ( Figure 5B). In addition, the ratio of BAX/Bcl-2 increased time-dependently in the AKR1B1 group but did not significantly change in the control group ( Figure 5C). Regarding the protein expression of BAX and Bcl-2 in AKR1B1-expressing T98G cells, the same effect was observed ( Figure 5D). Apoptosis-associated proteins such as BAX and Bcl-2 were measured using Western blot analysis. Data are presented as mean ± SEM from three independent experiments; * p < 0.05 and ** p < 0.01.

AKR1B1 Activated Caspase-3/7 in Glioma Cells
Considering that caspase-3/7 plays a major role in cell apoptosis [46][47][48][49], we next examined whether caspase-3/7 could be activated in AKR1B1-expressing glioma cells. We transfected the T98G cells with the control plasmid or 0.05/0.1/0.2 μg of the AKR1B1-expressing plasmid and then measured caspase-3/7 activity at 24 and 48 h after the transfection. The caspase-3/7 activity elevated, as the dose of AKR1B1 plasmid increased ( Figure  6A,B). The induction of apoptotic cell death by AKR1B1 expression was examined using the Annexin V/PI assay. As depicted in Figure 6C, T98G cells transfected with AKR1B1 expression plasmids for 24 h displayed Annexin V-FITC staining on the surface of apoptotic cells (in green), while PI staining highlighted the nuclei of apoptotic or dead cells (in red). Therefore, apoptosis could be a major contributor to the antitumor effect of AKR1B1 on glioma cells. (D) Apoptosis-associated proteins such as BAX and Bcl-2 were measured using Western blot analysis. Data are presented as mean ± SEM from three independent experiments; * p < 0.05 and ** p < 0.01.

AKR1B1 Activated Caspase-3/7 in Glioma Cells
Considering that caspase-3/7 plays a major role in cell apoptosis [46][47][48][49], we next examined whether caspase-3/7 could be activated in AKR1B1-expressing glioma cells. We transfected the T98G cells with the control plasmid or 0.05/0.1/0.2 µg of the AKR1B1expressing plasmid and then measured caspase-3/7 activity at 24 and 48 h after the transfection. The caspase-3/7 activity elevated, as the dose of AKR1B1 plasmid increased ( Figure 6A,B). The induction of apoptotic cell death by AKR1B1 expression was examined using the Annexin V/PI assay. As depicted in Figure 6C, T98G cells transfected with AKR1B1 expression plasmids for 24 h displayed Annexin V-FITC staining on the surface of apoptotic cells (in green), while PI staining highlighted the nuclei of apoptotic or dead cells (in red). Therefore, apoptosis could be a major contributor to the antitumor effect of AKR1B1 on glioma cells.

Discussion
This study demonstrated that AKR1B1 was significantly decreased in glioma tissues compared with that in the adjacent tissues. AKR1B1 also decreased in glioma cell lines (T98G and 8401) in comparison with that in human astrocytes. The MTT assays showed that AKR1B1 overexpression inhibited glioma cell proliferation whereas AKR1B1 knockdown increased it. Nonetheless, AKR1B1-induced p38 MAPK phosphorylation and SB203580 could reverse this inhibitory effect on cell proliferation. Furthermore, AKR1B1 overexpression increased the BAX protein and mRNA expression levels and the Bcl-2/BAX ratio but decreased the Bcl-2 protein and mRNA expression levels. AKR1B1 also induced caspase-3/7 activity.
Yamada et al. found that AKR1B1 is significantly hypermethylated and decreases in hepatocellular carcinoma tumors compared with that in normal liver tissues [9]. In adrenocortical carcinomas and prostate cancer, AKR1B1 expression is decreased, but the mechanism and function remain unknown [6,11]. In the present study, AKR1B1 expression was significantly decreased in glioma tissues compared with that in adjacent normal tissues. The AKR1B1 level was also decreased in glioma cell lines such as T98G and 8401 in comparison with that in human astrocytes ( Figure 1A,B). Based on our observations, we speculate that the low expression of AKR1B1 may involve glioma progression.
Moreover, p38 MAPK signaling plays an important role in GBM. The kinase p38 induces apoptosis in GBM cells while inhibiting p38 phosphorylation prevents apoptosis [50]. Earlier research suggests that the activation of p38 MAPK may have a positive impact on certain aspects of glioma therapy. Yao et al. discovered that the anti-proliferative effect of β-elemene on glioblastoma cells was dependent on the activation of p38 MAPK, and the inhibition of p38 MAPK reversed the anti-proliferative effect of β-elemene [51]. Therefore, p38 MAPK may be a potential target for glioma therapy.
Our data showed that AKR1B1 overexpression can induce p38 MAPK phosphorylation and inhibit glioma cell proliferation (Figures 3 and 4). However, the inhibitory effect of AKR1B1 on cell proliferation can be reversed by a p38 MAPK inhibitor (SB203580) ( Figure 4B). Therefore, targeting p38 MAPK may be an underlying mechanism by which AKR1B1 inhibits GBM proliferation. Reactive oxygen species (ROS) are potent oxidative agents that can serve as second messengers to directly or indirectly modulate the activation of the p38 MAPK pathway when their intracellular concentrations escalate. The activation of the p38 MAPK pathway augments cellular responses to ROS and participates in regulating apoptosis, inflammation, and other stress responses [52]. Previous investigations have revealed that arsenic trioxide stimulates ROS generation, activates the p38 MAPK signaling pathway and promotes apoptosis in cancer cells [53]. AKR1B1 plays a role in the metabolism and reduction of bioactive aldehydes, which can impact the intracellular redox balance and consequently alter intracellular ROS levels [14]. Therefore, we hypothesize that AKR1B1-induced phosphorylation of p38 MAPK may transpire through an elevation in intracellular ROS concentrations. AKR1B1 metabolizes glucose into sorbitol via the polyol pathway [54]. Sorbitol has been found to possibly possess antitumor properties. One study demonstrated that sorbitol induces apoptosis in colorectal cancer cells by increasing the phosphorylation of p38 MAPK, upregulating the expression of BAX and cleaved caspase-3 while downregulating the expression of Bcl-2 [55]. Another study showed that sorbitol induces apoptosis in gastric cancer cells by regulating PKC activity [56]. Furthermore, it has been demonstrated that sorbitol can induce p38 MAPK phosphorylation and enhance chemosensitivity in T98G glioma cells [57]. Therefore, AKR1B1-induced p38 MAPK phosphorylation through sorbitol in glioma cells is the focus of our future research.
BAX, a member of the Bcl-2 family, is a core regulator of the intrinsic pathway of apoptosis [58]. Silencing of p38 MAPK by si-RNA-blocked streptococcal pyrogenic exotoxin B induces BAX expression and apoptosis in A549 cells [59]. The treatment of PC12 cells with rotenone significantly induces apoptosis with the p38/p53/BAX signaling axis [37]. Conversely, the anti-apoptotic Bcl-2 proteins inhibit cell death by binding and inhibiting pro-apoptotic Bcl-2 proteins [58]. In HCT116 and SW480 colorectal cancer cells, inhibiting phosphorylated p38 MAPK reduces the Bcl-2 expression [60]. In our study, AKR1B1 overexpression in T98G cells induced BAX and reduced Bcl-2 expression, but these effects may be regulated by p38 MAPK signaling. In addition, AKR1B1 inhibited T98G cell proliferation and significantly increased the BAX/Bcl-2 ratio ( Figure 5).
Caspase-3, which plays an integral role in apoptosis, is a primary target for cancer therapy. Activated caspase-3 stimulates death protease and initiates protein breakdown, leading to apoptosis [49]. The report highlights the high expression of miR-155-5p and miR-221-3p in glioma cells, which inhibit the expression of caspase-3. The use of peptide nucleic acids targeting these two miRNAs has been shown to induce apoptosis in temozolomideresistant T98G glioma cells by enhancing caspase-3 protein expression [61]. The BAX/Bcl-2 mRNA and protein ratios reportedly correlate with caspase-3 expression [62]. In our study, AKR1B1 overexpression increased the caspase-3/7 activity of glioma cells, suggesting that AKR1B1 is a potential therapeutic effect of gliomas ( Figure 6A,B). Caspase-3 plays a pivotal role in cellular apoptosis, and its activity exhibits a positive correlation with the extent of apoptosis. During the initial phase of apoptosis, phosphatidylserine (PS) in the cell membrane undergoes translocation from the inner to the outer leaflet. As apoptosis progresses to the late stage, cells exhibit increased membrane permeability, allowing propidium iodide (PI) to enter and stain the DNA, eventually leading to the disintegration of cellular structures and formation of apoptotic bodies [44]. Our findings demonstrate that AKR1B1 significantly promotes apoptosis in T98G cells, as evidenced by the elevated percentages of Annexin V-positive and PI-positive cells observed in the Annexin V-FITC/PI double-staining assay ( Figure 6C). This observation substantiates that AKR1B1 augments caspase-3 activity and initiates downstream pathways.
In conclusion, AKR1B1 has an antitumor effect on glioma cells by inducing the phosphorylated levels of p38 MAPK and thereby increasing the BAX/Bcl-2 ratio and caspase-3/7 activity. Therefore, AKR1B1 may be a promising candidate for glioma treatment. Informed Consent Statement: All subjects involved in the study provided informed consent. The patients provided written informed consent for publication of this paper.