15227278, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/tox.23658 by Fu Jen Catholic University, Wiley Online Library on [14/01/2023].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Received: 27 May 2022 Revised: 23 August 2022 Accepted: 30 August 2022
DOI: 10.1002/tox.23658

RESEARCH ARTICLE

AKG induces cell apoptosis by inducing reactive oxygen

species-mediated endoplasmic reticulum stress and by
suppressing PI3K/AKT/mTOR-mediated autophagy in renal
cell carcinoma

Fan Wu 1 | Xuexia Xie 1 | Guoliang Li 1 | Dongping Bao 1 | Haomin Li 1 |

Guohao Wu 2 | Yiqi Lai 3 | Yaping Xing 4 | Peng Ouyang 5 | Guo Chen 2,6,7 |
8 1,2
Zhifeng Wang | Caiyong Lai

1
Department of Urology, The First Affiliated
Hospital of Jinan University, Guangzhou, China Abstract
2
Department of Urology, The Sixth Affiliated Background: Alpha-ketoglutarate (AKG) or 2-oxoglutarate is a key substance in the
Hospital of Jinan University, Dongguan, China
3
tricarboxylic acid cycle (TCA) and has been known to play an important role in can-
University of South China, Hengyang, China
4
Shenzhen Hospital of University of Hong
cerogenesis and tumor progression. Renal cell carcinoma (RCC) is the most common
Kong, Shenzhen, China type of kidney cancer, and it has a high mortality rate. Autophagy is a phenomenon
5
Department of General Surgery, The First of self-digestion, and its significance in tumor genesis and progression remains debat-
Affiliated Hospital of Jinan University,
Guangzhou, China able. However, the mechanisms underlying how AKG regulates autophagy in RCC
6
Department of Medical Biochemistry and remain unknown. Thus, the purpose of this study was to assess the therapeutic effi-
Molecular Biology, School of Medicine, Jinan
cacy of AKG and its molecular mechanisms.
University, Guangzhou, China
7
School of Biopharmacy, China Pharmaceutical
Methods: RCC cell lines 786O and ACHN were treated with varying doses of AKG
University, Nanjing, China for 24 h. CCK-8, Transwell, and scratch wound healing assays were utilized to evalu-
8
Department of Urology, Henan Provincial ate the role of AKG in RCC cells. Autophagy protein and PI3K/AKT/mTOR pathway
People's Hospital, Zhengzhou University
People's Hospital, Henan University People's protein levels were analyzed by Western blot.
Hospital, Zhengzhou, China
Results: AKG inhibited the proliferation of RCC cells 786O and ACHN in a dose-
Correspondence dependent manner according to the CCK-8 assay. In addition, flow cytometry and
Zhifeng Wang, Department of Urology, Henan
Western blot analysis revealed that AKG dose-dependently triggered apoptosis and
Provincial People's Hospital, Zhengzhou
University People's Hospital, Henan University autophagy in RCC cells. By promoting cell apoptosis and autophagy, AKG dramati-
People's Hospital, Zhengzhou, China.
cally suppressed tumor growth. Mechanistically, AKG induces autophagy by promot-
Email: china.shengyiwzf@163.com
ing ROS generation and inhibiting the PI3K/AKT/mTOR pathway.
Caiyong Lai, Department of Urology, The sixth
Affiliated Hospital of Jinan University, Conclusions: The anti-tumor effect of AKG promotes autophagy in renal cancer cells
Dongguan, China.
via mediating ROS-PI3K/Akt/mTOR, and may be used as a potential anticancer drug
Email: lcy2015@jnu.edu.cn
for kidney cancer.
Funding information
National Natural Science Foundation of China,
KEYWORDS
Grant/Award Numbers: 82073042, 81460386;
alpha-ketoglutarate, kidney cancer, PI3K/AKT/mTOR, ROS
Guangdong Basic and Applied Basic Research
Foundation, Grant/Award Number:
2022B1515020105; Science and Technology
Project of Health Bureau of Yangjiang City,
Grant/Award Numbers: 2021036, SF2021212

Fan Wu and Xuexia Xie contributed equally to this work.

Environmental Toxicology. 2023;38:17–27. wileyonlinelibrary.com/journal/tox © 2022 Wiley Periodicals LLC. 17


18
1 | I N T RO DU CT I O N
1.1 | Renal cell carcinoma (RCC)
RCC is the most widespread kind of kidney cancer, accounting for
85% of kidney malignancies. It is more common in men than in
1
women (1.7:1) and is more common in older adults. Despite break-
throughs in surgical methods, the 5-year survival rate for patients with
early localized renal cancer undergoing partial nephrectomy and radi-
cal nephrectomy has dramatically increased throughout the last
10 years. However, the general prognosis remains dismal. Since the
symptoms of RCC are often nonspecific in the early stages, 20%–30%
of patients exhibit distant metastases at the first diagnosis.2,3 These
patients often miss the opportunity for surgical treatment, and the
5-year survival rate is dismal. RCC is a kind of tumor that is relatively
insensitive to radiotherapy and chemotherapy.2-4 Although new tar-
geted medicines, including vascular endothelial growth factor (VEGF)
inhibitors and rapamycin (mTOR) inhibitors, have been developed, the
results fall far short of expectations. Therefore, more effective treat-
ment strategies are needed to improve the treatment of RCC.5-7
1.2 | Alpha-ketoglutarate (AKG) or 2-oxoglutarate
Alpha-ketoglutarate (AKG) or 2-oxoglutarate is a vital component in
the tricarboxylic acid cycle (TCA).8 It is located after isocitrate and
before succinyl-CoA in the cycle. It plays a vital role in cell energy
metabolism and can inhibit ATP synthetase.9,10 AKG is a cofactor and
substrate of numerous enzymes, including hypoxia-inducible factor
(HIF) prolyl hydroxylase (PHD), and is involved in DNA demethylation
in metabolic and regulatory activities inside the cell.11 AKG has a sub-
stantial capacity to control metabolism,8 activate autophagy,12 exert
anti-inflammatory effects,13 delay aging12 and prolong life.14 Due to
these characteristics, previous studies have reported that AKG is
15 16
effective in treating human cancers, such as osteosarcoma, breast
cancer,17 and colon cancer.18 However, AKG's function in renal cancer
has not been reported.
1.3 | Autophagy
Cell death can be divided into apoptosis, autophagy, ferroptosis, and
19
pyroptosis. Autophagy is an evolutionarily conservative process first
proposed by Christian de Duff in 1963. It is responsible for transfer-
ring damaged organelles, misfolded proteins, and other macromolecu-
lar substances to the lysosome, for degradation and recycling.20,21
Autophagy is a complex process that plays a role in cancer progression
and therapy.20 It can be used as a mechanism to protect cancer cells
from apoptosis induced by many anticancer drugs. Alternatively,
excessive autophagy can result in cell death and halt the progression
of cancer. 22,23
Autophagy plays an important anticancer role in renal
cancer.24 Several studies have demonstrated that autophagy regula-
tors may modulate the PI3K/Akt/mTOR signaling pathways in RCC to
produce therapeutic benefits.25,26 In addition, there is much evidence
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WU ET AL.
to suggest that anticancer drug-induced renal cell autophagy is caused
by the excessive accumulation of ROS.27-29
In this study, we found that AKG can inhibit the development of
renal carcinoma cells in a dose-dependent manner. Mechanistically,
AKG promotes autophagy in renal cancer cells by mediating the ROS-
PI3K/Akt/mTOR signaling pathway. Therefore, AKG can be used as a
potential anticancer drug for kidney cancer.
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Cell culture
Procell (China) supplied both the 786O and ACHN cell lines. The cells
were grown in RPMI 1640 medium (Gibco, USA) supplemented with
10% Fetal Bovine Serum, FBS (Excell, Shanghai, China), and Penicillin–
Streptomycin (10X) Solution (100 U/ml, Vivacell, Shanghai, China) in a
humid incubator containing 5% CO2 at 37 C.
Dimethyl-alpha-ketoglutarate (DM-AKG, a cell-permeable analog
of AKG, Sigma) was used in the experiments.
2.2 | Cell counting kit-8 (CCK-8)
96-well plate inoculated with 3000 cells per well. When the RCC cells
were in the logarithmic growth phase, DM-AKG was added at differ-
ent concentrations. After 24 h, 10 μl CCK-8 (Dojindo, Japan) was
added to per 100 μl of medium and incubated for 1 h. The OD was
measured at 450 nm using an enzyme marker.
2.3 | Transwell assays
For the Transwell assays, Transwell chambers with Matrigel-coated
8-μm membranes (Corning) were plated in the wells of a 24-well plate.
The lower chamber was seeded with 600 μl of 10% FBS medium (with
or without DM-AKG), and 786O or ACHN cells (5  104 cells/well)
entered the top chamber in a medium devoid of serum. After 24 h of
incubation at 37 C, the upper and lower chamber media were dis-
carded, and a cotton swab was used to remove the cells that couldn't
cross the membrane. Crystal violet was used to dye the migrating cells
for 30 min after they had been fixed with 4% paraformaldehyde.
Three fields of view of each specimen were randomly selected and
photographed under the microscope. The number of stained cells per
field was recorded.
2.4 | Wound healing assays
First, the RCC cell lines were cultured in a 6-well plate until conflu-
ence and starved 24 h before scratching. The tip of a 200 μl pipet was
quickly passed across the center of the well. Images were taken with a
microscope at 0/24 h after the cell scratch, and the wound healing
area was measured in each of the three wells by ImageJ software.

WU ET AL.
2.5 | Cell apoptosis analysis
786O and ACHN cells (1  105 cells/ml) were grown in 6-well plates
for 24 h. The cells in the 6-well plates were then treated with differ-
ent concentrations of DM-AKG (0, 5, 7.5, and 10 mM). The Cells were
washed with PBS buffer and then resuspended in 100 L of binding
buffer and stained for 15–20 min in the dark with 5 L Annexin V-FITC
and 5 L propidium iodide (BD, FACSCalibur, USA). Then, 400 μl of
binding buffer was utilized in a flow tube to resuspend the stained
cells. The rate of apoptosis was examined using flow cytometry. There
were three repetitions of the experiment.
2.6 | Cell cycle analysis
786O and ACHN cells were grown in 6-well plates with DM-AKG
(0, 5, 7.5, and 10 mM). After 24 h of treatment, the cells were fixed
overnight with 70% ethanol at 20 C. Next, PBS washed the cells,
and they were then stained with PI for 30 min at room temperature in
the dark, using BD Biosciences' PI/RNase Staining Buffer. Then, a
flow cytometer was utilized to measure the cells.
2.7 | JC-1
786O and ACHN cells were grown in 6-well plates, and different DM-
AKG treatments were applied (0, 5, 7.5, and 10 mM). JC-1 (Beyotime,
Shanghai, China) was added to the cells according to the instructions and
incubated for 30 min. Flow cytometry was used to examine the cells.
According to the color intensity, the ratio of JC-1 aggregates (red) to
monomers (green) was determined. FlowJo 7.6 was used for data analysis.
2.8 | Evaluation of ROS production
Briefly, the cells were cultured at 2.0  105 cells/well in 6-well plates
for 24 h. Next, treatment of the cells with DM-AKG (0, 5, 7.5, and
10 mM) for 24 h, and then followed the instructions of the ROS kit
(Beyotime, Shanghai, China) by flow cytometry to detect ROS produc-
tion. FlowJo 7.6 was used for data analysis.
F I G U R E 1 Cell counting kit-8
assay was used to detect the
inhibition of cell proliferation.
(A and B) Cell viability was
evaluated after treatment with
DM-AKG (0, 1.25, 2.5, 5, 10,
25, and 50 mM) for 24 h. Data
are represented as the mean
± SD of three separate
experiments. *p < .05; **p < .01;
***p < .001 versus 0 group
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19
2.9 | Western blot
Total protein was extracted from the indicated cells with RIPA buffer
containing PMSF (Beyotime, China) and phosphatase inhibitors
(Targetmol, China). The protein bands were distinguished using 6%–
15% SDS–PAGE and then transferred to a polyvinylidene fluoride
(PVDF) membrane. After 1 h of blocking with 5% skim milk containing
1% Tween 20 phosphate buffer (PBST), the PVDF membrane was
treated overnight at 4 C with primary antibodies. Anti-P21, anti-
Cyclin D1, anti-B-cell lymphoma-2 (Bcl-2), anti-Bax, anti-Cytochrome-
C, anti-Cox-IV, anti-p-mTOR, and anti-mTOR were purchased from
Abcam (Cambridge, USA). Anti-LC3B, anti-Cleaved Caspase-3, anti-p-
PI3K, anti-PI3K, anti-p-AKT, and anti-AKT were purchased from Cell
Signaling Technology (MA, USA). The β-actin was purchased from
Santa Cruz (CA, USA). After three washes, the PVDF were incubated
for 1 h in the corresponding secondary antibody. Finally, the mem-
branes were developed using an enhanced chemiluminescence kit
(ECL, Beyotime, China).
2.10 | Statistical analysis
At least three independent experiments are represented by the data
presented, and normally distributed data are presented as the mean
± SD. Two-sided t-test to test the statistical significance of differ-
ences between groups. p < .05 was considered statistically significant
for differences.
3 | RE SU LT S
3.1 | AKG inhibits the viability of RCC cells
To investigate whether AKG could be an effective antitumor agent, the
cell survival rate was detected by CCK-8 assay. It revealed that AKG
significantly inhibited the survival of 786O and ACHN cells in a dose-
dependent manner. In addition, the IC50 values of AKG in 786O and
ACHN cells were 17.7 mM and 13.56 mM, respectively (Figure 1A,B).
Thus, we chose 786O and ACHN cells treated with DM-AKG at con-
centrations of 0, 5, 7.5, and 10 mM for the next experiments.
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20 WU ET AL.

3.2 | AKG induces cell cycle arrest in the G1 phase
in RCC cells
To investigate the antiproliferative activity of AKG in RCC cells, flow
cytometry was used to examine the distribution of cell cycle stages in
the two RCC cell lines after treatment with 0, 5, 7.5, and 10 mM DM-
AKG for 24 h. Flow cytometry indicated that AKG resulted in signifi-
cant accumulation in the G1 phase in a dose-dependent manner in
786O and ACHN cells. The percentage of 786O cells in the G1 phase
increased to 46.90 ± 0.56%, 50.90 ± 3.87%, and 59.93 ± 8.93% at
doses of 5, 7.5, and 10 mM DM-AKG after treatment for 24 h. Fur-
thermore, the percentage of ACHN cells in the G1 phase increased to
53.00 ± 3.85%, 64.60 ± 4.2098%, and 70.37 ± 3.77% at doses of
5, 7.5, and 10 mM DM-AKG following inducement for 24 h
(Figure 2A,B). Since AKG caused cell cycle arrest in the G1 phase,
more research was undertaken to explore the expression of cyclin D1

F I G U R E 2 The effect of AKG on the cell cycle distribution of 786O and ACHN. (A) The cell cycle distribution of 786O and ACHN cells
treated with DM-AKG (0, 5, 7.5, and 10 mM) for 24 h was examined by flow cytometry. (B) Bar graphs show the quantification of cell cycle
distribution in 786O and ACHN cells. (C) 786O and ACHN cells were treated with DM-AKG (0, 5, 7.5, and 10 mM) for 24 h and western blotted
for cyclin D1 and p21. β-actin served as a control. *p < .05; **p < .01; ***p < .001
 15227278, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/tox.23658 by Fu Jen Catholic University, Wiley Online Library on [14/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WU ET AL. 21

and P21 proteins related to the transition from the G1 phase to the S while boosting the creation of p21 protein (Figure 2C). These studies
phase of the cell cycle in DM-AKG-treated cells. Our findings showed demonstrated that AKG exhibited potent anticancer action by arrest-
that AKG considerably suppressed the expression of cyclin D1 protein ing 786O and ACHN cancer cells in G1 phase.

F I G U R E 3 AKG induces
apoptosis in 786O and ACHN cells.
(A) 786O and ACHN cells were
treated with DM-AKG (0, 5, 7.5,
and 10 mM) as indicated. Cell
apoptosis was analyzed by Annexin
V staining at 24 h after treatment.
(B) Mitochondrial potential (DYm)
decreases with increasing DM-
AKG. DM-AKG (0, 5, 7.5, and
10 mM) treated cells were stained
with JC-1 for 30 min and examined
by flow cytometry. Changes in
Dym were detected by the ratio of
red fluorescence (JC-1 aggregates)
to green fluorescence (JC-1
monomers; up) and the data were
statistically compared (down).
(C) 786O and ACHN cells were
treated with DM-AKG (0, 5, 7.5,
and 10 mM) for 24 h and western
blotted for Bcl2, Bax and Cleaved
Caspase 3. β-actin was used as a
control. (D) 786O and ACHN cells
were treated with DM-AKG (0, 5,
7.5, and 10 mM) for 24 h. Then,
subcellular fractionation was
subsequently performed and the
cytochrome C levels in
mitochondria and cytoplasm were
analyzed by western blot. β-actin
and cox-IV were used as a control.
*p < .05; **p < .01; ***p < .001
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22 WU ET AL.

3.3 | AKG induced the apoptosis of RCC cells
To measure the effect of AKG on apoptosis, flow cytometry was per-
formed using annexin V-FITC/PI. The number of apoptotic cells rose
in a dose-dependent manner in cells treated with DM-AKG (5, 7.5,
and 10 mM) versus control for 24 h (Figure 3A). To further investigate
the mechanism by which AKG triggered apoptosis in cells, western
blotting showed a substantial decrease in Bcl-2 expression and a sub-
stantial increase in Bax and Cleaved caspase 3 expression in a dose-
dependent manner in 786O and ACHN cells compared to control
cells. (Figure 3C). We then observed a significant decrease in
mitochondrial cytochrome C levels and an increase in cytosol cyto-
chrome C following AKG treatment, suggesting that AKG can induce
translocation of cytochrome C from the mitochondria to the cell mem-
brane. (Figure 3D). Because early apoptosis is accompanied by mito-
chondrial membrane damage, we examined the effect of AKG on the
alteration of mitochondrial membrane potential (DYm). In 786O and
ACHN cells treated with various doses of DM-AKG for 24 h, green
fluorescence was increased, whereas red fluorescence was decreased
in a dose-dependent manner, indicating mitochondrial malfunction
(Figure 3B). These findings imply that the presence of AKG induces
intrinsic apoptosis by disrupting mitochondrial function.

F I G U R E 4 AKG inhibits the migration and invasiveness of 786O and ACHN cells. (A) Transwell experiments with Matrigel-coated inserts
were used to determine RCC cell invasion. Scale bars, 200 μm. (B) 786O and ACHN cells were treated with DM-AKG (0, 5, 7.5, and 10 mM) as
indicated. To evaluate the migratory ability of the designated RCC cells, wound healing experiments were conducted. The dashed lines indicate
the edge of migrating cells. Scale bars, 200 μm. **p < .01; ***p < .001

WU ET AL.
3.4 | AKG inhibits RCC cell migration and invasion
Since RCC is classified as a metastatic tumor, we tested 786O and
ACHN cells migration and invasion with wound healing and Transwell
experiments. These tests showed that DM-AKG treatment inhibited
cell migration and invasion in a dose-dependent manner in both cell
(Figure 4A,B). These data demonstrated that AKG reduces the malig-
nant characteristics of RCC cells.
3.5 | AKG activates the autophagy pathway in
RCC cells
To assess whether AKG could induce autophagy in RCC cells, we cul-
tured 786O and ACHN cells with 10 mM AKG for 24 h and observed
the morphological changes of the specified cells with an inverted
microscope. A significant accumulation of cytoplasmic vacuoles was
observed in RCC cells treated with DM-AKG (Figure 5A). LC3B is
widely used as an autophagosome marker. Thus, we treated 786O
and ACHN cells with different concentrations (0, 5, 7.5, and 10 mM)
of DM-AKG and detected the expression of LC3B by western blot-
ting. The findings demonstrated that AKG could upregulate LC3B-II in
F I G U R E 5 AKG activated the autophagy pathway in RCC cells.
(A) Morphological changes in 786O and ACHN cells. Twenty-four
hours after being treated with 10 mM DM-AKG, RCC cells were
observed under an inverted microscope. Arrows point to cytoplasmic
vacuoles. Scale bars, 50 μm. (B) 786O and ACHN cells were treated
with DM-AKG (0, 5, 7.5, and 10 mM) for 24 h, and the expression of
LC3B was detected using western blot analysis. (C) 786O and ACHN
cells were pretreated with 3-MA (2 mM) for 1 h, followed by
treatment with DM-AKG (10 mM) for 24 h, and western blot analysis
detected LC3B protein levels. β-actin was used as a control
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23
a dose-dependent manner (Figure 5B). Moreover, the autophagic
inhibitor 3-MA was employed to further demonstrate the autophagic
activation impact of AKG in 786O and ACHN cells. The results
showed that the increase in the autophagy-related protein LC3B-II
with AKG treatment was attenuated when 3-MA was added in 786O
and ACHN cells (Figure 5C). Autophagy might be induced in RCC cells
by AKG, as demonstrated by these studies.
3.6 | AKG activated the autophagy pathway via
ROS in RCC cells
Previous reports have shown that autophagy is induced by increasing
ROS levels in RCC cells. Therefore, cells were treated with DM-AKG
(0, 5, 7.5, and 10 mM) for 24 h and analyzed using flow cytometry. In
786O and ACHN cells, we discovered that AKG dramatically boosted
intracellular ROS accumulation in a dose-dependent manner (Figure 6A).
To further evaluate whether enhanced ROS accumulation plays a major
role in AKG-induced autophagy, 786O and ACHN cells were pretreated
with 4 mM NAC (a ROS scavenger) for 1 h before AKG treatment, and
we found that pretreatment with NAC effectively prevented AKG-
induced increases in the autophagy-related protein LC3-II in 786O and
ACHN cells (Figure 6B). According to our findings, AKG-induced cell
autophagy is linked to ROS formation in 786O and ACHN cells.
3.7 | AKG inhibits the PI3K/AKT/mTOR pathway
in RCC cells
AKT governs cell cycle progression, proliferation, and apoptosis in
cancer cells as a downstream effector of PI3K. The PI3K/AKT/mTOR
signaling pathway has been studied in the context of human RCC in
the past. To determine the molecular mechanisms underlying AKG-
induced autophagy in 786O and ACHN cells, Western blot analysis
was used to assess the expression of PI3K/AKT/mTOR pathway com-
ponents. The results demonstrated that the ratios of p-PI3K/PI3K, p-
AKT/AKT, and p-mTOR/mTOR decreased dose-dependently in DM-
AKG-treated cells (0, 5, 7.5, and 10 mM) and untreated cells (p < .05)
(Figure 7A). To shed light on whether AKG-induced autophagy is con-
nected with the PI3K/AKT/mTOR signaling pathway, we assessed the
ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in DM-
AKG/3-MA-cotreated cells. We found that the ratios of p-PI3K/PI3K,
p-AKT/AKT, and p-mTOR/mTOR were markedly downregulated with
cotreatment versus AKG alone (p < .05) (Figure 7B). These results
demonstrate that AKG might activate autophagy in human RCC 786O
and ACHN cells via modulating the ROS/PI3K/AKT/mTOR pathway.
4 | DI SCU SSION
One of the main reasons for cancer-related deaths globally is RCC.30
Mortality in patients with advanced renal cell carcinoma increases sig-
nificantly due to insensitivity to radiochemotherapy and distal metas-
tasis.31 AKG is a key TCA cycle molecule that plays a crucial role in
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24 WU ET AL.

F I G U R E 6 AKG activated the autophagy pathway by ROS in RCC cells. (A) Flow cytometry was used to measure ROS levels in 786O and
ACHN cells treated with DM-AKG (0, 5, 7.5, and 10 mM) during 24 h. Bar graphs indicate the quantification of ROS. (B) 786O and ACHN cells
were pretreated with NAC (4 mM) for 1 h, followed by treatment with DM-AKG (10 mM) for 24 h, and western blot was used to measure LC3B
protein levels. β-actin was used as a control. **p < .01; ***p < .001

lipid production, oxidative stress, protein modification, autophagy,
and cell death.10,32 It was reported that AKG can shape the epigenetic
landscape of chromatin by regulating the TCA cycle to modulate DNA
and histone methylation.33,34 Epigenetic modifications are vital in reg-
ulating DNA repair, replication, and damage, which have an impact on
the expression of genes that drive tumor development and
35,36
upkeep. AKG supplementation also modulates the accumulation
of succinate, ferredoxin, and other carcinogenic metabolites and
inhibits the secretion of angiogenic factors.14,37 In addition AKG can
directly regulate signaling pathways associated with tumorigenesis,
making it a potential anticancer agent.38 A study revealed that AKG
impedes the development of colon adenocarcinoma.18 Another study
indicated that AKG inhibits cell growth by inducing cyclin D1 protea-
somal degradation; inhibition of cell migration and invasion was
observed in human osteosarcoma cells treated with AKG, and this
treatment led to decreased activation of extracellular signal-regulated
kinase (ERK1/2).16 Although the inhibitory effect of AKG on the pro-
liferation and apoptosis of various tumor cells has been widely
reported, the effect of AKG on RCC cells and its mechanism are still
unclear. Our findings demonstrated that AKG significantly reduced
 15227278, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/tox.23658 by Fu Jen Catholic University, Wiley Online Library on [14/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WU ET AL. 25

F I G U R E 7 AKG inhibited the PI3K/AKT/mTOR pathway. (A) Western blot analysis of PI3K, AKT, and mTOR phosphorylation levels in 786O
and ACHN cells treated with DM-AKG (0, 5, 7.5, and 10 mM) during 24 h. (B) Phosphorylation levels of PI3K, AKT, and mTOR in 786O and
ACHN cells after treatment DM-AKG, 3-MA, or DM-AKG + 3-MA treatment for 24 h by western blot. The relative expression levels were
determined using ImageJ software. Data are represented as the mean ± SD of three separate experiments. *p < .05; **p < .01; ***p < .001, versus
control group. #p < .05, versus AKG group

RCC cell proliferation by inducing ROS accumulation, leading to apo-
ptosis and autophagy.
39
Apoptosis is the main type of programmed cell death. AKG was
discovered to trigger apoptosis in SAOS-2 and HOS osteosarcoma
cells via JNK- and caspase 9-dependent pathways.16 In addition,
α-ketoglutaric acid treatment reduced tumor growth and induced apo-
40
ptosis in a phD3-dependent and HIF1α-independent manner. In this
study, we found by flow cytometry that AKG significantly induced
apoptosis in a dose-dependent manner. In addition, AKG increased
expression of the apoptotic proteins Bax and Cleaved Caspase 3, and
decreased expression of the antiapoptotic protein Bcl-2. During cell
apoptosis, cytochrome c is released into the cytoplasm. We observed
that AKG could induce the translocation of cytochrome C from mito-
chondria to the cell membrane. The decrease in mitochondrial mem-
brane potential is considered to be a landmark event of early
apoptosis; JC-1 was detected by flow cytometry, and the results were
consistent with previous results indicating that AKG can induce apo-
ptosis in RCC cells.
Autophagy is a cellular mechanism that eliminates damaged
organelles and is connected with carcinogenic consequences and

26
cancer therapy sensitivity.41 Autophagy plays a dual role in cancer
according to distinct cell types and genetic variables.42 To endure
metabolic and therapeutic strain, tumor cells can activate autophagy
by limiting tumor necrosis and minimizing genetic damage, thus
improving anticancer strategies by blocking autophagy.42-44 In recent
years, it has been demonstrated that a range of medicines regulate
29,45
autophagy via distinct molecular processes. For example, thymo-
quinone induces autophagy through the AMPK/mTOR signaling path-
way in RCC.45 Costunolide induces autophagy by activating the
ROS/MAPK pathway in RCC.29 The study's findings indicated that
AKG-induced autophagy was demonstrated by an increase in LC3
transformation in RCC cells. In addition, 3-MA reduced AKG-induced
autophagy, suggesting that AKG-induced autophagy was involved in
cell death. These results suggest that AKG induces proapoptotic
autophagy in 786O and ACHN cells. ROS have been recognized as
significant molecules that control cell death or survival.46 Low concen-
trations of ROS are involved in cell signaling, while excessive ROS
damage proteins and DNA in cells, ultimately leading to autophagy or
47-49
cell death. This study showed that AKG induced a significant
increase in ROS levels, but NAC pretreatment significantly reversed
AKG-related autophagy, suggesting that AKG exerts autophagy
effects by producing ROS in 786O and ACHN cells.
Additionally, we studied other pathways involved in the suppres-
sion of kidney cancer cells by AKG. Previous studies have linked
PI3K/Akt/mTOR signaling to malignancies and aging illnesses.51
Moreover, the PI3K/Akt/mTOR signaling pathway may control apo-
ptosis and autophagy in cancer cells.52 Therefore, inhibiting PI3K sig-
naling is a possible treatment for several disorders and a therapeutic
research target. A study reported that AKG extends lifespan by down-
regulating the mTOR signaling pathway in Drosophila.13 In our
research, AKG considerably inhibited the PI3K/Akt/mTOR signaling
pathway. Moreover, the PI3K/AKT/mTOR signaling pathway was sug-
gested as the principal pathway involved in the initiation and control
of autophagy.26,53 Several studies have shown that AKT inhibition
stimulates autophagy,54 whereas AKT activation inhibits autophagy.55
Inhibiting the PI3K/AKT/mTOR pathway increases autophagy in cells.
In our investigation, AKG enhanced the expression of the autophagy-
related protein LC3B in 786O and ACHN cells in a dose-dependent
manner. As mentioned above, AKG is capable of inducing autophagy
in cells. According to a recent study, the issue of whether autophagy
promotes death or survival is still under debate.56 Nonetheless, exces-
sive autophagy may result in autophagic cell death. Inhibiting PI3K/
Akt/mTOR increased autophagy signaling and tumor cell death
(Figure 7). Cotreatment of cells with AKG and 3-MA attenuated the
AKG-induced decrease in tumor cell PI3K/Akt/mTOR activity. In
accordance with these findings, our data indicated that AKG inhibits
the PI3K/Akt/mTOR pathway and is involved in autophagy.
5 | C O N CL U S I O N
In summary, our findings demonstrate that AKG treatment exerts apo-
ptotic and autophagic effects via ROS/PI3K/AKT/mTOR pathway
15227278, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/tox.23658 by Fu Jen Catholic University, Wiley Online Library on [14/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WU ET AL.
signaling; therefore, AKG could serve as a novel potential anticancer
treatment for RCC.
AUTHOR CONTRIBU TIONS
Zhifeng Wang and Caiyong Lai designed and managed the project.
Xuexia Xie and Fan Wu conducted experiments and did data analysis.
Fan Wu wrote the manuscript. Guoliang Li and Dongping Bao partici-
pated in cell culture and in vitro experiments. Haomin Li and Guohao
Wu helped with data analysis. Yiqi Lai and Yaping Xing helped with
graphs. Peng Ouyang and Guo Chen made suggestions on the manu-
script. The final manuscript was reviewed and approved by all authors.
FUNDING INF ORMATI ON
This research was supported by the Science and Technology Project
of the Health Bureau of Yangjiang City (2021036 to C.Y.L.,
SF2021212 to C.Y.L.), National Natural Science Foundation of China
(82073042 to G. Chen), and Guangdong Basic and Applied Basic
Research Foundation (2022B1515020105 to G. Chen).
CONFLIC T OF INT ER E ST
The authors declare no potential conflict of interest.
OR CID
Caiyong Lai https://orcid.org/0000-0003-4679-0944
RE FE RE NCE S
1. Singh D. Current updates and future perspectives on the manage-
ment of renal cell carcinoma. Life Sci. 2021;264:118632.
2. Ljungberg B, Bensalah K, Canfield S, et al. EAU guidelines on renal cell
carcinoma: 2014 update. Eur Urol. 2015;67(5):913-924. doi:10.1016/
j.eururo.2015.01.005
3. Capitanio U, Montorsi F. Renal cancer. Lancet. 2016;387(10021):
894-906.
4. Pierorazio PM, Johnson MH, Patel HD, et al. Management of renal
masses and localized renal cancer: systematic review and meta-analy-
sis. J Urol. 2016;196(4):989-999.
5. Jonasch E, Gao J, Rathmell WK. Renal cell carcinoma. BMJ. 2014;349:
g4797.
6. Fang P, Zhou L, Lim LY, Fu H, Yuan Z-X, Lin J. Targeting strategies for
renal cancer stem cell therapy. Curr Pharm Des. 2020;26(17):1964-1978.
7. Barata PC, Rini BI. Treatment of renal cell carcinoma: current status
and future directions. CA Cancer J Clin. 2017;67(6):507-524.
8. Rini BI, Battle D, Figlin RA, et al. The society for immunotherapy of
cancer consensus statement on immunotherapy for the treatment of
advanced renal cell carcinoma (RCC). J Immunother Cancer. 2019;
7(1):354.
9. Xiao D, Zeng L, Yao K, Kong X, Wu G, Yin Y. The glutamine-alpha-
ketoglutarate (AKG) metabolism and its nutritional implications. Amino
Acids. 2016;48(9):2067-2080.
10. Liu S, He L, Yao K. The antioxidative function of alpha-ketoglutarate
and its applications. Biomed Res Int. 2018;2018:3408467-3408466.
11. Legendre F, MacLean A, Appanna VP, Appanna VD. Biochemical
pathways to α-ketoglutarate, a multi-faceted metabolite. World J
Microbiol Biotechnol. 2020;36(8):123.
12. Wu N, Yang M, Gaur U, Xu H, Yao Y, Li D. Alpha-ketoglutarate: physio-
logical functions and applications. Biomol Ther (Seoul). 2016;24(1):1-8.
13. Su Y, Wang T, Wu N, et al. Alpha-ketoglutarate extends lifespan by
inhibiting mTOR and activating AMPK. Aging (Albany NY). 2019;
11(12):4183-4197.

WU ET AL.
14. Liu S, Yang J, Wu Z. The regulatory role of α-ketoglutarate metabo-
lism in macrophages. Mediators Inflamm. 2021;2021:5577577.
15. Bayliak MM, Lushchak VI. Pleiotropic effects of alpha-ketoglutarate
as a potential anti-ageing agent. Ageing Res Rev. 2021;66:101237.
16. Morris JP, Yashinskie JJ, Koche R, et al. α-Ketoglutarate links p53 to
cell fate during tumour suppression. Nature. 2019;573(7775):595-599.
17. Kaławaj K, Sławin ska-Brych A, Mizerska-Kowalska M, et al. Alpha
ketoglutarate exerts in vitro anti-osteosarcoma effects through inhibi-
tion of cell proliferation, induction of apoptosis via the JNK and cas-
pase 9-dependent mechanism, and suppression of TGF-β and VEGF
production and metastatic potential of cells. Int J Mol Sci. 2020;
21(24):9406.
18. Atlante S, Visintin A, Marini E, et al. α-ketoglutarate dehydrogenase
inhibition counteracts breast cancer-associated lung metastasis. Cell
Death Dis. 2018;9(7):756.
19. Rzeski W, Walczak K, Juszczak M, et al. Alpha-ketoglutarate (AKG)
inhibits proliferation of colon adenocarcinoma cells in normoxic con-
ditions. Scand J Gastroenterol. 2012;47(5):565-571.
20. Kist M, Vucic D. Cell death pathways: intricate connections and dis-
ease implications. EMBO J. 2021;40(5):e106700.
21. Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer.
Nat Rev Cancer. 2017;17(9):528-542.
22. Mizushima N, Levine B. Autophagy in human diseases. N Engl J Med.
2020;383(16):1564-1576.
23. Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular
mechanisms. J Pathol. 2010;221(1):3-12.
24. Onorati AV, Dyczynski M, Ojha R, Amaravadi RK. Targeting autop-
hagy in cancer. Cancer. 2018;124(16):3307-3318.
25. Li X, He S, Ma B. Autophagy and autophagy-related proteins in can-
cer. Mol Cancer. 2020;19(1):12.
26. Xu Z, Han X, Ou D, et al. Targeting PI3K/AKT/mTOR-mediated
autophagy for tumor therapy. Appl Microbiol Biotechnol. 2020;104(2):
575-587.
27. He Y-H, Tian G. Autophagy as a vital therapy target for renal cell car-
cinoma. Front Pharmacol. 2020;11:518225.
28. Lopes TZ, de Moraes FR, Tedesco AC, Arni RK, Rahal P, Calmon MF.
Berberine associated photodynamic therapy promotes autophagy and
apoptosis via ROS generation in renal carcinoma cells. Biomed Phar-
macother. 2020;123:109794.
29. Gao L, Loveless J, Shay C, Teng Y. Targeting ROS-mediated crosstalk
between autophagy and apoptosis in cancer. Adv Exp Med Biol. 2020;
1260:1-12.
30. Fu D, Wu D, Cheng W, et al. Costunolide induces autophagy and apo-
ptosis by activating ROS/MAPK signaling pathways in renal cell carci-
noma. Front Oncol. 2020;10:582273.
31. Capitanio U, Bensalah K, Bex A, et al. Epidemiology of renal cell carci-
noma. Eur Urol. 2019;75(1):74-84.
32. Li P, Wong Y-N, Armstrong K, et al. Survival among patients with
advanced renal cell carcinoma in the pretargeted versus targeted
therapy eras. Cancer Med. 2016;5(2):169-181.
33. Asadi Shahmirzadi A, Edgar D, Liao C-Y, et al. Alpha-ketoglutarate, an
endogenous metabolite, extends lifespan and compresses morbidity
in aging mice. Cell Metab. 2020;32(3):447-456.e6.
34. Salminen A, Kaarniranta K, Hiltunen M, Kauppinen A. Krebs cycle
dysfunction shapes epigenetic landscape of chromatin: novel insights
into mitochondrial regulation of aging process. Cell Signal. 2014;26(7):
1598-1603.
35. Martínez-Reyes I, Chandel NS. Mitochondrial TCA cycle metabolites
control physiology and disease. Nat Commun. 2020;11(1):102.
_ cell apoptosis by inducing reactive oxygen species-mediated
36. Zdzisinska B, Zurek A, Kandefer-Szerszen  M. Alpha-ketoglutarate as a
molecule with pleiotropic activity: well-known and novel possibilities
of therapeutic use. Arch Immunol Ther Exp (Warsz). 2017;65(1):21-36.
37. Carey BW, Finley LWS, Cross JR, Allis CD, Thompson CB. Intracellular
α-ketoglutarate maintains the pluripotency of embryonic stem cells.
Nature. 2015;518(7539):413-416.
15227278, 2023, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/tox.23658 by Fu Jen Catholic University, Wiley Online Library on [14/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
27
38. Gyanwali B, Lim ZX, Soh J, et al. Alpha-ketoglutarate dietary supple-
mentation to improve health in humans. Trends Endocrinol Metab.
2022;33(2):136-146.
39. Greilberger J, Herwig R, Greilberger M, Stiegler P, Wintersteiger R.
Alpha-ketoglutarate and 5-HMF: a potential anti-tumoral combina-
tion against leukemia cells. Antioxidants (Basel). 2021;10(11):1804.
40. Bertheloot D, Latz E, Franklin BS. Necroptosis, pyroptosis and apo-
ptosis: an intricate game of cell death. Cell Mol Immunol. 2021;18(5):
1106-1121.
41. Tennant DA, Gottlieb E. HIF prolyl hydroxylase-3 mediates alpha-
ketoglutarate-induced apoptosis and tumor suppression. J Mol Med
(Berl). 2010;88(8):839-849.
42. Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in
cancer: therapeutic implications. Mol Cancer Ther. 2011;10(9):1533-
1541.
43. Silva VR, Neves SP, Santos LS, Dias RB, Bezerra DP. Challenges and
therapeutic opportunities of autophagy in cancer therapy. Cancers
(Basel). 2020;12(11):3461.
44. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in
cancer. Nat Rev Cancer. 2007;7(12):961-967.
45. Vessoni AT, Filippi-Chiela EC, Menck CF, Lenz G. Autophagy and
genomic integrity. Cell Death Differ. 2013;20(11):1444-1454.
46. Zhang Y, Fan Y, Huang S, et al. Thymoquinone inhibits the metastasis
of renal cell cancer cells by inducing autophagy via AMPK/mTOR sig-
naling pathway. Cancer Sci. 2018;109(12):3865-3873.
47. Villalpando-Rodriguez GE, Gibson SB. Reactive oxygen species (ROS)
regulates different types of cell death by acting as a rheostat. Oxid
Med Cell Longev. 2021;2021:9912436-9912417.
48. Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB. Oxidative
stress induces autophagic cell death independent of apoptosis in
transformed and cancer cells. Cell Death Differ. 2008;15(1):171-182.
49. Srinivas US, Tan BWQ, Vellayappan BA, Jeyasekharan AD. ROS and
the DNA damage response in cancer. Redox Biol. 2019;25:101084.
50. Gan Y, Yang D, Yang S, Wang J, Wei J, Chen J. Di-2-ethylhexyl
phthalate (DEHP) induces apoptosis and autophagy of mouse GC-1
spg cells. Environ Toxicol. 2020;35(2):292-299.
51. Xu F, Na L, Li Y, Chen L. Roles of the PI3K/AKT/mTOR signalling
pathways in neurodegenerative diseases and tumours. Cell Biosci.
2020;10(1):54.
52. Porta C, Paglino C, Mosca A. Targeting PI3K/Akt/mTOR signaling in
cancer. Front Oncol. 2014;4:64.
53. Heras-Sandoval D, Pérez-Rojas JM, Hernández-Damián J, Pedraza-
Chaverri J. The role of PI3K/AKT/mTOR pathway in the modulation
of autophagy and the clearance of protein aggregates in neurodegen-
eration. Cell Signal. 2014;26(12):2694-2701.
54. Quan C, Wang C, Duan P, et al. Bisphenol a induces autophagy and
apoptosis concurrently involving the Akt/mTOR pathway in testes of
pubertal SD rats. Environ Toxicol. 2017;32(8):1977-1989.
55. Degtyarev M, De Mazière A, Orr C, et al. Akt inhibition promotes
autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.
J Cell Biol. 2008;183(1):101-116.
56. Ravanan P, Srikumar IF, Talwar P. Autophagy: the spotlight for cellular
stress responses. Life Sci. 2017;188:53-67.
How to cite this article: Wu F, Xie X, Li G, et al. AKG induces
endoplasmic reticulum stress and by suppressing PI3K/AKT/
mTOR-mediated autophagy in renal cell carcinoma.
Environmental Toxicology. 2023;38(1):17‐27. doi:10.1002/tox.
23658