N-acetylcysteine (NAC) Inhibits ROS-induced Ferroptosis in CTNS

Knockdown β-cells in Vitro

Hongrui Liang

1,*

, Jingyu Xu

, Yanxu Chen

, Jingjie Yu

and Ruoxun Peng

China Agricultural University, Haidian District, Beijing 100089, China

Shanghai Foreign Language School, Shanghai 200083, China

Shanghai Starriver Bilingual School, Shanghai 201108, China

The Village School, Houston, TX, U.S.A.

International Department, The Second High School Attached to Beijing Normal University, Beijing 100192, China

Keywords: CTNS, Cystine, Ferroptosis, NAC, Insulin Secretion.

Abstract: CTNS silence or mutation in the human body leads to cystinosis, an autosomal recessive lysosomal storage

disease. Ferroptosis is an iron-dependent form of programmed cell death. N-acetylcysteine (NAC), a common

antioxidant, is the acetylated precursor of L-cysteine. There are no studies about the relationship between

cystinosis and ferroptosis yet. Therefore, in this paper, we aimed to 1) find out the relationship between CTNS

knockdown and ferroptosis in β-cells and 2) verify that NAC is a potential agent to protect CTNS knockdown

β-cells and is also a potent ferroptosis inhibitor. Since we do not have the access to do the experiments in a

laboratory, all the following results and conclusions are hypothetical or from existing papers: CTNS-targeting

siRNA inhibit cystinosin expression at mRNA and protein level; CTNS knockdown induces ferroptosis in β-

cells and NAC attenuates ferroptosis; NAC attenuates oxidative stress in CTNS knockdown β-cells; NAC

restores energy level and glucose-stimulated insulin secretion in CTNS knockdown β-cells. We conclude that,

in vitro, the NAC pretreatment can effectively rescue CTNS knockdown β-cells from ferroptosis by elevating

the GPX4 mRNA and its protein level.

1 INTRODUCTION

Cystinosin is a protein transporter that excretes

cystine combined with a proton from the lysosome to

the cytosol, which is encoded by the CTNS gene.

Mutations in the human CTNS gene will hinder the

efflux of lysosomal cystine and cause lysosomal

cystine accumulation, leading to an autosomal

recessive inheritance disease, that is, cystinosis

(Gahl, Thoene, Schneider 2009). Some researchers

discovered that lysosomal cystine accumulation in

cystinosin-deprived cells changes the cytosolic redox

milieu to higher oxidized status by limitation of

glutathione (GSH) synthesis (L. E, de G.-H. A, W. M,

van den H. L, M. L, B. H 2005, S. R, M. B, N. P, M.

T 2016, B. F et al 2010). Furthermore, abundant

research indicated that β-cell is more sensitive to

oxidative stress due to low expression of catalase and

peroxidase and low GSH level compared to other

tissues (N. S, S. H, A. R, T. T, and Y. T 2008, S. K et

al 2003, L. S, D. J, and T. M 1996). Former reports

showed that intracellular cysteine level, reactive

oxygen species (ROS) level, and energy production

correlate with insulin secretion (N. S, S. H, A. R, T.

T, and Y. T 2008, S. K et al 2003), which is

demonstrated in CTNS-knockdown β-cells (M. B, S.

R, S. C, M. T, and N. P 2015). The elevated cystine

and ROS level concomitant with stymied ATP

production in cytosol and mitochondria caused

attenuated insulin secretion. This conclusion at the

cell level keeps aligned with the summary of

cystinosis complication (N. G, G. W 2008).

Ferroptosis is an iron-dependent form of regulated

cell death. It is related to iron-dependent lipid

peroxidation metabolism and regulates cell death

through NADPH/H

, polyunsaturated fatty acids,

glutamine catabolism, and other signal pathways.

Ferroptosis demonstrates cellular atrophy and high-

density mitochondria in morphology. The cystine

antiporter system mediates the production of GSH,

which is an important ferroptosis inhibitor (X. Y et al

2015, S. BR et al. 2017). Cysteine is the rate-limiting

metabolite for GSH biosynthesis, so cysteine

depletion leads to the lowering of intracellular GSH

Liang, H., Xu, J., Chen, Y., Yu, J. and Peng, R.

N-acetylcysteine (NAC) Inhibits ROS-Induced Ferroptosis in CTNS Knockdown -Cells in Vitro.

DOI: 10.5220/0011390800003443

In Proceedings of the 4th Inter national Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1235-1243

ISBN: 978-989-758-595-1

1235

levels. GSH depletion triggers the inactivation of

GPX4 which is an enzyme that specifically reduces

phospholipid hydroperoxides using GSH as a

cofactor (Y. WS et al. 2013). Cystine starvation

impairs GPX4 protein expression by inhibiting

mTORC1/4E-BP1-mediated protein translation (Z. Y

et al. 2021). The inactivation of GPX4 leads to lipid

peroxidation which causes the accumulation of ROS

(Y. WS et al. 2013). ROS can react with

polyunsaturated fatty acids of lipid membranes and

induce lipid peroxidation on membranes. Also,

intracellular cysteine is used for the biosynthesis of

coenzyme A and subsequently CoQ10, a vital

metabolite for preventing membrane lipid

peroxidation and ferroptosis cell death (D. S et al.

2019, B. K et al. 2019, B. MA et al. 2020). Therefore,

the cyst(e)ine level is very essential for regulating

redox status and ferroptosis in cells.

N-acetylcysteine (NAC), the acetylated precursor

of L-cysteine, is a common antioxidant used for

clinical practice and biomedicine research. For

example, with its potent antioxidant ability, NAC

effectively prevents hemin-induced ferroptosis and

ROS/MAPK and p53-mediated ferroptosis and

rescues neuron cells having ferroptosis-like

phenotype (G. G et al. 2020, L. Y et al. 2020, K. SS

et al. 2018). And a clinical trial showed that

additional NAC given to nephropathic cystinosis

patients with routine cysteamine treatment for three

months could reduce oxidative stress and

significantly improve renal function without side-

effects (P. de F. G. L et al. 2014). However, there is

no more cell-level research related to NAC and

cystinosis, leaving the relation between NAC and

lysosomal cystine accumulation unknown.

Currently, the relationship between cystinosis and

ferroptosis has not been studied yet. According to the

results from Bernadette's research (M. B, S. R, S. C,

M. T, and N. P 2015), the CTNS-knockdown β-cells

with attenuated insulin secretion bore the characters

of high ROS, cyst(e)ine, and oxidized GSH (GSSG)

levels. Though the cyst(e)ine level is higher than

normal cells, oxidative stress still occurs. What's

more, β-cells are more sensitive to oxidative stress

than other types of tissues and cells in the human

body, which embodies in terms of insulin secretion

(N. P, R. E, A. F, K. M, C. A, C. R 2012). Combining

the aforementioned information, here we hypothesize

that ROS is a potent factor to cause ferroptosis in β-

cells regardless of the cyst(e)ine. Therefore, CTNS-

knockdown β-cells will undergo ROS-induced

ferroptosis and NAC could inhibit ferroptosis and

reverse the adverse effects caused by lack of

cystinosin.

2 MATERIALS AND METHODS

The big picture of our research shows in Figure 1. The

Mock means the β-cells underwent all of the siRNA

transfection steps without siRNA. β-cells were

transfected by non-targeting siRNA (NT) or CTNS-

targeting siRNA (CT) for 18 h. CT+ cells were

cultured with 10 mM NAC for 12 h and transferred to

normal culture for the next 12 h after siRNA

transfection, while other groups lived in normal

culture for 24 h post-transfection. In a word, after 24

h post-transfection, all groups were detected by real-

time PCR to justify whether siRNA interference was

successful or not. Then the subsequent experiments

were practiced for competent groups after 48 h

stabilization in normal culture. The specific

experiment steps are as follows.

Figure 1: Schematic research design.

The BRIN-BD11 β-cells line was processed and

divided into four groups: Mock, non-targeting siRNA

transfection (NT), CTNS-targeting siRNA

transfection (CT), and CTNS-targeting siRNA

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1236

transfection with NAC treatment (CT+). After

transcription-level verification of real-time PCR, all

groups were cultured in normal culture for 48 h and

then used for subsequent experiments.

2.1 Cell Culture and Process

Here we use the same materials, cell culture protocol,

and gene knockdown protocol of Bernadette's

research (M. B, S. R, S. C, M. T, and N. P 2015).

Briefly, we used ON-TARGETplus SMARTpool

technology by GE Dharmacon (Lafayette, CO, USA).

BRIN-BD11 cells were seeded and allowed to adhere

overnight. CTNS-targeting siRNA pool (80 pmol/μl;

CT) was transiently transfected into the cells for 18 h

using DharmaFECT 1, according to the instructions

of the manufacturer. Mock transfection involved the

transfection in the absence of siRNA, while the

negative control was the non-targeting siRNA pool

(NT). Following the NAC addition of Zhang's paper

(L. Y et al. 2020), half part of the CT cells is

pretreated with NAC (10 mM, CT+) for 12 h and then

cultured in normal culture for 12 h before real-time

PCR.

2.2 sIRNA Gene Knockdown

Verification

The real-time PCR and western blot were practiced

demonstrating CTNS knockdown at transcriptional

and expressional levels. The real-time PCR was

practiced 24 h after siRNA transfection and western

blot was used at 72 h post-transfection. Experiment

steps are strictly repeated according to Bernadette's

research (M. B, S. R, S. C, M. T, and N. P 2015).

2.3 Intracellular Iron Level

Intracellular iron levels were assessed using an

optimized FerroZine™, an iron-based assay

developed by Reimer (R. J, H. HH, C. H, R. SR, and

D. R, 2004). Before testing, each sample tube and the

standard tube is filled with 6.5 mM FerroZine™, 6.5

mM neocuproine, 2.5 M ammonium acetate, and 1 M

ascorbic acid dissolved in water. An iron detection

reagent is then being added to each tube. After that,

incubation will be performed for half an hour at room

temperature, which should allow researchers to

observe color development. Then, a microplate

reader measures the absorbance at 550 nm when 280

μl from both standard and sample tubes was added in

duplicate into wells of a 96-well plate. Finally, the

BCA assay will be carried out to determine

intracellular iron concentration. The concentration is

determined by the amount of dye in the blue ionic

from the measurement of the absorbance of the

solution.

2.4 GPX4 Level

We use the Glutathione peroxidase 4 (GPX4) kit

(Runyu biotechnology co. LTD, Shanghai, China) to

detect the GPX4 level in treated cell lines according

to Zhou's work (Z. Y 2020). The GPX4 gene level can

be detected via Real-Time Quantitative Polymerase

Chain Reaction (RT-qPCR). RNeasy Mini Kit

(Qiagen) was used for the extraction of total RNAs

according to the manufacture’s protocols. M-MLV

Reverse Transcriptase (Promega) was used for DNA

synthesis. GAPDH served as the internal reference.

The 2

−ΔΔCT

method was used for the calculation of the

relative gene transcription level. The GPX4 protein

level is detected using western blot following Li's

method (L. D et al. 2020).

2.5 MDA Level

The measure of MDA levels is performed by TBARS

assay. Glacial acetic acid is used to reconstitute

thiobarbituric acid (TBA) since regular acetic acid

affects TBA stability by its high-water content.

During sample preparation, butylated

hydroxytoluene is used in lysis buffer to prevent

further peroxidation while processing. After that,

TBA solution is added to the sample and then the

sample will be incubated at 95°C for 60 minutes.

Following that, the samples will cool down to room

temperature by using an ice bath for 10 minutes.

Finally, a microplate reader is used to measure the

output.

2.6 ROS Level

The examination of qualitative ROS level is referring

to Li (L. D et al. 2020). The ROS probes solution was

diluted to the required concentration in the

Phosphate-buffered saline (PBS) buffer and then

incubated at room temperature for 45 min. Then the

cells were washed with PBS 3 times and excited with

green light under a fluorescent microscope to observe

and shoot red emission images of the cells, which

represent ROS positive cells.

Referring to Bernadette's method (M. B, S. R, S.

C, M. T, and N. P 2015), the quantitative ROS level

was detected using ROS probe, 5-(and-6)-carboxy-

2’,7’-difluorodihydro fluorescein diacetate (carboxy-

DFFDA). Cells were incubated with 20μM

carboxy-H

DFFDA for 45 min, and the cell

N-acetylcysteine (NAC) Inhibits ROS-Induced Ferroptosis in CTNS Knockdown -Cells in Vitro

1237

suspension was analyzed immediately using the BD

Accuri C6 flow cytometer.

2.7 Intracellular GSH and GSSG

Levels

According to Zhou (Z. Y 2020), the levels of GSH

and GSSG were detected by GSH/GSSG Ratio

Detection Assay II (ab205811, Abcam), which is

used for the measurement of GSH, GSSG, and

GSH/GSSG. The GSH and GSSG levels were

normalized for protein content.

2.8 Cell Viability

Cells incubated in 96-well plates were treated as

indicated and cell proliferation was assessed by CCK-

8 assay (SAB biotech. College Park, MD, USA) at 24

h after CTNS knockdown and at 24 h after NAC

treatment following the manufacturer’s instruction.

Optical density (OD) was recorded at 450 nm.

2.9 Intracellular ATP Level

Following Bernadette's method (M. B, S. R, S. C, M.

T, and N. P 2015), cells were detached using 0.05%

trypsin–EDTA and washed with ice-cold PBS. The

cell pellets were resuspended in 100 μl ice-cold PBS.

The cell suspension was then diluted 25-fold and the

ATP content was assessed using the ATP

Bioluminescence Assay Kit HSII (Roche

Diagnostics, Mannheim, Germany), according to the

instructions of the manufacturer. The protein

concentration of the cell lysates was used to

normalize the ATP content.

2.10 Insulin Secretion

The chronic and acute glucose-stimulated insulin

secretion was repeated following Bernadette's

method(M. B, S. R, S. C, M. T, and N. P 2015). At 72

h post-transfection, the cell supernatant was removed

and used to determine chronic insulin release. The

cells were then washed with PBS and acute insulin

secretion was stimulated after cells were starved for

40 min with Krebs Ringer Buffer (KRB), pH 7.4,

containing 1.1 mM D-glucose. The cells were then

stimulated with KRB containing 16.7 mM glucose

plus 10 mM alanine for 20 min at 37 °C. The KRB

was collected and insulin release was determined

using the Mercodia ultra-sensitive rat insulin ELISA

kit (Uppsala, Sweden), according to the instructions

of the manufacturer.

2.11 Statistic Analysis

This research is based on hypotheses and speculations

instead of real data in the lab. Therefore, the data

presented in the following sections are mock data

from existing research and are modified due to our

prediction. Data are presented as the means of three

independent experiments ± SEM and were analyzed

with the GraphPad Prism 9.2 software (GraphPad

Software Inc., San Diego, CA, USA). Data were

analyzed by Student’s t-test and differences at a P-

value of <0.05 were considered significant. One, two,

three, four asterisks respectively represent P-value of

<0.05, <0.01, <0.001, and <0.0001.

3 HYPOTHESIS AND RESULT

SPECULATION

Here we hypothesize that CTNS knockdown will

induce oxidative stress in β-cells BRIN-BD11 and

express the phenotype of ferroptosis, while NAC can

attenuate ferroptosis by changing redox status in

cells. The mock results are listed as follows.

3.1 CTNS-Targeting sIRNA Inhibit

Cystinosin Expression at mRNA

and Protein Level

Our speculation thoroughly depends on the CTNS

knockdown in β-cells BRIN-BD11. Therefore, we

presume that we successfully silence the CTNS gene.

The CTNS mRNA levels in CT and CT+ are

significantly decreased by more than 80 % compared

to Mock and NT at 24 h post-transfection (Fig. 2A).

Combining mRNA level decrease, cystinosin level in

CT and CT+ is also decreased by approximately 50

% at 72 h post-transfection (Fig. 2B).

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1238

Figure 2: Evaluation of CTNS-targeting siRNA knockdown by real-time PCR and Western blot.

BRIN-BD11 cells were transiently transfected

with a CTNS-targeting siRNA pool (CT) for 18 h

using DharmaFECT 1 transfection reagent, according

to the manufacturer’s instructions. A part of CT

underwent 10 mM NAC pretreatment for 12 h after

transfection (CT+). Mock transfection (Mock) was

performed in the absence of siRNA, while the

negative control contained a scrambled non-targeting

siRNA pool (NT). CTNS mRNA and cystinosin levels

were assessed by real-time PCR (A) and Western blot

(B) at 24 and 72 h post-transfection, respectively.

Lanes from 1 to 4 of the Western blot analysis

correspond to Mock, NT, CT, and CT+, respectively.

Densitometry analysis of Western blots is expressed

as a ratio of cystinosin to α-tubulin expression and is

presented as mean fold change relative to Mock ±

SEM of three or more independent experiments.

***

****

show the CT's statistically significant difference

from Mock or NT, as indicated, at P<0.001 and

P<0.0001, respectively. Mock, mock transfection;

NT, non-targeting siRNA; CT, CTNS-targeting

siRNA; CT+, CTNS-targeting siRNA and NAC

treatment.

3.2 CTNS Knockdown Induces

Ferroptosis In β-cells and NAC

Attenuates Ferroptosis

From artificial Figure 3, we could know that the

CTNS knockdown β-cells get apparently damaged

with strong ferroptosis phenomenons: elevated

intracellular iron and MDA level and decreased

GPX4 mRNA and protein level. However, NAC

could potently attenuate negative effects caused by

CTNS knockdown.

Figure 3: Effect of CTNS knockdown and NAC on

ferroptosis in β-cells

At 72 h post-transfection, the BRIN-BD11 cell

viability (A), iron level (B), MDA level (C), GPX4

mRNA level (D), and GPX4 protein level (E) of NT,

CT, and CT+ are determined. Data are presented as

means ± SEM of at least three independent

experiments.

***

, and

****

represent significantly

difference at P<0.05, 0.01, 0.001, and 0.0001,

respectively. NT, non-targeting siRNA treatment; CT,

CTNS-targeting siRNA treatment; CT+, CTNS-

targeting siRNA and NAC treatment.

3.3 NAC Attenuates Oxidative Stress

in CTNS Knockdown β-cells

We forecast that BRIN-BD11 cells are affected by

serious oxidative stress after CTNS knockdown (Fig.

4). Compared with NT, the ROS (Fig. 4B) and GSSG

N-acetylcysteine (NAC) Inhibits ROS-Induced Ferroptosis in CTNS Knockdown -Cells in Vitro

1239

(Fig. 4D) levels of CT are extremely elevated, while

the GSH level has not significantly changed (Fig.

4C). The NAC is a competent antioxidant to inhibit

oxidative stress (Fig. 4E). Interestingly, the total GSH

level will not be improved when the cysteine level is

normal (B. JM, V. A, and L. BH 1989). Due to the

known result that total cysteine in CTNS knockdown

cells is a bit more than normal cells (M. B, S. R, S. C,

M. T, and N. P 2015), we speculate that the total GSH

level will not be improved after NAC treatment.

Figure 4: Effect of CTNS knockdown and NAC on redox status in β-cells.

At 72 h post-transfection, the ROS level in BRIN-

BD11 cells was showed in qualitative form (A) by

using Dihydroethidium (DHE) probe and quantitative

form (B) by using ROS probe carboxy-H

DFFDA.

The total glutathione (GSH) (C) and oxidized

glutathione (GSSG) (D) levels in BRIN-BD11 cells

were determined. (E) The ratio of GSH and GSSG is

calculated. Data are presented as means ± SEM of at

least three independent experiments.

and

***

represent the significant difference at P<0.05 and

<0.001, respectively. NT, non-targeting siRNA

treatment; CT, CTNS-targeting siRNA treatment;

CT+, CTNS-targeting siRNA and NAC treatment.

3.4

NAC Restores Energy Level and

Glucose-Stimulated Insulin

Secretion in CTNS Knockdown

β-cells

The decreased total ATP level (Fig. 5A) and restricted

chronic (Fig. 5B) and acute (Fig. 5C) insulin secretion

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1240

are demonstrated in CTNS knockdown β-cells. Based

on our hypothesis that β-cells are mainly affected by

oxidative stress, 10 mM NAC treatment can

significantly increase the intracellular ATP level and

improve insulin secretion due to its antioxidant

property.

Figure 5. Effect of CTNS knockdown and NAC on ATP level and insulin secretion

A) total ATP concentration is determined in

BRIN-BD11 cells at 72 h post-transfection. B) the

chronic release of insulin was assessed in cells

between 24–72 h after transfection. Cells are initially

incubated with KRB containing 1.1 mM glucose for

40 min at 37°C before insulin release was stimulated

for 20 min using KRB containing 1.1 mM glucose

(basal) or 16.7 mM glucose and 10 mM alanine. C)

acute stimulated insulin secretion was determined in

BRIN-BD11 cells at 72 h post-transfection. Data are

presented as means ± SEM of at least three

independent experiments.

, and

***

represent

significantly difference at P<0.05, 0.01 and 0.001,

respectively. NT, non-targeting siRNA treatment; CT,

CTNS-targeting siRNA treatment; CT+, CTNS-

targeting siRNA and NAC treatment.

4 DISCUSSIONS

During ferroptosis, lipid peroxidation happens, which

N-acetylcysteine (NAC) Inhibits ROS-Induced Ferroptosis in CTNS Knockdown -Cells in Vitro

1241

is related to the high oxidative level in the cytosol.

Currently, researchers think that N-acetylcysteine can

inhibit ferroptosis due to its potent antioxidant ability

(X. Y et al 2015). However, the specific mechanism

of NAC inhibiting ferroptosis has not received much

attention. We forecast that NAC may not only

scavenge ROS but also restore other substances that

act as ferroptosis inhibitors.

According to current research about ferroptosis

(X. Y et al 2015), cyst(e)ine starvation can lower the

level of GSH, inactivate GPX4, and inhibit the

biosynthesis of coenzyme A and subsequent CoQ10

which can greatly prevent lipid peroxidation and

ferroptosis. GPX4 can prevent normal oxidative

stress and lipid peroxidation by using GSH. No

researchers, however, study whether ROS itself can

inactivate GPX4 at mRNA and protein level under

the condition of normal cyst(e)ine and GSH

concentration.

According to Bernadette's research (M. B, S. R, S.

C, M. T, and N. P 2015), the level of cystine and

cysteine in β-cells both increase after CTNS

knockdown, but the oxidative stress and the

accumulation of ROS still happens, lowering the

secretion of insulin and impairing cell viability. The

cyst(e)ine-mediated ferroptosis is inactivated because

of the sufficient intracellular cyst(e)ine under this

situation. Under the hypothesis we raised before, we

speculate that the GPX4 level will not be affected by

cyst(e)ine but by ROS aggregation. ROS can

independently inactivate GPX4 mRNA and protein

through a certain unknown mechanism and form

positive feedback which leads to more ROS

accumulation and inevitable ferroptosis. Following

the hypothesis, the mRNA and protein level of GPX4

and cell viability will significantly decrease, the

MDA level highly increase. Referring to the

discussion above, the NAC treatment is applied to

reduce ROS, which can restore the GPX4

transcription and expression. Whether NAC could

directly rehabilitate GPX4 in different ways is

valuable to study. By rehabilitation from ferroptosis,

the insulin secretion and intracellular level of β-cells

rise, though we cannot estimate this increase whether

is caused by ferroptosis inhibition or oxidative stress

inhibition.

5 CONCLUSIONS

After CTNS knockdown, the BRIN-BD11 β-cells

undergo ROS-induced ferroptosis with impaired

insulin secretion and energy production. The NAC

pretreatment effectively rescues CTNS knockdown β-

cells from ferroptosis by increasing GPX4 mRNA

and protein level.

Currently, there are no research focusing on the

relationship between ferroptosis and cystinosis. The

intracellular cyst(e)ine level of cystinosis patients is

higher than that of healthy people. Not a few

Researchers study the ferroptosis caused by low

cyst(e)ine in cells, that is, cyst(e)ine starvation. CTNS

knockdown β-cells with high oxidative stress and

intracellular cyst(e)ine level provide a good model to

discover whether ferroptosis occurs with enough

cyst(e)ine. We innovatively exclude the influence of

cyst(e)ine and study the relationship between

ferroptosis and ROS with CTNS knockdown cell

model. Our work to some extant offer a new

perspective of ferroptosis induction.

ACKNOWLEDGEMENT

The authors are grateful to Dr. Shu-bing Qian for his

enlightenment and guide of our research and Dr.

Huijie Han for her instruction of paper writing.

The author's contributions are listed here.

Experimental design, H. LIANG; Materials

organization, H. LIANG, J. XU; Methodology, H.

LIANG, J. YU, R. PENG, Y. CHEN; Hypothesis

speculation, H. LIANG, J. YU, J. XU, Y. CHEN, R.

PENG; Data visualization, H. LIANG; Paper writing,

H. LIANG, J. XU, Y. CHEN; Proofreading, Y.

CHEN. All the authors have read and agreed to the

final version of the report with no conflict of interest.

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