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N-O reduction and ROS-mediated AKT/FOXO1 and AKT/P53 pathways were involved in growth promotion and cytotoxicity of Cyadox Qianying Liu, Zhixin Lei, Kaixiang zhou, Huiru Yu, Shenhe liu, Qiliang Sun, Xu Wang, Menghong Dai, and Zonghui Yuan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00194 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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Chemical Research in Toxicology

1

N-O reduction and ROS-mediated AKT/FOXO1 and

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AKT/P53 pathways were involved in growth promotion and

3

cytotoxicity of Cyadox

4 5

Qianying Liu1†, Zhixin Lei2†, Kaixiang Zhou3, Huiru Yu3,

6

Shenhe Liu3, Qiliang Sun3, Xu Wang3, Menghong Dai3*, Zonghui Yuan1,2,3*

7 8

National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO

1

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Key Laboratory for Detection of Veterinary Drug Residues, 2MOA Laboratory for

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Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong

11

Agricultural University, Wuhan, Hubei 430070, China,

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Innovation Center for Animal Nutrition and Feed Safety, Wuhan, Hubei, China.

Hubei Collaborative

3

13 14 15 16 17 18

*Corresponding author:

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Associated Prof. Dr. Menghong Dai, [email protected] Tel:

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0086-27-87287187

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Prof. Dr. Zong-hui Yuan, D. V. M., Ph. D., Tel: 0086-27-87287186, Fax:

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0086-27-87672232, [email protected]

23

†These authors have contributed equally to this work.

24

1 Environment ACS Paragon Plus

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1

Abstract

2

Cyadox is a novel derivative of quinoxaline-1,4-dioxides (QdNOs) with the

3

potential to be developed as a feed additive. However, the pharmacological and

4

toxicological bioactive molecules of cyadox and the molecular mechanism of its

5

pharmacological and toxic actions remain unclear. In the present study, cyadox and its

6

main metabolites of cy1, cy4, cy6 and cy12 were selected; the growth promotion

7

characteristic was indicated by the mRNA level of EGF; the cytotoxicity of cyadox

8

was determined by methylthiazol tetrazolium bromide (MTT) assay, lactate

9

dehydrogenase (LDH) release and Annexin V-FITC/PI apoptosis detection kit with

10

flow cytometry. The intracellular ROS, cyclin D1 and Akt/P53/FOXO1 signaling

11

pathway were also be investigated. Our data suggested that cyadox showed relatively

12

higher activity than its metabolites and the ROS was generated from N-O reduction of

13

cyadox. Moreover, cyadox (2 μM) activated the Akt, increased the EGF, cyclin D1

14

and FOXO1 expression levels. Cyadox (100 μM) induced cytotoxicity in L02 cells in

15

a concentration- and time-dependent manner. Additionally, the activated P53 pathway,

16

hyperactivated Akt and apoptosis were found in L02 cells after incubated with 100μM

17

cyadox. Our data demonstrated that Akt promoted cell survival when it was mildly

18

activated by cyadox at 2μM; Akt leads to apoptosis when it was severely activated by

19

cyadox at 100μM. Thus, the present study revealed that N-O reduction of cyadox and

20

ROS-mediated AKT/FOXO1 and AKT/P53 pathways were involved in growth

21

promotion and cytotoxicity of cyadox.



1

Keywords: Cyadox; Bioactive molecules; Metabolites; Akt; ROS

2

1. Introduction

3

Quinoxaline-1,4-dioxides (QdNOs), a group of synthetic antibacterial agents,

4

were used in animal husbandry because of their strong antimicrobial activity.1, 2 It is

5

reported that QdNOs ameliorate the intestinal microflora, promote the utilization and

6

synthesis of protein in vivo.3,

7

(CBX) and mequindox (MEQ), belong to QdNOs.1-9 However, it was reported that

8

CBX, OLA and MEQ were genotoxic carcinogens,1, 7 and because of these adverse

9

effects, CBX and OLA were prohibited in food-producing animals.9-11 Cyadox, is a

10

relatively new synthetic quinoxaline derivative with a broad antimicrobial spectrum.

11

Cyadox has strong growth-promoting activity to poultry, fish, pigs and goats 12-15 with

12

lower toxicity than other QdNOs.2,

13

cyadox had no genotoxic toxicity and carcinogenicity.2, 3, 6 Therefore, cyadox has the

14

potential to be widely used as an antibacterial agent.

15

4

Quinocetone (QCT), olaquindox (OLA), carbadox

3, 16, 17

The previous studies demonstrated that

The previous studies revealed that antibiotics mediated - pig growth was associated

16

with the intestinal microbiota,18-20 growth hormone axis and growth factors.21-22 In fact,

17

the growth hormone axis mediated - cell function played a critical role in animal

18

growth. Cyadox was reported to improve pig performance by regulating peripheral

19

metabolic hormones and growth factor, including epidermal growth factor (EGF).22 In

20

a recent study of evaluating the effect of cyadox on liver gene expression, the

21

expression level of EGF was up-regulated in a dose-dependent manner, suggesting


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1

that hepatocyte mitotic pathway was linked with growth promotion of cyadox.23

2

Additionally,

3

(PI3K)-protein kinase B (Akt) signaling pathway was associated with the expression

4

of EGF and oxidative phosphorylation in pig progenitor hepatocytes and pig kidney

5

cells (PK-15) after incubation with cyadox (data not published). However, it still

6

remains unclear the role of a PI3K-Akt signaling pathway in the pharmacological and

7

toxicological effects of cyadox.

our

early

data

confirmed

that

phosphatidylinositol-3-kinase

8

The previous studies revealed that both the antibacterial and mutagenic activity

9

depended upon the same bacterial activation mechanism of N-O reduction of

10

QdNOs.24,

11

lower antibacterial and mutagenic activity than quinoxaline-di-N-oxide (quindoxin),

12

whereas quinoxaline, a completely reduced derivative of quindoxin, showed no

13

mutagenic or antibacterial activity.24, 26, 27 The N-O reduction was also accounted for

14

genotoxicity of QdNOs in human peripheral lymphocytes28,

15

However, apparently contradictory to this result was a report showing that

16

bidesoxy-carbadox, a completely reduced derivative of CBX, had strong genotoxicity

17

in bacterial and mammalian cells.31 These reports indicated that the N-O reduction and

18

metabolites were linked with the toxicity and antibacterial activity of QdNOs. It was

19

reported that the metabolites of cyadox had no antibacterial activity.32 Additionally,

20

the toxicity of cyadox was found decreased after incubated with S9.33 Therefore, the

21

metabolites and ROS that generated from N-O reduction group were thought to

25

For example, the partially reduced quinoxaline-N-oxide exhibited a


29

and Vero cells.30


1

participate in toxicity and antibacterial activity of QdNOs.1, 6, 25, 34 However, up to now,

2

the mechanism underlying these two factors in the pharmacological and toxicological

3

effects of cyadox, and the pharmacological and toxicological bioactive molecules of

4

cyadox still remain unknown.

5

The liver is the largest exocrine gland in an animal and regulates amino acid and

6

lipid metabolism. Some genes that expressed in the liver are candidates for traits

7

related to growth, performance, body composition and fitness.23,35 The present study is

8

the first to explore the mechanisms underlying the roles of ROS and metabolites in

9

growth promotion and cytotoxicity of cyadox in human normal liver cells (L02 cell).

10

The four major metabolites of cyadox, such as cy1, cy4, cy6, cy12 were selected in

11

the present study to investigate the role of N-O reduction in the pharmacological and

12

toxicological effects of cyadox (Fig. 1).

13

indicated by the mRNA level of EGF. The cytotoxicity of cyadox was determined by

14

methylthiazol tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH)

15

release and apoptosis detection kit. The mediation of superoxide anion radicals was

16

tested by dynamic detection of intracellular ROS. Furthermore, cyclin D1 and

17

Akt/P53/FOXO1 signaling pathway were also be investigated to elucidate the

18

pharmacological and toxicological mechanisms of cyadox.

19

32,36

The growth promotion characteristic was

< Insert Fig. 1 here >

20 21

2 Materials and Methods


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1

2.1 Chemicals and reagents

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Cyadox (C12H9N5O3) and its metabolites (cy1, purity 98.5%; cy4, purity 99%; cy6,

3

purity 99%; cy12, purity 99%) were synthesized at the Institute of Veterinary

4

Pharmaceuticals (Wuhan, P.R. China); All five compounds were dissolved in dimethyl

5

sulfoxide

6

2-yl)-2,5–diphenylte-trazolium bromide (MTT) was obtained from Biotech (USA).

7

Perifosine (Akt inhibitor, S103701, purity 99.18%) was from selleck Co. (USA).

8

LY294002 (PI3K inhibitor, S110501, purity 99.72%) was from selleck Co. (USA).

9

Antibodies for FOXO1 (ab150059), p53 (ab2433), p-Akt (ab11901) and GAPDH

10

(ab9485) were purchased from sigma Inc. (USA), and cyclin D1 (2926p) was

11

purchased from Cell Signal Technology inc. (Massachusetts, USA). Secondary

12

antibodies were purchased from cell signal technology Inc.(Massachusetts, USA).

13

N-Acetyl-L-cysteine (NAC), 2,7-Dichlorodih-ydrofluorescein diacetate (DCFH-DA)

14

and N-acetylcysteine were purchased from Sigma Chemicals Co. (St Louis, Missouri,

15

USA). LDH, GSH and EGF kits were obtained from Nanjing Jiancheng

16

Bioengineering Institute (Nanjing, P.R. China). Reverse transcription reagents and

17

SYBR were from TAKARA-Bio Inc (Japan). PVDF membrane and ECL staining

18

solution were obtained from Millipore Corporation (Billerica, MA, USA).

19

AnnexinV-FITC cell apoptosis detection reagents were purchased from Beyotime

20

Institute of Biotechnology (Nanjing, China).

21

2.2 Cell Culture and Drug Treatment

(DMSO,

Amresco,

USA).


3-(4,5-dimethylthiazol-


1

The L02 cell was obtained from the Chinese Academy of Sciences. Cells were

2

grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 100 U/ml

3

penicillin-streptomycin in 5% CO2 at 37 °C. The experiments were carried out 12 h

4

after cells were seeded. The cells were exposed to a designated concentration of

5

chemicals for the indicated time. H2O2 (500μM), NAC (10mM), LY294002 (50μM)

6

and Perifosine (50μM) were selected in this study. Control cells were treated with

7

DMSO. In the apoptosis assay, H2O2 (500μM) was chosen as a positive control. All

8

studies were carried out at least three repetitions. For inhibitor assays, GH3 cells were

9

pretreated with PI3K-Akt signaling pathway inhibitors, LY294002 (50μM) and

10

Perifosine (50μM) for 4h, respectively, followed by addition of cyadox.

11

2.3 MTT, LDH and apoptosis

12

Cytotoxic effects of the cyadox and its metabolites on L02 cells were evaluated by

13

MTT and LDH leakage assay. For MTT assay, cells were cultured in 96-well cell

14

plates (1×105 cells/ml) treated with different concentration of cyadox, cy1, cy4, cy6,

15

cy12 (20, 40, 80, 160, and 200 μM ) for 12, 24 and 48 h respectively. After incubation,

16

the cells were treated with 100 mL/well with MTT solution (0.5 mg/ml) for 4 h at

17

37 °C. After incubation, the MTT formazan was qualified by a microplate reader. Cell

18

viability was expressed as a percent of the control culture value.

19

For the LDH leakage assay, L02 cells were seeded in 6-well culture plates (5×105

20

cells/well) and were cultured for 24 h, and then incubation with 20, 40, 80 μM of

21

cyadox and its metabolites at 37 °C for 12 h. The LDH release was detected by a


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commercial kit.

2

Cell apoptosis study was carried out by an Annexin V-PI apoptosis detection kit.

3

Briefly, cells from 60 % to 70 % confluent plates were washed in D-hanks and

4

suspended (1×106 cells/ml) in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl,

5

2.5 mM CaCl2). A fraction (100 μL/1×105 cells) of the cell suspension was incubated

6

with 195 μL annexin V-FITC binding buffer and 5 μL Annexin V conjugated to FITC

7

for 10 min at 25°C in the dark. after centrifugation for 5 min, 190 μL of binding

8

buffer and 10 μL propidium iodide (PI) was added. Apoptotic cells were determined

9

by the percentage of cells that stained positive for annexin V-FITC while remaining

10

impermeable to PI (AV+/PI−).

11

2.4 ROS, GSH and EGF assays

12

The intracellular ROS production was assessed by oxidation sensitive DCFH-DA

13

fluorescent probe. This study was based on the oxidation of DCFH-DA by

14

intracellular ROS, leading to the production of the fluorescent compound. ROS prober

15

dye 2,7-DCFH-DA (10 M) was added and incubated for 30 min at 37 °C. Cells were

16

treated with different concentrations of cyadox and its metabolites. After incubation,

17

the fluorescence intensity was detected by a fluorescence microplate reader. ROS

18

level was expressed as the fluorescence of the treated samples compared to the

19

fluorescence of the control samples.

20

Assay of GSH level in L02 cells was carried out by commercial kit. Human EGF

21

was assayed by using a commercial ELISA kit. Data were analyzed according to the



1

manufacturer's instructions. Protein concentration in L02 cells was detected by

2

BCA protein assay kit.

3

2.5. Quantitative Real-Time PCR (qRT-PCR)

4

Total RNA was isolated from L02 cell using Trizol Reagent (Invitrogen, USA)

5

based on the manufacturer’ s instructions. The cDNA was amplified by PCR using the

6

PCR premix(Takara, Japan) with the primers (Table 1). Complementary computer

7

software was used to analyze data. Relative quantification of gene expression was

8

measured by the method of 2-ΔΔCt.

9 10

< Insert Table. 1 here > 2.6. Western Blot

11

Total protein was isolated in a preparation of RIPA lysis buffer. The BCA Protein

12

Assay Kit was used to determined the protein concentrations. Samples with equal

13

amounts of protein (30 µg) except P53 with equal amounts of protein (100 µg) were

14

loaded for 1-dimensional SDS-PAGE using 12% separating gel and 5% stacking gel.

15

The proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF)

16

(Minipore) membranes. The membranes were blocked with 5% nonfat milk in

17

Tris-buffered saline and immunoblotted with the primary antibody (anti-FOXO1A,

18

anti-p53, anti-p-Akt and anti-cyclin D1 at a dilution of 1:1000 in 5 % BSA).

19

Thereafter, membranes incubated with the HRP-labeled secondary antibody (1:2000

20

in 1% milk) and developed with Super Signal West Pico or Super Signal West Femto

21

Substrate. Membranes were stripped by Restore Plus Western blot stripping buffer.


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1

2.7. Statistical Analysis

2

The difference was analyzed by the one-way ANOVA and q-test with SPSS 16.0

3

software. Results were presented as mean ± SD. A probability of p < 0.05 was

4

considered significant.

5

3 Results

6

3.1 Growth Promotion

7

3.1.1 Effect of Cyadox and its Metabolites on EGF expression

8

The effect of cyadox and its metabolites on mRNA expression of EGF was shown in

9

Fig. 2 (A-E). When L02 cells were treated with cyadox at concentrations of 1 and 2

10

μM for 0.5, 1, 2, 4, 8 and 12 h, the EGF expression was up-regulated at 1 μM for 1, 2,

11

4 h, and at 2 μM for 0.5, 1, 2 and 4 h, respectively (Fig. 2A). When L02 cells were

12

treated with cyadox at a concentrations of 1 and 2 μM for 1 and 2 h, the EGF

13

expression was significantly increased (p < 0.01)(Fig. 2A). Based on these findings,

14

the 2 μM and 1 h of cyadox exposure were selected for growth promotion

15

experiments. When L02 cells were treated with metabolites of cyadox at 2 μM for 0.5,

16

1, 2, 4, 8, 12 h, the EGF expression was slightly increased at 2 μM for 1 h (Fig.

17

2(B-E)). The trend of EGF expression in cyadox group was similar to that in

18

metabolites of cyadox groups, suggesting that EGF expression was up-regulated in

19

L02 cells after exposure to cyadox and its metabolites at 2 μM for 1 h. Additionally,

20

the effects of cyadox and its metabolites (2 μM, 1 h) on EGF protein expression was

21

detected by using a commercial ELISA kit. When L02 cells was treated with cyadox



1

and its metabolites (2 μM, 1 h), the EGF expression was significantly up-regulated in

2

the groups of CYA, cy4, cy6, cy12 (p < 0.05 or p < 0.01) (Fig. 2E). The EGF

3

expression level in metabolites groups was dramatically lower than that in cyadox

4

group, indicating higher growth promotion of cyadox. The ranker order of

5

pharmacological activity was cyadox > cy12 > cy4 > cy6 >cy1.

6

7 8

3.1.2 Cyadox up-regulates the expression of EGF by ROS and activating Akt

9

Fig. 3A and Fig. 3D showed the effect of cydaox (2 μM) on ROS and GSH

10

production in L02 cells. The time intervals (15, 30, 45, 60, 75, 90, 105 and 120

11

minutes) and (15, 45, 75, 105 and 120 minutes) were selected for ROS and GSH,

12

respectively. A significant increase in ROS production was observed at 15 min and

13

lasted as long as 2 h, reaching its peak level (1.2 fold) in about 45 minutes. The

14

generation of ROS is an acute and transient process, which indicated that the ROS

15

was originated in the metabolism of cyadox. The trend of GSH was similar to ROS

16

release, suggesting that the antioxidant capacity increased with ROS in L02 cells after

17

treated with cyadox at 2 μM (Fig. 3D). A significant reduction in EGF expression was

18

observed on NAC pre-treatment (Fig. 3B). The results suggested that the generation

19

of ROS from the metabolism of cyadox promotes the EGF expression.

20

L02 cells were pre-treated with the PI3K and Akt pathways inhibitors LY294002

21

and Perifosine for 4h, respectively, and then treated with 2 μM cyadox for 1 h. As


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1

shown in Fig. 3B, these specific inhibitors reversed the upregulation of EGF gene

2

expression induced by the cyadox, indicating that PI3K and Akt can regulate the

3

expression of EGF. The extent of EGF up-regulation reversal induced by Akt inhibitor

4

(Perifosine) was higher than that caused by a PI3K inhibitor (LY294002), suggesting

5

that the Akt might play a critical role in EGF expression. Furthermore, compared with

6

metabolites, the higher increased levels of p-Akt protein expression was noted in

7

cyadox (Fig. 3C). These results indicated that cyadox induced Akt activation in L02

8

cells. Additionally, a significant reduction in p-Akt protein expression was observed

9

on co-treatment with cyadox and NAC (Fig. 3C), suggesting that the Akt pathway was

10

activated by ROS. Thus, cyadox up-regulates EGF expression by the ROS and

11

activating Akt signaling.

12

13 14

3.1.3 FOXO1 and Cyclin D1 involved in the growth promotion of cyadox

15

The mRNA expressions of FOXO1 and p53 were up-regulated by about 1.55 and

16

1.20 fold, respectively, after exposure of 2 μM cyadox for 4h (Fig. 4A, 4B). To further

17

explore the molecular mechanisms, we analyzed the roles of ROS and PI3K-Akt

18

pathway in the expressions of FOXO1 and p53. After incubated with Akt inhibitor

19

(perifosine), the mRNA level of FOXO1 was significantly increased to 14.20 fold (p
cy12 > cy4 > cy6 > cy1.

16

To explore the role of ROS in the cytotoxicity to L02 cell, the intracellular ROS

17

release after exposure of cyadox, cy12, cy4, cy6 and cy1 (20, 40, 60, 80, 100, 120,

18

140, 160, 180 and 200 μM, 30 min) was detected (Fig. 5C). Compared with the

19

control group, the intracellular ROS release increased to 2.15, 1.30, 1.20, 1.10, 1.02

20

fold for cyadox, cy12, cy6, cy1 and cy4, respectively, at 20 μM after 30 min of

21

incubation (Fig. 5C). Thus, cyadox can significantly promote the release of ROS



1

compared to its metabolites. From the general trend, the ROS release from cyadox

2

group is the highest and the ROS release from the cy1 group is the lowest. This result

3

was consistent with the above observations in MTT and LDH leakage assays,

4

suggesting that the ROS release might be contributed to the cytotoxicity to L02 cell

5

induced by cyadox and its metabolites.

6

As showed in Fig. 5C, cyadox can significantly promote the release of ROS at 20

7

μM and there was no significant increase in ROS release as concentrations increased,

8

which indicated that ROS release was an acute reaction that might be originated from

9

the metabolism of cyadox. Additionally, the ROS release in the cy12 group is highest

10

among the metabolites groups (Fig. 5C). As presented in Fig. 1, cy12 is a partially

11

reduced cyadox-N-oxide, and cy1, cy4 and cy6 were the completely N-oxide reduced

12

derivative cyadox. Therefore, our results revealed that ROS was produced by the N-O

13

reduction of cyadox.

14

15 16

3.2.2 Cyadox-induced apoptosis via ROS and Akt/p53 pathway

17

The mRNA expression of FOXO1 was decreased after incubated with cyadox at

18

100 μM for 4 h, and there was no obvious change when PI3K and Akt were inhibited

19

(Fig. 6A). After co-treatment with cyadox and NAC, the expression of FOXO1 was

20

increased while the expression of p53 was decreased, which indicated that FOXO1

21

was negatively regulated by ROS, while p53 was positive regulated by ROS (Fig. 6A,


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1

6B). The mRNA expression of p53 was dramatically increased after incubated with

2

100 μM cyadox, suggesting that the p53 pathway was activated (Fig. 6B). The level of

3

P53 was significantly decreased after incubated with Akt inhibitor (Perifosine), which

4

indicated that cyadox up-regulated p53 via activating Akt pathway. Additionally, the

5

protein expressions of p-Akt and p53 were increased after exposure to cyadox at 100

6

μM for 4 h, and the protein expressions of p-Akt and p53 were reduced after

7

co-treatment with cyadox and Akt inhibitor (Perifosine) (Fig. 6C, 6D). Thus, our

8

results demonstrated that Akt and p53 signal pathways were activated by cyadox, and

9

cyadox activated p53 via activating Akt pathway.

10

11 12

To further examine whether ROS mediated cytotoxicity was implicated in the

13

activation of p53 pathway, the apoptosis in L02 cells was investigated. It was revealed

14

that cyadox (100μM, 12h) induced a significant increase in the number of apoptosis in

15

L02 cells (Fig. 7). A significant decrease in the number of apoptotic cells was

16

observed on co-treatment with cyadox and NAC (Fig. 7), indicating that cyadox

17

induced apoptosis via ROS. Therefore, these results illustrated that ROS and Akt/p53

18

signal pathway were involved in the apoptosis induced by cyadox.

19

20 21

4 Discussion



1

Page 18 of 38

EGF plays a central role in regulating cell proliferation in many cell types including

2

hepatocytes, and it has been implicated in liver regeneration.62,

3

evaluating the influence of cyadox on liver gene expression, the EGF was

4

up-regulated in a dose-dependent manner.23 In addition to that, the L02 cells express

5

CYP enzymes, which reflect the in vivo metabolism of cyadox and its metabolites.

6

Therefore, the L02 cells was selected in this study.

7

63, 64

In a study of

It was reported that bacterial activation mechanism of N-O reduction was involved N-O reduction was

8

in both the antibacterial and mutagenic activity of QdNOs.24,

9

found as a main metabolic pathway of cyadox in swine ileal flora,61 and

10

accompanying which, ROS and metabolites emerged that might participate in the

11

pharmacological action and toxicity of QdNOs.1,

12

mechanism of cyadox, it discovered that cyadox produced a lot of ROS in E. coli.37

13

Xanthine oxidase, aldehyde oxidase and cytochrome P450 were involved in

14

metabolism of cyadox to generate the ROS, which was found inextricably linked with

15

the pharmacological effects of cyadox.38 In the present study, the time for the ROS

16

release to peak was short, indicating that the ROS release was an acute reaction. Cy12

17

is a partially reduced cyadox-N-oxide, while cy1, cy4 and cy6 were the completely

18

N-oxide reduced derivatives of cyadox. It was observed that the ROS release in the

19

cy12 group was the highest among other metabolites, while lower than that in cyadox

20

group. This result suggested that the ROS was originated from the N-O reduction of

21

cyadox. Additionally, for cyadox and its metabolites, the rank order of

2, 34


25

In a study of antibacterial

Page 19 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

pharmacological activity was same to toxicological activity: cyadox > cy12 > cy4 >

2

cy6 > cy1, which was consistent with their order of ROS release. Therefore, our

3

results illustrated that ROS was produced from the N-O reduction of cyadox and

4

played a critical role in the pharmacologic action and toxicity of cyadox.

5

The previous studies found that ROS can promote cell proliferation at low

6

concentration.39-42 EGF is an essential growth factor for epithelial cell proliferation by

7

inducing intracellular ROS production.43 EGF-induced ROS generation is associated

8

with activation of Akt and mitogen-activated protein kinases (MAPK) signaling

9

pathway.43 In the present study, the EGF in cyadox group was dramatically higher

10

than that in metabolites groups, suggesting the higher growth promotion of cyadox.

11

Additionally, it was noted that noncytotoxic dose of cyadox (2 μM) resulted in mRNA

12

expression of EGF, ROS generation and the protein expression of p-Akt, while a

13

significant reduction in EGF and p-Akt expressions were observed on co-treatment

14

with cyadox and free radical scavenger-NAC. The level of ROS induced under this

15

condition is likely low (about 1.2 fold of control), however, it might be enough to

16

alter the redox homeostasis transiently and subsequently, activating transcription

17

factors and triggering redox-sensitive signaling pathways. EGF exerts biological

18

activity by combining with epidermal growth factor receptor (EGFR). Akt is a

19

downstream target of EGFR signaling. Cyclin D1, a downstream of Akt signal and

20

cell cycle-related gene, was significantly increased after treated with cyadox (2 μM).

21

Thus, our data documented that the Akt signaling was activated by a low



1

concentration of ROS that generated from the N-O reduction of cyadox, which

2

promotes the cyclin D1 expression and cell proliferation, and thereby exerting growth

3

promotion function.

4

Redox signaling plays an important role in the lives of cells. The protein tyrosine

5

phosphatase activity was inhibited by ROS and regulated by reversible

6

reduction/oxidation of cysteine.44 ROS can activate protein kinases and inhibit protein

7

phosphatases to increase the phosphorylation level of key proteins includes Akt,

8

MAPK, p38, protein kinase C (PKC), extracellular signal-regulating kinases 1/2

9

(ERK) and c-Jun amino-terminal kinase (JNK).45 Akt is a core member of major

10

intracellular signaling cascades that regulated many metabolic events.46 In this study,

11

the Akt signaling was activated by ROS and the p-Akt level was increased after

12

incubated with cyadox. Akt was mildly activated by 2 μM cyadox, while severely

13

activated by 100 μM cyadox. Significant reduction in the protein expression of p-Akt

14

was observed on co-treatment with cyadox and NAC, suggesting that the

15

phosphorylation of Akt was inhibited by ROS. However, it is unclear whether it is a

16

direct interaction of ROS with the kinases of Akt or whether its activation is a

17

consequence of inhibition of phosphatases of Akt. The cell proliferation and apoptosis

18

were noted in 2 μM and 100 μM cyadox groups, respectively. Thus, it was suspected

19

that Akt promoted cell proliferation when it was mildly activated, while triggered

20

apoptosis after severely activated by ROS that originated from the metabolism of

21

cyadox.


Page 20 of 38

Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

FOXO1 was involved in cellular functions, including apoptosis and ROS

2

scavenging.47-50 Activity and nuclear localization of FOXO1 were regulated by

3

phosphorylation of its certain residues. These certain residues can be phosphorylated

4

by Akt, leading to translocating of FOXO1 from the nucleus to the cytoplasm and

5

deactivation of FOXO1.51, 52 The activity of FOXO1 was also regulated by ROS. It

6

was reported that oxidative stress caused nuclear translocation of FOXO1 in

7

cardiomyocytes.35,

8

expression of antioxidant genes like SOD and GADD45 to promote ROS scavenging

9

and prevent DNA damage.48,

53

In response to mild oxidative stress, FOXO1 increased the

54

FOXO1 is also linked with apoptosis and atrophy

10

during the irreversible stress response.48,

11

expression of FOXO1 was observed on co-treatment with cyadox and NAC, while the

12

expression of FOXO1 was significantly increased when the Akt was inhibited in 2μM

13

cyadox treated cells. Therefore, it appeared that Akt might be involved in promoting

14

cell survival in L02 cells through a FOXO1-dependent mechanism. This indicates a

15

strong focus on anti-apoptosis, pro-survival actions by Akt/FOXO1 pathway in L02

16

cells after incubation with 2μM cyadox.

54

In the present study, the decreased

17

Regulation of ROS levels by p53 represents opposite effects at its steady-state and

18

activated-state level.56, 57 Transient cellular stress activated the p53 pathway to initiate

19

cell repair mechanism.58 However, when cells are subjected to continuous stimulation

20

and failure to repair, the p53 will be overactivated, resulting in a permanent inhibition

21

of cell proliferation.57, 59 P53, a tumor suppressor gene, plays an important role in the



1

apoptosis. BCL-2 family was known as group of proteins that involved in cell death

2

and intrinsic apoptosis, and some of them were downstream targets of p53.60 In this

3

study, p53 was observed mildly up-regulated by 2 μM cyadox, while significantly

4

up-regulated by 100 μM cyadox. P53 was significantly down-regulated when Akt was

5

inhibited, which indicated that p53 pathway was activated and was regulated by Akt

6

under the stressed condition. Additionally, the apoptosis was noted in 100 μM cyadox

7

group and the reduced cell apoptosis was observed on co-treatment with cyadox and

8

NAC, suggesting that the excessive ROS lead to the cell apoptosis. Our results

9

illustrated that the apoptosis was induced by 100 μM cyadox in L02 cells via ROS

10

and Akt/p53 pathway (Fig. 8).

11

In conclusion, the current study provided clear evidence that the ROS was

12

generated from N-O reduction of cyadox and the N-O group is responsible for its

13

pharmacological action and toxicity (Fig. 8). The ROS level produced by the N-O

14

reduction of cyadox determines the state of the cells and the expression of related

15

genes. At low concentration of cyadox, the metabolism of cyadox produces a small

16

amount of ROS, which activated Akt slightly, and thereby promotes the secretion of

17

cyclin D1 and expression of EGF. Simultaneously, a small amount of ROS

18

up-regulated the expression of FOXO1 to scavenge the ROS in L02 cells. At high

19

dose of cyadox, the metabolism of cyadox produced a large amount of ROS,

20

activating P53 signaling pathway and causing Akt to be excessively activated. The

21

apoptosis was induced by cyadox in L02 cells via ROS and p53 activation. Therefore,


Page 22 of 38

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1

N-O reduction of cyadox and ROS-mediated AKT/FOXO1 and AKT/P53 pathways

2

were involved in growth promotion and cytotoxicity of cyadox.

3

4 5 6

Conflict of interest statement The authors declare that there are no conflicts of interest.

7 8

Acknowledgements

9

This work was supported by the National Key Research and Development Program

10

of China (2017YFD0501401) and the National Natural Science Foundation of China

11

(grant no.31772797).

12 13

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Table 1 PCR Primers Used in the Gene Expression Analysis

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Gene name

Description

Primer sequence (5’ - 3’)

GAPDH

mGAPDH-F

ACGGATTTGGTCGTCGTATTG

mGAPDH-R

GGAAGATGGTGATGGGATT

mEGF-F

GATTGCTTTCCTGGGTATG

mEGF-R

CCATAGTCTGTTCCGTCAA

mCyclin D1-F

GTCGCTGGAGACCGTGAAA

mCyclin D1-R

CGGATGGAGTTGTCAGTGTAG

mFOXO1-F

AATAGCGTGCCCTACTTC

mFOXO1-R

TCGGCTTCGGCTCTTAG

mP53-F

CCACCATCCACTACAACTACAT

mP53-R

AAACACGCACCTCAAAGC

EGF

Cyclin D1

FOXO1

P53

12 13 14


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1

Figure Legends

2

3 4

Figure 1. The structures of cyadox and its metabolites.

5

6 7

Figure 2. The effects of cyadox and its metabolites on EGF mRNA expression (A-E)



1

in L02 cells. The effects of cyadox and its metabolites (2 μM, 1h) on EGF

2

protein expression (F) in L02 cells. The results were presented as Mean ±

3

SD. (n=6). Significant differences were indicated by * p < 0.05, ** p

P53

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