Antioxidant Stress and Anticancer of Peptide Chelate Selenium Element in vitro

This contribution reports a facile synthesis of anticancer bioactive peptides (ACBP) - functionalized selenium particles (ACBP-S-Se) with enhanced anticancer activities and a detailed mechanistic evaluation of its regulation of oxidative stress in vitro. Structural and chemical characterizations were proved by ultraviolet absorption spectrum (UV), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), Nuclear magnetic resonance carbon (13C NMR) and hydrogen spectra (H NMR), scanning electron microscope (SEM), energy dispersive X-ray spectrom (EDX) and inductively coupled plasma mass spectrometry (ICP-MS). The results show that ACBP are effectively sulfhydrylation modication with S-acetylmercaptosuccinic anhydride (SAMSA) via chemical absorption. After the polypeptide was modied by sulfhydrylation, there were many sulfhydryl groups on the molecule, and the sulfhydryl group was used as the binding site of Se. A panel of selected human cancer cell lines demonstrated high susceptibility toward ACBP-S-Se and displayed signicantly reduced proliferative abilities. Finally, the results presented herein suggest bioactive peptide chelate selenium element effectively inhibited the proliferation of MKN-45 and MKN-74 cell in vitro, which in turns allowed the successful application of the ACBP-S-Se in highly complex human cell lines. The related the regulation of oxidative stress gene is CDKN1A, CCNB1, TXN and MAP3K5, while CDKN1A and TXN have ability to protecting cells to reduce oxidative stress and promoting cell growth arrest. Therefore, the great potential exhibited by ACBP-S-Se could make them an ideal candidate as a chemotherapeutic agent for human cancers, especially for gastric cancer.


Introduction
Oxidative stress refers to the aberrant production of active substances including reactive oxygen species (ROS) and nitrogen free radicals under various stress stimuli, which leads to unbalanced physiological status or oxidative stress-related damage via oxidation-reduction reactions 1 . Rigorous studies have revealed that reactive oxygen species (ROS) generated by cells under oxidative stress (OS) are involved in gastric tumorigenesis 2 .
Previous studies have con rmed that ROS is an actuation factor in gastric carcinogenesis both in clinical studies and in mouse models in vivo. The dysregulation of ROS promotes tumorigenesis and the development of abnormalities due to its ability to increase aberrant cell proliferation, survival, and cell migration in vivo 3 . Meanwhile, the aberrant production of ROS also induces DNA damage, leading to genetic disease, tumorigenicity and subsequent tumor progression 4 . If the accumulation of ROS overwhelms the death threshold, cellular apoptosis levels signi cantly increase, which enhance the antitumor effects of ROS production 5 . Denicola et al. con rmed that ROS alleviate certain speci c cancers and that the redox state in cells is an important factor for the subsequent tumor formation potential, so the redox states of cells are likely to be potential therapeutic targets of cancers 6 . Finally, reactive oxygen species (ROS) may contribute to the regulation of apoptosis. Selenium (Se), with a narrow range between de ciency and toxic effects 7 , is an important oligoelement and one of the most important trace elements in organisms 8 . Meanwhile, Se is a key component of the antioxidant system 9 , which makes an indelible contribution to human health. The chemical forms of Se regulate its diverse biological effects, toxicity and bioavailability, and supplementation with Se improves the redox system, promotes proper immune system function and has anticarcinogenic effects 10 . Se de ciency leads to heart disease, hypothyroidism and a weakened immune system, but excess exposure to Se results in gastrointestinal disturbances, hair and nail changes and neurologic manifestations 11,12 .
The presence of Se enhances the functions of the immune system, improves the proliferation and biofunctions of immune system cells, and enhances the phagocytosis of immune system cells, thereby improving the body's immune function and fundamentally improving its ability to resist disease 13,14 . In addition, at higher concentrations, Se has pro-oxidant properties and is essential for the antioxidant activities involved in anticarcinogenic actions 15 . Meanwhile, Se inhibits hepatocyte necrosis and DNA damage by inhibiting CTX-induced oxidative stress 16 .
Previous studies have con rmed that Se is a promising therapy for cancer 17,18 via the formation of an internal environment that inhibits cancer proliferation. After cancer operation, Se supplementation obviously controls cancer deterioration, reduces the toxicity and side effects of chemotherapy and radiotherapy, and helps anticancer drugs improve the curative effect 19,20 .
Furthermore, Se, as an essential component of selenocysteine-containing proteins, is involved in most aspects of cell biochemistry and functions 21,22 . Younis et al. reported that supplementation with insulin transferrin selenium (ITS) to semen extenders bene ts the cryopreservation of chimpanzee (Pan troglodytes) spermatozoa 23 .
Selenoproteins are essential for life, and several selenoproteins have been characterized as antioxidant enzymes, protecting against damage caused by free radicals, as already stated 24 . Further studies on selenoprotein gene expression and cytokine content in the chicken thymus have shown that a signi cant decrease in the expression levels of selenoproteins could result in oxidative stress in chicken thymus 25 . In addition, Se-containing molecules exert antioxidant properties associated with tumor growth, metastasis, angiogenesis, and drug resistance 26 . One of the more exciting areas of research in drug design is the synthesis of so-called polypeptide selenium chelate, with the added advantage of cancer stem cell line inhibition and antioxidant activity 27 . Here, the anticancer bioactive peptide (ACBP) is a low-molecularweight active substance that was extracted from goat liver and was produced by the Clinical Medicine Research Center, the A liated Hospital at Inner Mongolia Medical University 28 . Our former studies found that ACBP has superior antineoplastic activities and inhibits tumor growth in nude mice with Dutch gallbladder carcinoma. It also increases the chemotherapeutic sensitizing effect and reduces the side effects associated with chemotherapy 29 . A series of nonnatural selenium-containing amino acid derivatives and peptides have been synthesized in a exible and modular strategy to establish an antigastric cancer system for treating diseases 30 . On the other hand, selenocarbohydrates, selenoamino acids, and selenopeptides take part in the synthesis of a large number of biological compounds. However, the related oxidative stress genes and potential antitumor e cacy of the as-synthesized ACBP chelate selenium have not been previously demonstrated. Furthermore, the molecular mechanism of the antioxidant regulatory system of the peptide chelate selenium is very important to reveal the regularity of tumorigenesis and drug resistance.
Here, sulfhydrylation modi cations of ACBP were performed with S-acetylmercaptosuccinic anhydride (S-AMSA) via chemical absorption. After sulfhydrylation, there were many sulfhydryl groups on the molecule, and the binding site of Se was used to link the sulfhydryl group. The molecular mechanism of the antioxidant regulatory system and antitumor effect of ACBP-S-Se on tumor cell lines were investigated in vitro. MKN-45 cells were cultured in RPMI1640 growth medium (61870036, Invitrogen, Thermo Fisher Scienti c, Shanghai, China) supplemented with 10% FBS and 100 U/mL P/S at 37°C in a humidi ed chamber containing 5% CO 2 .

Synthesis of ACBP-S-Se
MOPS (2.08 g) was dissolved in ddH 2 O (200 mL) to prepare a 3-morpholine-propanesulfonic acid (MOPS)-buffered solution. ACBP (1.0 g) was dissolved in a MOPS solution with a pH of 7.4 to obtain the ACBP solution. S-AMSA was added to the ACBP solution and incubated for 2 h, during which the pH was maintained at 7.4. Next, the sample was stored in nitrogen to avoid air oxidation products. Then, 0.05 mol/L hydroxylamine hydrochloride was added for further reaction for 3 h. After the isolation of the sulfhydrylated ACBP, puri ed ACBP-S was obtained by gel chromatography. Here, 0.66 g sodium selenite was added to the ACBP-S solutions for 6 h to obtain the raw product of ACBP-S-Se, and then gel chromatographic column puri cation was performed again to produce a pure ACBP-S-Se solution.
After vacuum freeze drying the ACBP-S-Se solution, the ACBP-S-Se particles were obtained for chemical structure characterization.

FT-IR spectroscopic analysis
FT-IR spectra were recorded using a Nicolet FT-IR 5700 spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA) under ambient conditions. The characterization of the four components (ACBP and ACBP-S-Se) was performed, and the samples were triturated with KBr at a ratio of 1:100 and pressed to form pelleted samples for FT-IR spectroscopic analysis at 500-4000 cm -1 .

X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron spectroscopy (XPS; Thermo Fisher, ESCALAB 250Xi, USA) was used to determine the elemental composition of ACBP and ACBP-S-Se, for which full (pass energy, 100 eV) and high-resolution spectra (pass energy, 20 eV) were recorded.

Particle morphology
The particle morphology of the ACBP-S-Se particles was examined by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). ACBP-S-Se was carefully lyophilized to maintain the surface structure. ACBP-S-Se was mounted, sputter-coated with gold, and observed under a Hitachi S-570 SEM microscope (Hitachi, Tokyo, Japan).

Carbon nuclear magnetic resonance (13C NMR) and hydrogen nuclear magnetic resonance (H NMR) spectra
H-NMR and 13C-NMR spectra were recorded in deuteroxide (D 2 O) at 17°C using a Bruker DRX 500 spectrometer. For each spectrum, approximately 1500 transients were collected using the following acquisition parameters: 3 s delays between pulses, 90° pulse of 5.4 µs, and contact time of 1 ms. Data were acquired and processed using Topspin 2.1 (Bruker, Rheinstetten, Germany).

SDS-Polyacrylamide gel electrophoresis
The molecular weights of ACBP, ACBP-S and ACBP-S-Se were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 16% separating gel and a 8% stacking gel according to previous studies 31 . The ACBP, ACBP-S and ACBP-S-Se powders were mixed in 0.1 M Tris-HCl buffer (pH = 6.8) containing 5% β-mercaptoethanol, 2% SDS, 0.02% bromophenol blue and 10% glycerin, with a sample concentration of 2 mg/mL. The mixed solutions were heated at boiling temperature for 5 min, while 15 µL of the prepared samples was injected into each well and then ran to the bottom of the gel at 100 V. The gel was stained using the Coomassie Blue (R-250) staining method, and the molecular weight was determined by comparison with the MW standard markers (BIO-RAD) (MW 10.0~180.0 kDa). Images were analyzed by Image Lab software version 5.0 (2000 Alfred Nobel Drive, Bio-Rad Laboratories Inc., WDC, USA).

ICP-MS
The selenium content of ACBP-S-Se was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies 7900ICP, Beijing Tai Chi Flying Co., Ltd.). After ignition, the tested elements and internal standard (10 bbp TuneA) were set up under standard model. The parameters were set up as follows: H 2 /He was used as a collision gas with a ow velocity of 5.5 mL/min, the Co sensitivity was adjusted to 20000 CPS/ppb, the U signal maximum was adjusted, and then the Se in ACBP-S-Se was measured. The amount of selenium bound to ACBP was determined by the following formula:

Drug treatments
Before drug treatments, three cell lines were preplated in 6-well culture dishes (Corning, China) at a density of 5×10 5 cells/well and randomly divided into the following groups based on the ACBP-S-Se supplement to the basic culture medium. When the cell con uence reached 70%, drug treatment was carried out. The optimization of the ACBP-S-Se concentration applied in this study was performed according to our former studies of BAPT 32 .

Cell proliferation
The proliferation ability of MKN-45 cells or MKN-74 cells after ACBP-S-Se treatments was determined by the IncuCyte Live Cell Analysis System according to our previous studies. Brie y, MKN-45 and MKN-74 cells were inoculated into a 96-well plate at a density of 5×10 3 /well and cultured for 24 h. After ACBP-S-Se treatment, the 96-well plates were placed in the designated location of the IncuCyte live cell workstation, and cell proliferation was recorded for 24 h at intervals of 2 h. Here, for MKN-45 and MKN-74 cells, the control group was not treated with any additional ACBP-S-Se (0 mg/mL), while the experimental group was treated with different concentrations of ACBP-S-Se (5 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL and 80 mg/mL). After the assay, the cell fusion degree was counted, and the cell proliferation rate was calculated.

Cell migration
The cell migration of MKN-45 cells and MKN-74 cells after ACBP-S-Se treatments was determined by the IncuCyte live cell workstation according to our previous studies. Brie y, MKN-45 and MKN-74 cells were inoculated into a 96-well plate at a density of 5×10 4 /well. After scratching, the cells were treated with ACBP-S-Se. Here, for MKN-45 and MKN-74 cell the control group was not treated with any additional ACBP-S-Se (0 mg/mL), while the experimental group was treated with different concentrations of ACBP-S-Se (1 mg/mL and 5 mg/mL). The 96-well plate with drug treatment after the scratch was placed in the IncuCyte live cell workstation, and cell proliferation was recorded for 36 h at intervals of 2 h. After the assay, the original data were analyzed according to the scratch wound healing protocol.

Functional enrichment of oxidative stress-related genes
The screening and enrichment analysis of oxidative stress-related genes, network diagram of functional enrichment and association of genes with oxidative stress were evaluated by biological information analysis.

Reverse transcription PCR and Real-time PCR Analyses
After drug treatments for 24 h, the total RNA was extracted with TRIzol reagent (79306, Gibco, Shanghai, China), and then cDNA was synthesized according to the manufacturer's instructions (Prime ScriptTM RT reagent kit with gDNA Eraser, RR047A, Takara, Dalian, China). For PCR ampli cation, speci c primers were designed on the NCBI website and commercially synthesized by Invitrogen (Shanghai, China). Realtime PCR was performed within a Thermo Pikoreal machine with commercial kits (RR820A, Premix Taq™, Takara, Dalian, China).
The PCRs started with an initial melting cycle for 5 min at 95°C, followed by 35 cycles of melting at 95°C for 30 s, annealing at 58°C for 30 s and expansion at 72°C for 30 s. The ubiquitously expressed β-actin gene was used as an internal control. The qualities of the PCRs were con rmed by melting curve analyses, and all experiments were performed in triplicate. The relative mRNA expression was calculated using the 2 -ΔΔCt method 33 . The primers for reverse transcription PCR and real-time PCR analyses are shown in Table 1.

Determination of the chemical structure of ACBP-S-Se by 13C NMR and H NMR
In the H NMR spectra, the absorption peaks of -OH, -NH and -SH can be found in the attachment of δ2.31, δ6 ~ 8.2 and δ1.43, respectively (Fig. 1D). At the same time, there are different absorption peaks and different chemical shifts. In the 13C NMR spectra, in the attachment of δ180, there are absorption peaks of CHO and -C-S at δ100 and absorption peaks of different degrees and different chemical shifts at the same time. The results showed that there were different amounts of binding between bioactive polypeptides and selenium in different proportions, so the molecular structure of ACBP-S-Se was inferred.

Determination of the chemical structure of ACBP-S-Se by XPS
As observed in Figure. Table 2). As seen from the XPS spectra of nitrogen, the binding energy decreased from 399.65 eV in ACBP to 399.02 eV in ACBP-S-Se. However, as seen from the XPS spectra of sulfur, the binding energy increased from 166.8 eV in ACBP to 167.57 eV in ACBP-S-Se.
Additionally, the binding energy of Se formed at 56.2 eV in ACBP-S-Se, the new -SH = Se bond in ACBP-S-Se was formed, the electronegativity of Se 3d increased, and the binding energy also increased.

SEM-EDX observation and ICP-MS of ACBP-S-Se
As shown in Fig. 3A, the morphology of ACBP is a coarse brous structure. From the morphology of the ACBP-S-Se in Fig. 3C, the particle structure has an average uneven grain of 5 µm in diameter. Compared with ACBP, in ACBP-S-Se, the distribution of the elements C, N and O decreased, while the distribution of elements Se and S increased.
As indicated in Fig. 3D, the selenium content was up to 0.28%. The selenium content was 829.030 [He]/PPb when the ratio of ACBP and selenium was 2:1 ( Table 3). The content of selenium in the ACBP molecule was 2.1 per mole.

The inhibitory effect of ACBP-S-Se on tumor cell lines
As shown in Fig. 4 (A and B), the growth of MKN-45 and MKN-74 cells was inhibited after treatment with ACBP-S-Se, and the rates of apoptotic cells were increased in a dose-dependent manner. The inhibition of MKN-45 and MKN-74 cell proliferation was not obvious with increasing concentrations of ACBP-S-Se from 5 mg/mL to 80 mg/mL.
The wound healing ability of MKN-45 and MKN-74 cells was detected in the control group and after treatment with different concentrations of ACBP-S-Se and was increased with increasing ACBP-S-Se concentrations, especially after treatment with 1 mg/mL ACBP-S-Se ( Figure C and D). The results indicated that from 1 mg/mL to 5 mg/mL ACBP-S-Se has the ability to inhibit cell migration.

Functional Gene Selection Principles
The results of functional gene analyses showed 911 upregulated genes and 1122 downregulated genes after ACBP-S-Se treatment in MKN-45 cells. The screening results and enrichment analysis of oxidative stress-related genes are shown in Table 4. Table 4 Screening and enrichment analysis of oxidative stress-related genes. From the functional enrichment results of oxidative stress-related genes, a total of 75 genes with signi cant differences were selected (Fig. 5A). From the gene interaction mapping, CDKN1A, CCNB1, TXN and MAP3K5 were extracted due to the role of most genes associated with them and the signi cantly enriched pathways, which can be used as downstream molecules (Fig. 5B).
In Fig. 6A, according to the interaction of genes related to oxidative stress and lncRNA, networks of oxidative stress genes and lncRNA interactions (lncRNA FPKM > 5) were constructed via lncRNA trans and cis analysis (TRANS_ENERGY<-50). The results showed that the network contained 55 genes related to oxidative stress and 37 lncRNAs.
The selected lncRNAs included MSTRG.13268.2, ENST00000508832, ENST00000580180, ENST00000454068, ENST00000607476, ENST00000504539, ENST00000326677, ENST00000448718, ENST00000531126 and MSTRG.5031.1. As shown in Fig. 6B-E and Table 5, the expression of oxidative stress-related genes (the 4 selected genes) in TCGA and the expression of CDKN1A increased after treatment with ACBP-S-Se, but the expression of this gene was lower in tumor samples than in paracancerous samples. The expression of the other three genes decreased after treatment with ACBP-S-Se but was lower in tumor samples than in paracancerous samples.

Discussion
Gastric cancer (GC) is one of the major threats to human health worldwide. Previous studies have demonstrated that ACBP combined with oxaliplatin (OXA) signi cantly inhibited the growth of MKN-45 cells. Recently, the replacement of peptides with chemical modi cations to achieve the same therapeutic effects as natural peptide counterpart in combined chemotherapeutics has been rapidly developed. Furthermore, the incorporation of the Se atom in amino acids and peptides is mostly restricted to selenocysteine derivatives, and there is still a need for further developments toward the synthesis of amino acid-derived chiral selenium compounds, including potential candidates for biological evaluations.
Here, ACBP was modi ed by sulfhydrylation to combined Se to form the ACBP selenium chelate, and its properties and inhibitory effect on GC cells in vitro was investigated. The polypeptide has high glutathione peroxidase (GPX) activity and can clear the free radicals in human body 37 . Although several functions of selenoproteins are unknown, many disorders are related to alterations in selenoprotein expression or activity. Selenium insu ciency and polymorphisms or mutations in the genes encoding selenoproteins and synthesis cofactors are involved in the pathophysiology of many diseases, including immune dysfunctions and cancer 24 . Considering the perspective of oxidative stress to explore the ceRNA mechanism of ACBP-S-Se in the treatment of GC, it is necessary to determine the changes in the expression of oxidative stress-related genes after ACBP-S-Se treatment. By carefully studying oxidative stress-related pathways and genes, functional genes were detected.
From all these genes, the ones that were consistently regulated were the selenoprotein genes.
Furthermore, the Se-speci c effects noted were caused primarily by Se de ciency and not high Se 38 . A total of 75 genes with signi cant differences were selected. From the gene interaction mapping, CDKN1A, CCNB1, TXN and MAP3K5 were selected. The cyclin-dependent kinase inhibitor 1 A (CDKN1A, also known as p21), a cell-cycle-dependent kinase suppressor molecule, has been shown to protect cells from oxidative stress damages 39 . Cyclin B1 (CCNB1), also inhibits certain speci c factors in ubiquitinproteasome 40 . For example, hydrogen peroxide prevents ubiquitin (Ub) from binding to late-promoting complex (APC/C) substrates, which also prevents the degradation of CCNB1 and thus promotes cell growth arrest caused by oxidative stress. Increased CCNB1 expression has been observed in several cancers; for example, the expression of CCNB1 in gastric cancer tissues was higher than that in normal gastric tissues 41 . However, ACBP-S-Se treatment decreased the expression of CCNB1 in MKN-45 and MKN-74 cells. The thioredoxin protein gene (TXN) plays an important role in cellular antioxidant defense 42,43 . Mitogen-activated protein kinase kinase kinase 5 (MAP3K5), also known as apoptotic signal-regulated kinase 1, regulates the biological and physiological processes of apoptosis, immunity and gastric emptying by regulating the ow of apoptotic kinases 44 . Furthermore, MAP3K5, activated in response to stress signals, plays an important role in oxidative stress regulation, cell proliferation, differentiation and death, and immune response 45 . It has been shown that a molecular target of reactive oxygen species (ROS), thioredoxin (TXN/Trx), is an inhibitor of MAP3K5 46 . Moreover, oxidation via ROS disrupts the binding of Trx to MAP3K5, resulting in apoptosis 47 . The results show that the formation of ACBP-S-Se negatively regulates MAP3K5 activity, thus attenuating the proapoptotic signal in MKN-74 cells. Inhibition of apoptosis via this mechanism would result in increased proliferation and survival, increasing the probability of tumorigenesis and/or metastases. Therefore, oxidative stress is closely related to the occurrence and development of cancer. Oxidative stress results in cell apoptosis/death, stressors alter the miRNA expression pro le, and miRNAs play a role in the cell response to stress.

Conclusion
In summary, the application of sulfhydrylation and deacetylation is an effective method to enhance the chelating e ciency of ACBP and Se. According to the UV, FT-IR and 13C NMR, H NMR, XPS, EDX, and ICP-MS analyses, Se was chelated into ACBP via sulfhydrylation; the sulfhydryl group was used as the binding site of Se and increased the chelating rate. Finally, the results presented herein suggest ACBP-S-Se effectively inhibited the MKN-45 and MKN-74 cell growth and migration in vitro, which in turns suggests that ACBP-S-Se could be applied in the clinic.

Declarations
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