Peroxiredoxin I deficiency increases keratinocyte apoptosis in a skin tumor model via the ROS-p38 MAPK pathway

Ying-Hao Han a, **, 1, Yong-Qing Zhang a, 1, Mei-Hua Jin a, 1, Ying-Hua Jin b, Mei-Yu Qiu a, Wei-Long Li a, Chao He a, Li-Yun Yu a, Jin Won Hyun c, Jiyon Lee d, e, Do-Young Yoon d, Hu-Nan Sun a, ***, Taeho Kwon e, *

Abstract

MAPK signaling Keratinocyte hyperproliferation is an essential link in skin cancer pathogenesis. Peroxiredoxin I (Prx I) is known to regulate cancer cell proliferation, differentiation, and apoptosis, but its role in skin cancer remains unclear. This study aimed to elucidate the role and mechanism of Prx I in skin cancer pathogenesis. Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were used to create a skin tumor model of the initiation/promotion stage of cancer. The role of Prx I in H2O2induced keratinocyte apoptosis was also investigated. After DMBA/TPA treatment, Prx I deficiency was significantly associated with less skin tumors, lower Bcl-2 expression, and higher p-p38 and cleaved caspase-3 expressions in Prx I knockout tumors than in wild-type controls. H2O2 stimulation caused more cellular apoptosis in Prx I knockdown HaCaT cells than in normal HaCaT cells. The signaling study revealed that Bcl-2, p-p38, and cleaved caspase-3 expressions were consistent with the results in the tumors. In conclusion, the deletion of Prx I triggered the DMBA/TPA-induced skin tumor formation in vivo and in vitro by regulating the reactive oxygen species (ROS)-p38 mitogen-activated protein kinase (MAPK) pathway. These findings provide a theoretical basis for treating skin cancer.

Keywords:
Peroxiredoxin I
DMBA/TPA
Apoptosis
ROS-p38

1. Introduction

Skin cancer is one of the major causes of cancer-related deaths [1]. Over the years, the incidence of squamous cell carcinoma (SCC) has increased from 3% to 10% annually, accounting for one-third of new annual cancer cases in North America [2]. About 16% of SCCs develop into metastatic diseases [3]. Despite the recent progress in SCC diagnosis and treatment, the survival of SCC patients has not been significantly improved due to the lack of biomarkers for early diagnosis and targeted treatment [4].
Multistage mouse skin tumor models using dimethylbenz[a] anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) are widely used models for studying SCCs [5e8]. DMBA can bind to aryl hydrocarbon receptors (AHRs) on the cell’s surface [9e11]. When this happens, the AHR breaks away from the complex, travels into the nucleus, activates xenobiotic response elements, and causes the expression of metabolism-related genes [12]. The intermediate metabolites of DMBA are transported out of the cells and act on epidermal stem cells in the basal epidermis [13], causing gene mutation in epidermal stem cells [11,14]. The target gene of the main mutation at the initial stage is the HRAS I gene, which is also the main target of DMBA metabolites [15]. DMBA metabolites mainly induce A-T mutation at the 61st position of the HRASI locus [14,16]. In the promotion phase, the most commonly used chemical is TPA [17]dan activator of protein kinase C and a tumor promoter [18,19]. TPA can induce tissue to produce a large number of reactive oxygen species (ROS) and recruit inflammatory cells [20,21]. As a result, mutated cells develop into prominent sarcomas known as papillomas [22].
Peroxiredoxin 1 (Prx I), a member of the peroxidase reductase family, protect cells by scavenging ROS and acting as molecular chaperones, thereby reducing oxidative stress on cells [23,24]. Prx I can regulate cell proliferation, differentiation, and apoptosis and play an important role in cancer development [25e28]. Prx I has various roles in different tissues and diverse mechanisms of action [29e31]. However, the role and mechanism of Prx I in skin tumors remain unclear. This study aimed to investigate how Prx I affects skin cancer pathogenesis in wild-type (WT) vs. Prx I gene knockout mice treated with DMBA/TPA.

2. Materials and methods

2.1. Animal acquisition and treatment

Prx I gene knockout mice were generated by the same method described previously [32]. The sequence from exon 1 to exon 6 of a Prx I gene was replaced with the neo gene and the homologous recombination was confirmed by polymerase chain reaction (PCR) [32]. WT and Prx I gene knockout (Prx I/) 129/SVJ mice were kindly provided by the Dr. Dae-Yeul Yu ([email protected], Korea Research Institute of Bioscience and Biotechnology, Republic of Korea), mice were maintained in a pathogen-free facility, where the temperature at 20e22C, the humidity 50e60% and the 12-h-dark/ light cycles were maintained. Eight-week-old WT and Prx I gene knockout (Prx I/) 129/SVJ mice were divided into control and DMBA/TPA treatment groups. The back skin of mice was carefully shaved 2 days before treatment. During treatment, 0.2 mL 0.5 mM DMBA was applied to the back skin 3 times a week. After 1 week, 0.2 mL 0.05 mM TPA was applied to the same area 3 times weekly for 20 weeks. Tumor diameter was measured using 25 mm highprecision calipers.

2.2. Cell culture

HaCaT cells were obtained from Bogoo Biological Technology (Shanghai, China) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Solarbio, Beijing, China), 2 mM L-glutamine, and 100 U/mL penicillin and streptomycin (Solarbio), at 37 C in a 5% CO2 humidified incubator (Sanyo, Osaka, Japan). Cells were cultured in 100 mm cell culture dishes (NEST Biotechnology, Wuxi, China).

2.3. Establishing the Prx I knockdown stable cell line

Prior to viral infection, HaCaT cells were seeded in 6-well plates (NEST Biotechnology) at 3 105 cells/well and grown to 70e80% confluence. To construct the lentiviral Prx I shRNA packaging, scrambled control small hairpin RNAs (shRNAs) (negative control sequence: 50-TTCTCCGAACGTGTCACGTTTC-30) and shRNAs (sequence: 50-GGAGGACTGGGACCCATGA-3) specifically targeting Prx I were synthesized and inserted into the pGLV3/H1/GFP þ Puro lentiviral vector (GenePharma, Shanghai, China). To produce lentiviral particles, 0.5 mg/mL expression plasmids (Prx I- pGLV3/H1/ GFP þ Puro and negative control plasmid) and 1.5 mg/mL packaging vectors (pGag/Pol, pRev, and pVSV-G) (GenePharma) were cotransfected into 293T cells (30e40% confluence) using the RNAiMate transfection kit (GenePharma) according to manufacturer instructions. Seventy-two hours after transfection, the supernatant containing lentiviral particles was collected and centrifuged at 1500 g for 4 min at 4 C and filtered with 0.45 mm cellulose acetate filters (Sartorius, Goettingen, Germany) to eliminate cell debris. The lentiviral particles were ultracentrifuged at 48,400 g for 2 h at 4 C. Thereafter, viral stock containing shPrx I at a multiplicity of infection of 150 and 5 mg/mL polybrene (GenePharma) were added to the HaCaT cells. After 24 h, cells were inoculated in fresh culture media. Transduced HaCaT cells were incubated in 10% FBS DMEM containing 2 mg/mL puromycin (Solarbio) for selection. After two selections, transduced HaCaT cells were incubated in 10% FBS DMEM without puromycin. The population of HaCaT cells transduced was confirmed via Western blot.

2.4. Hematoxylin and eosin staining

The dorsal skin was excised and fixed in formalin (SigmaAldrich, St. Louis, MO) overnight. The skin was washed with running water for 90 min and dehydrated in a tissue dehydrator (Dimensional Biotechnology Co., Ltd., Jiangsu, China). The skin was then embedded in paraffin (Leica, Wetzlar, Germany); 5 mm sections were stained with hematoxylin and eosin (Beyotime Biotech, Jiangsu, China).

2.5. Western blot analysis

To isolate protein from mouse skin, the dorsal skin was excised. After fat removal, remaining skin tissues were immediately placed in liquid nitrogen and pulverized in a mortar. The pulverized skin was lysed in 1 mL ice-cold cell extraction buffer [20 mM HEPES (pH 7.0), 50 mM NaCl, 10% Triton X-100, 10% glycerol, 1 mM b-mercaptoethanol, and 1 tablet of protease inhibitor cocktail (Roche, Mannheim, Germany)] for 30 min. Lysates were centrifuged at 13,200 g at 4 C for 20 min, and total protein was extracted and quantified. Gel electrophoresis was performed on 12% sodium dodecyl sulfate and polyacrylamide gel (SDS-PAGE; Sigma-Aldrich) to separate 20 mg of total protein. The proteins were transferred to nitrocellulose membranes (Merck Millipore, Billerica, MA) via electro-imprinting. Each blot was sealed with 5% skim milk (BD, Franklin Lakes, NJ) for 1 h and rinsed 5 times with TBST buffer [15 mM NaCl, 0.05% Tween-20, and 10 mM Tris HCl (pH 7.0)] for 5 min. The membranes were incubated for 6 h at 4 C with 1:2000 dilution of COX-2, NOS-2, p38, phospho-p38, Bcl-2, cleaved caspase-3 (c-C3), Prx I, and b-actin polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Each blot was rinsed 3 times with PBST buffer (phosphate-buffered saline with Tween detergent) for 5 min, incubated with 1:5000 dilution of horseradish peroxide-conjugated secondary antibody (ZSGB-Bio, Beijing, China), and washed again 3 times with TBST buffer. The transferred proteins were visualized with a chemiluminescence detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

2.6. Cell viability assay

Mock and shPrx I HaCaT cells were seeded into 96-well plates at 5 103 cells/well and treated with H2O2 (Sigma-Aldrich) at 25, 50,100, and 150 mM. Twenty-four hours after, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) was added to each well; the well was incubated at 37 C for 4 h at 5% CO2. The supernatant was removed, and DMSO (Quanrui Reagent Co., Ltd., Liaoning, China) was added to dissolve formazan. Finally, a UV Max kinetic microplate reader (Tecan, M€annedorf, Switzerland) was used to measure absorbance at 570 nm.

2.7. Flow cytometry analysis

The Annexin-V-PE Kit (Beyotime, Shanghai, China) was used to measure HaCaT cell apoptosis. Mock and shPrx I HaCaT cells were seeded into 6-well plates at 3 106 cells/well. After being grown to 70e80% confluence, cells were treated with 50 mM H2O2 at 0, 3, 6, and 12 h and washed with PBS afterward. Annexin-V-PE staining solution was added to each well, and cells were incubated for 15 min at room temperature without light. The cells were washed twice with PBS and analyzed by flow cytometry (BD).

2.8. Statistical analysis

A two-way analysis of variance was used to analyze timedependent changes and differences between groups. Tukey’s range test was used for post-hoc testing (a ¼ 0.05). P-values of <0.05, <0.01, and <0.001 were considered statistically significant.

3. Results

3.1. Prx I gene deletion enhanced H2O2-induced death of HaCaT cells

To explore the molecular mechanism of Prx I, the Prx I gene was knocked down with lentivirus vectors in HaCaT cells in vitro. On Western blot analysis, Prx I expression was significantly lower in shPrx I HaCaT cells than in mock cells (Fig. 1A). With H2O2 stimulation, cell viability was significantly decreased in both cell types, with shPrx I cells exhibiting more cytotoxicity than mock cells (Fig. 1B). Prx I knockdown accelerated H2O2-induced intracellular ROS accumulation in HaCaT cells (Fig. 1C).

3.2. Prx I deletion enhanced ROS-induced apoptosis of HaCaT cells through the p38 pathway

To define the regulatory mechanism of decreased cell viability after Prx I deletion, apoptosis and intracellular signal changes were evaluated after HaCaT cells were stimulated with H2O2. The flow cytometry analysis showed that apoptosis was significantly higher in shPrx I cells than in mock cells (Fig. 2A). Changes in levels of proteins (such as p38, Bcl2, and c-C3) affected by cellular ROS were also evaluated. Bcl-2 expression decreased while c-C3 and p-p38 expressions increased upon H2O2 stimulation (Fig. 2B).

3.3. Prx I deletion inhibited DMBA/TPA-induced skin tumor formation

To explore the role of Prx I in skin tumor formation, DMBA and TPA were used to stimulate the back skin of mice (Fig. 3A). DMBA/ TPA treatment efficiently induced skin tumors in both WT and Prx I/ mice (Fig. 3B). At 8 weeks, both groups began to develop tumors, and with extended TPA treatment, tumors gradually increased in number, with WT mice having significantly more tumors than Prx I/ mice (Fig. 3C). Prx I/ mice had less number of tumors <4 mm in diameter than WT mice, while there was no significant difference for tumors >4 mm in diameter (Fig. 3D). Therefore, Prx I had no significant effect on the later stage of tumor development. On histological analysis, skin thickness was significantly lower in Prx I/ mice after the 20-week TPA treatment (Fig. 3E), further suggesting that Prx I may affect DMBA/TPAinduced skin tumor formation but not the later development of the tumor.

3.4. Prx I deletion increased epidermal cell apoptosis after DMBA/TPA treatment

The apoptosis of mutated cells can limit tumor development [33]. This study assessed the number of apoptotic cells in the basal epidermis of WT and Prx I/ mice. Polymorphonuclear and mononuclear inflammatory cells were identified in skin tissue layers after DMBA/TPA treatment (Fig. 4A). Numerous cells with nuclear pyknosis and deeply stained apoptotic cells were also identified in the basal epidermis, with Prx I/ mice having more apoptotic cells than WT mice (Fig. 4B). On Western blot analysis, Bcl-2 expression was lower while c-C3 and p-p38 expressions were higher after DMBA/TPA treatment in Prx I/ mice than in WT mice. However, there was no significant difference in iNOS and COX-2 expressions between both groups (Fig. 4C).

4. Discussion

In the early stage of DMBA/TPA-induced skin tumor formation, complex changes occur in skin tissue [34]. Short-term TPA stimulation can also produce a large number of ROS in cells [35], causing damage and apoptosis. In most cases, the rate of apoptosis is far slower than that of cell proliferation [36,37]. DMBA metabolites can lead to Ras gene mutation in some basal epidermal keratinocytes [11,13,14], and such mutation can influence several important signaling pathways, such as the mitogen-activated protein kinase (MAPK) and Akt pathways [38e40]. Ras mutation can also lead to abnormal expression of many apoptosis-related proteins [41,42]. Therefore, Ras-mutated cells are more vulnerable to ROS attack and apoptosis than normal cells. In this study, numerous cells with nuclear pyknosis and deeply stained apoptotic cells were produced in the basal epidermis after TPA treatment (Fig. 4A). Although no difference in skin thickness was observed between knockout and WT mice after 10 days of TPA treatment, skin thickness was significantly lower in knockout mice after 20 weeks of TPA treatment (Fig. 3E). Therefore, the changes in epidermal thickness due to keratinocyte apoptosis become more obvious in the long term.
In the TPA-induced promotion stage of cancer, inflammation is the most important event, and neutrophils and macrophages play an important role [43,44]. In this study, no significant difference in inflammatory response was observed in the early stage of TPAinduced promotion, suggesting that Prx I was not involved in TPA-induced inflammatory response (Fig. 4A). On Western blot analysis, Prx I influenced the expressions of inflammatory-related proteins NOS-2 and COX-2, which were induced by TPA (Fig. 4C). However, after TPA treatment, the expression of c-C3 was higher, that of Bcl-2 was lower, and that of p-p38 was higher in Prx Ideficient mice than in controls (Fig. 4C), indicating that Prx I was involved in keratinocyte apoptosis by regulating ROS levels.
The dual role of Prx I as a tumor suppressor and tumor promoter had sparked interest among researchers in the past. Prx I-deficient mice spontaneously produced various tumors and were more susceptible to other induced cancers. Prx I was also positively correlated with multiple cancers. In cancer cells, hydrogen peroxide levels are extremely high. In this case, Prx I is oxidized, altering its function from being a peroxidase reductase to a molecular chaperone involved in cancer development.
P38 MAPK, an MAPK subtype, is involved in cell proliferation, differentiation, death, migration, and invasion [45]. The P38 MAPK pathway is activated in response to various stimuli and can regulate cell survival and death [46]. The MAPK pathway can be activated by ROS [47]. In this study, the phosphorylation level of p38 was significantly higher in Prx I/ mice than in WT mice. In vitro, the expression levels of ROS and p38 in shPrx I HaCaT cells stimulated by H2O2 increased significantly. These results indicate that Prx I may regulate ROS-mediated phosphorylation of p38 to, in turn, regulate keratinocyte apoptosis.
In conclusion, Prx I could inhibit p38 phosphorylation and enhance the anti-apoptotic activity of keratinocytes by regulating ROS levels in epidermal cells, resulting in mutant keratinocytes accumulating in the tumor microenvironment and proliferating in large numbers to form tumor tissues. The findings provide a theoretical basis for treating skin tumors.

References

[1] S. Zhou, S.D. da Silva, P.M. Siegel, A. Philip, CD109 acts as a gatekeeper of the epithelial trait by suppressing epithelial to mesenchymal transition in squamous cell carcinoma cells in vitro, Sci. Rep. 9 (2019), https://doi.org/10.1038/ s41598-019-50694-z, 16317.
[2] A. Wadhera, M. Fazio, G. Bricca, O. Stanton, Metastatic basal cell carcinoma: a case report and literature review. How accurate is our incidence data? Dermatol. Online J. 12 (2006) 7.
[3] L. Jennings, C.D. Schmults, Management of high-risk cutaneous squamous cell carcinoma, Journal of Clinical & Aesthetic Dermatology 3 (2010) 39e48.
[4] M. Peter Marinkovich, Tumour microenvironment: laminin 332 in squamouscell carcinoma, Nat. Rev. Canc. 7 (2007) 370e380.
[5] D.M. Owens, F.M. Watt, Contribution of stem cells and differentiated cells to epidermal tumours, Nat. Rev. Canc. 3 (2003) 444e451.
[6] J. Perez-Losada, A. Balmain, Stem-cell hierarchy in skin cancer, Nat. Rev. Canc. 3 (2003) 434e443.
[7] E.L. Abel, J.M. Angel, K. Kiguchi, J. Digiovanni, Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications, Nat. Protoc. 4 (2009) 1350e1362.
[8] C.J. Kemp, Multistep skin cancer in mice as a model to study the evolution of cancer cells, Semin. Canc. Biol. 15 (2005) 460e473.
[9] M.G. Borland, K. Prasad, L. Christina, P.P. Albrecht, W. Shan, M.T. Bility, C.B. Marcus, J.M. Lin, A. Shantu, F.J. Gonzalez, Modulation of aryl hydrocarbon receptor (AHR)-dependent signaling by peroxisome proliferator-activated receptor b/d (PPARb/d) in keratinocytes, Carcinogenesis (2014) 7.
[10] C. Esser, I. Bargen, H. Weighardt, T. Haarmann-Stemmann, J. Krutmann, Functions of the aryl hydrocarbon receptor in the skin, Semin. Immunopathol. 35 (2013) 677e691.
[11] A. Uhmann, I. Heß, A. Frommhold, S. Konig, S. Zabel, F. Nitzki, K. Dittmann,€ F. Lühder, H. Christiansen, J. Reifenberger, W. Schulz-Schaeffer, H. Hahn, DMBA/TPA treatment is necessary for BCC formation from patched deficient epidermal cells in ptchflox/floxCD4Creþ/ mice, J. Invest. Dermatol. 134 (2014) 2620e2629, https://doi.org/10.1038/jid.2014.157.
[12] E. van den Bogaard, J. Bergboer, M. Vonk-Bergers, I. Vlijmen-Willems, S. Hato, P. van der Valk, J.-M. Schroder, I. Joosten, P. Zeeuwen, J. Schalkwijk, Coal tar€ induces AHR-dependent skin barrier repair in atopic dermatitis, J. Clin. Invest. 123 (2013), https://doi.org/10.1172/JCI65642.
[13] B.F.-H. Karsten Boehnke, Hans-Jürgen Stark, P. Boukamp*, Stem cells of the human epidermis and their niche: composition and function in epidermal regeneration and carcinogenesis, Carcinogenesis 33 (2012) 1247e1258.
[14] I. Verbinnen, S. Boens, M. Ferreira, K. Szeker, L. Van Wijk, A. Van Eynde, M. Bollen, Enhanced DNA-repair capacity and resistance to chemically induced carcinogenesis upon deletion of the phosphatase regulator NIPP1, Oncogenesis 9 (2020) 30, https://doi.org/10.1038/s41389-020-0214-3.
[15] J. Goldstein, E. Roth, N. Roberts, R. Zwick, S. Lin, S. Fletcher, A. Tadeu, C. Wu, A. Beck, C. Zeiss, Loss of endogenous Nfatc1 reduces the rate of DMBA/TPAinduced skin tumorigenesis, Mol. Biol. Cell 26 (2015) 3606e3614.
[16] R. Sanchez-Ortiz, D. Vaughn, Renal, testicular, and penile cancer, Penn Clinical Manual of Urology (2007) 571e615, https://doi.org/10.1016/B978141603848-1.10016-6.
[17] F. Wang, H. Ma, Z. Liu, W. Huang, X. Xu, X. Zhang, a-Mangostin inhibits DMBA/ TPA-induced skin cancer through inhibiting inflammation and promoting autophagy and apoptosis by regulating PI3K/Akt/mTOR signaling pathway in mice, Biomed. Pharmacother. 92 (2017) 672e680, https://doi.org/10.1016/ j.biopha.2017.05.129.
[18] H. Steinbrenner, M. Ramos, D. Stuhlmann, D. Mitic, H. Sies, P. Brenneisen, Tumor promoter TPA stimulates MMP-9 secretion from human keratinocytes by activation of superoxide-producing NADPH oxidase, Free Radic. Res. 39 (2005) 245e253, https://doi.org/10.1080/10715760500053487.
[19] Y. Matsuzaki, Y. Takaoka, T. Hitomi, H. Nishino, T. Sakai, Activation of protein kinase C promotes human cancer cell growth through downregulation of p18INK4c, Oncogene 23 (2004) 5409e5414, https://doi.org/10.1038/ sj.onc.1207702.
[20] W.S. Wu, The signaling mechanism of ROS in tumor progression, Canc. Metastasis Rev. 25 (2006) 695e705.
[21] H.-C. Hsu, W.-C. Lin, P.-J. Chang, C.-Z. Hong, C.-H. Chen, Propyl gallate inhibits TPA-induced inflammation via the nuclear factor-kB pathway in human THP1 monocytes, Experimental & Therapeutic Medicine 5 (2013) 964e968.
[22] E. Roemer, T.H. Ottmueller, H.J. Urban, C. Baillet-Mignard, SKH-1 mouse skin painting: a short-term assay to evaluate the tumorigenic activity of cigarette smoke condensate, Toxicol. Lett. 192 (2010) 155e161, https://doi.org/10.1016/j.toxlet.2009.10.019.
[23] S. Immenschuh, E. Baumgart-Vogt, Peroxiredoxins, oxidative stress, and cell proliferation, Antioxidants Redox Signal. 7 (2005) 768e777, https://doi.org/ 10.1089/ars.2005.7.768.
[24] H.H. Jang, K.O. Lee, H.C. Yong, B.G. Jung, S.K. Park, H.P. Jin, J.R. Lee, S.S. Lee, J.C. Moon, J.W. Yun, Two enzymes in one: two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function, Cell 117 (2004), 0-635.
[25] Malignant astrocytomas of elderly patients lack favorable molecular markers: an analysis of the NOA-08 study collective, Neuro Oncol. (2013) 8.
[26] J. Cao, J. Schulte, X. Knight, N. Leslie, A. Zagozdzon, R. Bronson, Y. Manevich, C. Beeson, C. Neumann, Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity, EMBO J. 28 (2009) 1505e1517, https://doi.org/10.1038/ emboj.2009.101.
[27] L. Yan, W. Yang, S. Lei, B. Zhou, Z. Qiu, K. Wang, Z. Xia, Inhibition of HDAC6 activity alleviates myocardial ischemia/reperfusion injury in diabetic rats: potential role of peroxiredoxin 1 acetylation and redox regulation, Oxidative Medicine & Cellular Longevity (2018) 1e15, 2018.
[28] Q.M. Wang, Y. Cai, D.R. Tian, H. Yang, J.S. Han, Peroxiredoxin1, A potential obesity-related factor in the hypothalamus, Medical Science Monitor International Medical Journal of Experimental & Clinical Research 16 (2010). BR321-326.
[29] B. Ha, E.-K. Kim, J.-H. Kim, H. Lee, K. Lee, S.Y. Lee, H.H. Jang, Human peroxiredoxin 1 modulates TGF-?1-induced epithelial-mesenchymal transition through its peroxidase activity, Biochem. Biophys. Res. Commun. 421 (2012) 33e37, https://doi.org/10.1016/j.bbrc.2012.03.103.
[30] S. Jacek, T.-C. Justyna, P. Ewa, C. Krzysztof, N. Dominika, Peroxiredoxin-1 as a prognostic factor in patients with ovarian cancer, Annals of Agricultural & Environmental Medicine Aaem 26 (2019).
[31] Y.L. Sun, J.Q. Cai, F. Liu, X.Y. Bi, L.P. Zhou, X.H. Zhao, Aberrant expression of peroxiredoxin 1 and its clinical implications in liver cancer, World J. Gastroenterol. (2015) 146e158.
[32] Y.H. Han, T. Kwon, S.U. Kim, H.L. Ha, T.H. Lee, J.M. Kim, E.K. Jo, B.Y. Kim, D.Y. Yoon, D.Y. Yu, Peroxiredoxin I deficiency attenuates phagocytic capacity of macrophage in clearance of the red blood cells damaged by oxidative stress, BMB Rep 45 (2012) 560e564, https://doi.org/10.5483/ bmbrep.2012.45.10.082.
[33] H.S. Chin, M.X. Li, I.K.L. Tan, R.L. Ninnis, B. Reljic, K. Scicluna, L.F. Dagley, J.J. Sandow, G.L. Kelly, A.L. Samson, S. Chappaz, S.L. Khaw, C. Chang, A. Morokoff, K. Brinkmann, A. Webb, C. Hockings, C.M. Hall, A.J. Kueh, M.T. Ryan, R.M. Kluck, P. Bouillet, M.J. Herold, D.H.D. Gray, D.C.S. Huang, M.F. van Delft, G. Dewson, VDAC2 enables BAX to mediate apoptosis and limit tumor development, Nat. Commun. 9 (2018) 4976, https://doi.org/10.1038/ s41467-018-07309-4.
[34] E. Abel, J. Angel, K. Kiguchi, J. DiGiovanni, E.L. Abel, J.M. Angel, K. Kiguchi, J. DiGiovanni, Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications, Nat Protoc 4: 1350-1362, Nature protocols 4 (2009) 1350e1362, https://doi.org/10.1038/nprot.2009.120.
[35] D.R. Tasat, R. Mancuso, S. O’Connor, B. Molinari, Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages*, Aging Cell 2 (2003) 159e164, https://doi.org/10.1046/j.14749728.2003.00051.x.
[36] P.P. Roger, S. Reuse, P. Servais, B. Van Heuverswyn, J.E. Dumont, Stimulation of 7,12-Dimethylbenz[a]anthracene cell proliferation and inhibition of differentiation expression by tumorpromoting phorbol esters in dog thyroid cells in primary culture, Canc. Res. 46 (1986) 898e906.
[37] P. Friederich, B.W.H.V. Heumen, I.D. Nagtegaal, M. Berkhout, J.H.J.M.V. Krieken, W.H.M. Peters, F.M. Nagengast, Increased epithelial cell proliferation in the ileal pouch mucosa of patients with familial adenomatous polyposis, Virchows Archiv An International Journal of Pathology 451 (2007) 659e667.
[38] L.L. Santarpia, S. Lippman, A. Elnaggar, Targeting the mitogen-activated protein kinase RAS-RAF signaling pathway in cancer therapy, Expert Opin. Ther. Targets 16 (2012) 103.
[39] E. Castellano, J. Downward, RAS interaction with PI3K: more than just another effector pathway, Genes Cancer 2 (2011) 261e274, https://doi.org/10.1177/ 1947601911408079.
[40] L.S. Steelman, W.H. Chappell, S.L. Abrams, R.C. Kempf, J. Long, P. Laidler, S. Mijatovic, D. Maksimovic-Ivanic, F. Stivala, M.C. Mazzarino, M. Donia, P. Fagone, G. Malaponte, F. Nicoletti, M. Libra, M. Milella, A. Tafuri, A. Bonati, J. B€asecke, L. Cocco, C. Evangelisti, A.M. Martelli, G. Montalto, M. Cervello, J.A. McCubrey, Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging, Aging (Albany NY) 3 (2011) 192e222, https://doi.org/10.18632/ aging.100296.
[41] A.D. Cox, C.J. Der, The dark side of Ras: regulation of apoptosis, Oncogene 22 (2003) 8999e9006, https://doi.org/10.1038/sj.onc.1207111.
[42] J.J. Chen, W.P. Bozza, X. Di, Y. Zhang, W. Hallett, H-Ras regulation of TRAIL death receptor mediated apoptosis, Oncotarget 5 (2014) 5125e5137.
[43] T. Bald, J. Landsberg, P. Jansen, E. Gaffal, T. Tüting, Phorbol ester-induced neutrophilic inflammatory responses selectively promote metastatic spread of melanoma in a TLR4-dependent manner, OncoImmunology 5 (2015), 00-00.
[44] T.H. Gasparoto, C. Oliveira, L. Freitas, C. Pinheiro, R. Ramos, A. Silva, G. Garlet, J. Silva, A. Campanelli, Inflammatory events during murine squamous cell carcinoma development, J. Inflamm. 9 (2012) 46, https://doi.org/10.1186/ 1476-9255-9-46.
[45] H.K. Koul, M. Pal, S. Koul, Role of p38 MAP kinase signal transduction in solid tumors, Genes Cancer 4 (2013) 342e359.
[46] M. James, Olson, R. Andrew, Hallahan, p38 MAP kinase: a convergence point in cancer therapy, Trends Mol. Med. (2004).
[47] J.A. McCubrey, M.M. LaHair, R.A. Franklin, Reactive oxygen species-induced activation of the MAP kinase signaling pathways, Antioxidants Redox Signal.8 (2006) 1775e1789.