Prx1 is positively associated with colitis and colon cancer. Two-dimensional agarose gel electrophoresis (2-DE) followed by mass spectrometry analysis of proteins isolated from biopsies (sigmoid colon) in two patients with active ulcerative colitis (UC), two patients with inactive ulcerative colitis, and four healthy subjects showed that Prx1 is upregulated in active UC compared to inactive UC and healthy controls ^[1][36]. Oxidized Prx1 protein levels were higher in healthy and inactive UC groups while reduced Prx1 level was higher in the active UC group. Immunohistochemical (IHC) staining of patient samples confirmed that Prx1 increased with increasing inflammation in mucosal crypts ^[1][36]. Furthermore, IHC staining of Prx1 in 22 normal mucosae, 6 UC-associated low-grade dysplasias, 5 high-grade dysplasias, and 5 UC-associated carcinomas detected increasing Prx1 expression in dysplasia and carcinoma ^[1][36]. Further studies are warranted to establish a causal relationship between Prx1 and colon tumorigenesis in inflammation-associated sporadic colorectal cancer (CRC) as well as hereditary CRC models.

In breast cancer, loss of Prx1 due to reduced zinc (Zn) intake is linked to tumor formation ^[2][37]. Bostanci et al. treated offspring of nulliparous mice fed control (ZA, 30 mg Zn/kg) or a marginal Zn diet (ZD, 15 mg Zn/kg) with corn oil or 7,12-dimethylbenz(a)anthracene (DMBA, 1 mg/wk) for 4 weeks. Mice fed ZD had shorter tumor latency and greater incidence of non-palpable tumors. Mechanistic studies showed reduced protein levels of Prx1 and p53 and higher oxidative DNA damage in mammary tissue of mice fed ZD. The authors propose that Zn deficiency compromises the antioxidant capacity of mammary cells, leading to higher oxidative stress and carcinogenesis ^[2][37]. This points to the need to delineate the role of diet components, such as Zn, in transcriptional and translational regulation of Prxs in normal physiology and cancers. In addition, Prx1 inhibits the cancer-associated fibroblast-like phenotype in breast cancer ^[3][38]. Primary mammary fibroblasts (MFs) isolated from Prx1 knockout mice had increased α-SMA, collagen, and Vimentin compared to Prx1 wildtype MFs. Mechanistic studies revealed that Prx1 knockdown MFs had increased oxidation of PTEN and phosphorylation of JNK when treated with H₂O₂. JNK1 binds to reduced Prx1 but not to overoxidized Prx1. Thus, Prx1 prevents corrupt activation of MFs ^[3][38].

Prx2 enhances intestinal tumorigenesis induced by APC mutation ^[4][39]. Prx2 homozygous knockout mice developed significantly fewer small intestine and colon tumors and had longer survival compared to Prx2 heterozygous and Prx2 wildtype groups in an APC^Min/+ mouse model. Prx2 knockdown increased H₂O₂ accumulation and decreased total β-catenin protein levels in APC-mutant HT-29 and SW480 cells. β-catenin reduction could be blocked by inhibition of proteasomes and GSK3β. Immunoprecipitation (IP) assay revealed that Prx2 increased Axin1 complexes by blocking PARylation/ubiquitination of Axin1 ^[4][39]. In an in vitro PARP assay, the authors found that Prx2 loss impaired tankyrase activity in HT-29 and SW480 cell lines. In summary, Prx2 promotes intestinal tumorigenesis by inhibiting β-catenin degradation.

In a urethane-induced lung cancer model, human Prx4-expressing transgenic mice developed larger tumors than non-transgenic control mice ^[5][40]. IHC staining of extracted tumors showed increased cell proliferation, decreased oxidative DNA damage and apoptosis, and increased microvascular permeability and macrophage infiltration in Prx4 overexpressing tumors. Western blot analysis of tumor tissues showed increased p-c-Jun and p-p65 in Prx4-overexpression tumors, suggesting the involvement of NF-κB and AP-1 pathways ^[5][40]. Additionally, Prx4 knockout FVB/N mice developed a reduced number and size of tumors compared to wildtype FVB/N mice in azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colorectal cancer and urethane-induced non-small cell lung cancer (NSCLC) ^[6][7][41,42]. Loss of Prx4 reduced tumor cell proliferation in the lung cancer model and increased tumor cell death in the colorectal cancer model. The studies also report novel functions of Prx4 in promoting immune infiltration into the tumors as well as regulating cytokine secretion from the immune cells ^[6][7][41,42]. Thus, Prx4 promotes tumor formation in lung cancer and colorectal cancer.

Prx6 also promotes lung tumorigenesis in animal models ^[8][43]. Presenilin 2 (PS2) N141I transgenic mice developed significantly lower spontaneous lung cancer compared to wildtype transgenic mice. The authors found that mutant PS2 transgenic mice tumors had over 500 times lower Prx6 expression compared to wildtype ^[8][43]. Accordingly, both peroxidase and phospholipase activities were lowered in mutant PS2 transgenic mice compared to their wildtype transgenic counterpart. IHC staining of Prx6 in human lung cancer tissue array showed overexpression of Prx6 in tumors compared to normal tissues. In addition, the authors discovered a 50% increase in PLA₂ activity in cancer tissues compared to normal tissues. In IP assay, Prx6 and PS2 co-localization was increased in PS2 mutant skin fibroblasts AG09908 cells compared to non-mutated epithelial cells A431 cells ^[8][43]. Immunofluorescence analysis proved that this co-localization could be reversed by treatment of γ-secretase inhibitor L685,458. Prx6 and PS2 co-localization was increased in urethane-induced lung tumors isolated from mutant PS2 transgenic mice compared to wildtype. IP analysis of A549 and NCIH460 also demonstrated that, compared to wildtype PS2, the mutant PS2 had a higher affinity for Prx6 ^[8][43]. Transfection of mutant PS2 plasmid into A549 and H460 cells inhibited Prx6 expression and cell viability and increased PLA₂ cleavage and γ-secretase activity compared to wildtype PS2 transfection or vector transfection. Thus, PS2 mutation inhibits the PLA₂ activity of Prx6 to suppress lung tumor development ^[8][43].

In lung cancer, Prx1 protects cells against apoptosis and promotes invasion in vitro. Knockdown of Prx1 in A549 cells upregulated E-cadherin at the protein level and suppressed TGF-β-induced cell migration ^[9][44]. Using a luciferase activity assay, it was shown that catalytic Cys51 of Prx1 was critical in regulation of E-cadherin expression. The mechanism of how Prx1 peroxidase activity is used to regulate E-cadherin is not understood. In a different study, Prx1 overexpression increased anchorage-dependent colony formation and Matrigel invasion of A549 cells ^[10][45]. In A549 cells, inhibition of Prx1 with the small molecule AMRI-59 caused apoptosis ^[11][46]. AMRI-59 treatment activated both mitochondria- and apoptosis signal-regulated kinase-1-mediated signaling pathways, resulting in cell death. This could be prevented by Prx1 overexpression or N-acetyl cysteine (NAC) pretreatment. AMRI-59 was later discovered to act as a radiosensitizer in non-small cell lung cancer cells ^[12][47]. In a clonogenic assay of H460 and H1299, pretreatment with AMRI-59 increased sensitivity of these cells to irradiation. Western blot analysis showed an increase in cleaved caspase 3 upon combined treatment with ionizing radiation (IR) and AMRI-59, and cell survivability could be rescued by pan-caspase inhibitor z-Vad-Fmk. Similarly, combined treatment with IR and AMRI-59 induced ROS production (measured using DCFDA assay) and oxidative DNA damage (measured using γH2AX immunofluorescence staining), both of which could be rescued by NAC ^[12][47]. Subcutaneous injection of these NSCLC cell lines in BALB/c nu mice followed by various modes of treatments showed the combination of IR and AMRI-59 to be the most effective approach. Western blot analysis of cells in vitro showed that the combined effect of IR and AMRI-59 could be further increased by CREB-1 inhibitor ^[12][47]. Thus, inhibition of Prx1 is a novel approach to overcome radioresistance in NSCLC.

Prx1 protects hepatoma cells against apoptosis in vitro ^[13][48]. Knockdown of Prx1 in the HCC cell line decreased cell proliferation and increased apoptosis. This was associated with upregulation of Bax protein level and activation of mitochondrial fission as indicated by elevated Drp1, Fis1, and Dyn2 protein levels. Prx1 also contributes to epithelial–mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC): Long non-coding RNA LINC00460, which enhances HNSCC cell proliferation and metastasis, physically interacts with Prx1 and facilitates Prx1 entry into the nucleus ^[14][49]. Prx1, in turn promotes the transcription of LINC00460, forming a positive feedback loop. In addition, overexpression of Prx1 upregulated Zeb1, Zeb2, Vimentin, and N-cadherin at mRNA and protein levels. Using quantitative real-time polymerase chain reaction (qRT-PCR) of paired HNSCC and adjacent normal tissues, the authors also demonstrated that high levels of LINC00460 and Prx1 expression were positively associated with lymph node metastasis and tumor size in HNSCC patients ^[14][49].

Prx1 expression in stromal cells of the breast tumor microenvironment is associated with inhibition of cancer progression. Loss of Prx1 prompts collagen remodeling known to promote breast cancer development ^[15][59]. Knockdown of Prx1 in mammary fibroblasts followed by injection into mammary fat of BALB/c mice resulted in an enrichment of intratumoral collagen in the shPrx1 group. In vitro studies indicated that Prx1-depleted mammary fibroblasts had higher α collagen, β collagen, and β/α collagen ratio ^[15][59]. Conditioned media derived from MDA-MB-231 cells caused Y194 phosphorylation of Prx1 (known to inactivate peroxidase activity), and this could be reversed by co-treatment with Src inhibitor PP1. shPrx1 mammary fibroblasts had increased secretion of lysyl oxidase (LOX). Per IP assay of LOX in HEK293T cells, endogenous Prx1 interacts with LOX, but this interaction was decreased when Prx1 was phosphorylated, resulting in increased extracellular LOX accumulation and collagen remodeling ^[15][59]. Another study suggested that Prx1 mediates tumor suppressor activity of cytoskeletal protein transgelin-2 (TAGLN2) ^[16][60]. Knockdown of TAGLN2 in MDA-MB-231 increased cell migration in vitro and increased lung metastasis in a tail-vein injection mouse model. IP of TAGLN2 in MDA-MB-231 lysates followed by mass spectrometry analysis showed that TAGLN2 binds to Prx1. Knockdown of TAGLN2 in MDA-MB-231 caused downregulation of the Prx1 protein. Accordingly, DCFDA assay demonstrated that TAGLN2 knockdown resulted in higher ROS production ^[16][60]. Thus, interaction of Prx1 with LOX and TAGLN2 plays anti-tumorigenic role. The direct or indirect mechanism of how TAGLN2 upregulates Prx1 needs to be examined. Furthermore, the importance of peroxidase function in Prx1 in its interaction with TAGLN2 remains to be seen.

Prx2 promotes progression of non-small cell lung cancer (NSCLC). Western blot analysis showed that the expression of Prx2 in NSCLC cell lines is higher than in normal bronchial epithelial cells (BEAS-2b) ^[17][63]. Knockdown of Prx2 in A549 cells reduced cell proliferation, migration, and invasion. Subcutaneous injection of Prx2 knockdown A549 cells resulted in slower tumor growth compared to control cells. IHC staining of extracted tumors revealed a decrease in cell proliferation in the shPrx2 group. Tail-vein injection of A549 cells resulted in fewer metastatic nodules in Prx2 knockdown group compared to control, and this was associated with higher E-cadherin and lower Vimentin and Slug expression ^[17][63]. Jing et al. have reported similar findings in A549 and H1299 cell lines. They also discovered that loss of Prx2 reduced the phosphorylation of AKT and mTOR ^[18][64]. In addition, Prx2 promoted the stemness of drug-resistant cancer stem cells. Knockdown of Prx2 reduced colony formation and sphere formation, increased ROS (DCFDA assay) and apoptosis, and reduced migration and invasion of gefitinib-resistant A549 (A549/GR) CD133⁺ cells ^[19][65]. The authors validated that microRNA miR-122 targets Prx2 and showed that overexpression of miR-122 also suppressed proliferation, migration, and invasion of A549/GR CD133⁺ cells. In mechanistic studies, the authors used Western blot analysis to show that miR-122-mediated downregulation of Prx2 resulted in reduced activation of the Hedgehog, Notch, and Wnt/β-Catenin signaling pathways in A549 cells ^[19][65]. Finally, loss of Prx2 activity resulted in death of lung cancer cells. S-nitrosoglutathione (GSNO) nitrosylates Prx2 on Cys51 and Cys172, resulting in H₂O₂ accumulation and apoptosis in A549 and NCI-H1299 cells ^[20][66]. GSNO-induced H₂O₂ increased phosphorylation of AMPK and inhibited deacetylation activity of SIRT1, leading to cell death. Thus, Prx2 aids survival and malignancy of NSCLC through a variety of pathways.

Prx2 increases growth and progression of CRC. Knockdown of Prx2 using shRNAs reduced proliferation of HCT116 and LoVo cell lines ^[21][67]. Flow cytometry analysis proved that Prx2 knockdown caused increased cell cycle arrest in G2/M phase in HCT116 and G1 phase in LoVo cells. There was no difference in p53 mRNA levels after Prx2 knockdown, but cycloheximide treatment showed an increased half-life of p53 in shPrx2 cell lines ^[21][67]. The authors discovered through IP and mass spectrometry that ribosomal protein RPL4 binds to Prx2. Ubiquitination assays were used to confirm RPL4 interaction with MDM2 and show that Prx2 increases ubiquitination of p53. Subcutaneous injection of control and shPrx2 cell lines resulted in higher tumor growth and the larger tumor volume in the control group. IHC analysis showed shPrx2 tumors had a higher expression of p53 ^[21][67]. Thus, Prx2 causes colorectal cancer growth in vitro, likely by facilitating degradation of p53.

Prx2 loss inhibits autophagy in CRC ^[22][77]. Analysis of RNA-Seq data of HT-29 and SW480 control and siPrx2 cell lines followed by KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis indicated enrichment of the FOXO pathway. Knockdown of Prx2 resulted in an increase of p21 and p27 proteins in Western blot analysis. shPrx2 cells had lower LC3B-GFP staining than non-targeting control cells ^[22][77]. Western blot analysis was used to show reduced LC3B II/LC3B I ratio and Beclin 1 along with increased Sqstm1/p62 in Prx2 knockdown cells compared to control. This indicates that Prx2 inhibits autophagosome formation. Western blot analysis also showed reduced p-p38 in shPrx2 cells. Treatment with 1 μM dehydrocorydaline chloride (DHC, a p38 MAPK activator) for 24 h rescued p-p38 to some extent in Prx2 cells. In addition, DHC also caused a decrease in p21 proteins to similar levels as non-targeting controls. Subcutaneous injection of control and shPrx2 cells into nude mice resulted in smaller tumor formation in the shPrx2 group ^[22][77]. Another study reported that oxiconazole (Oxi), an antifungal compound derived from imidazole, downregulates Prx2 in CRC cells to initiate autophagy and inhibit autolysosome formation by downregulating Rab7a ^[23][78]. When nude mice were subcutaneously injected with HCT116 cells followed by control or Oxi treatment (50 mg/kg/day), the Oxi treatment group developed significantly smaller tumors. Annexin V staining showed increased apoptosis in HCT116 and RKO after Oxi treatment. Oxi treatment increased cellular ROS levels as measured by active oxygen analysis kit. This increase in ROS and apoptosis could be inhibited by co-treatment with N-acetyl cysteine, suggesting that Oxi promotes ROS production to induce apoptosis ^[23][78]. Through immunofluorescence staining, the authors found that Oxi treatment increased autophagosome formation but not autolysosome formation in HCT116 and RKO cells. In Oxi-treated xenograft tissue, IHC showed stronger staining for LC3 than in the control group. Co-treatment with 3-mA (an autophagy inhibitor) rescued the decrease in cell viability caused by Oxi ^[23][78]. Oxi treatment also decreased Prx2 expression in a dose-dependent manner. Accordingly, Prx2 was found to be depleted in an Oxi-treated mouse xenograft model. Western blot analysis was used to examine lysosome–autophagosome fusion proteins and the authors found that Oxi decreased Rab7a expression. This could be partially reversed by Prx2 overexpression. Rab7a expression was also lower in the Oxi-treated mouse xenograft. Tandem monomeric mRFP-GFP tagged LC3 immunofluorescence assay in cells suggested that Oxi inhibits autolysosome formation through downregulation of Rab7a ^[23][78]. 

Prx3 promotes survival and proliferation of lung adenocarcinoma cells in vitro. Prx3 was upregulated at mRNA and protein levels in 36 human lung adenocarcinoma (LUAD) samples compared to adjacent normal tissue ^[24][79]. Overexpression of tumor suppressor DACH1 resulted in downregulation of Prx3 transcript and protein in LUAD cell lines LTEP-α-2 and A549. DACH1-mediated downregulation of Prx3 resulted in reduced cell proliferation and anchorage-dependent colony formation in both cell lines. Similarly, downregulation of Prx3 increased susceptibility of NSCLC cells to radiation ^[25][80]. Knockdown of oncogene TP53-regulated inhibitor of apoptosis 1 (TRIAP1) in A549 and H460 cells sensitized these cells to irradiation. TRIAP1 knockdown cells had increased apoptosis and decreased cell invasion upon irradiation compared to wildtype cells. Irradiation of A549 and H460 increased transcript and protein levels of TRIAP1 and impaired the radiation-induced increase of antioxidants including Prx3, Prx4, and Prx6 ^[25][80]. Downregulation of Prx3 also increased the susceptibility of NSCLC cells to thiosemicarbazones. Myers group have reported that tridentate iron chelator triapine (Tp) (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) oxidizes Prx3 in lung cancer lines (A549, H23, and H1703) and A2780 ovarian cancer cells ^[26][81]. Cytotoxicity of Tp correlated with Prx3 oxidation in the clonogenic survival of lung cancer lines. Knockdown of Prx3 further sensitized A549 cells to Tp ^[26][81].

Downregulation of Prx3 reduces viability of breast cancer cells in vitro. Knockdown of B7-H4 (also called VTCN1) decreased cell viability of MCF-7 and T47D cells ^[27][82]. This was associated with depletion of Prx3. Silencing Prx3 using siRNA caused increased intracellular ROS and decreased cell viability, similar to B7-H4 knockdown ^[27][82]. Thus, Prx3 likely protects breast cancer cells from oxidative stress-induced cell death.

Prx3 promotes stemness and survival of colon cancer cells ^[28][83]. qRT-PCR analysis showed increased Prx3 expression in CD133⁺ CSCs freshly isolated from eight patients with colon cancer compared to non-cancer stem cells. mRNA levels of Prx3 and CD133 in CSCs isolated from patient tissues showed significant positive correlation. Prx3 knockdown resulted in a decrease in the size of the CD133⁺ CSC population and sensitized the CSCs to 5-FU-induced cell death through mitochondrial dysfunction. Mice subcutaneously injected with CD133⁺ cells sorted from HT-29 shPrx3 showed reduced tumor volume and enhanced 5-FU-induced cell death compared with HT-29 shControl-injected mice ^[28][83]. Depletion of Prx3 resulted in a significant reduction in liver metastasis, colon metastasis, and local invasion in an orthotopic xenograft model produced by the injection of colon CSCs into the spleen and cecum of SCID mice. Chromatin immunoprecipitation assays showed that FOXM1 transcriptionally activates CD133 and Prx3 by binding to the promoter region of these genes. Overexpression of FOXM1 increased Prx3 and CD133 protein levels and expanded CD133⁺ population ^[28][83]. Thus, Prx3 supports CRC stem cells.

Prx4 increases proliferation and survival of prostate cancer cells. IHC staining of Prx4 in a human tissue microarray showed that Prx4 is upregulated in prostate adenocarcinoma compared to normal prostate ^[29][84]. Treatment of the LNCaP cell line with synthetic androgen R1881 led to a dose-dependent increased expression of Prx4 and PSA. In a LNCaP xenograft model, castration suppressed tumor growth. Western blot analysis of tumor lysates showed a significant downregulation of Prx4 and PSA in the castration group compared to control. Knockdown of Prx4 in LNCaP and DU145 cell lines reduced cell proliferation, migration, and invasion in vitro ^[29][84]. Phosphokinase array revealed that Prx4 depletion reduced activation of AKT and GSK3α/β signaling pathways. Prx4 depletion also sensitized LNCaP and DU145 cells to irradiation-induced cell death due to increased ROS accumulation (DCFDA assay) and DNA damage (indicated by γH2AX foci formation). Subcutaneous injection of control and Prx4 knockout DU145 cells resulted in smaller tumor volumes in the Prx4 knockout group. Similarly, irradiation of control and Prx4 knockout subcutaneous xenograft tumors suppressed tumor growth to a significantly higher extent in the Prx4 knockout group ^[29][84]. Thus, loss of Prx4 sensitizes prostate cancer cells to irradiation.

Prx4 is suggested to promote bone metastasis of prostate cancer and breast cancer ^[30][85]. Prx4 was depleted in MDA-MB-231 and PC3 cell lines, and conditioned media was collected to treat RAW264.7 cells. In vitro osteoclastogenesis assay showed suppression of osteoclast formation by knockdown of Prx4 compared to control cells, likely by reducing ERK phosphorylation and nuclear translocation of NFATc1. In an animal model, shPrx4 PC3 cells induced significantly smaller osteolytic lesions in tibia of CD-1 immunodeficient nude mice compared to control cells ^[30][85]. Little else is known about the mechanism of Prx4 secretion or the function of secreted Prx4 in metastasis of tumor cells.

Prx4 also promotes the progression of NSCLC. Knockdown of Prx4 decreases anchorage-independent colony formation and Matrigel invasion of A549 cells ^[10][45]. Phosphokinase array and Western blot analysis showed that loss of Prx4 represses c-Jun-mediated AP-1 activation. Subcutaneous injection and tail-vein injection of A549 cells into SCID mice resulted in smaller tumor volumes and fewer lung metastatic nodules in a Prx4 knockdown group compared to control ^[10][45]. Finally, Prx4 also promotes progression of CRC. IHC staining of 59 CRC patient samples showed that Prx4 is upregulated in CRC samples compared to adjacent normal samples, and Prx4 upregulation is positively correlated with infiltration depth, lymph node metastasis, and Dukes’ stage ^[31][86]. In 2-DE and mass spectrometry analysis of eight Stage 1 and eight Stage 4 CRC samples, significant upregulation of Prx4 in Stage 4 samples was confirmed ^[32][87]. Knockdown of Prx4 reduced cell proliferation and migration of DLD1 cells in vitro and subcutaneous tumor growth in vivo. Western blot analysis showed reduced phosphorylation of EGFR, RhoA, PKCα, and ERK in DLD1 shPrx4 cells compared to controls ^[32][87].

Prx5 promotes proliferation and EMT phenotype in gastric cancer cells ^[33][88]. Five-year survival data analyzed via the log-rank test indicated that overexpression of Prx5 was correlated with poor survival of gastric cancer patients. Expression of Prx5 significantly correlated with tumor size, lymph node invasion, and metastasis (TNM) stage ^[33][88]. Both proliferation and anchorage-dependent colony formation were higher in SNU-216 Prx5 overexpression cells than in parental SNU-216 cells. In Western blot analysis, E-cadherin was decreased, while Snail and Slug were increased in Prx5 overexpression SNU-216 cells ^[33][88].

Prx5 also promotes survival and EMT in NSCLC. Administration of non-thermal plasma therapy using plasma-activated medium (PAM) induced apoptosis in cancer cells by increasing ROS levels ^[34][89]. PAM was developed by treating A549 cell culture medium with low temperature plasma at 16.4 kV for 0, 60, 120, or 180 s. Knockdown of Prx5 enhanced ROS production, cytotoxicity, and inhibition of migration in A549 cells caused by PAM ^[35][90]. Western blot analysis showed that Prx5 knockdown in A549 reduced p-ERK and BCL2 and increased p-JNK and BAD proteins to promote apoptosis. Besides contributing to survival, Prx5 also promotes NSCLC growth and progression through its interaction with Nrf2 and Stat3. Prx5 could be pulled down using anti-Nrf2 antibody and vice versa in NSCLC and non-tumor lung tissues as well as in H1229 and A549 cell lysates ^[36][91]. Knockdown of Prx5 induced by H₂O₂ treatment decreased NQO1 protein levels in A549 and H1299 cells. Similarly, knockdown of Prx5 or NQO1 reversed the increase in cell proliferation of A549 and H1299 cells that was induced by H₂O₂ treatment. In analysis of patient samples, the authors found strong correlation of Prx5 mRNA with Nrf2 and NQO1 ^[36][91]. Subcutaneous injection of A549 cells into flanks of nude mice followed by no treatment or intra-tumoral injection of Nrf2 or Prx5 shRNA led to significantly reduced tumor growth in the shRNA treated group. Prx5 interaction with Stat3 was also reported by the Xue research group. qRT-PCR of 121 paired NSCLC tumor and adjacent normal samples revealed that 65% of the samples contained Prx5 promoter demethylation, and Prx5 promoter demethylation was associated with higher TNM stage ^[37][92]. Overexpression of Stat3 in H1299 cells pretreated with 100 µM H₂O₂ increased the Prx5 protein level, whereas knockdown of Stat3 decreased Prx5, suggesting that Stat3 is at least partially responsible for regulation of Prx5 expression. Overexpression of Prx5 in H1299 cells pretreated with 100 µM H₂O₂ increased in vitro migration and invasion. This was associated with a decrease in E-cadherin and increases in Vimentin, Nrf2, and NQO1, shown through Western blot analysis ^[37][92]. Thus, Prx5 plays a pro-tumorigenic role in NSCLC.

Prx5 promotes EMT phenotype in SW480 colon cancer cells ^[38][93]. Prx5 overexpression in SW480 cells increased cell proliferation, migration, and invasion rates in vitro. Western blot analysis showed that Prx5 overexpressing cells had lower E-cadherin and higher Vimentin, Slug, and Snail ^[38][93]. Knockdown of Prx5 using siRNA reversed the increase in cell proliferation, migration, invasion, and E-cadherin expression seen upon Prx5 overexpression. Prx5 also protects colon cancer cells from ROS-induced apoptosis ^[39][94]. β-lapachone, a compound extracted from the South American lapacho tree, is known to have anti-cancer activity. The authors performed bioinformatics analysis using the GEPIA website to show that Prx5 was upregulated in colon cancer compared to normal tissue. Increasing concentrations of β-lapachone treatment for 24 h showed SW480 shPrx5 cells to be more sensitive to this compound, whereas SW480 HisPrx5 cells were more resistant, compared to mock SW480 cells ^[39][94]. This was shown using a cell viability assay and annexin V staining. β-lapachone treatment decreased BCL2 (pro-apoptotic protein) expression in SW480 mock and shPrx5 cells. Dihydroethidium (DHE) staining indicated that β-lapachone treatment increased ROS levels in SW480 mock and shPrx5 cells ^[39][94]. In addition to ROS scavenging, Prx5 also regulates the Wnt/β-catenin pathway in response to β-lapachone. According to Western blot analysis, the ratio of p-Gsk3-β/Gsk3-β was higher and p-β-catenin/β-catenin was lower in Prx5-His cells than in mock and shPrx5 SW480 cells. The group also reported that Prx5 protects HCT116 and HT-29 cells from ROS-induced apoptosis ^[40][95]. Shikonin, a natural compound purified from the Lithospermum erythrorhizon plant, was previously reported to induce ROS in cancer cells. HCT116 and HT-29 cells were treated with increasing concentrations of Shikonin. JC-1 staining demonstrated that mitochondrial ROS increased in a dose-dependent manner. Similarly, DCFDA staining demonstrated a dose-dependent increase ^[40][95]. Using Western blot analysis, the authors found that Shikonin did not affect expression of proteins Prx2, Prx3, and Prx6, but Prx1 was increased and Prx5 was decreased with higher concentrations of Shikonin. Increasing concentrations of Shikonin resulted in a dose-dependent decrease of p-mTOR/mTOR ratio in HT-29 cells ^[40][95]. In an MTT assay, HisPrx5 HT-29 cells had higher viability than control mock HT-29 cells with increasing Shikonin concentrations. This increased resistance to cell death was also confirmed by DHE staining and annexin V flow cytometry analysis. Finally, HisPrx5 overexpression prevented a decrease in p-mTOR/mTOR ratio upon Shikonin ^[40][95]. Thus, Prx5 plays a potential role in suppressing apoptosis in colon cancer.

Prx6 promotes NSCLC growth and survival. Overexpression of Prx6 increased cell proliferation, invasion, and migration of A549 cells; these were decreased by knockdown of Prx6 ^[41][96]. Western blot analysis indicated that Prx6 overexpression promotes EMT by downregulating E-cadherin and upregulating Vimentin, Twist, β-catenin, and c-Myc. In a subcutaneous A549 xenograft model, Prx6 overexpression increased tumor growth while Prx6 knockdown suppressed tumor growth ^[41][96]. A positive correlation between CD133 and Prx6 protein expression in NSCLC patient samples is reported ^[42][97]. Knockdown of Prx6 decreased the CD133⁺/ABCG2⁺ population in H1299 and A549 cells as well as the sphere formation ability of these cancer stem-like cells. Knockdown of Prx6 also reduced the IC₅₀ of cisplatin for H1299 and A549 CSCs by 50% ^[42][97]. Another study also reported a positive association between Prx6 and drug resistance. Two-dimensional gel electrophoresis of six pairs of pretreatment fresh primary lung adenocarcinoma tumors with varied chemotherapy responses revealed that Prx6 was upregulated in chemo-resistant tumors ^[43][98]. Furthermore, Prx6 promotes growth of NSCLC. Withangulatin A (WA) is a small molecule isolated from Physalis angulata var. villosa and is reported to reduce proliferation of cancer cells. Stable isotope labeling by amino acids in cell culture and activity-based protein profiling in H1975 cells identified Prx6 as a direct target of WA ^[44][99]. WA covalently binds to Prx6 to inhibit its function and increases the production of ROS as indicated by DCFDA assay in H1975 cells. Subcutaneous injection of wildtype and Prx6 knockout (Prx6 KO) H1975 cells resulted in significantly lower tumor volume in the Prx6 KO group. WA treatment had no significant effect on proliferation, GPx activity, and PLA₂ activity in H1975 Prx6 KO cells in vitro, or in growth of Prx6 KO tumors in vivo, confirming that WA acts through Prx6 ^[44][99].

Prx6 also contributes to the progression of CRC. IHC staining of Prx6 in a CRC patient tissue microarray showed that Prx6 was upregulated in node-positive CRC compared to node-negative CRC ^[45][100]. Stable knockdown of Prx6 reduced cell migration and invasion in HCT116 cells. Prx6 knockdown also caused downregulation of N-cadherin, CDK1, and Twist1 in HCT116. This was associated with reduced phosphorylation of PI3K, AKT, p38, and p50, as indicated by Western blot analysis ^[45][100]. Treatment of HCT116 with NAC, PI3K/AKT inhibitor wortmannin, and p38 MAPK inhibitor SB203580 resulted in a decreased trimethylation of histone H3 lysine 4 (H3K4me3) of Prx6 promoter ^[45][100]. This suggests a role for PI3K/AKT pathway in upregulation of Prx6 in CRC.