**4. Peroxiredoxin-4**

#### **4.1 Peroxiredoxin-4 and its function**

H2O2 is produced during the disulfide bond formation in protein synthesis and from a side effect of cell biology mechanism located in the ER lumen and extracellular matrix [54]. PRDX4 is localized in the endoplasmic reticulum and highly expressed in the pancreas, liver, and heart but is low in blood and brain leukocytes [5]. PRDX4 plays a role as an antioxidant by scavenging H2O2 and is a chaperone molecule to activate ER stress pathway [55]. PRDX4 is important for cell protection by reducing H2O2 to water in order to reduce oxidative stress.

In addition, PRDX4 has a role in regulating the activation of NF-ĸB transcription factor and TNF-related apoptosis-inducing ligand (TRAIL) [56]. TRAIL is a transmembrane protein involved in apoptosis extrinsic pathway by binding to receptors DR4 or DR5. A reduction in PRDX4 expression leads to TRAIL activation and apoptosis induction, whereas an increase in PRDX4 expression inhibits the TRAIL activation and leads to cell survival [57]. The expression of PRDX4 in the cytosol increases NF-ĸB activity through phosphorylation of IκB-α [58]. Meanwhile, the expression of PRDX4 in the endoplasmic reticulum reduces NF-ĸB activity. NF-ĸB plays a role in cell proliferation and survival. A study has shown that PRDX4 is bound to the endothelium and is secreted when there is a redox change in the extracellular matrix [59]. This study is supported with the presence of high PRDX4 in the serum that indicates membrane leakage due to tissue destruction and cell apoptosis [60].

#### **4.2 Mechanism of peroxiredoxin-4 as an antioxidant**

PRDX4 triggers H2O2 signaling and protects cells from oxidative stress by oxidizing thioredoxin (Trx) proteins. Trx is a group of co-factor proteins with chaperone activity to control cell homeostasis and inhibit H2O2 signaling pathway [61]. PRDX4 requires Trx peroxidase activity as an electron donor. The oxidized Trx is recycled by Trx reductase and NADPH as the primary electron donor. The main target of H2O2 molecules is two cysteine residues on PRDX4 to form catalytic peroxide mechanisms [62]. PRDX4 has two cysteine residues known as peroxidatic cysteine residue (CysP-) and resolution cysteine residue (CysR-). These two cysteine subunits have peroxidatic properties to oxidize H2O2.

When the oxidative stress level is high, CysP- acts on peroxide to form sulfenic acid (CysP-SOH). CysP- is one of the cysteine units located near the N-terminal of PRXD4. CysP-SOH then reacts with another systemic subunit known as CysR- to form a stable disulfide bond, and water molecule is produced. Trx enzyme reduces the disulfide bond and completes the PRDX4 catalytic cycle by detoxifying the peroxide and producing an active thiol. If the oxidative stress keeps increasing, –SOH is

**109**

*Silencing of Peroxiredoxin-4 in Anticancer Activity of Gamma-Tocotrienol*

oxidized to –SO2H [63]. PRDX4 in -SO2H is reduced and re-oxidized by sulfiredoxin (Srx) through the reduction of ATP to ADP. This process is reversible [1]. However, in high oxidation state, –SO2H is continuously oxidized to –SO3H, and PRDX4 will undergo hyperoxidation. This will cause inhibition of oxidation, and PRDX4 will activate other pathways to counteract the oxidation state. In conclusion, PRDX4 antioxidant activity is inhibited in high oxidation state and indirectly activates the

Studies have shown that the ER produces higher level of H2O2 than the mitochondria [65]. The main source of H2O2 in the ER lumen is the reaction of oxidase-1 in the ER (Ero1) with disulfide isomerase protein (PDI) for disulfide bond formations during protein synthesis. The disulfide bonds are important to prevent the protein from degrading after it is excreted from the ER lumen. Disulfide bonds formation involves high oxidative processes, and the unfolded bonds on damaged proteins may cause unfolded protein response (UPR). Unfolded proteins are degraded by dissolution of disulfide bonds before the protein is bound to the cytosol membrane [66]. UPR is the action of cells to balance the increasing oxidative stress and repair the function of or degrade the unfolded protein. The UPR will activate chaperone proteins of multiple pathways to balance the oxidative stress [67]. High oxidative stress in the ER will induce UPR and activate Ero1 to oxidize PDI catalytic enzymes by using oxygen molecules as the electron receiver [68]. The UPR action will form three intermediate sensors which are ATF6, IRE1α, and PERK and at the same time increase Ero1 and PRDX4 expressions as homeo-

**5. The effect of viability, apoptosis rate, and reactive oxygen species production in HepG2 cell silenced with peroxiredoxin-4 and treated** 

Gamma-tocotrienol has been shown to have high antitumor activity [12] that exhibits cell cycle arrest and apoptosis in alveolar adenocarcinoma epithelial cells, A549 [70], and colorectal cancer cell HT-29 [71]. Its action is specific and causes the cancer cells to be more susceptible and sensitive to treatment [72]. A previous study by Sazli et al. [7] reported that PRDX4 was upregulated when HepG2 cells were treated with GTT. In this study, we silenced *PRDX4* gene and treated with 70 μM of GTT to determine the role of PRDX4 in anticancer activity of GTT. Finding has shown that GTT treatment reduces HepG2-shRNA-PRDX4 cell viability significantly (*p* < 0.01) compared to its control (HepG2-shRNA-PRDX4) and also compared to HepG2 treated with GTT (*p* < 0.05) (**Table 1**). This viability assay is the first line of observation to show that GTT treatment does have an effect toward the

To investigate further the cause of reduction in cell viability, an apoptosis assay was done. The findings showed that the apoptosis rate was significantly increased (*p* < 0.05) in HepG2 treated with GTT compared to HepG2 control. Besides that, apoptosis rate increased in HepG2-shRNA-PRDX4-GTT (*p* < 0.05) compared to HepG2-shRNA-PRDX4 and HepG2-GTT (*p* < 0.05). GTT is capable to induce intrinsic and extrinsic apoptotic pathways in cancer cells such are breast cancer cell line, MCF-7, and MDA-MB-231 by increasing biomarker stress in the endoplasmic reticulum [73]. It is suggested that the main reason for the apoptosis in GTTtreated group is high level of ROS production. GTT has triggered ROS production through PRDX4 activity. Studies reported that one of the anticancer activities of GTT is apoptosis induction through increases of ROS production [74]. We have postulated that GTT might trigger the ROS production through PRDX4 activity.

*DOI: http://dx.doi.org/10.5772/intechopen.88813*

ER-pressure pathway [64].

static chaperones [69].

**with GTT**

silenced *PRDX4* gene.

*Synthetic Biology - New Interdisciplinary Science*

and protein expression of targeted gene.

**4.1 Peroxiredoxin-4 and its function**

by reducing H2O2 to water in order to reduce oxidative stress.

**4.2 Mechanism of peroxiredoxin-4 as an antioxidant**

subunits have peroxidatic properties to oxidize H2O2.

**4. Peroxiredoxin-4**

cell apoptosis [60].

the plasmid carried by the lentivirus has no effect on the cell. Although shRNA functions are at the mRNA level, only changes in protein expression may contribute to phenotypic changes. Therefore, validation of the mRNA and protein expression of the targeted proteins should be carried out to ensure that the gene expression is suppressed [53]. In addition, it is essential to validate the silencing effect by using more than two sequences of targeted gene shRNA. Every segment of gene sequence has a different effect in silencing the gene. The best sequence is selected based on the higher silencing rates shown through reduction of mRNA

H2O2 is produced during the disulfide bond formation in protein synthesis and from a side effect of cell biology mechanism located in the ER lumen and extracellular matrix [54]. PRDX4 is localized in the endoplasmic reticulum and highly expressed in the pancreas, liver, and heart but is low in blood and brain leukocytes [5]. PRDX4 plays a role as an antioxidant by scavenging H2O2 and is a chaperone molecule to activate ER stress pathway [55]. PRDX4 is important for cell protection

In addition, PRDX4 has a role in regulating the activation of NF-ĸB transcription factor and TNF-related apoptosis-inducing ligand (TRAIL) [56]. TRAIL is a transmembrane protein involved in apoptosis extrinsic pathway by binding to receptors DR4 or DR5. A reduction in PRDX4 expression leads to TRAIL activation and apoptosis induction, whereas an increase in PRDX4 expression inhibits the TRAIL activation and leads to cell survival [57]. The expression of PRDX4 in the cytosol increases NF-ĸB activity through phosphorylation of IκB-α [58]. Meanwhile, the expression of PRDX4 in the endoplasmic reticulum reduces NF-ĸB activity. NF-ĸB plays a role in cell proliferation and survival. A study has shown that PRDX4 is bound to the endothelium and is secreted when there is a redox change in the extracellular matrix [59]. This study is supported with the presence of high PRDX4 in the serum that indicates membrane leakage due to tissue destruction and

PRDX4 triggers H2O2 signaling and protects cells from oxidative stress by oxidizing thioredoxin (Trx) proteins. Trx is a group of co-factor proteins with chaperone activity to control cell homeostasis and inhibit H2O2 signaling pathway [61]. PRDX4 requires Trx peroxidase activity as an electron donor. The oxidized Trx is recycled by Trx reductase and NADPH as the primary electron donor. The main target of H2O2 molecules is two cysteine residues on PRDX4 to form catalytic peroxide mechanisms [62]. PRDX4 has two cysteine residues known as peroxidatic cysteine residue (CysP-) and resolution cysteine residue (CysR-). These two cysteine

When the oxidative stress level is high, CysP- acts on peroxide to form sulfenic acid (CysP-SOH). CysP- is one of the cysteine units located near the N-terminal of PRXD4. CysP-SOH then reacts with another systemic subunit known as CysR- to form a stable disulfide bond, and water molecule is produced. Trx enzyme reduces the disulfide bond and completes the PRDX4 catalytic cycle by detoxifying the peroxide and producing an active thiol. If the oxidative stress keeps increasing, –SOH is

**108**

oxidized to –SO2H [63]. PRDX4 in -SO2H is reduced and re-oxidized by sulfiredoxin (Srx) through the reduction of ATP to ADP. This process is reversible [1]. However, in high oxidation state, –SO2H is continuously oxidized to –SO3H, and PRDX4 will undergo hyperoxidation. This will cause inhibition of oxidation, and PRDX4 will activate other pathways to counteract the oxidation state. In conclusion, PRDX4 antioxidant activity is inhibited in high oxidation state and indirectly activates the ER-pressure pathway [64].

Studies have shown that the ER produces higher level of H2O2 than the mitochondria [65]. The main source of H2O2 in the ER lumen is the reaction of oxidase-1 in the ER (Ero1) with disulfide isomerase protein (PDI) for disulfide bond formations during protein synthesis. The disulfide bonds are important to prevent the protein from degrading after it is excreted from the ER lumen. Disulfide bonds formation involves high oxidative processes, and the unfolded bonds on damaged proteins may cause unfolded protein response (UPR). Unfolded proteins are degraded by dissolution of disulfide bonds before the protein is bound to the cytosol membrane [66]. UPR is the action of cells to balance the increasing oxidative stress and repair the function of or degrade the unfolded protein. The UPR will activate chaperone proteins of multiple pathways to balance the oxidative stress [67]. High oxidative stress in the ER will induce UPR and activate Ero1 to oxidize PDI catalytic enzymes by using oxygen molecules as the electron receiver [68]. The UPR action will form three intermediate sensors which are ATF6, IRE1α, and PERK and at the same time increase Ero1 and PRDX4 expressions as homeostatic chaperones [69].
