**1. Introduction**

Peroxiredoxin-4 (PRDX4) is one of the unique isomers of peroxiredoxin (PRDX) and is located in the endoplasmic reticulum. It has a peroxidase site to oxidize hydrogen peroxide (H2O2) and thus functions as an antioxidant, increases cell proliferation, and is involved in regulating multiple signaling transduction pathways [1, 2]. Double cysteine residues at the peroxidase site of PRDX4 oxidize H2O2 and form water molecule. Oxidation is one of the processes that control H2O2 homeostasis in the cells and thus indirectly reduces the risk of high level of oxidative stress [3]. The increase in reactive oxygen species (ROS) production in the tumor cells has been linked to increased PRDX4 expression, which makes the cell susceptible toward apoptosis [4]. Besides that, PRDX4 is a potential tumor biomarker [5] because of its high expression in most of the tumor tissues as it plays a role in tumor

growth and progression. The pattern of *PRDX4* gene expression is different among cancer cells and depends on its role. *PRDX4* gene may be involved in increasing the immune system of a normal cell to kill the cancer cells or reducing the level of oxidative stress to make the cancer cell more insusceptible toward the anticancer activity. *PRDX4* gene expression is high in hepatocellular, pancreas, colon, and prostate cancer cells, whereas its expression is low in the lung, kidney, and thymus cancer cells [6]. Recent findings have shown that *PRDX4* gene is highly expressed in HepG2 liver cancer cells treated with gamma-tocotrienol (GTT) [7] and reduces the HepG2 viability [8]. However, currently, there are no studies yet that focus on the role of PRDX4 in GTT treatment.

GTT is one of the tocotrienol isomers of vitamin E. It has an unsaturated and short phenyl chain and is differentiated based on the methyl group located on the chromanol ring [9]. This structure enables GTT to pass through the saturated lipid bilayer membrane and to be absorbed by the cells efficiently. GTT and other tocotrienol isomers are renowned as antioxidants [10]. Besides that, GTT has also been proven to have the capability as a signaling molecule to induce anticancer mechanism through activation of multiple pathways [11]. Previous studies have shown that GTT has an effective antitumor activity compared to other tocotrienol isomers [12]. GTT as an anticancer agent is selective on malignant cells, is capable of targeting multiple signaling pathways simultaneously, and has a synergistic effect with chemotherapy [13, 14]. Hence, various studies have been done to identify GTT capabilities and its mechanism in anticancer activities.

To determine the role of PRDX4 in anticancer activity of GTT, *PRDX4* gene in HepG2 cells was silenced using lentivirus particles that carry the RNA sequence of *PRDX4* gene (shRNA-PRDX4). The shRNA-PRDX4 is complementary with the *PRDX4* gene sequence in the HepG2 cell. The *PRDX4* gene and protein expression was reduced and indirectly caused the genotypic and phenotypic changes in the cell [15]. The silenced HepG2 cells were resistant toward puromycin and also encoded green fluorescent protein (GFP). Puromycin resistance enabled the culture of only silenced cell in the media. The presence of GFP as a reporter gene that exhibits bright green fluorescence when exposed to ultraviolet light was used as a visual tag for the expression of other genes. To silence the gene using lentivirus, there are several critical factors to be considered to avoid false positive and to increase silencing efficiency [16]. In this study, the silenced HepG2 cells were treated with GTT for 48 h. Then, cell viability, apoptosis rate, and ROS production were determined. Protein profiling was done to further confirm the proteins involved in the pathways.

## **2. Gene silencing**

Gene silencing is used to study the role of specific genes by introducing antisense RNA to block the translation of messenger RNA (mRNA) and inhibit gene expression or translation. This biological process is known as RNA interference (RNAi). RNA plays a role as a mediator in regulating gene expression. In relation to this, synthetic RNAi is developed to mimic the targeted gene and reduce its expression [17]. The silencing possibly occurs during the transcription or translation phase. When the targeted gene is silenced, its expression is reduced to 70% without eliminating the whole expression [18]. This technique offers one step forward for the therapeutic strategy of specialized medication for patients to undergo treatment for cancer or infectious diseases [19].

There are three types of RNAi which are small interference RNA (siRNA), microRNA (miRNA), and small hairpin RNA (shRNA). siRNA is a double-stranded RNA comprising 20–25 nucleotides. siRNA sequences are coupled with a polymer

**105**

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

Thus, it can be applied in cell and animal model [27].

in the culture media to select for the silenced cells.

**3. Mechanism of gene silencing**

degrade the mRNA of the targeted gene.

and liposome carrier to enter the cell by using exogenous mechanism to silence the gene [20]. This process is known as transfection. shRNA is encoded specific RNA transcription which comprises 19–29 nucleotides. These nucleotides form a bridge for small hairpin of nine nucleotides [21]. shRNA silencing mechanism is known as transduction. The delivery of shRNA to the cell is through vectors such as plasmid, adenovirus, lentivirus, or retrovirus with a U6 promoter to regulate shRNA expression [22]. The vector will ensure that shRNA is expressed to silence the targeted gene. The silenced gene is inherited by the daughter cells [23]. miRNA is singlestranded RNA (ssRNA) which comprises 21–23 nucleotides. This type of RNAi is complementary with mRNA molecule and can be used to silence the gene [24]. In vivo studies have shown that RNAi silences the targeted gene without affecting other cellular activities such as interferon action which may inhibit protein synthesis [25]. siRNA can only be used in actively dividing cells and the silencing effect is temporary. This is because siRNA concentration decreases when the silenced cell divides. siRNA is also limited to cells with low susceptibility toward a foreign molecule [26]. In relation to this, the improvement of transfection method is required by introducing shRNA, which is able to silence the targeted gene in a more specific and effective way. In comparison to siRNA, shRNA and its lentivirus vector have the ability to stably integrate into the host genome. The silencing effect is passed to the daughter cell, resulting in permanent gene silencing. It increases the potential of the targeted gene to be silenced in nondividing cells, and the silencing effect can be delivered to cells that have low susceptibility toward lipid penetration.

Both siRNA and shRNA have the same silencing mechanism; however, the choice of RNAi method to be used depends on the cell type, the time required to silence the gene, and the duration to silence the gene whether temporarily or permanently. The lentivirus plasmid used in this study has a GFP sequence, which functions as reporter gene, puromycin-resistance sequence for silenced cell selection, and a shRNA sequence that is antisense to the *PRDX4* sequence. Thus, the percentage of transduced cells can be determined by viewing the cells under a fluorescent microscope and culturing the cells with an optimal dose of puromycin

The purpose of gene silencing is to regulate gene expression by degrading the targeted gene's product or reducing its mRNA translation through the delivery of RNAi agents into the cytoplasm [28]. Gene silencing mechanism involves both exogenous and endogenous pathways. The silencing mechanism of synthetic RNAi agents such as siRNA, shRNA, and miRNA is through the exogenous pathway, whereas the silencing mechanism for miRNA that exists naturally in the cell is through the endogenous pathway [29]. The mechanism of these three synthetic RNAi agents depends on the RNA-induced silencing complex (RISC) to cleave or

Further process of siRNA silencing occurs in the cytoplasm, whereas for shRNA and pre-miRNA, DNA integration occurs in the nucleus prior to the changes of pri-miRNA/pri-shRNA to the pre-miRNA/pre-shRNA [30]. The siRNA pathway is activated when dsRNA, together with the carrier complex, penetrates the cell membrane. Then, an endogenous dicer enzyme identifies the dsRNA sequence and splits it into small fragments of siRNA. The RISC complex binds to the siRNA fragments and causes RNA splitting for gene silencing to occur [31]. Plasmid carried by the lentiviral vector encodes the shRNA sequence of the

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

#### *Silencing of Peroxiredoxin-4 in Anticancer Activity of Gamma-Tocotrienol DOI: http://dx.doi.org/10.5772/intechopen.88813*

*Synthetic Biology - New Interdisciplinary Science*

role of PRDX4 in GTT treatment.

capabilities and its mechanism in anticancer activities.

growth and progression. The pattern of *PRDX4* gene expression is different among cancer cells and depends on its role. *PRDX4* gene may be involved in increasing the immune system of a normal cell to kill the cancer cells or reducing the level of oxidative stress to make the cancer cell more insusceptible toward the anticancer activity. *PRDX4* gene expression is high in hepatocellular, pancreas, colon, and prostate cancer cells, whereas its expression is low in the lung, kidney, and thymus cancer cells [6]. Recent findings have shown that *PRDX4* gene is highly expressed in HepG2 liver cancer cells treated with gamma-tocotrienol (GTT) [7] and reduces the HepG2 viability [8]. However, currently, there are no studies yet that focus on the

GTT is one of the tocotrienol isomers of vitamin E. It has an unsaturated and short phenyl chain and is differentiated based on the methyl group located on the chromanol ring [9]. This structure enables GTT to pass through the saturated lipid bilayer membrane and to be absorbed by the cells efficiently. GTT and other tocotrienol isomers are renowned as antioxidants [10]. Besides that, GTT has also been proven to have the capability as a signaling molecule to induce anticancer mechanism through activation of multiple pathways [11]. Previous studies have shown that GTT has an effective antitumor activity compared to other tocotrienol isomers [12]. GTT as an anticancer agent is selective on malignant cells, is capable of targeting multiple signaling pathways simultaneously, and has a synergistic effect with chemotherapy [13, 14]. Hence, various studies have been done to identify GTT

To determine the role of PRDX4 in anticancer activity of GTT, *PRDX4* gene in HepG2 cells was silenced using lentivirus particles that carry the RNA sequence of *PRDX4* gene (shRNA-PRDX4). The shRNA-PRDX4 is complementary with the *PRDX4* gene sequence in the HepG2 cell. The *PRDX4* gene and protein expression was reduced and indirectly caused the genotypic and phenotypic changes in the cell [15]. The silenced HepG2 cells were resistant toward puromycin and also encoded green fluorescent protein (GFP). Puromycin resistance enabled the culture of only silenced cell in the media. The presence of GFP as a reporter gene that exhibits bright green fluorescence when exposed to ultraviolet light was used as a visual tag for the expression of other genes. To silence the gene using lentivirus, there are several critical factors to be considered to avoid false positive and to increase silencing efficiency [16]. In this study, the silenced HepG2 cells were treated with GTT for 48 h. Then, cell viability, apoptosis rate, and ROS production were determined. Protein profiling was done to further confirm the proteins involved in the pathways.

Gene silencing is used to study the role of specific genes by introducing antisense RNA to block the translation of messenger RNA (mRNA) and inhibit gene expression or translation. This biological process is known as RNA interference (RNAi). RNA plays a role as a mediator in regulating gene expression. In relation to this, synthetic RNAi is developed to mimic the targeted gene and reduce its expression [17]. The silencing possibly occurs during the transcription or translation phase. When the targeted gene is silenced, its expression is reduced to 70% without eliminating the whole expression [18]. This technique offers one step forward for the therapeutic strategy of specialized medication for patients to undergo treatment

There are three types of RNAi which are small interference RNA (siRNA), microRNA (miRNA), and small hairpin RNA (shRNA). siRNA is a double-stranded RNA comprising 20–25 nucleotides. siRNA sequences are coupled with a polymer

**104**

**2. Gene silencing**

for cancer or infectious diseases [19].

and liposome carrier to enter the cell by using exogenous mechanism to silence the gene [20]. This process is known as transfection. shRNA is encoded specific RNA transcription which comprises 19–29 nucleotides. These nucleotides form a bridge for small hairpin of nine nucleotides [21]. shRNA silencing mechanism is known as transduction. The delivery of shRNA to the cell is through vectors such as plasmid, adenovirus, lentivirus, or retrovirus with a U6 promoter to regulate shRNA expression [22]. The vector will ensure that shRNA is expressed to silence the targeted gene. The silenced gene is inherited by the daughter cells [23]. miRNA is singlestranded RNA (ssRNA) which comprises 21–23 nucleotides. This type of RNAi is complementary with mRNA molecule and can be used to silence the gene [24].

In vivo studies have shown that RNAi silences the targeted gene without affecting other cellular activities such as interferon action which may inhibit protein synthesis [25]. siRNA can only be used in actively dividing cells and the silencing effect is temporary. This is because siRNA concentration decreases when the silenced cell divides. siRNA is also limited to cells with low susceptibility toward a foreign molecule [26]. In relation to this, the improvement of transfection method is required by introducing shRNA, which is able to silence the targeted gene in a more specific and effective way. In comparison to siRNA, shRNA and its lentivirus vector have the ability to stably integrate into the host genome. The silencing effect is passed to the daughter cell, resulting in permanent gene silencing. It increases the potential of the targeted gene to be silenced in nondividing cells, and the silencing effect can be delivered to cells that have low susceptibility toward lipid penetration. Thus, it can be applied in cell and animal model [27].

Both siRNA and shRNA have the same silencing mechanism; however, the choice of RNAi method to be used depends on the cell type, the time required to silence the gene, and the duration to silence the gene whether temporarily or permanently. The lentivirus plasmid used in this study has a GFP sequence, which functions as reporter gene, puromycin-resistance sequence for silenced cell selection, and a shRNA sequence that is antisense to the *PRDX4* sequence. Thus, the percentage of transduced cells can be determined by viewing the cells under a fluorescent microscope and culturing the cells with an optimal dose of puromycin in the culture media to select for the silenced cells.
