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

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 silenced *PRDX4* gene.

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.


*The data is presented as the mean ± standard deviation. Each group consists of technical triplicate and three biological replicates. GTT, gamma-tocotrienol; PRDX4, peroxiredoxin-4; HepG2-shRNA-PRDX4, HepG2 cell with PRDX4 gene silenced.*

*a A significant difference compared to HepG2 group (p < 0.05).*

*b A significant difference compared to HepG2-shRNA-PRDX4 group (p < 0.01).*

*c A significant difference compared to HepG2 GTT-treated group (p < 0.05).*

#### **Table 1.**

*The effect of GTT treatment on HepG2 and HepG2-shRNA-PRDX4 cell viability, apoptosis rate, and free radical production.*

Hence, the level of ROS in each group was measured. The result showed that GTT increased ROS production (*p* < 0.05) in HepG2 group compared to the control but reduced ROS production in HepG2-shRNA-PRDX4 group. In HepG2-shRNA-PRDX4, ROS production was even higher compared to other groups but causes no effect on the cell viability. This proves that the function of PRDX4 is to reduce ROS level and oxidative stress. GTT either increases ROS production [75] to stimulate apoptosis pathway in HepG2 group or directly activate the apoptosis pathway in HepG2-shRNA-PRDX4.

To investigate further, protein profiling was done using LCMS machine to detect proteins expressed in HepG2-shRNA-PRDX4 group treated with GTT compare with HepG2-shRNA-PRDX4 as its control group. Protein profiling has shown a total of 3413 proteins expressed in HepG2-shRNA-PRDX4 and 3659 proteins expressed in HepG2-shRNA-PRDX4 treated with GTT. There are 2121 similar proteins expressed in both HepG2-shRNA-PRDX4 control group and HepG2-shRNA-PRDX4 treated with GTT. Statistical analysis has been done to differentiate protein which significantly expressed HepG2-shRNA-PRDX4 treated with GTT compared to HepG2 shRNA-PRDX4 control group using Fisher Exact test. The proteins are significantly expressed if the p value is less than 0.00398. Then, the significantly expressed protein is filtered and selected based on their functional processes that are involved in anticancer activity using UniProt and Reactome database.

There are 6 upregulated protein expressions that are CLU, NDRG1, NUDT2, PRDX5, RALB, and SLC25A6 (**Table 2**) and 14 downregulated proteins expression (**Table 3**) in HepG2-shRNA-PRDX4 group treated with GTT compared to HepG2-shRNA-PRDX4 control group. The downregulated protein expressions are EEF1A1, DHX9, PRDX1, RPS27, HIST2H2AA3, UBA52, UTP20, GSTP1, HSPB1 NPM1, PRDX2, PRDX6, PRKDC, and TXN—the significant different expressed proteins involved in multiple anticancer mechanism targeted pathway. Most of the upregulated proteins are involved in apoptotic pathway and DNA damage, whereas for the 14 downregulated proteins are involved in carcinogenesis pathway, anti-apoptotic, and cell cycle arrest. NDRG1, NUDT2, and PRDX5 expressions resulted from cellular response on ROS production causes on the downstream action. Those proteins trigger cell cycle arrest due to DNA damage and apoptosis [76]. This situation worsens the cell conditions; thus, GTT has increased pro-apoptotic proteins to induce apoptosis. The apoptotic pathway is regulated by the expression of RALB, SLC25A6, and CLU which mediate the releases of cytochrome c from mitochondria [77].

**111**

protein expression.

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

Clusterin CLU 0.0013 1.0 Release of

Protein NDRG1 NDRG1 0.0001 3.4 DNA damage

*P***-value Fold** 

**change**

NUDT2 0.00056 8.5 Apoptotic process,

PRDX5 0.0023 1.6 Apoptotic

RALB 0.0020 4.8 Apoptotic process,

SLC25A6 0.0003 1.4 Apoptotic process Pro-

**Functional process Mechanism**

Proapoptosis

DNA damage

Proapoptosis

Proapoptosis

Proapoptosis

apoptosis

cytochrome c from mitochondria

response, signal transduction by p53 class mediator, cellular response to hypoxia

cellular response to oxidative stress

process, cell redox homeostasis, cellular response to reactive oxygen species

cell cycle

**Accession name (Swiss Prot)**

The treatment of GTT on HepG2-shRNA-PRDX4 causes downregulated protein expression that is involved in cell cycle arrest, carcinogenesis pathway, proteins resulted in the ER stress, and anti-apoptotic. The level of ROS production was reduced in HepG2-shRNA-PRDX4 group treated with GTT, while the apoptosis activity was induced. EEF1A1, PRDX 1, PRDX2, PRDX6, and TXN are the proteins that function to reduce ROS level and thus become negative regulator for cell apoptosis [78]. Thus, GTT has shown to reduce the ROS accumulation in HepG2 shRNA-PRDX4. GTT also suppressed the HIST2H2AA3, UBA52, and UTP20 protein expressions that are involved in cell cycle arrest. The reduction of ROS level promotes cell proliferation [79], and this justifies the downregulation of protein expression for cell cycle arrest. The expression of DHX9 is downregulated, and it plays a role in regulating DNA repair. GTT in silenced cells reduces the ROS level

*List of upregulated protein expression in HepG2-shRNA-PRDX4 treated with GTT compared to HepG2-*

GTT also suppressed PRDX1 and RPS27 expression that are involved in carcinogenesis. PRDX1 is a positive regulator of stress-activated MAPK cascade, whereas protein RPS27 is involved in JNK cascade and Wnt signaling pathway. The activation of ER stress response leads to the expression of protein that involves the MAPK pathway through the activation of JNK as mediator [80]. Most of the downregulated proteins are involved in direct negative regulator of apoptosis or negative regulator of oxidative stress-induced proteins. The proteins are GSTP1, HSPB1, NPM1, PRDX2, PRDX6, PRKDC, TXN, and UBA52. Protein profiling of HepG2-shRNA-PRDX4 showed that GTT induces apoptosis by reducing oxidative stress in the endoplasmic reticulum and upregulated pro-apoptotic

and stimulates an apoptotic pathway to cause cell death.

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

**Functional cluster/ protein name**

Bis(5′-nucleosil) tetraphosphatase

Peroxiredoxin-5, mitochondrial

Ras-related protein

*shRNA-PRDX4 control group.*

Ral-B

**Table 2.**

ADP/ATP translocase 3


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

#### **Table 2.**

*Synthetic Biology - New Interdisciplinary Science*

*A significant difference compared to HepG2 group (p < 0.05).*

*A significant difference compared to HepG2-shRNA-PRDX4 group (p < 0.01).*

*A significant difference compared to HepG2 GTT-treated group (p < 0.05).*

HepG2-shRNA-PRDX4.

HepG2-shRNA-PRDX4 with 70 μM GTT

*PRDX4 gene silenced.*

*radical production.*

*a*

*b*

*c*

**Table 1.**

Hence, the level of ROS in each group was measured. The result showed that GTT increased ROS production (*p* < 0.05) in HepG2 group compared to the control but reduced ROS production in HepG2-shRNA-PRDX4 group. In HepG2-shRNA-PRDX4, ROS production was even higher compared to other groups but causes no effect on the cell viability. This proves that the function of PRDX4 is to reduce ROS level and oxidative stress. GTT either increases ROS production [75] to stimulate apoptosis pathway in HepG2 group or directly activate the apoptosis pathway in

*The effect of GTT treatment on HepG2 and HepG2-shRNA-PRDX4 cell viability, apoptosis rate, and free* 

**Cell groups Cell viability (%) Apoptosis rate (%) Free radical production** 

63 ± 10.38b,c 92.65 ± 6.58b,c 1.61 ± 0.01b

HepG2 100 ± 0.08 13.26 ± 1.97 1.00 ± 0.01 HepG2-shRNA-PRDX4 98 ± 12.33 18.91 ± 2.90 3.59 ± 0.33a HepG2 with 70 μM GTT 90 ± 8.98 76.09 ± 4.89a 2.32 ± 0.09a

*The data is presented as the mean ± standard deviation. Each group consists of technical triplicate and three biological replicates. GTT, gamma-tocotrienol; PRDX4, peroxiredoxin-4; HepG2-shRNA-PRDX4, HepG2 cell with* 

**(arbitrary unit)**

To investigate further, protein profiling was done using LCMS machine to detect proteins expressed in HepG2-shRNA-PRDX4 group treated with GTT compare with HepG2-shRNA-PRDX4 as its control group. Protein profiling has shown a total of 3413 proteins expressed in HepG2-shRNA-PRDX4 and 3659 proteins expressed in HepG2-shRNA-PRDX4 treated with GTT. There are 2121 similar proteins expressed in both HepG2-shRNA-PRDX4 control group and HepG2-shRNA-PRDX4 treated with GTT. Statistical analysis has been done to differentiate protein which significantly expressed HepG2-shRNA-PRDX4 treated with GTT compared to HepG2 shRNA-PRDX4 control group using Fisher Exact test. The proteins are significantly expressed if the p value is less than 0.00398. Then, the significantly expressed protein is filtered and selected based on their functional processes that are involved

There are 6 upregulated protein expressions that are CLU, NDRG1, NUDT2, PRDX5, RALB, and SLC25A6 (**Table 2**) and 14 downregulated proteins expression (**Table 3**) in HepG2-shRNA-PRDX4 group treated with GTT compared to HepG2-shRNA-PRDX4 control group. The downregulated protein expressions are EEF1A1, DHX9, PRDX1, RPS27, HIST2H2AA3, UBA52, UTP20, GSTP1, HSPB1 NPM1, PRDX2, PRDX6, PRKDC, and TXN—the significant different expressed proteins involved in multiple anticancer mechanism targeted pathway. Most of the upregulated proteins are involved in apoptotic pathway and DNA damage, whereas for the 14 downregulated proteins are involved in carcinogenesis pathway, anti-apoptotic, and cell cycle arrest. NDRG1, NUDT2, and PRDX5 expressions resulted from cellular response on ROS production causes on the downstream action. Those proteins trigger cell cycle arrest due to DNA damage and apoptosis [76]. This situation worsens the cell conditions; thus, GTT has increased pro-apoptotic proteins to induce apoptosis. The apoptotic pathway is regulated by the expression of RALB, SLC25A6, and CLU which mediate the

in anticancer activity using UniProt and Reactome database.

releases of cytochrome c from mitochondria [77].

**110**

*List of upregulated protein expression in HepG2-shRNA-PRDX4 treated with GTT compared to HepG2 shRNA-PRDX4 control group.*

The treatment of GTT on HepG2-shRNA-PRDX4 causes downregulated protein expression that is involved in cell cycle arrest, carcinogenesis pathway, proteins resulted in the ER stress, and anti-apoptotic. The level of ROS production was reduced in HepG2-shRNA-PRDX4 group treated with GTT, while the apoptosis activity was induced. EEF1A1, PRDX 1, PRDX2, PRDX6, and TXN are the proteins that function to reduce ROS level and thus become negative regulator for cell apoptosis [78]. Thus, GTT has shown to reduce the ROS accumulation in HepG2 shRNA-PRDX4. GTT also suppressed the HIST2H2AA3, UBA52, and UTP20 protein expressions that are involved in cell cycle arrest. The reduction of ROS level promotes cell proliferation [79], and this justifies the downregulation of protein expression for cell cycle arrest. The expression of DHX9 is downregulated, and it plays a role in regulating DNA repair. GTT in silenced cells reduces the ROS level and stimulates an apoptotic pathway to cause cell death.

GTT also suppressed PRDX1 and RPS27 expression that are involved in carcinogenesis. PRDX1 is a positive regulator of stress-activated MAPK cascade, whereas protein RPS27 is involved in JNK cascade and Wnt signaling pathway. The activation of ER stress response leads to the expression of protein that involves the MAPK pathway through the activation of JNK as mediator [80]. Most of the downregulated proteins are involved in direct negative regulator of apoptosis or negative regulator of oxidative stress-induced proteins. The proteins are GSTP1, HSPB1, NPM1, PRDX2, PRDX6, PRKDC, TXN, and UBA52. Protein profiling of HepG2-shRNA-PRDX4 showed that GTT induces apoptosis by reducing oxidative stress in the endoplasmic reticulum and upregulated pro-apoptotic protein expression.


**113**

**6. Conclusion**

*shRNA-PRDX4 control group.*

**Table 3.**

**Acknowledgements**

**Conflict of interest**

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

Thioredoxin TXN 0.0001 0.01 Negative

*P***-value Fold** 

UBA52 0.0011 0.70 Signal

UTP20 0.00010 0.20 Negative

**change**

**Functional process**

regulation of hydrogen peroxideinduced cell death

transduction by p53 class mediator resulting in cell cycle arrest, negative regulation of apoptotic process

regulation of cell proliferation **Mechanism**

Anti-apoptosis

Cell cycle arrest and Anti-apoptosis

Cell cycle arrest

Gene silencing is a technique to prevent the expression of certain genes. This technique is very useful to study biochemical pathway or produce therapeutics to treat cancer and diseases. Optimizing on the basic criteria for gene silencing is very important to achieve efficient silencing. GTT treatment reduces cell viability and causes apoptosis in both silenced HepG2-shRNA-PRDX4 and non-silenced HepG2 groups, but ROS production was increased non-silenced cells. The silencing of *PRDX4* gene in HepG2 cells caused ROS accumulation but did not cause cell death. Proteomic technique showed that GTT caused HepG2 cell death through activation of multiple pathways. It either triggers the apoptosis pathway directly in silenced cell or increases ROS production through PRDX4 activity, thus increasing pro-

*List of downregulated protein expression in HepG2-shRNA-PRDX4 treated with GTT compared to HepG2-*

The authors would like to express gratitude to all researchers and staff of the Biochemistry Department, Faculty of Medicine, the National University of Malaysia. This study was funded by the National University of Malaysia Medical Centre grant (FF-2016-063) and the Ministry of Higher Education under the Fundamental Research Grant Scheme (FRGS/1/2013/SKK01/UKM/02/2).

apoptotic and reducing anti-apoptotic protein expressions.

The authors declare no conflict of interest.

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

**Accession name (Swiss Prot)**

**Functional cluster/protein name**

Ubiquitin-60S ribosomal protein L40

Small subunit processome component 20 homolog


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

#### **Table 3.**

*Synthetic Biology - New Interdisciplinary Science*

**Accession name (Swiss Prot)**

*P***-value Fold** 

DHX9 0.0015 0.80 Positive

EEF1A1 0.0001 0.80 Response to

GSTP1 0.0001 0.60 Negative

HIST2H2AA3 0.0001 0.70 Negative

HSPB1 0.0001 0.70 Negative

Nucleophosmin NPM1 0.0018 0.80 Negative

Peroxiredoxin-1 PRDX1 0.0001 0.60 Regulation

Peroxiredoxin-2 PRDX2 0.0001 0.70 Negative

Peroxiredoxin-6 PRDX6 0.0001 0.60 Negative

PRKDC 0.0001 0.80 Negative

RPS27 0.0025 0.30 JNK cascade,

**change**

**Functional process**

regulation of DNA repair

endoplasmic reticulum stress

regulation of extrinsic apoptotic signaling pathway

regulation of cell proliferation

regulation of oxidative stress-induced intrinsic apoptotic signaling pathway

regulation of apoptotic process

of stressactivated MAPK cascade, response to oxidative stress

regulation of apoptotic process, response to oxidative stress

apoptosis regulation by regulating reactive oxygen species

regulation of apoptotic process

Wnt signaling pathway

**Mechanism**

Carcinogenesis

ER stress

Anti-apoptosis

Cell cycle arrest

Anti-apoptosis

Anti-apoptosis

Carcinogenesis

Anti-apoptosis

Anti-apoptosis

Anti-apoptosis

Carcinogenesis

**Functional cluster/protein name**

ATP-dependent RNA helicase A

Cluster of elongation factor 1-alpha 1

Glutathione S-transferase P

Cluster of histone H2A type 2-A

Heat shock protein beta-1

**112**

DNA-dependent protein kinase catalytic subunit

Cluster of 40S ribosomal protein S27

*List of downregulated protein expression in HepG2-shRNA-PRDX4 treated with GTT compared to HepG2 shRNA-PRDX4 control group.*

### **6. Conclusion**

Gene silencing is a technique to prevent the expression of certain genes. This technique is very useful to study biochemical pathway or produce therapeutics to treat cancer and diseases. Optimizing on the basic criteria for gene silencing is very important to achieve efficient silencing. GTT treatment reduces cell viability and causes apoptosis in both silenced HepG2-shRNA-PRDX4 and non-silenced HepG2 groups, but ROS production was increased non-silenced cells. The silencing of *PRDX4* gene in HepG2 cells caused ROS accumulation but did not cause cell death. Proteomic technique showed that GTT caused HepG2 cell death through activation of multiple pathways. It either triggers the apoptosis pathway directly in silenced cell or increases ROS production through PRDX4 activity, thus increasing proapoptotic and reducing anti-apoptotic protein expressions.

#### **Acknowledgements**

The authors would like to express gratitude to all researchers and staff of the Biochemistry Department, Faculty of Medicine, the National University of Malaysia. This study was funded by the National University of Malaysia Medical Centre grant (FF-2016-063) and the Ministry of Higher Education under the Fundamental Research Grant Scheme (FRGS/1/2013/SKK01/UKM/02/2).

#### **Conflict of interest**

The authors declare no conflict of interest.

*Synthetic Biology - New Interdisciplinary Science*
