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ORIGINAL ARTICLE
Year : 2022 | Volume
: 18
| Issue : 7 | Page : 1994-2000
PRDX3 promotes resistance to cisplatin in gastric cancer cells
Hao Yan1, Xinyu Cai1, Shanshan Fu2, Xiubin Zhang2, Jianna Zhang2
1 Department of Gastroenterology, The First Affiliated Hospital of Shandong First Medical University and Shandong Provincial Qianfoshan Hospital; Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong Province, China
2 Department of Gastroenterology, The First Affiliated Hospital of Shandong First Medical University and Shandong Provincial Qianfoshan Hospital, Jinan, Shandong Province, China
Correspondence Address:
Jianna Zhang
No. 16766, Jingshi Road, Jinan, Shandong Province - 250014
China
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/jcrt.jcrt_970_22
Objective: This study aims to investigate peroxiredoxin 3 (PRDX3) expression in gastric cancer tissue and its effects on cisplatin resistance in gastric cancer cells and its possible mechanism.
Methods: PRDX3 expression in human gastric cancer tissue microarrays was detected via immunohistochemistry. The PRDX3 small interfering RNA (siPRDX3 group) and the negative control siNC (siNC group) were transfected into AGS and MKN-74 cell lines, respectively, whereas a blank control group was set up. Each group was treated with different cisplatin concentrations (0, 5, 10, 15, 20, 25, and 30 μg/ml), and the half-inhibitory concentration (IC50) of each group of the two cell lines was calculated using the CCK8 assay. The corresponding IC50 concentration of the siPRDX3 group in the two cell lines was used to treat cells of each group. Flow cytometry was used to detect cell apoptosis, and Western blotting was used to detect the expression levels of cleaved caspase-3 and Bax in each group.
Results: PRDX3 was overexpressed in gastric adenocarcinoma tissue compared with adjacent noncancer tissue (P = 0.0053). After cisplatin treatment, the IC50 in the siPRDX3 group of AGS cells (5.91 ± 0.18 μg/ml) and the siPRDX3 group of MKN-74 cells (3.48 ± 0.30 μg/ml) was significantly lower than in the corresponding siNC groups (10.01 ± 0.99 and 6.39 ± 0.70 μg/ml; P = 0.0022 and 0.0027, respectively). AGS cells (38.81% ± 1.69%) and MKN-74 cells (25.03% ± 2.80%) in the siPRDX3 group showed significantly higher apoptosis rates than in the corresponding siNC groups (23.17% ± 1.43% and 16.7% ± 1.39%; P = 0.0003 and 0.0099, respectively). The expression levels of cleaved caspase-3 and Bax were significantly higher in the siPRDX3 group of both cell lines than in the siNC group (P < 0.0001).
Conclusion: PRDX3 increases the gastric cancer cell resistance to cisplatin by reducing apoptosis and thus may serve as a target to overcome cisplatin resistance.
Keywords: Apoptosis, cisplatin resistance, gastric cancer, peroxiredoxin 3, reactive oxygen species
According to the Global Cancer Statistics report, gastric cancer is the fifth most common malignancy and the third most common cause of cancer-related deaths worldwide.[1] To prolong the recurrence-free and overall survival times, chemotherapy is still needed even after surgical treatment for advanced gastric cancer. Palliative chemotherapy is often required for patients with unresectable locally advanced gastric cancer and distant metastasis.[2],[3] Cisplatin (cis-diamminedichloroplatinum II) is one of the commonly used chemotherapy drugs, but cisplatin resistance, including intrinsic and acquired resistance, is one of the main factors limiting the efficacy of cisplatin.[4]
After cisplatin enters the cell, highly reactive monohydrate or dihydrate cisplatin is formed, and aquated cisplatin reacts with DNA to generate intrastrand and interstrand adducts. If cisplatin adducts cannot be repaired by the nucleotide excision repair (NER) or mismatch repair system, the cell cycle is blocked, or apoptosis occurs.[5] The formation of adducts between cisplatin and nuclear DNA is not the only mechanism by which cisplatin induces cytotoxicity, and cisplatin also has significant toxicity to enucleated cells.[6],[7] Some studies have shown that oxidative stress is related to cisplatin-induced cytotoxicity, and cisplatin induces intracellular reactive oxygen species (ROS) increase, thus inducing apoptosis.[8],[9],[10] Low cisplatin-induced ROS levels in cancer cells are related to drug resistance.[11]
Oxidative stress is the relative excess of intracellular ROS compared with antioxidants. The intracellular antioxidant system includes nonenzymatic small molecules and enzymatic antioxidants. Glutathione, as a typical nonenzymatic small molecule, can directly remove ROS. Enzymatic antioxidants include superoxide dismutase (SOD), catalase, glutathione peroxidase, and peroxiredoxins (PRDXs).[12] Affecting the expression of antioxidant enzymes can affect the efficacy of cisplatin. SOD1 expression is elevated in cisplatin-resistant human bladder urothelial cancer cells, which reduces cisplatin-induced ROS and apoptosis.[13] SOD1 inhibition in cisplatin-resistant ovarian cancer cells enhances the sensitivity of cisplatin-resistant cells.[14]
PRDXs are a family of thiol-based peroxidases that reduce peroxides with a conserved cysteine residue, which are classified into six different, structurally discernable members (PRDX1–PRDX6). In mammals, PRDX3 and PRDX5 are within the mitochondrial matrix.[15],[16] Among them, PRDX3 can remove 90% of hydrogen peroxide (H2O2) in the mitochondrial matrix because of its high efficiency in enzyme kinetics.[17] Studies have shown that PRDX3 expression in cisplatin-resistant human ovarian cancer cells is significantly higher than in cisplatin-sensitive groups.[18] However, PRDX3 expression in gastric cancer and its effects on cisplatin resistance remain unclear.
> Materials and MethodsImmunohistochemistry
Human gastric cancer tissue microarrays (Shanghai Biochip Co., Ltd.) were dewaxed, hydrated, subjected to high-temperature and high-pressure antigen retrieval, endogenous peroxidase-blocked, and incubated overnight at 4°C using PRDX3 monoclonal antibody (Abcam). After the addition of horseradish peroxidase-labeled goat antirabbit IgG polymer (Servicebio), the color was developed using 3,3′-diaminobenzidine (Servicebio) and counterstained with hematoxylin (Servicebio). Tissue microarray sections were scanned after immunohistochemical staining using a tissue microarray scanner (3DHISTECH, Pannoramic MIDI), and H-score scoring was performed using Quant Center analysis software. H-score = Σ(propidium iodide (PI) × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3), where PI is the percentage of positive cells in each section and I is the staining intensity. Our approval from the ethics committee is obtained and the date of the approval is 2016-02-16.[19]
Cell culture and transfection
Human gastric cancer cell lines AGS and MKN-74 were obtained from the American Type Culture Collection. AGS cells were seeded into Dulbecco's modified Eagle's medium (LIFE) containing 10% fetal bovine serum (Gibco), and MKN-74 cells were seeded into RPMI-1640 medium (LIFE) containing 10% fetal bovine serum, both cultured in a 37°C, 5% CO2 incubator. AGS and MKN-74 cells were seeded into six-well plates, and transfection was performed when cells were cultured to ~50% confluence. PRDX3 interfering RNA (siPRDX3 group) and negative control (siNC group) were transfected according to the instructions of the siTran1.0 transfection kit (Origene), and the blank control group was not transfected. The medium was replaced after 16 to 24 h transfection, and cells were collected for subsequent experiments. The siRNA sequences were as follows: siPRDX3-1 5′-CCATCTTGCCTGGATAAAT-3′, siPRDX3-2 5′-GCGTTCCAGTATGTAGAAA-3′, siPRDX3-3 5′-GGTTATTTGTAGAAGGCAA-3′, siPRDX3-4 5′-GGAAACACTCTTCTTTCTT-3′, and siNC 5′-TTCTCCGAACGTGTCACGT-3′.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells in each group using the Trizol kit (Kangwei), and the concentration and quality of the extracted RNA were determined using a Nanodrop 2000/2000C spectrophotometer (Thermo Fisher Scientific). PCR amplification was conducted after cDNA synthesis using the HiFiScript cDNA synthesis kit (Kangwei). The reaction system was 25 μL: 1 μL each of upstream and downstream primers, 1 μL cDNA template, 12.5 μL SYBR Premix Ex Taq, and double-distilled water supplemented to 25 μL. The PCR conditions are as follows: denaturation at 95°C for 15 s and annealing/extension at 60′ for 1 min. Reactions were run for 40 cycles, and the relative expression of the target gene was calculated using (2−ΔΔCt). The primer sequences are as follows: PRDX3-forward 5′-GTTGTCGCAGTCTCAGTGGA-3′, PRDX3-reverse 5′-AACAGCACACCGTAGTCTCG-3′, β-actin-forward 5′-AGACCTGTACGCCAACACAG-3′, and β-actin-reverse 5′-CGGACTCGTCATACTCCTGC-3′.
Protein extraction and Western blotting
Cells in each group were collected and lysed with RIPA buffer for 15 min on ice. After centrifugation, the supernatant was taken for quantification, and protein electrophoresis buffer was added for denaturation at 100°C for 10 min. A 20 μg denatured protein was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skimmed milk in Tris-buffered saline-Tween 20 (TBST) for 1 h at room temperature, the primary antibody and the internal reference protein glyceraldehyde 3-phosphate dehydrogenase (Proteintech) were added overnight at 4°C, respectively. The primary antibodies were PRDX3 (Abcam), cleaved caspase-3 (Affinity), and Bax (ABclonal). After washing with TBST, the membranes were incubated with antirabbit (ABclonal) or antimouse (ABclonal) secondary antibodies for 2 h at room temperature and developed using an enhanced chemiluminescence kit (Amersham). The protein band gray values were analyzed using ImageJ.
Cell proliferation assay
After transfection of AGS and MKN-74 cells with siPRDX3 and siNC, respectively, for 24 h, cells were collected and seeded into 96-well plates. Seven concentrations (0, 5, 10, 15, 20, 25, and 30 μg/ml) were set for each group with three replicate wells. Each well was incubated with 10 μg CCK8 (MCE) solution for 1 to 2 h. Absorbance at 450 nm was measured with a microplate reader, and the survival rate was calculated. Cell viability = [(absorbance of experimental wells − absorbance of blank wells)/(absorbance of control wells − absorbance of blank wells)] × 100%. The half-inhibitory concentration (IC50) was calculated by curve fitting with the cell survival rate as the ordinate and the concentration as the abscissa.
Apoptosis detection
As described previously, after transfection of AGS and MKN-74 cells with siPRDX3 and siNC, respectively, cells were treated with the corresponding cisplatin concentrations. After 48 h, cells were collected, and the apoptosis rate was detected using the Annexin V-fluorescein isothiocyanate (FITC)/PI kit (KeyGEN) according to the manufacturer's instructions.
Statistical analysis
All data were analyzed using GraphPad Prism8. All quantified data were expressed as mean ± standard deviation (x ± s), and t-tests were used to compare the two groups. The IC50 values were calculated by nonlinear regression (curve fitting). P < 0.05 was regarded as statistically significant.
> ResultsPRDX3 expression is higher in gastric cancer tissue than in adjacent noncancer tissue
Immunohistochemical analysis was conducted to examine PRDX3 expression in gastric cancer tissue microarray. Positive PRDX3 staining was observed predominantly in the cytoplasm of normal epithelial and cancer cells. Immunostaining results indicated that PRDX3 expression in gastric cancer tissue (n = 31) was significantly higher than in adjacent noncancer tissue (n = 31; P = 0.0053; [Figure 1]).
Figure 1: PRDX3 expression level in adjacent noncancer and gastric cancer tissue (original magnification, ×400). (a) Immunohistochemistry images of normal gastric tissue. (b) Immunohistochemistry images of moderately differentiated adenocarcinoma. (c) Immunohistochemistry images of poorly differentiated adenocarcinoma. (d) PRDX3 expression in gastric cancer tissue was significantly higher than in adjacent noncancer tissue. **P < 0.01PRDX3 knockdown increases the sensitivity of AGS and MKN-74 gastric cancer cells to cisplatin
To examine the effects of PRDX3 on the sensitivity of AGS and MKN-74 cells to cisplatin, four siRNAs (siPRDX3-1, siPRDX3-2, siPRDX3-3, and siPRDX3-4) were used to downregulate PRDX3 expression levels in the two cell lines, respectively. Based on the qRT-PCR results, S2 and S4 were selected as the siRNAs used for subsequent experiments with AGS and MKN-74, respectively. qRT-PCR and Western blotting results showed that PRDX3 was significantly downregulated at the mRNA (P < 0.0001) and protein (P < 0.0001) levels in AGS cells. In MKN-74 cells, PRDX3 was also significantly downregulated at the mRNA (P = 0.0032) and protein (P < 0.0001) levels [Figure 2]. CCK8 assays showed that the sensitivity of cells to cisplatin increased after PRDX3 knockdown in AGS and MKN-74 cells, whereas the IC50 was significantly lower than in the siNC group [Figure 3].
Figure 2: PRDX3 is downregulated by siRNA in AGS and MKN-74 cells. (a) Relative mRNA expression of PRDX3 in AGS after transfection with siNC and four siRNAs. (b) Relative mRNA expression of PRDX3 in MKN-74 after transfection with siNC and four siRNAs. (c) Relative PRDX3 protein expression in AGS after transfection with siNC and siPRDX3. (d) Relative PRDX3 protein expression in MKN-74 after transfection with siNC and siPRDX3. (e) Western blot bands of PRDX3 protein in AGS and MKN-74 after transfection with siNC and siPRDX3
Figure 3: IC50 values of each group after PRDX3 knockdown in AGS and MKN-74 cells. (a) Control (CON) group of AGS. (b) siNC group of AGS. (c) siPRDX3 group of AGS. (d) CON group of MKN-74. (e) siNC group of MKN-74. (f) siPRDX3 group for MKN-74. (g) Comparative histogram of IC50 in each group in AGS cells. (h) Comparative histogram of IC50 in each group in MKN-74 cells. **P < 0.01Downregulation of PRDX3 expression increased the rate of cisplatin-induced apoptosis in gastric cancer cells
To further identify the effects of PRDX3 on cisplatin-induced gastric cancer cell apoptosis, flow cytometry analysis of gastric cancer cells was conducted after cisplatin treatment using Annexin V-FITC/PI double staining. Results showed that PRDX3 knockdown significantly increased the rate of cisplatin-induced gastric cancer cell apoptosis in AGS (P = 0.0003) and MKN-74 (P = 0.0099) cells [Figure 4]. Western blotting results indicated that the related proteins Bax (P < 0.0001) and cleaved caspase-3 (P < 0.0001) in the apoptotic pathway in AGS cells after cisplatin treatment were significantly higher in the siPRDX3 group than in the siNC group. The Bax (P < 0.0001) and cleaved caspase-3 (P < 0.0001) levels were also significantly higher in the siPRDX3 group of MKN-74 cells than in the siNC group [Figure 5]. Thus, downregulation of PRDX3 expression in the two gastric cancer cell lines increased the sensitivity of gastric cancer cells to cisplatin by inducing apoptosis.
Figure 4: Apoptosis results of AGS and MKN-74 cells treated with the siNC, siPRDX3, and control groups after cisplatin treatment. Lower left (Q1-LL), live cells; lower right (Q1-LR), early apoptosis; upper right (Q1-UR), late apoptosis; upper left (Q1-UL), necrotic/dead cells. (a) CON group of AGS cells. (b) siNC group of AGS cells. (c) siPRDX3 group of AGS cells. (d) CON group of MKN-74 cells. (e) siNC group of MKN-74 cells. (f) siPRDX3 group for MKN-74 cells. (g) Comparative histogram of apoptosis in each group in AGS cells. (h) Comparative histogram of apoptosis in each group in MKN-74 cells
Figure 5: Expression level of the related proteins in the apoptosis pathway in AGS and MKN-74 cells transfected with siPRDX3 after cisplatin treatment. ***P < 0.001. (a) Western blot bands. (b) Relative expression of cleaved caspase-3 protein in each group of AGS cells. (c) Relative expression of Bax protein in each group of AGS cells. (d) Relative expression of PRDX3 protein in each group of AGS cells. (e) Relative expression of cleaved caspase-3 protein in each group of MKN-74 cells. (f) Relative expression of Bax protein in each group of MKN-74 cells. (g) Relative expression of PRDX3 protein in each group of MKN-74 cells > Discussion Oxidative stress is one of the signature events of cancer cells mainly due to its different metabolic and signal transduction processes from normal cells.[20] Because of its definite genotoxicity, mildly elevated ROS not only plays a role in the transformation of malignant tumors but also promotes the proliferation and metastasis of cancer cells through signal transduction cascades during tumor progression.[21] During tumor initiation, development, and metastasis, ROS levels in cancer cells gradually increase, and the redox state of cells tends to be oxidized.[22] As the degree of oxidative stress within cancer cells increases, antioxidant mechanisms need to be enhanced accordingly to maintain intracellular ROS levels below the threshold that triggers apoptosis and necrosis.
ROS is mainly produced in the mitochondria, and PRDX3, the main peroxidase in the mitochondria, removes 90% of H2O2 in the mitochondrial matrix. PRDX3 uses its conserved cysteine residue as the peroxide oxidation site. The peroxidative Cys (CP) of PRDX3 reacts with H2O2 to form a sulfonic acid intermediate, which condenses with the resolving Cys of the adjacent PRDX3 molecule to form an intermolecular dimer.[15] Thioredoxin 2 (Trx2) transfers electrons to PRDX3 for reduction, whereas thioredoxin reductase 2 in the mitochondria can reduce Trx2 in turn.[23]
PRDX3 expression increases in lung,[24] prostate,[25] endometrial,[26] and liver[27] cancers. In this study, PRDX3 expression was significantly higher in gastric cancer than in adjacent tissue.
ROS-mediated apoptosis is one of the anticancer mechanisms of cisplatin. Intracellular oxidative stress can induce mitochondrial outer membrane permeabilization, resulting in the release of proapoptotic proteins, such as cytochrome c, Smac/DIABLO (second mitochondrial activator of caspase/direct IAP-binding protein with low PI), and HTRA2 (HtrA serine peptidase 2) into the cytoplasm, and activation of effector caspases.[28],[29] Therefore, in the application of cisplatin, at the same time, interventions that increase the ROS levels directly or reduce ROS scavenging can further increase intracellular ROS levels and enhance the sensitivity of cancer cells to cisplatin. This study also showed that PRDX3 downregulation increased the sensitivity of gastric cancer cells to cisplatin by increasing cisplatin-induced apoptosis.
One of the mechanisms leading to the increase of ROS in cancer cells is that cisplatin leads to mitochondrial DNA damage. Because of the lack of the NER system in the mitochondria, mitochondrial DNA adducts formed by cisplatin cannot be removed, interfering with protein synthesis related to the electron transport chain encoded by mitochondrial DNA. Consequently, the respiratory chain function is impaired, leading to ROS production.[8]
Meanwhile, cisplatin causes nuclear and mitochondrial DNA damage, which activates peroxisome proliferator-activated receptor-γ coactivator-1α and increases transcription factor A (TFAM) expression. TFAM transposition to the mitochondria leads to mitochondrial biosynthesis and activation, thereby increasing mitochondrial ROS and subsequent apoptosis of cancer cells.[30]
In summary, this study showed that PRDX3 expression was significantly elevated in gastric cancer tissue. Downregulation of its expression in vitro increased the sensitivity of gastric cancer cells to cisplatin by increasing cisplatin-induced apoptosis. Therefore, PRDX3 may be a target for overcoming cisplatin resistance.
> ConclusionsIn this study, PRDX3 was significantly higher expressed in gastric cancer tissue than in paracancerous tissue. PRDX3 also increased the resistance of gastric cancer cells to cisplatin by reducing apoptosis. These findings suggested that PRDX3 may be a potential candidate as a target for overcoming cisplatin resistance. More research is needed to determine its clinical guidance in improving the efficacy of chemotherapeutic agents for gastric cancer.
Financial support and sponsorship
This project was granted by the Key Research and Development Project of Shandong Province, China (2018GSF118145) and Medical and Health Science and Technology Development Plan Project of Shandong Provincial, China (2017WS084).
Conflicts of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
> References
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