Downregulation of MUTYH contributes to cisplatin‑resistance of esophageal squamous cell carcinoma cells by promoting Twist‑mediated EMT
- Authors:
- Published online on: October 1, 2019 https://doi.org/10.3892/or.2019.7347
- Pages: 2716-2727
Abstract
Introduction
Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal carcinoma, is one of the most refractory cancer types with a high mortality rate in the world (1,2). Although various treatment regimens have been adopted to improve curability, the overall 5-year survival rate for patients with ESCC over the past 30 years is still poor at ~20% (2). At present, surgical treatment combined with radiotherapy and chemotherapy is the main clinical therapeutic method of ESCC, and cisplatin (CDDP) is still used as one of the pivotal chemotherapeutic drugs in the front-line therapeutic regimen, playing an irreplaceable role in prevention of recurrence and metastasis of ESCC (3). However, satisfactory chemotherapeutic effects have rarely been achieved, as CDDP-based therapies always just obtain initial chemotherapeutic success and eventually induce CDDP resistance in ESCC (4,5).
It has been proposed that several molecular mechanisms can drive chemoresistance to CDDP, which may be unique to different types of cancer (4,5). Prominent CDDP-resistance mechanisms involve various biological regulatory processes, including blockade of DNA damage response signal, activation of DNA repair pathways, epithelial-mesenchymal transition (EMT), thiols and metallothionein-mediated detoxification, and efflux of CDDP from cancer cells (4). Despite the progress in identifying mechanisms responsible for resistance to CDDP, the underlying mechanisms of resistance in ESCC are not entirely understood and the clinical treatment of CDDP-resistant ESCC remains a critical obstacle. Therefore, the present study aimed to further elucidate mechanisms behind CDDP resistance in ESCC and novel biomarkers, which may be used to predict prognosis and chemosensitivity.
Accumulating evidence has suggested that CDDP-induced nuclear DNA damage alone is not enough to illustrate its high-level of effectiveness or cytotoxicity (6,7). Several recent studies have demonstrated that, independent of CDDP-DNA adduct generation, the mechanism of CDDP-stimulated production of reactive oxygen species (ROS) and subsequent oxidative damage of the DNA base also have a crucial involvement in CDDP cytotoxicity (7–9). One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine, which can induce G:C to T:A transversion mutations. The mutations are recognized and repaired by mutY homolog (MUTYH) (10,11). MUTYH is a key component of the system of base excision repair (BER), which is able to remove the oxidized bases paired erroneously, thereby ensuring the maintenance of DNA integrity (10,11). It has been confirmed by experimental evidence that MUTYH has an important role in various cancer types; it was reported that modification of the tissue expression of MUTYH is associated with the elevated risk of development of various cancer types, such as colorectal cancer, lung cancer, esophageal carcinoma, thyroid cancer and head and neck cancer (12–17). Mutations in MUTYH reduce BER effectiveness and predisposition to cancer (17,18). Recent research also suggests that patients with colorectal cancer have lower MUTYH expression in cancer tissues compared with normal tissues (19). Meanwhile, the MUTYH gene was found to present reduced expression in more advanced stages of colorectal cancer, and lower MUTYH expression may contribute to worse patient prognosis (12). In the present study, the association of MUTYH with CDDP resistance in ESCC cells was analyzed. The present data demonstrated that downregulation of MUTYH induced the EMT process by regulating both the transcription and degradation of Twist, which eventually contributed to resistance to CDDP.
Materials and methods
Establishment of a CDDP-resistant ESCC cell line
The human ESCC cell line EC109 was obtained from The Cell Bank of the Chinese Academy of Sciences and was maintained in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (HyClone; GE Healthcare Life Sciences). The CDDP-resistant cell line EC109/CDDP was selected by culturing EC109 cells in increasing concentrations of CDDP (Selleck Chemicals) starting from 0.1 µM to a final concentration of 5.0 µM. The cells were maintained in a humidified incubator with 5% CO2 at 37°C.
Cell viability and cell death analysis
The inhibitory effect of CDDP on cell viability in EC109 and EC109/CDDP cells was determined by an MTT (Sigma-Aldrich; Merck KGaA) colorimetric assay using a Thermo Fisher Multiskan microplate reader (Thermo Fisher Scientific, Inc.).
Apoptosis induced by CDDP in EC109 and EC109/CDDP cells was tested using an Annexin V-FITC/Propidium Iodide Apoptosis Detection kit, according to the manufacturer's protocol (BD Biosciences). A flow cytometer (FACSCaliber; BD Biosciences) was used for fluorescence quantification.
Proteasome activity assay
The proteasome substrates Suc-LLVY-AMC (N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), Bz-LLE-AMC (benzyloxycarbonyl-L-leucyl-leucyl-glutamyl-methylcoumarylamide) and Bz-VGR-AMC (Bz-Val-Gly-Arg-7-amino-4-methylcoumarin) from Enzo Life Sciences, Inc., were used to detect activities of chymotrypsin-like (ChT-L), trypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) of proteasomes in cells, respectively. Whole cell lysates were extracted with lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40 and 2 mM DTT], and incubated at 37°C for 40 min with the substrates in the assay buffer. Proteasome activities were determined by measuring fluorescent activities of the substrates using a Mithras LB-940 reader (Berthold Technologies).
Transwell Matrigel invasion assay
Cell invasion was measured by a Transwell Matrigel invasion assay using Growth Factor Reduced Matrigel Invasion Chambers from BD Biosciences. In total, 0.5×106 cells were resuspended in RPMI-1640 medium supplemented with 1% FBS serum into the upper chamber with Matrigel matrix added (BD Biosciences). RPMI-1640 medium supplemented with 10% serum, serving as a chemoattractant, was added to the bottom well. After 48 or 72 h, cells that migrated to the bottom chamber were stained with Giemsa (Bejing Solarbio Science & Technology Co., Ltd.) and were imaged under a bright-field microscope (Nikon Corp.; Magnification, ×200). The stained cells were counted using ImageJ software (National Institutes of Health).
Wound healing assay
A wound healing assay was performed to examine cell migration. When cells grew to approximately 95% confluence in 6-well plates, a scratch (wound) was introduced using a pipette tip. Then, the cells were washed three times with PBS to remove detached cells and cultured continually for 24 or 48 h. The wound was imaged under a bright-field microscope (Nikon Corp.; magnification, ×100) at 0, 24 and 48 h. The wound gaps were digitally quantified using ImageJ software (National Institutes of Health). The equation for the ‘relative wound area’ is: Relative wound area=wound area of different group/wound area of EC109 cells at 0 h.
ROS measurement
The production of ROS was monitored using the fluorescent dye hydroethidine 2′,7′-dichlorodihydrofluorescein diacetate [H(2)DCFDA; Sigma-Aldrich; Merck KGaA]. After treatment as indicated, cells were incubated with 10 µM H(2)DCFDA at 37°C for 30 min. Then, the cells were washed with PBS and subjected to ROS measurement by flow cytometry. In the experiments, to test the scavenging effect of the antioxidant N-acetylcysteine (NAC; Sigma-Aldrich; Merck KGaA) on ROS generation, cells were treated with 2 mM NAC for 24 h before ROS measurement.
Western blot assay
A western blot assay was performed, as previously described (20). Whole cell lysates were prepared with RIPA buffer, according to the manufacturer's protocol (Beyotime Institute of Biotechnology). Proteins were quantified using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). Blots were incubated with primary antibodies against MUTYH (cat. no. 19650-1-AP; rabbit polyclonal antibody; dilution 1:500), E-cadherin (cat. no. 60335-1-lg; mouse monoclonal antibody; dilution 1:2,000), vimentin (cat. no. 60330-1-lg; mouse monoclonal antibody; dilution 1:2,000), Twist (cat. no. 25465-1-AP; rabbit polyclonal antibody; dilution 1:500) and ubiquitin (cat. no. 10201-1-AP; rabbit polyclonal antibody; dilution 1:500) (all from ProteinTech Group, Inc.) overnight at 4°C, followed by incubation with appropriate peroxidase-conjugated secondary antibodies (anti-mouse IgG (H+L) peroxidase-labeled polyclonal antibody, cat. no. 074-1806; dilution 1:5,000; anti-rabbit IgG (H+L) peroxidase-labeled polyclonal antibody; cat. no. 074-1506; dilution 1:5,000; purchased from KPL, Inc.; SeraCare Life Sciences, Inc.). GAPDH (cat. no. sc-47724; mouse monoclonal antibody; dilution 1:3,000; Santa Cruz Biotechnology, Inc.) served as a protein loading control. Immunocomplexes were visualized using chemiluminescence (EMD Millipore) and exposed to X-ray film.
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was prepared using an RNAiso Plus Kit (Takara Bio, Inc.). cDNA was synthesized using a PrimeScript RT Reagent Kit (Takara Bio, Inc.) and RT-qPCR was carried out using the Eppendorf RT-qPCR System (Mastercycler ep realplex; Eppendorf). The PCR reaction conditions for all assays were as follows: 95°C for 30 sec, followed by 40 cycles of amplifcation (95°C for 5 sec, 58°C for 30 sec and 72°C for 30 sec). Changes in the mRNA levels were normalized to the level of GAPDH and calculated using the 2−ΔΔCq method (21). The primer sequences are summarized in Table I.
Transient transfection of plasmids and small interfering RNAs (siRNAs)
The pcDNA3.1-MUTYH plasmid was constructed to induce overexpression of MUTYH (Shanghai Integrated Biotech Solutions Co., Ltd.). Empty vector pcDNA3.1 served as a control. Knockdown of MUTYH was performed by transiently transfecting siRNA duplex oligonucleotides targeting MUTYH (BioSune). The MUTYH siRNA sequences were as follows: Sense, 5′-GGAGGCAGAAGCAUGCUAATT-3′ and antisense, 5′-UUAGCAUGCUUCUGCCUCCTT-3′. The scramble siRNA sequences were as follows: Sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. Transfection of the overexpression plasmid or siRNA was performed using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.
Statistical analysis
Data are presented as the mean ± SD and were analyzed using GraphPad Prism software (GraphPad Software, Inc.). The statistical significance of mean differences between the treated and control groups was determined using a two-tailed Student's unpaired t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
Evaluation of the CDDP resistance of the established CDDP-resistant ESCC cell line
The aim of the present study was to test the sensitivity of the established EC109/CDDP cells and the parental EC109 cells to CDDP. As shown in Fig. 1A, EC109/CDDP cells showed markedly higher cell viability than EC109 after 48 h treatment with CDDP at concentrations of 2.5–100 µM and the concentrations that induced cellular death at 50% (IC50) were calculated. EC109/CDDP cells were resistant to CDDP, with a relatively marginally increased IC50 value at ~50 µM compared with ~10 µM in EC109 cells. The resistant index (RI) of EC109/CDDP cells for CDDP was ~5 at 48 h. Fig. 1B demonstrates that the CDDP-resistance of EC109/CDDP cells was more prominent at 72 h of CDDP incubation, with an RI at ~7. Flow cytometry was performed to detect the apoptotic cells exposed to CDDP in EC109/CDDP cells. The present results demonstrated that CDDP at 5, 10, 20 and 40 µM caused significant increases in the proportion of apoptotic EC109 cells, ~14.25, 28.58, 43.81 and 56.00%, respectively. By comparison, the apoptotic EC109/CDDP cell fractions were 10.51, 12.72, 18.12 and 34.45% respectively, showing a significant reduction (Fig. 1C and D). Therefore, the present data demonstrated that EC109/CDDP cells were resistant to CDDP when compared with parental EC109 cells.
Downregulation of MUTYH contributes to CDDP resistance in ESCC cells
In the process of identifying potential targets involved in CDDP resistance, MUTYH, the crucial gene for excision repair of oxidized bases, was noted for its pronounced downregulation in the resistant cells. As shown in Fig. 2A, RT-qPCR analysis demonstrated that the mRNA expression of MUTYH decreased ~5-fold in resistant EC109/CDDP cells compared with that noted in the parental cells. Similar to the mRNA level, the resistant cells presented a significant decrease in the corresponding protein level of MUTYH (Fig. 2B).
To further investigate the effect of MUTYH in CDDP resistance, MUTYH was either overexpressed with a MUTYH expression plasmid in resistant cells or ablated using siRNA in parental cells, and the cell proliferation in response to CDDP was determined. The present results demonstrated that the MUTYH expression level was significantly enhanced by transfecting cells with a MUTYH expression plasmid (Fig. 2C and D), and ectopic expression of MUTYH significantly re-sensitized resistant cells to CDDP treatment at concentrations of 10–80 µM for 48 h (Fig. 2E). Conversely, it was observed that the MUTYH-targeted siRNA reduced the expression of MUTYH from the basal level in sensitive EC109 cells (Fig. 2F and G), and the result of the cell viability assay demonstrated that knockdown of MUTYH in sensitive EC109 cells resulted in an attenuated response to CDDP (Fig. 2H). Therefore, these data demonstrated that downregulation of MUTYH, at least in part, contributes to the CDDP-resistant phenotype.
CDDP-resistant ESCC cells exhibit an EMT-like phenotypic change
Based on the significant effect of MUTYH on CDDP-resistance, the signaling mechanisms underlying MUTYH-mediated CDDP resistance in resistant cells were investigating. By comparing the differential phenotype between the resistant and parental cells, it was observed that EMT was markedly enhanced in resistant cells, which may be associated with MUTYH downregulation. EMT plays an important role in various cancer-associated biological events, which can contribute to invasion, metastasis as well as drug resistance of cancer (22,23). As shown in Fig. 3A, in contrast to parental cells, EC109/CDDP cells displayed morphological characteristics of EMT, as observed by the transition from cobblestone-like cells into slender spindle-shaped cells with increased detachment.
Enhanced capacities of migration and invasion are common along with resistance acquisition in cancer cells. Wound healing assays showed that EC109/CDDP cells displayed significantly higher migratory potential, as demonstrated by more rapid and complete wound healing than parental cells (Fig. 3B). Wound areas in parental cells were ~2- and ~2.5-fold at 24 and 48 h, respectively, as large as those in resistant cells (Fig. 3C). Additionally, Transwell invasion analysis revealed that the invasiveness through Matrigel was also significantly increased in the resistant cells, 2–3-fold compared with that noted in the parental cells (Fig. 3D and E). To eliminate the interference of cell proliferation on migration and invasion, proliferation rates of the two cell lines were also compared. The present results showed that the proliferation rate of resistant cells decreased ~13, ~28 and ~52% at 24, 48 and 72 h, respectively, compared with the parental cells (Fig. 3F). Therefore, the enhanced wound healing and invasiveness through Matrigel of the EC109/CDDP cell line were not caused by proliferation, which further demonstrated its increased migratory and invasive capacities.
Moreover, the expressions of various genes associated with migration and invasion in both cells were detected. The results of RT-qPCR showed that epithelial marker E-cadherin (encoded by CDH1 gene) was significantly downregulated (~14-fold) while mesenchymal markers N-cadherin (encoded by CDH2 gene) and vimentin (encoded by VIM gene) were significantly upregulated (~10-fold and ~6-fold, respectively) in the resistant cells compared with levels in the parental cells (Fig. 3G). Notably, Twist (encoded by TWIST1 gene), a key transcription factor of EMT that transcriptionally regulates the expression of E-cadherin, N-cadherin and vimentin, was significantly enhanced in the EC109/CDDP cells, ~8-fold increase in resistant cells compared with parental cells (Fig. 3G). In contrast, the upregulation of Slug (encoded by SNAI2 gene) and Snail (encoded by SNAI1 gene), other transcription factors of EMT, was only ~1.6-fold and 1.4-fold, respectively (Fig. 3G), much lower than Twist (~8-fold). Expression changes in Twist, E-cadherin and vimentin were further validated; the protein levels were similar, as determined by western blotting (Fig. 3H). In addition, matrix metalloproteinase (MMP)7 and MMP9 (encoded by MMP7 and MMP9 genes, respectively), two core MMPs, are positively correlated with invasion and were also markedly upregulated in CDDP-resistant cells (Fig. 3G). Expression of Claudin1 (encoded by CLDN1 gene), another important epithelial-related factor, was also markedly decreased in CDDP-resistant cells (Fig. 3G). Taken together, these data demonstrated an EMT phenotypic conversion in CDDP-resistant EC109/CDDP cells, which was largely in a Twist-dependent manner.
Downregulation of MUTYH contributes to EMT via enhancement of Twist in resistant ESCC cells
Subsequently, it was further assessed whether there was an association between MUTYH downregulation and induction of EMT in resistant cells. The functional involvement of MUTYH in EMT was studied by overexpressing MUTYH in resistant cells. As shown in Fig. 4A, RT-qPCR results showed that ectopic expression of MUTYH prominently suppressed the mRNA level of Twist in EC109/CDDP cells by ~5-fold, suggesting a role of MUTYH in the transcriptional regulation of Twist. Correspondingly, cells transfected with the MUTYH expression plasmid demonstrated a significant induction of E-cadherin (~1.4-fold) and Claudin-1 (~2.5-fold), and a pronounced suppression of N-cadherin (~5-fold), vimentin (~5-fold), MMP7 (~3-fold) and MMP9 (~17-fold). Similar to the mRNA levels, restoration of MUTYH expression in resistant cells significantly elevated the protein level of E-cadherin and caused a reduction in the protein expression of vimentin and Twist to different levels (Fig. 4B).
Furthermore, it was analyzed whether knockdown of MUTYH led to induction of EMT in the parental cells. As shown in Fig. 4C, following MUTYH knockdown, the parental cells presented an EMT phenotypic conversion as demonstrated by induction of Twist, N-cadherin, vimentin, MMP7 and MMP9, as well as the inhibition of E-cadherin and Claudin-1. Concordantly, the expression changes of these genes in response to depletion of MUTYH were further confirmed by their protein levels, as observed in Fig. 4D. Therefore, these data suggested that downregulation of MUTYH contributed to induction of EMT by enhancing Twist and subsequently regulating its downstream targets, including E-cadherin, N-cadherin and vimentin in CDDP-resistant EC109/CDDP cells.
Downregulation of MUTYH blocks Twist degradation via reduced ROS and proteasome activity in resistant cells
Due to the essential role of Twist in EMT mediated by downregulation of MUTYH, we aimed to ascertain whether MUTYH affects the degradation of Twist in resistant cells. As MUTYH is associated with ROS and previous studies have found that ROS exert a regulatory effect on proteasome activity (24,25), the present study aimed to investigate whether MUTYH affects the proteasomal degradation of Twist, which also contributed to an increase in Twist in EC109/CDDP cells. The results in Fig. 5A and B demonstrated lower levels of ROS in resistant cells compared with parental cells. Additionally, a lower level of proteasome activity was identified, as demonstrated by reduced ChT-L and PGPH activities (Fig. 5C) as well as increased ubiquitinated proteins (Fig. 5D) in EC109/CDDP cells, suggesting that a blockade of proteasomal degradation of Twist occurred in resistant cells.
The effect of MUTYH on ROS and proteasome activity was additionally investigated. As shown in Fig. 6A and B, ectopic expression of MUTYH in resistant cells led to an increase in the level of ROS. Furthermore, as a consequence of MUTYH overexpression, the resistant cells demonstrated downregulation of ubiquitinated proteins, suggesting that MUTYH suppression attenuated proteasomal degradation of proteins, including Twist (Fig. 6C).
To determine whether MUTYH is involved in EMT via modulation of ROS, NAC, an antioxidant agent, was used. As shown in Fig. 6D, NAC significantly reduced the amount of ROS induced by overexpression of MUTYH. Furthermore, following the blocking of ROS generation by NAC, decreases in ubiquitinated proteins (Fig. 6E) and Twist (Fig. 6F) as well as changes of EMT markers (Fig. 6F) in response to overexpression of MUTYH, were predominantly rescued under conditions where ROS was blocked by NAC in EC109/CDDP cells. Correspondingly, ectopic expression of MUTYH led to fewer morphological characteristics of EMT in resistant cells while this reversing effect was markedly abolished when scavenging of ROS occurred from the addition of NAC (Fig. 6G). Therefore, the present data demonstrated that downregulation of MUTYH was able to reduce the degradation of Twist through ROS, which could function together with the increase in Twist transcription to accelerate EMT.
Discussion
MutY homolog (MTUYH) was initially thought to play a prominent role in the BER system of oxidized DNA bases in response to reactive oxygen species (ROS). Notably, recent studies have identified that MTUYH is associated with tumorigenesis or development, including in esophageal carcinoma. In the present study, the exact role of MTUYH in the cisplatin (CDDP) resistance of esophageal squamous cell carcinoma (ESCC) was further investigated. Based on the establishment of a CDDP-resistant cell model EC109/CDDP, MUTYH was initially identified to be downregulated in resistant ESCC cells. It was identified for the first time, to the best of our knowledge, that downregulation of MUTYH contributed to CDDP resistance of ESCC. Further experiments revealed that downregulation of MUTYH stimulated EMT by enhancing both mRNA and protein levels of Twist in CDDP-resistant EC109/CDDP cells; moreover, downregulation of MUTYH contributed to low ROS levels and subsequently inhibited proteasome activity, which in turn led to suppression of Twist degradation. The present data suggested that, as a consequence of these events, a low level of MUTYH might, at least partially, confer Twist-mediated EMT and drug resistance.
Although CDDP alone or in combination with other chemotherapeutic drugs is recommended as an effective front-line strategy to treat advanced ESCC, occurrence of resistance to CDDP is the main challenge in chemotherapy. The CDDP-resistance mechanisms are multi-factorial and may be unique to different types of cancer (4,5). Further understanding concerning the CDDP-resistance mechanisms at a molecular level remains a critical goal for cancer therapy. In the present study, a CDDP-resistant ESCC cell line EC109/CDDP was established from parental EC109 cells. The CDDP-resistant cell line was characterized with decreased sensitivity to CDDP and increased EMT phenotypes compared with parental cells. Notably, a significant downregulation of MUTYH was observed in resistant cells. The causal association between MUTYH downregulation and CDDP-resistance acquisition was demonstrated by transfecting an expression vector or RNA interference. Overexpression of MUTYH restored CDDP sensitivity, while MUTYH inhibition made cells resistant to CDDP. To the best of our knowledge, this is the first study to demonstrate that downregulation of MUTYH contributed to acquisition of CDDP-resistance in ESCC.
Epithelial-mesenchymal transition (EMT) has been now widely recognized as a vital process that contributes to cancer progression and drug resistance (22,23). It has been identified that EMT is executed by a relatively small number of master regulators, mainly including the transcription factors Twist, Snail and Slug (22,23). Accumulating evidence has demonstrated that EMT activation confers multi-drug-resistance to cancer cells (22,23,26). EMT-induced multidrug resistance involves various mechanisms, including a lower level of proliferation, increased expression of anti-apoptotic proteins as well as ATP-binding cassette transporters (23,26,27). In the present study, EC109/CDDP cells underwent EMT, as characterized by the changes in cell morphology and the expression of EMT markers, displaying upregulation of two mesenchymal markers, N-cadherin and Vimentin, and reduction of an epithelial marker E-cadherin. Notably, the master regulator of EMT, Twist, also presented a marked increase in expression in CDDP-resistant cells, demonstrating that the occurrence of EMT in EC109/CDDP cells was largely in a Twist-dependent manner. Based on these events mentioned, it was hypothesized that downregulation of MUTYH was associated with Twist-mediated EMT and in turn conferred CDDP resistance to ESCC. In support of this hypothesis, MUTYH suppression enhanced the Twist level and stimulated cells to acquire a higher potential for EMT in parental cells; conversely, restoring MUTYH in CDDP-resistant cells reduced the Twist level and inhibited the EMT process.
Moreover, in addition to transcriptionally regulating Twist, it was also observed that MUTYH affected the degradation of Twist. Twist is a labile protein, rapidly polyubiquitinated and degraded via the ubiquitin-proteasome pathway (UPP) (28). UPP is regarded as the primary cytosolic proteolytic machinery for protein degradation (29,30). In the process of UPP, multiple ubiquitin molecules firstly tag substrate proteins, which are then degraded by the 26S proteasome. Therefore, the UPP is a major pathway responsible for the protein quality control mechanism (29,30). Changes in UPP capacity of degrading proteins can occur in response to cellular environmental insults, including oxidative stress (24,25). It has been demonstrated that exposure to various forms of oxidative stress leads to increased intracellular protein degradation, which results from the elevation of the rate of ubiquitin conjugation by increasing substrate availability and enhancement of activities of ubiquitin-conjugating enzymes (24,25,31), thereby elevating ubiquitination and degradation capacities. In the present study, it was determined that compared with parental cells, resistant E109/CDDP cells presented a lower level of ROS and reduced activity of UPP, which led to the hypothesis that MUTYH modulates ROS and UPP. The present data demonstrated that MUTYH downregulation contributed to reduced ROS and UPP activity, thereby leading to an inhibition of Twist degradation. The addition of an antioxidant agent NAC significantly abolished the effect of MUTYH on UPP and the EMT phenotype, further validating this hypothesis. Nevertheless, the underlying mechanisms of these signaling pathways require further investigation.
In summary, the present study provided novel insight into the role of MUTYH in the acquisition of resistance to CDDP in ESCC cells and suggested a novel mechanism in that downregulation of MUTYH conferred EMT-mediated drug resistance via an increased level of Twist. Regulation of the signaling response may provide a practical therapeutic strategy to strengthen the effect of CDDP-based chemotherapy in ESCC cells.
Acknowledgements
Not applicable
Funding
This study was supported by the National Natural Science Foundation of China (81603140), Shandong Provincial Natural Science Foundation, China (2015GSF118037, ZR2017BH108, ZR2017PH033), Jinan Science and Technology Development Program (201704087) and the Jining Key Research and Development Plan (2018SMNS001).
Availability of data and materials
The datasets used during the present study are available from the corresponding author upon reasonable request.
Authors' contributions
CP, YL and YG designed the research. YG, YL, YJ, SW, DG, LZ and ZL performed the experiments. FK, NL and SW contributed new reagents and analytic tools in conducting the experiments. YG and YJ analyzed the data. YG and YL wrote the paper. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
Not applicable
Patient consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests
References
|
Murphy G, McCormack V, Abedi-Ardekani B, Arnold M, Camargo MC, Dar NA, Dawsey SM, Etemadi A, Fitzgerald RC, Fleischer DE, et al: International cancer seminars: A focus on esophageal squamous cell carcinoma. Ann Oncol. 28:2086–2093. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Liu J, Wei Z, Zhang J, Hu W, Ma Z and Liu Q: Which factors are associated with extremely short-term survival after surgery in patients with esophageal squamous cell carcinoma? Asia Pac J Clin Oncol. 12:308–313. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Baba Y, Watanabe M, Yoshida N and Baba H: Neoadjuvant treatment for esophageal squamous cell carcinoma. World J Gastrointest Oncol. 6:121–128. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, Castedo M and Kroemer G: Molecular mechanisms of cisplatin resistance. Oncogene. 31:1869–1883. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Kartalou M and Essigmann JM: Mechanisms of resistance to cisplatin. Mutat Res. 478:23–43. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
De Koning P, Neijt JP, Jennekens FG and Gispen WH: Evaluation of cis-diamminedichloroplatinum (II) (cisplatin) neurotoxicity in rats. Toxicol Appl Pharmacol. 89:81–87. 1987. View Article : Google Scholar : PubMed/NCBI | |
|
Marullo R, Werner E, Degtyareva N, Moore B, Altavilla G, Ramalingam SS and Doetsch PW: Cisplatin induces a mitochondrial-ROS response that contributes to cytotoxicity depending on mitochondrial redox status and bioenergetic functions. PLoS One. 8:e811622013. View Article : Google Scholar : PubMed/NCBI | |
|
Santos NA, Catão CS, Martins NM, Curti C, Bianchi ML and Santos AC: Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Arch Toxicol. 81:495–504. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Jiang Y, Guo C, Vasko MR and Kelley MR: Implications of apurinic/ apyrimidinic endonuclease in reactive oxygen signaling response after cisplatin treatment of dorsal root ganglion neurons. Cancer Res. 68:6425–6434. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Markkanen E, Dorn J and Hübscher U: MUTYH DNA glycosylase: The rationale for removing undamaged bases from the DNA. Front Genet. 4:182013. View Article : Google Scholar : PubMed/NCBI | |
|
Ruggieri V, Pin E, Russo MT, Barone F, Degan P, Sanchez M, Quaia M, Minoprio A, Turco E, Mazzei F, et al: Loss of MUTYH function in human cells leads to accumulation of oxidative damage and genetic instability. Oncogene. 32:4500–4508. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Nascimento EFR, Ribeiro ML, Magro DO, Carvalho J, Kanno DT, Martinez CAR and Coy CSR: Tissue expresion of the genes MUTYH and OGG1 in patients with sporadic colorectal cancer. Arq Bras Cir Dig. 30:98–102. 2017.(In English, Portuguese). View Article : Google Scholar : PubMed/NCBI | |
|
Dong J, Wang X, Yu Y, Yan X and Cui JW: Association of base excision repair gene polymorphisms with the response to chemotherapy in advanced non-small cell lung cancer. Chin Med J (Engl). 131:1904–1908. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Santos LS, Branco SC, Silva SN, Azevedo AP, Gil OM, Manita I, Ferreira TC, Limbert E, Rueff J and Gaspar JF: Polymorphisms in base excision repair genes and thyroid cancer risk. Oncol Rep. 28:1859–1868. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Kong F, Han XY, Luan Y, Qi TG, Sun C, Wang J, Hou HY, Jiang YH, Zhao JJ and Cheng GH: MUTYH Association with esophageal adenocarcinoma in a Han Chinese population. Asian Pac J Cancer Prev. 14:6411–6413. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Sliwinski T, Markiewicz L, Rusin P, Pietruszewska W, Olszewski J, Morawiec-Sztandera A, Mlynarski W and Majsterek I: Polymorphisms of the DNA base excision repair gene MUTYH in head and neck cancer. Exp Oncol. 31:57–59. 2009.PubMed/NCBI | |
|
Przybylowska K, Kabzinski J, Sygut A, Dziki L, Dziki A and Majsterek I: An association selected polymorphisms of XRCC1, OGG1 and MUTYH gene and the level of efficiency oxidative DNA damage repair with a risk of colorectal cancer. Mutat Res. 745-746:6–15. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, Hodges AK, Davies DR, David SS, Sampson JR and Cheadle JP: Inherited variants of MYH associated with somatic G:C->T:A mutations in colorectal tumors. Nat Genet. 30:227–232. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Slyskova J, Naccarati A, Pardini B, Polakova V, Vodickova L, Smerhovsky Z, Levy M, Lipska L, Liska V and Vodicka P: Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls. Mutagenesis. 27:225–232. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Liu YQ, Wang SK, Xu QQ, Yuan HQ, Guo YX, Wang Q, Kong F, Lin ZM, Sun DQ, Wang RM and Lou HX: Acetyl-11-keto-β-boswellic acid suppresses docetaxel-resistant prostate cancer cells in vitro and in vivo by blocking Akt and Stat3 signaling, thus suppressing chemoresistant stem cell-like properties. Acta Pharmacol Sin. 40:689–698. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Smith BN and Bhowmick NA: Role of EMT in metastasis and therapy resistance. J Clin Med. 5:E172016. View Article : Google Scholar : PubMed/NCBI | |
|
Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, Wu CC, LeBleu VS and Kalluri R: Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature. 527:525–530. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Shang F and Taylor A: Ubiquitin–proteasome pathway and cellular responses to oxidative stress. Free Radic Biol Med. 51:5–16. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Dudek EJ, Shang F, Valverde P, Liu Q, Hobbs M and Taylor A: Selectivity of the ubiquitin pathway for oxidatively modified proteins: Relevance to protein precipitation diseases. FASEB J. 19:1707–1709. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Shibue T and Weinberg RA: EMT, CSCs, and drug resistance: The mechanistic link and clinical implications. Nat Rev Clin Oncol. 14:611–629. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Saxena M, Stephens MA, Pathak H and Rangarajan A: Transcription factors that mediate epithelial-mesenchymal transition lead to multidrug resistance by upregulating ABC transporters. Cell Death Dis. 2:e1792011. View Article : Google Scholar : PubMed/NCBI | |
|
Díaz VM, Viñas-Castells R and García de Herreros A: Regulation of the protein stability of EMT transcription factors. Cell Adh Migr. 8:418–428. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Goldberg AL: Protein degradation and protection against misfolded or damaged proteins. Nature. 426:895–899. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Glickman MH and Ciechanover A: The ubiquitin–proteasome proteolytic pathway: Destruction for the sake of construction. Physiol Rev. 82:373–428. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Shang F, Gong X and Taylor A: Activity of ubiquitin-dependent pathway in response to oxidative stress. Ubiquitin-activating enzyme is transiently upregulated. J Biol Chem. 272:23086–23093. 1997. View Article : Google Scholar : PubMed/NCBI |
