Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in the base excision repair pathway. It also has redox activity and maintains various transcription factors in an active reduced state. APE1 may be associated with chemoresistance. In the present study, we first investigated the expression level of APE1 protein and its correlation with oncologic outcomes of oxaliplatin-based chemotherapy in patients with stage III colon cancer. Further, we investigated the effects of human APE1 siRNA on the sensitivity of oxaliplatin in SNU-C2A colon cancer cells.
Tissue specimens from tumor and normal colon of 33 patients with stage III colon cancer were obtained from 2006 to 2009. The patients received at least eight cycles of oxaliplatin-based chemotherapy. APE1 expression was analyzed by immunohistochemistry and Western blotting using a cultured SNU-C2A cell line. Cell viability and apoptosis were determined by Cell Counting Kit-8 and caspase-3 cleavage using Western blotting.
All the colon cancer tissues showed APE1 staining in the nucleus, whereas all the normal colon tissues were negative for APE1 staining in the cytoplasm. The group with a higher expression of APE1 demonstrated poorer prognosis than the group with low expression (P=0.026 for overall survival and P=0.021 for disease-free survival). Treatment with oxaliplatin resulted in a dose-dependent increase in APE1 expression in SNU-C2A cells. APE1 siRNA significantly enhanced oxaliplatin-induced growth inhibition, and also increased oxaliplatin-induced apoptosis in SNU-C2A cells.
APE1 could be considered a prognostic factor in colon cancer patients treated with oxaliplatin-based chemotherapy.
Colon cancer is the third most common cancer and the fourth leading cause of cancer-related deaths in the world [
Surgical resection is pivotal in the treatment of colon cancer. However, frequent recurrence is observed even after complete resection of the tumor [
Oxaliplatin is a third-generation of platinum-based drug having
The human apurinic/apyrimidinic endonuclease 1 (APE1, also known as APEX1 or REF
We reviewed the medical records of the patients who underwent curative resection of colon cancer and received adjuvant chemotherapy in our institute. Among the patients reviewed, a total of 33 patients from July 2006 to June 2009 were included in this study. These patients were treated by curative resection and adjuvant FOLFOX chemotherapy. The data included patients’ characteristics, operative findings, histopathology reports, and follow-up data. Preoperative evaluations consisted of history taking, physical examination, estimation of carcinoembryonic antigen (CEA) level, peripheral blood test, colonoscopy, and computed tomography. The primary colon cancer was characterized by TNM staging, pathological type, and tumor location. The postoperative disease staging was determined according to the criteria defined by the American Joint Committee on Cancer (8th edition). The patients having lymph node involvement (N1 or N2) were included in this study. The present study was reviewed and approved by the Institutional Review Board (IRB) of Chungnam National University Hospital (IRB No. CNUH 201302003) and the informed consent was waived by IRB.
The median age of the patients at the time of operation was 63 years (range, 32–80 years). No chemotherapy or radiotherapy was administered to the patients before surgery of colon cancer. All the patients received >8 cycles of FOLFOX chemotherapy.
The expression of APE1 was evaluated by immunohistochemistry of paraffin-embedded tissue sections obtained from colon cancer and normal colonic mucosa (collected more than 2 cm far from the cancerous lesion). Sections from paraffin blocks having a thickness of 3 μm were used for immunohistochemical staining. Mouse anti-human APE1 monoclonal antibody (Novus, Littleton, CO, USA) was diluted (1:4,000) with a background-reducing diluent (Dako, Carpinteria, CA, USA) and the tissue sections were incubated in the mixture for 30 minutes. All the immunostaining processes were performed using the Ventana Discovery system (Ventana, Tucson, AZ, USA) according to the manufacturer’s instructions. The negative controls were treated identically but without the primary antibody. The entire stained sections of each sample were scanned by a light microscope (×20 or ×40). The slides were reviewed by a pathologist who was unaware of any information related to the enrolled patients. Immunochemical scoring was performed according to the percentage of cell staining and the intensity of staining. Low expression was defined as a weak intensity of staining with positive cell percentage <50% or moderate intensity of staining with positive cell percentage <25%.
Human colon cancer cell line was used in the present study. SNU-C2A cells were purchased from Korean Cell Line Bank (Seoul, Korea) and cultured in Dulbecco’s modified Eagle’s medium (Thermo Scientific HyClone, Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum and penicillin (100 U/mL)-streptomycin (100 μg/mL) obtained from GIBCO/BRL Life Technologies (Grand Island, NY, USA). The cells were maintained in a humidified incubator at 37°C in an atmosphere containing 5% CO2 and 95% air.
The cells were seeded at 5×103 cells/mL in 96-well microplates and allowed to attach for 24 hours. After drug treatment, cell cytotoxicity and/or proliferation was assessed by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, a highly water-soluble tetrazolium salt, WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), produced an orange-colored water-soluble product called formazan. The amount of formazan dye produced by dehydrogenases present in the cells was directly proportional to the number of living cells. CCK-8 (10 μL) was added to each well and incubated for 3 hours at 37°C. The cell proliferation and cytotoxicity were assessed by estimating the absorbance at 450 nm using a microplate reader. Three replicated wells were used for each experimental condition.
The cells were incubated with drugs for 24 hours and washed twice in cold phosphate buffered saline (PBS). The cells were lysed with lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% TritonX-100, 0.5% NP-40, 1 mM propidium iodide, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) and placed on ice for 1 hour with occasional vortexing. It was followed by centrifugation followed at 10,000 rpm for 10 minutes. Each cell lysates (50 μg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was transferred on to polyvinylidene difluoride membrane. The blots were blocked with 5% skimmed milk in PBS containing 0.05% Tween-20 for 1 hour at 25°C. The blots were then incubated with primary antibody, followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody IgG, and finally visualized with enhanced chemiluminescence.
Two siRNAs against APE1 and negative control siRNA were transfected using siRNA Reagent System (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) according to the manufacturer’s instructions (siRNA Transfection Protocol). The down-regulation of APE1 gene expression was analyzed by immunofluorescence staining.
All statistical analyses were performed using SPSS version 24.0 (IBM Corp., Armonk, NY, USA). The correlation between APE1 expression and clinicopathologic parameters was evaluated using chi-square analysis. A survival curve was plotted using the Kaplan-Meier method, and the log-rank test was used to determine the statistical difference. A forward stepwise selection of covariates was used in the Cox proportional hazard model, and a P-value of <0.1 was defined as the threshold for covariate inclusion. All experiments were repeated three times and are expressed as the mean±standard deviation. Mann-Whitney test was performed and a P-value of <0.05 was considered statistically significant.
We have investigated the expression of APE1 in 33 colon cancer and normal colon tissue (collected more than 2 cm away from the cancerous lesion) using immunohistochemistry. All the colon cancer tissues showed APE1 staining in the both the nucleus and cytoplasm, whereas all the normal colon tissues showed negative APE1 staining in the cytoplasm. Of the normal colon tissue specimens, 11 specimens (33.3%) showed negative APE1 staining in both the nucleus and cytoplasm (
The median follow-up period for all the patients was 51.1 months (range, 13.6–72.7 months) in the present study. During the follow-up period, recurrence occurred in 11 patients (33.3%). The 5-year overall survival and disease-free survival rates were 76.1% and 76.8%, respectively.
By the immunohistochemistry results and survival curve analysis of stage III colon cancer patients, overexpression of APE1 was found to be associated with poor oncological outcomes. From this data, we postulated that APE1 overexpression might be correlated with the resistance of oxaliplatin-based chemotherapy. To determine whether oxaliplatin chemotherapy associated with the expression of APE1, Western blotting was performed with SNU-C2A cells treated with oxaliplatin. APE1 was found to be strongly expressed in SNU-C2A cells treated with ascending doses of oxaliplatin for 24 hours. The higher expression of APE1 with ascending doses of oxaliplatin statistically different (P<0.05 as compared to control) (
To silence the expression of APE1 specifically, SNU-C2A cells were transfected with siRNA-targeting APE1. APE1 expression was determined by confocal microscopy after immunofluorescence staining. As shown in
We have determined that APE1 siRNA inhibited APE1 overexpression in oxaliplatin-induced SNU-C2A cells. To determine whether targeting inhibition of APE1 enhanced the activity of oxaliplatin in SNU-C2A cells, the cells were pretreated with APE1 siRNA or control siRNA, and then treated with oxaliplatin at a concentration of 30 μg/mL for 24 hours. The cell viability was determined by CCK-8 assay.
To investigate the effects of APE1 siRNA on apoptosis induction by oxaliplatin in vitro, we estimated the pro-caspase-3/GAPDH ratio by Western blot analysis using caspase-3 antibody (
APE1 is a ubiquitous and multifunctional protein and its overexpression is often observed in several human tumors [
Adjuvant chemotherapy following surgical resection with curative intent in patients with stage III colon cancer can be attributed to a high rate of recurrences. Therefore, authoritative societies such as ASCO and NCCN recommend the use of adjuvant chemotherapy in all patients with stage III colon cancer. An international, multicenter, prospective, randomized clinical trial, conducted by the Multicenter International Study of Oxaliplatin/5-Fluorouracil/Leucovorin in the Adjuvant Treatment of Colon Cancer (MOSAIC) investigators demonstrated that oxaliplatin-based chemotherapy (FOLFOX) improved survival among patients with stage II or III colon cancer [
Oxaliplatin is a diaminocyclohexane (DACH) derivative of cisplatin. This third-generation of platinum-based drug showed in vitro and in vivo antitumor activities against colorectal cancer [
APE1 is a key enzyme involved in the base excision repair pathway, which takes part in DNA repair after oxidative and alkylating damage. Therefore, APE1 is important for the protection of tumor cells against the DNA damaging effects of anti-cancer agents. As mentioned, different types of cancers express APE1 [
The MOSAIC trial demonstrated that 72.2% of the patients with stage III colon cancer after adjuvant FOLFOX chemotherapy had a 3-year disease-free survival [
Previous studies have reported that APE1 protein knockdown using siRNA could result in decreased activity of APE1 and increased sensitivity to ionizing radiation, alkylating, and oxidizing DNA damaging agents [
In addition to DNA base excision repair, APE1 also functions as a redox factor activating various transcription factors, such as HIF-1-alpha, p53, NK-κB, CREB, and AP-1, in an active reduced state [
In conclusion, we have reported that the APE1 expression can predict the oncological outcomes and the sensitivity to oxaliplatin-based chemotherapy in stage III colon cancer patients. The in vitro assay demonstrated that the APE1 knockdown could enhance the sensitivity of human colon cancer cells to oxaliplatin. These results suggest that determination of APE1 expression in colon cancer cells before initiating chemotherapy could be a predictor of effective oxaliplatin-based adjuvant chemotherapy in patients with colon cancer.
Jin Soo Kim is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.
Immunohistochemical staining for APE1 protein (×100). (A) Negative expression of APE1 in normal colon tissue. (B) High level of APE1 expression in normal colon tissue. Normal colonic mucosa was stained more densely in the nucleus than in the cytoplasm. (C) Low level of expression of APE1 in the adenocarcinoma tissues. The tumor cells were stained more densely in the nucleus than cytoplasm. (D) High level of expression of APE1 in the adenocarcinoma tissues. The tumors cells were densely stained both in the nucleus and cytoplasm. APE1, apurinic/apyrimidinic endonuclease 1.
(A) Overall survival and (B) disease-free survival of stage III colon cancer patients who were treated with curative resection and oxaliplatin-based adjuvant chemotherapy according to the results of APE1 immunohistochemical staining. APE1, apurinic/apyrimidinic endonuclease 1.
Oxaliplatin-induced APE1 expression in a dose-dependent manner in SNU-C2A cells. The cells were treated with different concentrations of oxaliplatin for 24 hours. Western blot analysis was performed with antibodies for APE1, and GAPDH served as a loading control. APE1, apurinic/apyrimidinic endonuclease 1. a)P<0.05 as compared to control.
Knockdown effects of APE1 siRNA in SNU-C2A cells. The cells were treated with oxaliplatin (30 μg/mL) with or without APE1 siRNA. The APE1 expression (green) was assessed by confocal microscopy after immunofluorescence staining. APE1, apurinic/apyrimidinic endonuclease 1.
APE1 siRNA enhanced oxaliplatin-induced growth inhibition in SNU-C2A cells. After knockdown with APE1 siRNA, the cells were treated with oxaliplatin (30 μg/mL) for 24 hours. The cell viability was determined by Cell Counting Kit-8 assay. APE1, apurinic/apyrimidinic endonuclease 1. a)P<0.05.
APE1 siRNA enhanced oxaliplatin-induced apoptosis in SNU-C2A cells. After knockdown with APE1 siRNA, the cells were treated with oxaliplatin (30 μg/mL) for 24 hours. Apoptosis was measured by the pro-caspase-3/GAPDH ratio with Western blot analysis using caspase-3 antibody. APE1, apurinic/apyrimidinic endonuclease 1. a)P<0.05 as compared to control.
Clinical characteristics and APE1 expression
Characteristics | APE1 low expression (n=13) | APE1 high expression (n=20) | P-value |
---|---|---|---|
Age (yr) | 1.000 | ||
≤65 | 9 (69.2) | 14 (70.0) | |
>65 | 4 (30.8) | 6 (30.0) | |
| |||
Sex | 0.493 | ||
Male | 6 (46.2) | 12 (60.0) | |
Female | 7 (53.8) | 8 (40.0) | |
| |||
Location | 1.000 | ||
Right side colon | 4 (30.8) | 5 (25.0) | |
Left side colon | 9 (69.2) | 15 (75.0) | |
| |||
CEA level (ng/mL) | 0.169 | ||
≤5 | 5 (38.5) | 13 (65.0) | |
>5 | 8 (61.5) | 7 (35.0) | |
| |||
Histologic type | 0.263 | ||
WD, MD | 11 (84.6) | 13 (65.0) | |
PD | 2 (15.4) | 7 (35.0) | |
| |||
Pathologic T stage | 0.136 | ||
T1, T2 | 0 | 4 (20.0) | |
T3, T4 | 13 (100) | 16 (80.0) | |
| |||
Pathologic N stage | 1.000 | ||
N1 | 8 (61.5) | 11 (55.0) | |
N2 | 5 (38.5) | 9 (45.0) |
Values are presented as number (%).
APE1, apurinic/apyrimidinic endonuclease 1; CEA, carcinoembryonic antigen; WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated.
Univariate analysis for the prognostic factors influencing 5-year survival in colon cancer
Variable | No. of patients | Overall survival | Disease-free survival | ||
---|---|---|---|---|---|
|
| ||||
Rate (%) | P-value | Rate (%) | P-value | ||
Age (yr) | 0.718 | 0.868 | |||
≤65 | 23 | 77.2 | 90.0 | ||
>65 | 10 | 78.8 | 80.0 | ||
| |||||
Sex | 0.892 | 0.999 | |||
Male | 18 | 74.7 | 74.3 | ||
Female | 15 | 79.4 | 80.0 | ||
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Location | 0.460 | 0.425 | |||
Right side colon | 9 | 88.9 | 88.9 | ||
Left side colon | 24 | 71.3 | 72.2 | ||
| |||||
CEA level (ng/mL) | 0.067 | 0.060 | |||
≤5 | 18 | 88.5 | 87.8 | ||
>5 | 15 | 57.8 | 64.2 | ||
| |||||
Histologic type | 0.001 | <0.001 | |||
WD, MD | 24 | 90.8 | 90.8 | ||
PD | 9 | 33.9 | 0 | ||
| |||||
Pathologic T stage | 0.276 | 0.269 | |||
T1, T2 | 4 | 100.0 | 100.0 | ||
T3, T4 | 29 | 72.5 | 73.1 | ||
| |||||
Pathologic N stage | 0.037 | 0.046 | |||
N1 | 19 | 86.5 | 86.9 | ||
N2 | 14 | 62.3 | 62.5 | ||
| |||||
APE1 expression | 0.026 | 0.021 | |||
Low | 13 | 100.0 | 100.0 | ||
High | 20 | 62.3 | 60.0 |
CEA, carcinoembryonic antigen; WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; APE1, apurinic/apyrimidinic endonuclease 1.
Multivariate analysis for the prognostic factors influencing 5-year survival in colon cancer
Factor | No. of patients | Overall survival | Disease-free survival | ||
---|---|---|---|---|---|
|
| ||||
HR (95% CI) | P-value | HR (95% CI) | P-value | ||
Histologic type | 0.008 | 0.004 | |||
| |||||
WD, MD | 24 | 1.00 | 1.00 | ||
| |||||
PD | 9 | 9.45 (1.82–49.09) | 28.73 (2.92–281.99) |
HR, hazard ratio; CI, confidence interval; WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated.