Ann Surg Treat Res.  2017 Jan;92(1):15-22. 10.4174/astr.2017.92.1.15.

Clinical implications of APEX1 and Jagged1 as chemoresistance factors in biliary tract cancer

Affiliations
  • 1Department of Premedical Course, Chosun University School of Medicine, Gwangju, Korea.
  • 2Department of Urology, Chosun University Hospital, Gwangju, Korea.
  • 3Department of General Surgery, Chosun University Hospital, Gwangju, Korea.
  • 4Department of Otolaryngology Head and Neck Surgery, Chosun University Hospital, Gwangju, Korea.
  • 5Division of Hemato-Oncology, Department of Internal Medicine, Chosun University Hospital, Gwangju, Korea. sgpark@chosun.ac.kr

Abstract

PURPOSE
Biliary cancer is a highly malignant neoplasm with poor prognosis and most patients need to undergo palliative chemotherapy, however major clinical problem associated with the use of chemotherapy is chemoresistance. So far, we aimed at investigating clinical implications of apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and Jagged1 as chemoresistance factors in biliary tract cancer.
METHODS
We used 5 human biliary tract cancer cell lines (SNU-245, SNU-308, SNU-478, SNU-1079, and SNU-1196), and investigated the chemosensitivity of APEX1 and Jagged1 through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and Western blot. Alternately, the 10 patients of advanced biliary cancer consist of 2 group according to the chemotherapy response examined by immunohistochemistry using APEX1 and Jagged1 antibody, and protein expression level was scored for staining intensity and percent positive cell.
RESULTS
The result of MTT assay after APEX1 knockdown showed that strong coexpression of APEX1 and Jagged1 cell line (SNU-245, SNU-1079, and SNU-1196) showed a greater decrease in ICâ‚…â‚€ of chemotherapeutic agent (5-fluorouracil, gemcitabine and cisplatin). The Western blot analysis of APEX1 and Jagged1 expression in biliary cancer cell lines after APEX1 knockdown definitively demonstrated decreased Jagged1 expression. The APEX1 and Jagged1expression level of immunohistochemistry represented that chemorefractory patients had higher than chemoresponsive patients.
CONCLUSION
These results demonstrate that simultaneous high expression of APEX1 and Jagged1 is associated with chemoresistance in biliary cancer and suggest that is a potential therapeutic target for chemoresistance in advanced biliary cancer.

Keyword

APEX1 protein; Jagged1 protein; 5-FU; Gemcitabine; Cisplatin

MeSH Terms

Biliary Tract Neoplasms*
Biliary Tract*
Blotting, Western
Cell Line
Cisplatin
Drug Therapy
Fluorouracil
Humans
Immunohistochemistry
Prognosis
Cisplatin
Fluorouracil

Figure

  • Fig. 1 All biliary cancer cell lines showed apurinic/apyrimidinic endodeoxyribonuclease1 (APEX1) expression by western blot. APEX1 protein expression was detected by western blot analysis with α-tubulin as a loading control.

  • Fig. 2 MTT assay for chemotherapeutic drugs (A: 5-FU, B: cisplatin, C: gemcitabine) in biliary cancer cells. Cell plated in 96-well plate were treated cisplatin, 5-FU, or gemcitabine and the MTT assay was carried out on day three of the treatments. Result are shown as means ± standard deviation (n = 3). 5-FU, 5-fluorouracil; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

  • Fig. 3 Western blot analysis of apurinic/apyrimidinic endodeoxyribonuclease1 (APEX1) expression in biliary cancer cell lines after APEX1 knockdown. SNU-245, SNU-308, SNU-478, SNU-1079, and SNU-1196 cells were transfected with control or APEX1-targeted siRNAs. Forty-eight hours after transfection, whole cell lysates were probed with antibodies specific for APEX1 and α-tubulin.

  • Fig. 4 (A) All biliary cancer cell line showed apurinic/apyrimidinic endodeoxyribonuclease1 (APEX1) and Jagged1 expression by Western blot. APEX1 and Jagged1 protein expression was detected by western blot analysis with α-tubulin as a loading control. (B) Western blot analysis of APEX1 and Jagged1 expression in biliary cancer cell lines after APEX1 knockdown. Western blot of all biliary cancer cells treated with APEX1 siRNA. Samples were collected 48 hours after APEX1-siRNA treatment, and Western blot was done with APEX1 antibody and reproved with α-tubulin antibody as a loading control.

  • Fig. 5 Immunohistochemistry of advanced biliary cancer patients. Correlation between apurinic/apyrimidinic endodeoxyribonuclease1 (APEX1) and Jagged1 expression in biliary cancer patients. APEX1 and Jagged1 proteins in biliary cancer patients are shown by immunohistochemistry with anti-APEX1 and anti-Jagged1 antibodies. Brown staining indicates positive APEX1 or Jagged1 staining (P < 0.01, Pearson correlation test. Scale bars, 200 µm).

  • Fig. 6 Immunohistochemistry scoring of advanced biliary cancer patients. Apurinic/apyrimidinic endodeoxyribonuclease1 (APEX1) and Jagged1 expression levels assessed by immunohistochemistry scoring (see METHODS section).


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