Original Paper Pharmacology 2015;95:154–159 DOI: 10.1159/000381029
Received: November 14, 2014 Accepted after revision: February 16, 2015 Published online: April 1, 2015
Irinotecan Induces Cell Cycle Arrest, but Not Apoptosis or Necrosis, in Caco-2 and CW2 Colorectal Cancer Cell Lines Yoshiko Kaku Ayako Tsuchiya Takeshi Kanno Tomoyuki Nishizaki Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan
Abstract Irinotecan, a topoisomerase I inhibitor, is clinically used as an anticancer drug. The present study investigated the anticancer effect of irinotecan on p53-negative Caco-2 and p53-positive CW2 human colorectal cancer cell lines. Cell viability for both Caco-2 and CW2 cells was little affected by treatment with irinotecan at concentrations ranging from 0.3 to 30 μmol/l for 24–48 h. Irinotecan did not increase the number of TUNEL-positive cells and did not affect the population of propidium iodide (PI)-positive and annexin V-negative cells, corresponding to primary necrosis, or that of PI-positive and annexin-positive cells, corresponding to late apoptosis/secondary necrosis, in either of the two cell lines. In the cell cycle analysis, irinotecan significantly increased the proportions at the S and G2/M phases of cell cycling in parallel with a decreased population at the G1 phase in both cell lines. Irinotecan significantly inhibited tumor growth in mice inoculated with CW2 cells. Taken together, these results indicate that irinotecan induces cell cycle arrest, but not apoptosis or necrosis, both in Caco-2 and CW2 cells, leading to suppression of cell proliferation. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0031–7012/15/0954–0154$39.50/0 E-Mail [email protected] www.karger.com/pha
Introduction
Irinotecan is clinically used together with cetuximab, fluorouracil, and leucovorin for first-line chemotherapy of colorectal cancer [1]. Irinotecan is metabolized into SN-38 by carboxylesterase 1 and 2 [2]. Irinotecan is transferred into hepatocytes by solute carrier organic aniontransporter family member 1B1 [3]. SN-38, on the other hand, is transported into cells by ABC transporters such as ABCC1, ABCC2, ABCB1, and ABCG2 [4–7]. Inside the cells, SN-38 serves as an inhibitor of topoisomerase I, bearing DNA replication and transcription [8]. SN-38 is inactivated through uridine diphosphate glycosyltransferase 1A1-mediated glucuronidation [9]. SN-38, alternatively, is inactivated through cytochrome P450 3A subfamily-mediated oxidation [10]. Irinotecan has been shown to induce cell cycle arrest and apoptosis of colorectal cancer cells. The mechanism behind the interindividual difference in the anticancer actions of irinotecan on colorectal cancers, however, is still under investigation. In the present study, we carried out a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining, an apoptosis assay using propidium iodide (PI) and annexin V-FITC, and cell cycle analysis in p53-posiProf. Tomoyuki Nishizaki, MD, PhD Division of Bioinformation, Department of Physiology Hyogo College of Medicine 1-1 Mukogawa-cho, Nishinomiya 663-8501 (Japan) E-Mail tomoyuki @ hyo-med.ac.jp
Downloaded by: UCSF Library & CKM 128.218.166.245 - 5/13/2015 4:02:24 PM
Key Words Irinotecan · Anticancer drug · Colorectal cancer · Cell cycle arrest
tive Caco-2 and p53-negative CW2 human colorectal cancer cell lines and assessed the effect of irinotecan on tumor growth in mice inoculated with CW2 cells. We show here that irinotecan induces cell cycle arrest at the S and G2/M phases, but not apoptosis or necrosis, both in Caco-2 and CW2 cells, thereby preventing tumor growth.
Materials and Methods Animal Care All procedures have been approved by the Animal Care and Use Committee at Hyogo College of Medicine and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Cell Culture Caco-2 and CW2 cell lines were obtained from RIKEN cell bank (Ibaraki, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (final concentration, 100 U/ml), and streptomycin (final concentration, 0.1 mg/ml) in a humidified atmosphere of 5% CO2 and 95% air at 37 ° C.
TUNEL Staining TUNEL staining was performed to detect in situ DNA fragmentation as a marker of apoptosis using the In situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan). Briefly, fixed and permeabilized cells were reacted with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate (FITC)-deoxyuridine triphosphate for 90 min at 37 ° C. FITC signals were visualized with a confocal scanning laser microscope (LSM 510; Carl Zeiss Co., Ltd., Oberkochen, Germany).
Apoptosis Assay Cells were suspended in a binding buffer, stained with both PI and annexin V-FITC, and loaded on a flow cytometer (FACSCalibur; Becton Dickinson, Franklin Lakes, N.J., USA) available for FL1 (annexin V) and FL2 (PI) bivariate analysis. Data from 20,000 cells/sample were collected, and the quadrants were set according to the population of viable, unstained cells in untreated samples. The percentage of cells in the respective quadrants was calculated and analyzed using a BD CellQuest Pro software (Becton Dickinson). Cell Cycle Analysis Cells were harvested by trypsinization and fixed with 70% (v/v) ethanol at 4 ° C overnight. Fixed cells were incubated in phosphatebuffered saline containing 1.5 μg/ml RNase A for 1 h at 37 ° C, followed by staining with 5 μl of PI for 20 min on ice. Then, cells were collected on a nylon mesh filter (pore size, 40 μm), and cell cycles were assayed with a flow cytometer (FACSCalibur; Becton Dickinson) at an excitation of 488 nm and an emission of 585 nm, and analyzed using a BD CellQuest Pro software (Becton Dickinson).
Statistical Analysis Statistical analysis was carried out using Dunnett’s test, analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (PLSD) test, and unpaired t test. A p value of
Received: November 14, 2014 Accepted after revision: February 16, 2015 Published online: April 1, 2015
Irinotecan Induces Cell Cycle Arrest, but Not Apoptosis or Necrosis, in Caco-2 and CW2 Colorectal Cancer Cell Lines Yoshiko Kaku Ayako Tsuchiya Takeshi Kanno Tomoyuki Nishizaki Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan
Abstract Irinotecan, a topoisomerase I inhibitor, is clinically used as an anticancer drug. The present study investigated the anticancer effect of irinotecan on p53-negative Caco-2 and p53-positive CW2 human colorectal cancer cell lines. Cell viability for both Caco-2 and CW2 cells was little affected by treatment with irinotecan at concentrations ranging from 0.3 to 30 μmol/l for 24–48 h. Irinotecan did not increase the number of TUNEL-positive cells and did not affect the population of propidium iodide (PI)-positive and annexin V-negative cells, corresponding to primary necrosis, or that of PI-positive and annexin-positive cells, corresponding to late apoptosis/secondary necrosis, in either of the two cell lines. In the cell cycle analysis, irinotecan significantly increased the proportions at the S and G2/M phases of cell cycling in parallel with a decreased population at the G1 phase in both cell lines. Irinotecan significantly inhibited tumor growth in mice inoculated with CW2 cells. Taken together, these results indicate that irinotecan induces cell cycle arrest, but not apoptosis or necrosis, both in Caco-2 and CW2 cells, leading to suppression of cell proliferation. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0031–7012/15/0954–0154$39.50/0 E-Mail [email protected] www.karger.com/pha
Introduction
Irinotecan is clinically used together with cetuximab, fluorouracil, and leucovorin for first-line chemotherapy of colorectal cancer [1]. Irinotecan is metabolized into SN-38 by carboxylesterase 1 and 2 [2]. Irinotecan is transferred into hepatocytes by solute carrier organic aniontransporter family member 1B1 [3]. SN-38, on the other hand, is transported into cells by ABC transporters such as ABCC1, ABCC2, ABCB1, and ABCG2 [4–7]. Inside the cells, SN-38 serves as an inhibitor of topoisomerase I, bearing DNA replication and transcription [8]. SN-38 is inactivated through uridine diphosphate glycosyltransferase 1A1-mediated glucuronidation [9]. SN-38, alternatively, is inactivated through cytochrome P450 3A subfamily-mediated oxidation [10]. Irinotecan has been shown to induce cell cycle arrest and apoptosis of colorectal cancer cells. The mechanism behind the interindividual difference in the anticancer actions of irinotecan on colorectal cancers, however, is still under investigation. In the present study, we carried out a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining, an apoptosis assay using propidium iodide (PI) and annexin V-FITC, and cell cycle analysis in p53-posiProf. Tomoyuki Nishizaki, MD, PhD Division of Bioinformation, Department of Physiology Hyogo College of Medicine 1-1 Mukogawa-cho, Nishinomiya 663-8501 (Japan) E-Mail tomoyuki @ hyo-med.ac.jp
Downloaded by: UCSF Library & CKM 128.218.166.245 - 5/13/2015 4:02:24 PM
Key Words Irinotecan · Anticancer drug · Colorectal cancer · Cell cycle arrest
tive Caco-2 and p53-negative CW2 human colorectal cancer cell lines and assessed the effect of irinotecan on tumor growth in mice inoculated with CW2 cells. We show here that irinotecan induces cell cycle arrest at the S and G2/M phases, but not apoptosis or necrosis, both in Caco-2 and CW2 cells, thereby preventing tumor growth.
Materials and Methods Animal Care All procedures have been approved by the Animal Care and Use Committee at Hyogo College of Medicine and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Cell Culture Caco-2 and CW2 cell lines were obtained from RIKEN cell bank (Ibaraki, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (final concentration, 100 U/ml), and streptomycin (final concentration, 0.1 mg/ml) in a humidified atmosphere of 5% CO2 and 95% air at 37 ° C.
TUNEL Staining TUNEL staining was performed to detect in situ DNA fragmentation as a marker of apoptosis using the In situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan). Briefly, fixed and permeabilized cells were reacted with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate (FITC)-deoxyuridine triphosphate for 90 min at 37 ° C. FITC signals were visualized with a confocal scanning laser microscope (LSM 510; Carl Zeiss Co., Ltd., Oberkochen, Germany).
Apoptosis Assay Cells were suspended in a binding buffer, stained with both PI and annexin V-FITC, and loaded on a flow cytometer (FACSCalibur; Becton Dickinson, Franklin Lakes, N.J., USA) available for FL1 (annexin V) and FL2 (PI) bivariate analysis. Data from 20,000 cells/sample were collected, and the quadrants were set according to the population of viable, unstained cells in untreated samples. The percentage of cells in the respective quadrants was calculated and analyzed using a BD CellQuest Pro software (Becton Dickinson). Cell Cycle Analysis Cells were harvested by trypsinization and fixed with 70% (v/v) ethanol at 4 ° C overnight. Fixed cells were incubated in phosphatebuffered saline containing 1.5 μg/ml RNase A for 1 h at 37 ° C, followed by staining with 5 μl of PI for 20 min on ice. Then, cells were collected on a nylon mesh filter (pore size, 40 μm), and cell cycles were assayed with a flow cytometer (FACSCalibur; Becton Dickinson) at an excitation of 488 nm and an emission of 585 nm, and analyzed using a BD CellQuest Pro software (Becton Dickinson).
Statistical Analysis Statistical analysis was carried out using Dunnett’s test, analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (PLSD) test, and unpaired t test. A p value of
