1. Volume 134, Issue 1, Pages 131-144 (January 2008)
γ-Secretase Inhibitors Enhance Taxane-Induced Mitotic Arrest and Apoptosis in Colon Cancer Cells 
Takashi Akiyoshi, Masafumi Nakamura, Kosuke Yanai, Shuntaro Nagai, Junji Wada, Kenichiro Koga, Hiroshi Nakashima, Norihiro Sato, Masao Tanaka, Mitsuo Katano 
Gastroenterology 
Volume 134, Issue 1, Pages (January 2008)
DOI: /j.gastro
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2. Figure 1
TXL-induced apoptosis is enhanced by DAPT. (A) DAPT increases TXL-induced apoptosis. SW480 and DLD-1 cells were treated with 75 μg/mL (SW480) or 50 μg/mL (DLD-1) 5-FU for 48 hours, 4 μmol/L CPT for 24 hours, 40 μmol/L (SW480) or 100 μmol/L (DLD-1) cisplatin for 24 hours, 200 ng/mL (SW480) or 30 ng/mL (DLD-1) TRAIL for 24 hours, or 5 nmol/L (SW480) or 20 nmol/L (DLD-1) TXL for 24 hours in the absence (white bars) or presence (black bars) of 25 μmol/L DAPT. Cells were then stained with Hoechst and examined by fluorescence microscopy. Apoptotic cells were counted. (B) SW480 and DLD-1 cells were treated with the appropriate drug combinations for 24 hours, stained with Hoechst 33342, and photographed. Bars = 10 μm. (C) SW480 and DLD-1 cells were treated with appropriate drug combinations for 24 hours and stained with Hoechst (D) Soft agar colony formation assay. SW480 and DLD-1 cells were cultured with appropriate drug combinations for 12 days. Colonies were stained with crystal violet and counted.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

3. Figure 2
The combined use of TXL and DAPT increases sub-G1 and G2/M populations. (A) SW480 and DLD-1 cells were treated with TXL (5 nmol/L for SW480 and 20 nmol/L for DLD-1 cells) in the absence or presence of 10 μmol/L or 25 μmol/L DAPT for 24 hours, and the cell cycle was analyzed by flow cytometry. (B) SW480 cells were treated with appropriate drug combinations. Cells were harvested at the indicated time points and analyzed by flow cytometry. (C) SW480 cells were cultured with 5 nmol/L TXL and 10 μmol/L Compound E or 5 μmol/L L-685,458 for 24 hours and analyzed by flow cytometry. (D) Cells were treated with TXL (5 nmol/L for SW480, 20 nmol/L for DLD-1, 50 nmol/L for LoVo cells, and 2 nmol/L for MK-1, GCTM-1, NCI-N87, MDA-MB-231, SK-BR-3, and MCF-7 cells) in the absence (white bars) or presence (black bars) of 25 μmol/L DAPT for 24 hours (48 hours for LoVo). The cell cycle was analyzed by flow cytometry. Percentages of sub-G1 (left) and G2/M cells (right) are indicated.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

4. Figure 3
γ-Secretase inhibitors increase mitotic arrest in colon cancer cells. (A) SW480, DLD-1, and MCF-7 cells were treated with appropriate concentrations of TXL for 12 hours, followed by assay for cyclin B1/cdk1 kinase activity. No difference in cdk1 protein levels was detected by immunoblotting. (B) Increase in cyclin B1/cdk1 activity by DAPT in colon cancer cells but not in MCF-7 cells was seen. SW480, DLD-1, and MCF-7 cells were treated with TXL (5 nmol/L for SW480, 20 nmol/L for DLD-1, and 2 nmol/L for MCF-7 cells) in the absence or presence of 25 μmol/L DAPT or 25 μmol/L roscovitine for 12 hours and assayed for cyclin B1/cdk1 kinase activity as in A. (C) SW480 and DLD-1 cells were treated with appropriate combinations of drugs for 12 hours and assayed for cyclin B1/cdk1 kinase activity. (D) SW480 and DLD-1 cells were treated with TXL with or without 25 μmol/L DAPT or 10 μmol/L Compound E or 5 μmol/L L-685,458 for 12 hours and assayed for cyclin B1/cdk1 kinase activity. (E) SW480 cells were treated with 5 nmol/L TXL with or without 25 μmol/L DAPT and analyzed for MPM-2 reactivity by flow cytometry at the indicated time points. The percentage of MPM-2–positive cells is indicated for each sample. (F) SW480 cells were treated with 5 nmol/L TXL with or without 25 μmol/L DAPT and analyzed for expressions of MPM-2, cyclin B1, cdk1, survivin, p27, p21, and glyceraldehyde-3-phosphate dehydrogenase by immunoblotting at the indicated time points. Equal amounts of proteins from MCF-7 cells were loaded with the positive control for p27.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

5. Figure 4
Increased cdk1 activity is a consequence of TXL- and TXL plus DAPT–induced apoptoses. (A) SW480 cells were treated with the appropriate combinations of drugs (5 nmol/L TXL, 25 μmol/L DAPT, 25 μmol/L roscovitine) for 24 hours, followed by flow cytometry. (B) SW480 cells were treated with appropriate combinations of drugs for 12 hours and assayed for cyclin B1/cdk1 kinase activity. (C) Real-time RT-PCR showed that siRNA targeting CDC2 reduced its mRNA level by 87% 24 hours after transfection in SW480 cells. cdk1 protein expression also markedly decreased in SW480 cells 48 or 72 hours after transfection. (D) SW480 cells transfected with control or cdk1 siRNAs were harvested after 48 hours, treated with 5 nmol/L TXL with or without 25 μmol/L DAPT for 24 hours, and then examined by flow cytometry. (E) SW480 cells were treated with 150 μg/mL 5-FU for 48 hours or with indicated combinations of 5 nmol/L TXL and 25 μmol/L DAPT for 24 hours with or without 25 μmol/L zVAD-fmk, and caspase-3 activity was measured. (F) SW480 cells were treated as in E, and the cell cycle was analyzed by flow cytometry. The percentage of hypodiploid cells with sub-G1 DNA content from triplicate determinations is indicated.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
DAPT blocks Notch signaling in colon cancer cells. (A) RT-PCR was used to analyze the expression of a variety of Notch components in SW480, DLD-1, MCF-7, and human umbilical vein endothelial cells. (B) Expressions of Hes1, Hey1, and Hey2 mRNAs in paired normal human colon and colon tumor samples. Logarithmic ratios of tumor (T)/normal (N) tissue mRNA levels (Log T/N) of Hes1, Hey1, and Hey2 were determined by real-time RT-PCR. (C) SW480 and DLD-1 cells were treated with 10 μmol/L DAPT for 8 hours, followed by immunoblot analysis with anti-cleaved Notch-1 (NICD) antibody. Actin was used as loading control. (D) Total RNA was isolated from SW480 and DLD-1 cells treated with 10 μmol/L DAPT for 8 hours, and Hes1 mRNA was quantified by real-time RT-PCR.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Silencing of Notch1–3 does not enhance TXL-induced mitotic arrest and apoptosis. (A) Real-time RT-PCR showed that siRNAs targeting Notch1–3 reduced mRNA levels by 80% 48 hours after transfection in SW480 cells. (B) Notch1–3 protein expression also markedly decreased in SW480 cells 48 hours after transfection. (C) Silencing of Notch1–3 did not enhance TXL-induced mitotic arrest and apoptosis. SW480 cells transfected with control or Notch1–3 siRNAs were harvested after 48 hours, treated with TXL for 24 hours, and then examined by flow cytometry.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

8. Figure 7
The combination of TXL and a γ-secretase inhibitor as a novel antitumor treatment against colon cancer in vivo. (A) SW480 cells (5 × 106) were injected subcutaneously into the flanks of athymic nude mice and grown for 5 days (∼50–75 mm3) before initiation of treatment. Animals (n = 6 per group) were injected intraperitoneally with vehicle, DAPT alone (D, 50 mg/kg), TXL alone (T, 15 mg/kg), or a combination of TXL and DAPT at appropriate time intervals (arrows). DAPT was given for 2 successive days. For single-agent treatment, vehicle was given in place of DAPT or TXL with the same schedule. Black circles, vehicle; black squares, DAPT alone; black triangles, TXL alone; ×, combination of TXL and DAPT. *P = .0026, vehicle versus TXL alone; **P < .0001, vehicle versus TXL + DAPT. (B) SW480 xenograft tumors on day 16. Mean tumor size in mice treated with a combination of TXL and DAPT was smaller than that of other treatment groups.
Gastroenterology  , DOI: ( /j.gastro )
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