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. 2009 Apr 1;69(7):2757-65.
doi: 10.1158/0008-5472.CAN-08-3060. Epub 2009 Mar 24.

TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and induces apoptosis in pancreatic cancer: involvement of Notch-1 signaling pathway

Zhiwei Wang  1 , Asfar Sohail Azmi, Aamir Ahmad, Sanjeev Banerjee, Shaomeng Wang, Fazlul H Sarkar, Ramzi M Mohammad
Affiliations

TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and induces apoptosis in pancreatic cancer: involvement of Notch-1 signaling pathway

Zhiwei Wang et al. Cancer Res. .

Abstract

Overexpression of Bcl-2 family proteins has been found in a variety of aggressive human carcinomas, including pancreatic cancer, suggesting that specific agents targeting Bcl-2 family proteins would be valuable for pancreatic cancer therapy. We have previously reported that TW-37, a small-molecule inhibitor of Bcl-2 family proteins, inhibited cell growth and induced apoptosis in pancreatic cancer. However, the precise role and the molecular mechanism of action of TW-37 have not been fully elucidated. In our current study, we found that TW-37 induces cell growth inhibition and S-phase cell cycle arrest, with regulation of several important cell cycle-related genes like p27, p57, E2F-1, cdc25A, CDK4, cyclin A, cyclin D1, and cyclin E. The cell growth inhibition was accompanied by increased apoptosis with concomitant attenuation of Notch-1, Jagged-1, and its downstream genes such as Hes-1 in vitro and in vivo. We also found that down-regulation of Notch-1 by small interfering RNA or gamma-secretase inhibitors before TW-37 treatment resulted in enhanced cell growth inhibition and apoptosis. Our data suggest that the observed antitumor activity of TW-37 is mediated through a novel pathway involving inactivation of Notch-1 and Jagged-1.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The University of Michigan has filed a patent on TW-37, which has been licensed by Ascenta Therapeutics, Inc. The University of Michigan and S. Wang own equity in Ascenta. S. Wang also serves as a consultant for Ascenta and is the principal investigator on a research contract from Ascenta to The University of Michigan. The other authors disclosed no potential conflicts of interest.

Figures

Figure 1
Figure 1
Effect of TW-37 on pancreatic cancer cell growth. A, dose and time responses of TW-37 on growth of pancreatic cancer cells. Cells were seeded in 96-well plates at 5,000 per well and treated with varied concentrations of TW-37 for different times. After treatment, cell densities were determined by the WST assay. Columns, mean (n = 6) of three independent experiments; bars, SD. *P < 0.05, **P < 0.01, compared with the control. B and C, cell survival of the human pancreatic cancer cell lines BxPC-3 and Colo-357. Cells treated with varied concentrations of TW-37 for 72 h were evaluated by the clonogenic assay. Photomicrographic difference in colony formation in cells untreated and treated with TW-37 (B). There was a significant reduction in the colony formation in BxPC-3 and Colo-357 cells treated with TW-37 compared with control cells (C). P values represent comparisons between cells treated by TW-37 and control using the paired t test.
Figure 2
Figure 2
Effect of TW-37 on pancreatic cancer cell apoptotic death. A, BxPC-3 and Colo-357 cells were exposed to different concentrations of TW-37 for different times. Apoptosis was measured by histone DNA ELISA. Columns, mean; bars, SD. *P < 0.05, **P < 0.01, compared with the control. B, TUNEL was performed in BxPC-3 and Colo-357 cells treated with 500 nmol/L TW-37 for 72 h using an apoptosis detection kit. Propidium iodide stains both apoptotic and nonapoptotic cells red. Fluorescein-12-dUTP incorporation results in localized green fluorescence within the nucleus of apoptotic cells only. C, BxPC-3 and Colo-357 cells were treated with 500 nmol/L TW-37 for 48 h. After treatment, cells were washed with cold PBS and fixed in ethanol for 1 h. The cells were then stained with 5 µg/mL Hoechst for 30 min and visualized under a fluorescence microscope. Bright condensed, punctuate, or granular nuclei were considered apoptotic. We observed more bright condensed and granular stained nuclei in TW-37–treated cells compared with control. D, effect of TW-37 on cell cycle distribution. The 500 nmol/L TW-37–treated BxPC-3 and Colo-357 cells were harvested for cell cycle analysis using propidium iodide staining. X axis, DNA content; Y axis, the number of nuclei.
Figure 3
Figure 3
Effect of TW-37 on the expression of several known cell cycle regulatory factors. A, the protein levels of several cell cycle regulatory factors were detected by Western blotting in BxPC-3 and Colo-357 pancreatic cancer cells treated with TW-37 for 72 h. B and C, the mRNA levels of cell cycle regulatory factors were investigated by real-time RT-PCR in BxPC-3 and Colo-357 pancreatic cancer cells treated with TW-37 for 72 h.
Figure 4
Figure 4
Effect of TW-37 on Notch-1 expression in human pancreatic cancer cells. A, the expression of Notch-1, its ligand Jagged-1, and its target gene Hes-1 was detected by Western blotting. B, the Colo-357 pancreatic cancer cells treated with 500 nmol/L TW-37 for 72 h were subjected to immunofluorescent staining using anti–Notch-1 antibody and anti–Jagged-1 antibody. C, the Notch-1 mRNA level was detected in BxPC-3 and Colo-357 cell lines treated with TW-37 for 72 h as measured by real-time RT-PCR. D, top, GSI (L-685,458 and DAPT) significantly inhibited Colo-357 cell growth. TW-37 plus GSI inhibited Colo-357 cell growth to a greater degree compared with TW-37. Bottom, the expression of Notch-1 was detected by Western blotting to check the GSI efficacy of down-regulation of Notch-1.
Figure 5
Figure 5
Pancreatic cancer cell growth inhibition and cell death induced by GSI or Notch-1 siRNA and TW-37. Con, control; TW, TW-37; NS, Notch-1 siRNA; NS+TW, TW-37+Notch-1 siRNA; NP, Notch-1 plasmid; TW+NP, TW-37+Notch-1 plasmid. A, down-regulation of Notch-1 by siRNA significantly inhibited BxPC-3 and Colo-357 cell growth. TW-37 plus Notch-1 siRNA inhibited cell growth to a greater degree compared with TW-37 alone. B, pancreatic cancer cell death induced by Notch-1 siRNA and TW-37. Notch-1 siRNA–transfected cells were significantly more sensitive to spontaneous and TW-37–induced apoptosis. C, the expression of Notch-1 was detected by Western blotting to check the Notch-1 plasmid transfection efficacy. D, Notch-1 and Hes-1 expression was up-regulated by Bcl-2 cDNA. However, Notch-1 and Hes-1 expression was down-regulated by Bcl-2 siRNA.
Figure 6
Figure 6
TW-37 inhibits the expression of Notch-1, Jagged-1, and Hes-1 in vivo. A, the schedule for animal experiments. B, immunohistochemical demonstration of apoptosis by TUNEL in tissues harvested from tumor-bearing SCID mouse. Significant differences in the percentage of TUNEL-positive cells were noted in tumors derived from the TW-37 treatment group relative to control group. C, the expression of Notch-1, Jagged-1, Hes-1, p65, and PARP was detected by Western blotting of tumor tissue extracts. D, immunohistochemical staining of Colo-357 tumor xenografts. Tumors were resected and processed for routine histologic analysis, and the 5 µmol/L tissue sections were stained with antibodies to phospho-p65, Ki-67, and PCNA.
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