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, 123 (4), 958-66

TW-37, a Small-Molecule Inhibitor of Bcl-2, Inhibits Cell Growth and Invasion in Pancreatic Cancer

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TW-37, a Small-Molecule Inhibitor of Bcl-2, Inhibits Cell Growth and Invasion in Pancreatic Cancer

Zhiwei Wang et al. Int J Cancer.

Retraction in

  • Retraction
    Int J Cancer 139 (9), 2146. PMID 27534921. - Retraction of Publication
    This article has been retracted at the request of: Editor-in-Chief and Author 'TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and invasion in pancreatic …

Abstract

Bcl-2 family of proteins plays critical roles in human cancers, including pancreatic cancer, suggesting that the discovery of specific agents targeting Bcl-2 family proteins would be extremely valuable for pancreatic cancer therapy. We have previously reported the synthesis and characterization of TW-37, which seems to be a negative regulator of Bcl-2. In this investigation, we tested our hypothesis whether TW-37 could be an effective inhibitor of cell growth, invasion and angiogenesis in pancreatic cancer cells. Using multiple cellular and molecular approaches such as MTT assay, apoptosis enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, Western blotting, electrophoretic mobility shift assay for measuring DNA binding activity of NF-kappaB, migration, invasion and angiogenesis assays, we found that TW-37, in nanomolar concentrations, inhibited cell growth in a dose- and time-dependent manner. This was accompanied by increased apoptosis and concomitant attenuation of NF-kappaB, and downregulation of NF-kappaB downstream genes such as MMP-9 and VEGF, resulting in the inhibition of pancreatic cancer cell migration, invasion and angiogenesis in vitro and caused antitumor activity in vivo. From these results, we conclude that TW-37 is a potent inhibitor of progression of pancreatic cancer cells, which could be due to attenuation of Bcl-2 cellular signaling processes. Our findings provide evidence showing that TW-37 could act as a small-molecule Bcl-2 inhibitor on well-characterized pancreatic cancer cells in culture as well as when grown as tumor in a xenograft model. We also suggest that TW-37 could be further developed as a potential therapeutic agent for the treatment of pancreatic cancer.

Conflict of interest statement

Conflict of Interest: University of Michigan has filed a patent on TW-37, which has been licensed by Ascenta Therapeutics Inc. University of Michigan and Dr. Shaomeng Wang own equity in Ascenta. Dr. Shaomeng Wang also serves as a consultant for Ascenta and is the principal investigator on a research contract from Ascenta to University of Michigan.

Figures

Figure 1
Figure 1
(a,b), Chemical structure of TW-37 and TW-37a. (c), The level of Bcl-2 expression was compared between a panel of pancreatic cancer cell lines. The expression of protein was assayed by Western blot analysis. (d), NF-κB activation was evaluated by the EMSA between a panel of pancreatic cancer cell lines, including AsPC-1, BxPC-3, Colo-357, HPAC, L3.6pl, MIAPaCa and PANC-1 cell lines (lanes 1–7, respectively). Retinoblastoma protein level served as the nuclear protein loading control. Supershift assay showed that NF-κB band was shifted because of the formation of a bigger complex after addition of anti- NF-κB p65 antibody. This assay confirmed the specificity of NF-κB binding to the DNA consensus sequence. Lane 1, nonspecific antibody (anti-cyclin D1); lane 2, p65 antibody.
Figure 2
Figure 2
Effect of TW-37 on pancreatic cancer cell growth and apoptosis. (a) Dose and time responses of TW-37 on growth of pancreatic cancer cells. Cells were seeded in 96-well plates at 5,000 cells per well and treated with varied concentrations of TW-37 or for different time periods. After treatment, cell densities were determined by MTT assay. Each value represents the mean ± SD (n = 6) of 3 independent experiments. *p < 0.05, **p < 0.01, compared to the control. (b) Cell death assay for measuring apoptosis induced by TW-37.BxPC-3, HPAC and Colo-357 cells were cultured in RPMI containing 5% FBS and exposed to different dose TW-37 for 72 hr. Apoptosis was measured by Histone DNA ELISA. Values are reported as mean ± SD. *p < 0.05, **p < 0.01, compared to the control.
Figure 3
Figure 3
Inhibition of NF-κB activation and the expression of its target genes by TW-37. (a), EMSA analysis was done for pancreatic cancer cells. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. Retinoblastoma protein level served as the nuclear protein loading control. Left panel: over-expression of Bcl-2 by cDNA transfection increased NF-κB DNA-binding activity. However, down-regulation of Bcl-2 by siRNA inhibited the NF-κB DNA-binding activity in Colo-357 pancreatic cancer cells. 1, control siRNA; 2, Bcl-2 siRNA; 3, control plasmid; 4, Bcl-2 cDNA plasmid. Middle and right panel: inhibition of NF-κB DNA binding activity by 500 nM TW-37 in time-dependent manner and dose-dependent manner for 72 hr in Colo-357 and BxPC-3 pancreatic cancer cells, respectively. (b) Inhibition of NF-κB target gene expression by TW-37 treatment of Colo-357 pancreatic cancer cells for 72 hr. Western blot analysis showed that TW-37 inhibited the expression of Cyclin D1, Survivin, VEGF, MMP-9, and COX-2 genes in pancreatic cancer cells. (c), TW-37 inhibited the expression of IKKβ, pIκBα and p65 in BxPC-3 and Colo-357 cell lines. (d), Left panel: inhibition of NF-κB DNA-binding activity by p65 siRNA and TW-37 in Colo-357 cells tested by EMSA (1, control; 2, p65 siRNA; 3, 500 nM TW-37; 4, p65 siRNA and 500 nM TW-37). Right panel: the effect of p65 cDNA transfection and TW-37 on the NF-κB DNA-binding activity (1, control; 2, p65 cDNA; 3, 500 nM TW-37; 4, p65 cDNA and 500 nM TW-37).
Figure 4
Figure 4
(a) Left panel: Induction of apoptosis by p65 siRNA and TW-37 in Colo-357 cells tested by ELISA (con, control; si, p65 siRNA; TW, 500 nM TW-37; si + TW, p65 siRNA and 500 nM TW-37). Right panel: the effect of p65 cDNA transfection and TW-37 on the induction of apoptosis (con, control; p65, p65 cDNA; TW, 500 nM TW-37; p65 + TW, p65 cDNA and 500 nM TW-37). *p < 0.05, compared to the control. (b) TW-37 inhibited the expression and activity of MMP-9 in Colo-357 pancreatic cancer cells. Left panel: Real-time RT-PCR analysis of MMP-9 mRNA expression in pancreatic cancer cells treated with TW-37 for 72 hr. Right panel: MMP-9 activity assay showing that MMP-9 was inhibited by TW-37 treatment for 72 hr. *p < 0.05, compared to the control. (c) TW-37 inhibited the expression and activity of VEGF. Left panel: Real-time RT-PCR analysis of VEGF mRNA expression in Colo-357 pancreatic cancer cells treated with TW-37. Right panel: VEGF activity assay showing that VEGF level in the culture medium was inhibited by TW-37 treatment for 72 hours. *p < 0.05, compared to the control.
Figure 5
Figure 5
TW-37 decreased pancreatic cancer cell migration, invasion and reduced the HUVECs tube formation. (a) Left panel: Migration assay showing that 250 nM TW-37 decreased Colo-357 pancreatic cancer cell migration at 24 hr. Right panel: value of fluorescence from the migrated cells as presented in the left panel. *p < 0.05, compared to the control. (b) Left panel: invasion assay showing that 250 nM TW-37-treated cells resulted in low penetration through the Matrigel-coated membrane at 24 hr, compared to control cells. Right panel: value of fluores-cence from the invaded cells. The value indicated the comparative amount of invaded cells as shown in the left panel. *p < 0.05, compared to the control. (c) Left panel: conditioned media from 250 nM TW-37-treated Colo-357 cells were able to significantly reduce the tube formation of HUVECs in 6 hr incubation compared to the medium from control cells. Right panel: Image analysis of tubule/capillary length was carried out using software image analysis program Scion Image. Quantification of cumulative tube length of endothelial cells is also shown. *p < 0.05, compared to the control.
Figure 6
Figure 6
TW-37 inhibits tumor growth and the expression of NF-κB target genes in vivo. Colo-357 xenografts were generated by inoculating cells subcutaneously (s.c.) in SCID mice. Once transplanted, fragments developed into palpable tumors (about 80 mg), and groups of 9 animals were removed randomly and assigned to different treatment groups. Mice were injected with TW-37 at 20 mg/kg iv x3 days, for 2 cycles. The control group received vehicle only. (a,b) TW-37 retards the growth of Colo-357 tumor xenografts in nude mice. Tumor volumes in SCID mice were plotted against time (a) and total tumor weight at time of sacrifice. (b) *p < 0.05, compared to the control. (c) TW-37 inhibits NF-κB DNA-binding activity in vivo. Tumor xenografts were removed, and nuclear protein extracts were prepared. Binding of NF-κB consensus element with nuclear extracts was detected by EMSA. Retinoblastoma (Rb) protein level was used as a nuclear protein loading control. (d) The expression of NF-κB target genes was detected by Western blotting of tumor tissue extracts. TW-37 inhibited the expression of NF-κB target genes, including VEGF, MMP-9, COX-2, Cyclin D1 and Survivin in tumor tissue of control compared to TW-37 treated animals.

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