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. 2017 May 15;7(1):1917.
doi: 10.1038/s41598-017-02162-9.

PDE3 inhibitor and EGCG combination treatment suppress cancer stem cell properties in pancreatic ductal adenocarcinoma

Motofumi Kumazoe  1 , Mika Takai  1 , Shun Hiroi  1 , Chieri Takeuchi  1 , Maasa Yamanouchi  2 , Takashi Nojiri  3 , Hiroaki Onda  3 , Jaehoon Bae  1 , Yuhui Huang  1 , Kanako Takamatsu  1 , Shuya Yamashita  1 , Shuhei Yamada  1 , Kenji Kangawa  3 , Takashi Takahashi  4 , Hiroshi Tanaka  2 , Hirofumi Tachibana  5
Affiliations

PDE3 inhibitor and EGCG combination treatment suppress cancer stem cell properties in pancreatic ductal adenocarcinoma

Motofumi Kumazoe et al. Sci Rep. .

Abstract

Recurrence following chemotherapy is observed in the majority of patients with pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that cancer stem cells (CSCs) may be involved in PDAC recurrence and metastasis. However, an efficient approach to targeting pancreatic CSCs remains to be established. Here we show that in cancer cells overexpressing the 67-kDa laminin receptor (67LR)-dependent cyclic GMP (cGMP) inducer, epigallocatechin-3-O-gallate (EGCG) and a phosphodiesterase 3 (PDE3) inhibitor in combination significantly suppressed the Forkhead box O3 and CD44 axis, which is indispensable for the CSC properties of PDAC. We confirmed that the EGCG and PDE3 inhibitor in combination strongly suppressed tumour formation and liver metastasis in vivo. We also found that a synthesized EGCG analog capable of inducing strong cGMP production drastically suppressed the CSC properties of PDAC and extended the survival period in vivo. In conclusion, the combination treatment of EGCG and a PDE3 inhibitor as a strong cGMP inducer could be a potential treatment candidate for the eradication of CSCs of PDAC.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
PDE3A was overexpressed in CD44+ cells of PDAC. (a) Immunofluorescence staining for PDE3A of pancreatic cancer patient tissues (×10) (n = 5). (b–e) Panc-1, BxPC-3, and MIAPaCa-2 cells (×40), PDAC patient tissues (×60). (f–i) Correlation between CD44 expression and PDE3A expression in cells (Panc-1 cells n = 32, BxPC-3 cells n = 19, MIA PaCa-2 cells n = 8, Primary cancer cells n = 53). (j–m) Expression of PDE3A in CD44− cells and CD44+ cells ((j) CD44− cells n = 21, CD44+ cells n = 11, (k) CD44− cells n = 12, CD44+ cells n = 7, (l) CD44− cells n = 5, CD44+ cells n = 3, (m) CD44− cells n = 34, CD44+ cells n = 19). A Spearman rank test was used for correlations. All data are presented as means ± SEM.
Figure 2
Figure 2
PDE3 inhibitor synergistically potentiated the inhibitory effect of EGCG on CSCs. (a) Colony assay in the presence or absence of EGCG (5 μM) or trequinsin (2.5 μM) (n = 3). (b–d) Spheroid assay in the presence or absence of EGCG or trequinsin (2.5 μM) (n = 3). Scale bar: 50 μm. (e) Isobologram analysis revealed the synergism of EGCG plus trequinsin combination. (f) Panc-1 cells were pretreated with anti-67LR antibodies or control IgM antibodies for 3 h, then treated with EGCG (25 μM) for 21 days (n = 3). (g) Spheroid assay in the presence or absence of EGCG or trequinsin (n = 3). All data are presented as the means ± SEM.
Figure 3
Figure 3
PDE3 inhibitor synergistically potentiated the inhibitory effect of EGCG on the FOXO3 and CD44 axis. (a) FOXO3 (48 h) and (b) CD44 (72 h) levels measured by Western blotting after EGCG (5 μM) and trequinsin (2.5 μM) treatment in Panc-1 cells. (n = 3). (c) Intercellular cGMP level measured after treatment with EGCG (5 μM) and the PDE3 inhibitor, trequinsin (2.5 μM) for 3 h (n = 3).
Figure 4
Figure 4
PDE3 inhibitor and 67LR agonist in combination inhibited metastasis in vivo. (a–e) Nude mice implanted with Panc-1 cells were injected with EGCG (10 mg/kg/2 days i.p.), trequinsin (5 mg/kg/2 days i.p.), and gemcitabine (100 mg/kg/6 days i.p.). (a) The tumor volume was measured (n = 7), (b,c) liver metastasis (n = 7). (d) Representative immunofluorescence staining for CD44 and FOXO3 on a tumor segment (×40). Scale bar: 50 μm. (e) AST and ALT levels in the serum were measured as an evaluation of toxicity (n = 7). All data are presented as the means ± SEM.
Figure 5
Figure 5
cGMP induction could be a novel therapeutic strategy for PDAC. (a) Panc-1 cells were treated with EGCG or its analogs (n = 3). (a,b) The chemical structure of the 67LR agonist No. 19 that demonstrated the strongest inhibitory effect on spheroid formation. (c) 67LR agonist activity determined (n = 3). (d) Intercellular cGMP levels in EGCG (5 μM) or No. 19 (5 μM) treated Panc-1 cells (n = 3). (e) BxPC-3 cell implanted mice were used as a peritoneal dissemination model and injected with EGCG (10 mg/kg/2 days i.p.), trequinsin (5 mg/kg/2 days i.p.), and No. 19 (5 mg/kg/2 days i.p.). (Control n = 9, the others n = 8). Overall survival was analyzed using a Kaplan–Meier survival model with a log-rank test.
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References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65:5–29. doi: 10.3322/caac.21254. - DOI - PubMed
    1. Burris HA, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol. 1997;15:2403–2413. doi: 10.1200/JCO.1997.15.6.2403. - DOI - PubMed
    1. Conroy T, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 2011;364:1817–1825. doi: 10.1056/NEJMoa1011923. - DOI - PubMed
    1. Rao CV, Mohammed A. New insights into pancreatic cancer stem cells. World J Stem Cells. 2015;7:547–555. doi: 10.4252/wjsc.v7.i3.547. - DOI - PMC - PubMed
    1. Bailey JM, et al. DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer. Gastroenterology. 2014;146:245–256. doi: 10.1053/j.gastro.2013.09.050. - DOI - PMC - PubMed

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