N1-guanyl-1,7-diaminoheptane sensitizes bladder cancer cells to doxorubicin by preventing epithelial-mesenchymal transition through inhibition of eukaryotic translation initiation factor 5A2 activation

Abstract

Drug resistance greatly reduces the efficacy of doxorubicin-based chemotherapy in bladder cancer treatment; however, the underlying mechanisms are poorly understood. We aimed to investigate whether N1-guanyl-1,7-diaminoheptane (GC7), which inhibits eukaryotic translation initiation factor 5A2 (eIF5A2) activation, exerts synergistic cytotoxicity with doxorubicin in bladder cancer, and whether eIF5A2 is involved in chemoresistance to doxorubicin-based bladder cancer treatment. BIU-87, J82, and UM-UC-3 bladder cancer cells were transfected with eIF5A2 siRNA or negative control siRNA before incubation with doxorubicin alone or doxorubicin plus GC7 for 48 h. Doxorubicin cytotoxicity was enhanced by GC7 in BIU-87, J82, and UM-UC-3 cells. It significantly inhibited activity of eIF5A2, suppressed doxorubicin-induced epithelial-mesenchymal transition in BIU-87 cells, and promoted mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Knockdown of eIF5A2 sensitized bladder cancer cells to doxorubicin, prevented doxorubicin-induced EMT in BIU-87 cells, and encouraged mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Combination therapy with GC7 may enhance the therapeutic efficacy of doxorubicin in bladder cancer by inhibiting eIF5A2 activation and preventing epithelial-mesenchymal transition.

Keywords: Doxorubicin; drug resistance; epithelial-mesenchymal transition; eukaryotic translation initiation factor 5A; urinary bladder neoplasms.

Fig. 1

Determination of the effect of N1-guanyl-1,7-diaminoheptane (GC7) on cytotoxicity and inhibition of eukaryotic translation initiation factor 5A2 (eIF5A2) activity in bladder cancer cells. BIU-87 (a), J82 (b), and UM-UC-3 (c) cells were incubated with different concentrations of GC7 for 48 h. The CCK8 values of the treated bladder cancer cells were normalized to the control group (Ctrl), which was incubated without GC7. **P < 0.01; ***P < 0.001. Effects of GC7 (50 μM) on hypusine formation of eIF5A2 in BIU-87 (d), J82 (e), and UM-UC-3 (f) cells after incubation in the presence of ³H-labeled spermidine were measured by fluorography after SDS-PAGE separation. Western blot analyses showed eIF5A2 steady state protein expression.

Fig. 2

Cytotoxicity of doxorubicin or doxorubicin plus N1-guanyl-1,7-diaminoheptane (GC7) in bladder cancer cells. GC7 (50 μM) significantly enhanced the cytotoxicity of doxorubicin in BIU-87 (a), J82 (b), and UM-UC-3 (c) cells. Solid and dashed lines denote the best fit and 95% confidence intervals, respectively, of the different treatments. Photomicrographs and bar charts depict the 5-ethynyl-2′-deoxyuridine (EdU) staining pattern and relative EdU-positive ratio, respectively, of BIU-87 (d), J82 (e), and UM-UC-3 (f) cells after 48 h of treatment with doxorubicin or doxorubicin plus GC7. ***P < 0.001.

Fig. 3

N1-guanyl-1,7-diaminoheptane (GC7) alters the expression of doxorubicin-induced epithelial–mesenchymal transition (EMT) markers in bladder cancer cells. Phase-contrast microscopic images (a), Western blot analyses of expression of EMT markers (E-cadherin and vimentin), and EMT-associated transcription factors (Twist-1, Zeb-1, and snail) (b), and immunofluorescent images of EMT markers (c) in control bladder cancer cells and bladder cancer cells treated for 48 h with doxorubicin alone, GC7 alone, or doxorubicin plus GC7. (d) Activity of eukaryotic translation initiation factor 5A2 (eIF5A2) was measured by fluorography through detection of newly synthesized hypusinated eIF5A2 in bladder cancer cells treated with doxorubicin (Dox), doxorubicin plus GC7, or vehicle.

Fig. 4

Effects of Twist-1 siRNA on expression levels of epithelial–mesenchymal transition markers and sensitivity to doxorubicin treatment in bladder cancer cells. Phase-contrast microscopic images (a) and Western blot analyses (b) of expression of epithelial–mesenchymal transition markers in doxorubicin-treated Twist-1 or negative siRNA-transfected bladder cancer cells. (c) Cytotoxicity of doxorubicin in Twist-1 or negative siRNA-transfected bladder cancer cells. Solid and dashed lines denote the best fit and 95% confidence intervals, respectively, of the different treatments.

Fig. 5

Cytotoxicity of doxorubicin or doxorubicin plus N1-guanyl-1,7-diaminoheptane (GC7) in eukaryotic translation initiation factor 5A2 (eIF5A2) siRNA-transfected bladder cancer cells. Knockdown of eIF5A2 reduced the synergistic effect of GC7 plus doxorubicin in BIU-87 (a), J82 (b), and UM-UC-3 (c) cells. Solid and dashed lines denote the best fit and 95% confidence intervals, respectively, of the different treatments. Bonferroni's post-hoc test revealed no significant difference (P > 0.05 for doxorubicin versus doxorubicin plus GC7). Photomicrographs and bar charts depict the 5-ethynyl-2′-deoxyuridine (EdU) staining pattern and relative EdU-positive ratio, respectively, of eIF5A2 siRNA-transfected BIU-87 (d), J82 (e), and UM-UC-3 (f) cells after 48 h of treatment with doxorubicin or doxorubicin plus GC7.

Fig. 6

Knockdown of eukaryotic translation initiation factor 5A2 (eIF5A2) alters doxorubicin-induced epithelial–mesenchymal transition in BIU-87 cells and the mesenchymal phenotype of J82 and UM-UC-3 bladder cancer cells. Western blot analyses of expression of epithelial–mesenchymal transition markers (E-cadherin and vimentin) and E-cadherin repressor (Twist-1) in bladder cells transfected with eIF5A2 siRNA (+) or negative siRNA (−) and treated with doxorubicin for 48 h.