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. 2015 Jul 30;6(21):18445-59.
doi: 10.18632/oncotarget.4315.

YM155, a survivin suppressant, triggers PARP-dependent cell death (parthanatos) and inhibits esophageal squamous-cell carcinoma xenografts in mice

Nan Zhao  1 , Yousheng Mao  2 , Gaijing Han  1 , Qiang Ju  1 , Lanping Zhou  1 , Fang Liu  1 , Yang Xu  1 , Xiaohang Zhao  1
Affiliations
Free PMC article

YM155, a survivin suppressant, triggers PARP-dependent cell death (parthanatos) and inhibits esophageal squamous-cell carcinoma xenografts in mice

Nan Zhao et al. Oncotarget. .
Free PMC article

Abstract

Here we demonstrated that sepantronium bromide (YM155), a survivin suppressant, inhibited esophageal squamous-cell carcinoma (ESCC) growth in mice bearing human ESCC xenografts without affecting body weight. In cell culture, YM155 decreased survivin levels and caused PARP-1 activation, poly-ADP polymer formation, and AIF translocation from the cytosol to the nucleus. Genetic knockdown of PARP-1 or AIF abrogated YM155-induced parthanatos cell death. Furthermore, FOS, JUN and c-MYC gene transcription, which is stimulated by activated PARP-1, was increased following YM155 treatment. Our data demonstrate that YM155 did not trigger apoptosis, but induced parthanatos, a cell death dependent on PARP-1 hyper-activation, and support clinical development of YM155 in ESCC.

Keywords: PARP; chemotherapy; esophageal cancer; parthanatos.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of YM155 on esophageal cancer cell lines
A. The chemical molecular formula of YM155. B. Whole cell protein extract from each of eight esophageal cancer cell lines was subjected to analyzed by western blotting using antibodies against survivin and SLC35F2. Quantitative values of relative survivin and SLC35F2 levels normalized to actin (mean ± SD). C. Dose-response curve for KYSE410, KYSE150, and MEF cells after YM155 treatment for 24 h. Cells were treated with the indicated concentrations of YM155 for 24 h. The survival curves of KYSE 410, KYSE150 and MEF cells were constructed using the CCK-8 assay. D. necrosis was measured as LDH release after YM155 treatment for 24 and 48 h in both KYSE410 and KYSE150 cells. E. The long-term viability of cells was determined after YM155 treatment for 24 h in KYSE410 and KYSE150 cells using the colony-formation assay.
Figure 2
Figure 2. YM155 triggers non-apoptotic cell death in KYSE410 and KYSE150
A. The cell-cycle analysis of KYSE410 and KYSE150 cells was performed using PI staining and analyzed by flow cytometry after 12 h of YM155 treatment. B. A histogram illustrated the percentages of KYSE410 and KYSE150 cells in G1, S and G2/M phases. C. Apoptosis, as quantified by annexin V/PI staining. KYSE410 and KYSE150 cells were treated with YM155 (20 nM) for 24 h. Cells were stained by annexin V/PI and analyzed by flow cytometry. D. KYSE410 and KYSE150 cells were treated with 20 nM YM155 for 12 or 24 h. Total cell lysates were assayed for caspase-9 and caspase-3 cleavage and for survivin and XIAP protein expression by western blotting. Beta-actin was used as a loading control.
Figure 3
Figure 3. YM155 induces DNA damage and ROS production in both KYSE410 and KYSE150 cells
A. KYSE410 and KYSE150 cells were treated with 20 nM YM155 for 12 h, stained with Mito Tracker Red CMXRos, and analyzed by flow cytometry. B. KYSE410 and KYSE150 cells were treated as indicated for 12 h. Cells were stained with H2DCF-DA for 20 min, and ROS were measured by flow cytometry. All experiments were conducted at least three times. C. Localization of gamma-H2AX in KYSE410 and KYSE150 cells was determined with immunofluorescent analysis after 12 h YM155 treatment. Nuclei were stained with DAPI, as shown in blue. Scale bars: 10 μm. D. Total cellular protein extract from KYSE410 and KYSE150 after YM155 treatment for 12 and 24 h was subject to analysis by western blotting using antibodies against gamma-H2AX. Quantitative values of relative gamma-H2AX levels on the right are normalized to actin (mean ± SD).
Figure 4
Figure 4. YM155 induces PARP-1-dependent parthanatos
A. Nuclear accumulation of active PARP-1 in KYSE410 cells after 12 h of YM155 treatment was evaluated using immunofluorescent analysis. Nuclei were stained with DAPI, as shown in blue. Scale bars: 10 μm. B. Following treatment with YM155 for 12 h, cytosolic fractions were isolated from the treated cells and analyzed for PARP-1, PAR and AIF by western blotting. HSP60 and Lamin B protein were used as loading and fraction controls, respectively. C. Accumulation of the poly-ADP polymer in KYSE410 was evaluated using immunofluorescent analysis after YM155 treatment for 12 h. Nuclei were stained with DAPI, as shown in blue. Scale bars: 10 μm. D. Total KYSE410 cell protein extract was analyzed for phosphorylation of AKT and ERK, mTOR and phosphorylation of S6 and 4-EBP by western blotting. Beta-actin was used as a loading control. E and F. The effects of PARP and AIF gene siRNA knockdown were analyzed by western blot analysis. G and H. After treatment with YM155, the survival curve following PARP and AIF knockdown in KYSE410 cells was detected using the CCK-8 assay.
Figure 5
Figure 5. Clustering display of the microarray data after YM155 treatment
A. RNA was extracted using an RNA isolation kit for microarray analysis after YM155 treatment for 6 h in KYSE410 cells. Heatmap representing genes upregulated and downregulated in the micro-array analysis of KYSE410, demonstrating gene categories with altered expression after YM155 treatment. Data are represented as log2 fold change, n = 2 per group. B. The upregulated genes were functionally analyzed using the online tool STRING. C. The upregulated genes were functionally classified based on their biological process using the DAVID functional annotation clustering tool. D. The mRNA levels of cell death-associated and candidate genes in KYSE410 treated with 20 nM YM155 for 6 h was determined using real-time RT-PCR. Data represent the mean ± SEM of relative mRNA levels versus untreated cells. E. Transmission electron microscopy of KYSE410 cells after YM155 treatment. The integrity of the membrane was noted in the untreated cells, and the collapse of the membrane and the swelling of the cellular organelles were observed in cells treated with YM155.
Figure 6
Figure 6. Inhibition of tumor growth in vivo
A. Tumor volume was measured every 2 to 3 days after YM155 treatment. KYSE410 cells decreased the growth of the xenograft tumors compared to the untreated group (P < 0.001). B. Mouse body weight was measured over the course of 45 days. YM155 did not affect mouse body weight. C. Tumor weight was significantly reduced at study termination (P < 0.05). D. Xenograft tissue was subject to analysis by immunohistochemistry for survivin, Ki67 and PARP (× 200). Survivin, Ki67 and PARP localized mainly in the nuclei of esophageal cancer epithelial cells. E. Illustration of the signaling pathway for YM155-induced PARP-mediated parthanatos. Innate hyperactive PARP and AIF translocation are required for PARP-1-dependent parthanatos induced by YM155. The blue and red arrows represent the downregulated and upregulated genes, respectively.
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