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, 9 (3), e91094
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Convallatoxin, a Dual Inducer of Autophagy and Apoptosis, Inhibits Angiogenesis in Vitro and in Vivo

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Convallatoxin, a Dual Inducer of Autophagy and Apoptosis, Inhibits Angiogenesis in Vitro and in Vivo

Seung Ya Yang et al. PLoS One.

Abstract

Autophagy and apoptosis are important processes that control cellular homeostasis and have been highlighted as promising targets for novel cancer therapies. Here, we identified convallatoxin (CNT), isolated from Antiaris toxicaria, as a dual inducer of autophagy and apoptosis. CNT exerts cytotoxic effects on a number of cancer and normal cell lines and induces apoptosis by increasing caspase-3 and poly ADP ribose polymerase (PARP) cleavage. Moreover, dose- and time-dependent autophagic activity was detected in CNT-treated cells, and mammalian target of rapamycin (mTOR)/p70S6K signal pathway inhibition was observed. Notably, CNT inhibits human umbilical vein endothelial cell (HUVEC) growth and exerts anti-angiogenic activity in vitro and in vivo. Collectively, these results demonstrate that the naturally occurring compound, CNT, is a novel anti-angiogenic compound via dual inducing of autophagy and apoptosis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chemical structure and cytotoxic effect of CNT on HeLa cells.
(A) The chemical structure of CNT (C29H42O10, MW 550.64). (B) The effect of crude extract of Antiaris toxicaria on cell growth. (C) The effect of CNT on cell growth. (D) The effect of CNT on cell viability.
Figure 2
Figure 2. CNT-induced apoptosis in HeLa cells.
(A) Enhancement of sub G0/G1 cell population by CNT for 2 days, and analyzed for apoptosis by FACS. (B) Increased cleavage of PARP and caspase-3, in a dose-dependent manner after 24 h. (C) Increased cleavage of PARP and caspase-3 in a time-dependent manner with 10 nM CNT. Tubulin was used as an internal control. (D) Effect of apoptosis inhibitor on CNT-treated HeLa cells. (E) Detection of DNA fragmentation in CNT- or camptothecin-treated cells.
Figure 3
Figure 3. Autophagic activity of CNT.
(A) Detection of autophagic vacuoles by MDC staining in HeLa cells incubated for 24 h in the absence or presence of 10 nM CNT or 10 µM rapamycin (N: non-treatment, Rapa: rapamycin, CNT: convallatoxin). (B) Detection of CNT-induced autophagic vacuoles by LysoTracker Red. HeLa cells were treated with 10 nM CNT or 10 µM rapamycin for 24 h. Scale bars indicate 50 µm. (C) The conversion of LC3-I to LC3-II in CNT-treated HeLa cells after 24 h with different concentrations of CNT or 10 µM rapamycin. (D) The conversion of LC3-I to LC3-II following treatment with 10 nM CNT over time. Tubulin was used as an internal control.
Figure 4
Figure 4. Effect of CNT on the mTOR/p70S6K signaling pathway.
Tubulin was used as an internal control.
Figure 5
Figure 5. Anti-proliferative activity of CNT on HUVECs.
(A) Growth inhibitory activity of CNT on HUVECs. (B) The effect of CNT on HUVEC viability as determined by the trypan blue assay. (C) The conversion of LC3-I to LC3-II and increased cleavage of PARP following treatment with 10 nM CNT over time on HUVECs. Tubulin was used as an internal control. (D) Relative ratio of LC3 conversion and cleaved PARP was calculated as the percentage of LC3-I conversion to LC3-II and PARP to cleaved PARP, respectively.
Figure 6
Figure 6. Anti-angiogenic activity of CNT in vitro and in vivo.
(A) Effect of CNT on HUVEC tube-forming ability. Arrows indicate broken tubes formed by VEGF-stimulated HUVECs. (B) Inhibitory activity of CNT on HUVEC invasiveness. (C) Effect of CNT on in vivo angiogenesis assay, CAM (NT: EtOH control, RA: Retinoic acid (1 µg/egg), and CNT (0.5 ng/egg)). Arrows indicate compound-mediated inhibition of CAM neovascularization. The inhibition ratio was calculated as the percentage of inhibited eggs relative to the total number of eggs tested.
Figure 7
Figure 7. Anti-angiogenic effect of CNT decreased in autophagy- or apoptosis-inhibited cells.
(A) Atg5 and LC3 conversion levels with 5 nM CNT treatment in normal HeLa cells or Atg5-knocked down cells. (B) Cleaved PARP levels with 10 nM CNT treatment in normal HeLa cells or Z-VAD-FMK pretreated cells. β-actin was used as an internal control. (C) Quantitative analysis of in vitro chemoinvasion assay with conditioned media from 5 nM CNT-treated HeLa cells or CNT-treated in Atg5-knocked down HeLa cells. (D) Quantitative analysis of in vitro chemoinvasion assay with conditioned media from 10 nM CNT-treated HeLa cells or with Z-VAD-FMK pretreated cells.
Figure 8
Figure 8. Schematic summary of CNT mediated signaling pathway related with anti-angiogenic activity.

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Grant support

This study was partly supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; 2009-0092964, 2009-0083522, 2011-0006165, 2012M3A9D1054520), the Center for Food and Drug Materials of Agriculture Science & Technology Development (PJ0079772012), the Rural Development Administration, the National R&D Program, the Ministry of Health & Welfare (0620360-1), and the Brain Korea 21 Plus Project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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