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. 2015 Jun 20;6(17):15348-61.
doi: 10.18632/onco_target.3610.

Lycorine is a novel inhibitor of the growth and metastasis of hormone-refractory prostate cancer

Meichun Hu  1 , Shihong Peng  1 , Yundong He  1 , Min Qin  1 , Xiaonan Cong  1 , Yajing Xing  1 , Mingyao Liu  1   2 , Zhengfang Yi  1
Affiliations

Lycorine is a novel inhibitor of the growth and metastasis of hormone-refractory prostate cancer

Meichun Hu et al. Onco_target. .

Abstract

Lycorine, a natural alkaloid extracted from the Amaryllidaceae plant family, has been reported to exhibit a wide range of physiological effects, including the potential effect against cancer. However, the anti-prostate cancer (PCa) efficacy of Lycorine remains unrevealed. In this context, we figured out Lycorine's anti-proliferative and anti-migratory properties for PCa treatment. Lycorine inhibited proliferation of various PCa cell lines, induced cell apoptosis and cell death. Here we showed that Lycorine decreased proliferation, migration, invasion, survival and EMT of prostate cancer cell lines. Subcutaneous and orthotopic xenotransplantations by ectopic implantation of the human hormone-refractory PC-3M-luc cells were used to confirm in vivo anticancer effects of Lycorine. Lycorine inhibited both growth and metastasis in multiple organs (liver, lung, kidney, spleen and bone) in vivo and improved mice survival. Lycorine prevented EGF-induced JAK/STAT signaling. Importantly, anti-cancer effects of Lycorine were dependent on STAT expression. We suggest that Lycorine is a potential therapeutic in prostate cancer.

Keywords: STAT3 signaling; hormone-refractory PCa; lycorine; tumor growth and metastasis.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1. Effects of Lycorine on proliferation, migration and invasion of PCa cells in vitro
A. Chemical structure of Lycorine. B. PC-3M, LNCaP, 22RV1, DU145 and PNT1A were treated with indicated concentrations of Lycorine (0, 0.05, 0.1, 1, 5, 10, 20, 50 and 100 μM) for 48 hours. Cell viability was assessed by MTS assay (n = 3). C. PC-3M, LNCaP, 22RV1 and DU145 cells were treated with Lycorine with indicated concentrations (from 0 μM to 50 μM) and hours (from 24 h to 96 h) to test the time- and dose-dependent effects. Cell viability was assessed by MTS assay (n = 3). D. PC-3M cells were allowed to migrate cross a “wound” when treated with Lycorine (from 0 μM to 10 μM) for 12 hours. The number of migrated cells were calculated. E. PC-3M cells were seeded on the upper chamber of Transwell. After 5 to 7 hours incubation with Lycorine (from 0 μM to 10 μM), migrated cells were fixed, stained and counted. F. PC-3M cells were treated with Lycorine (from 0 μM to 10 μM) for 12 hours and seeded in the upper chamber of Transwell coated with Matrigel to invade for another 12 hours. Invaded cells were fixed, stained and counted. All data are represented as mean ± S.D. from triplicate wells. *p < 0.05, **p < 0.01, ***p < 0.001, as compared to control.
Figure 2
Figure 2. Effects of Lycorine on PCa cell colony formation and apoptosis
A. 4 PCa cells were treated with different concentrations of Lycorine (from 0 μM to 10 μM), respectively. On the 8th day, colonies were fixed, stained, and counted. Representative results of these 4 cell lines were taken by camera. B. PC-3M cells were treated with different concentrations of Lycorine (from 0 μM to 50 μM) for 48 h and stained with live/dead reagent. The cell-permeable fluorescent dye Calcein AM stains live cells (green) and the dead cells are stained by EthD-II (red). Stained live and dead cells are visualized by fluorescence microscopy. C. PC-3M cells were treated with indicated concentrations of Lycorine (from 0 μM to 50 μM) for 48 hours. Apoptosis was assessed by Annexin V/PI staining and flow cytometry (n = 3). D. Statistic result of apoptosis results of PC-3M, DU145 and LNCaP cells when treated with indicated concentrations of Lycorine (from 0 μM to 50 μM) for 48 hours. E. PC-3M cells were incubated with various concentrations of Lycorine (from 0 μM to 25 μM) for 48 hours. Effects on the expression of CL–caspase 3 and PARP were determined by Western blotting. Beta-actin served as a loading control.
Figure 3
Figure 3. In vivo anticancer effects of Lycorine on PCa mouse xenograft models
A. Representative images of PC-3M subcutaneous tumor xenografts after mice sacrificed. PC-3M cells were injected subcutaneously into the nude mice. The tumor model was established according to the steps described in Materials and Methods. B. Quantitative analysis of growing tumor volume in mice back subcutaneous every 2 days. C. Representative in vivo bioluminescence images of mice bearing orthotropic–injected PC-3M-luc cells. D. Quantization of whole-body bioluminescence (total photon flux) in control and treatment groups. E. Representative images of prostate of mice bearing orthotropic–injected PC-3M-luc cells after mice sacrificed. F. Graphic representation of mice survival curves during therapeutic administration. The means and 95% confidence intervals (error bars) were presented (***P < 0.001, **P < 0.01, *P < 0.05).
Figure 4
Figure 4. Effects of Lycorine on PCa metastasis in vivo
A. Representative photos of metastasis site (prostate, lymph node, liver, lung, kidney, spleen, bone) monitored by in vivo bioluminescence detection of PC-3M-luc cells in excised organ from control and treatment tumor-bearing nude mice. B. Survey of mets to the whole body of each mouse monitored by in vivo bioluminescence detection of PC-3M-luc cells in excised organ from control and treatment tumor-bearing nude mice. C. Primary tumors sections were excised and processed for H&E staining and immunohistochemical analysis to detect Ki-67, cleaved-caspase 3, p-STAT3 and E-cadherin. D. Quantitative analysis of Ki-67, cleaved-caspase 3, p-STAT3 and E-cadherin about the immunohistochemistry results. The means and 95% confidence intervals (error bars) were presented (***P < 0.001, **P < 0.01, *P < 0.05).
Figure 5
Figure 5. Reverse effect of Lycorine on EMT in PCa cells
A. PC-3M cells were treated with 10ng/ml EGF and indicated concentrations of Lycorine (0, 10 and 25 μM) for 48 hours, and then the cell morphology changes were photographed. B–C. RT-PCR analysis and Western blotting analysis of Twist, E-cadherin, N-cadherin, vimentin and fibronectin in PC-3M cells. Cells were treated with 10ng/ml EGF and indicated concentrations of Lycorine (0, 10 and 25 μM) for 48 hours. Cell lysates were subjected to RT-PCR analysis (B) and Western blotting analysis (C). Beta-actin served as a loading control. D. Western blotting analysis for p-STAT3, STAT3, MMP2 and MMP9 in PC-3M cells. Cells were treated with10ng/ml EGF and indicated concentrations of Lycorine (0, 10 and 25 μM) for 48 hours, and then the cells were subjected to Western blotting analysis. Beta-actin served as a loading control. E. Lycorine's effects on the expression levels of endogenous STAT & p-STAT3 in DU145, PC-3M and LNCaP cells. Cells were treated with or without 10 μM Lycorine for 48 hours, and then the cells were subjected to Western blotting analysis. F. Statistic result of Lycorine's effects on the migration of DU145, PC-3M and LNCaP cells. The Transwell migration assay was conducted as described in Materials and Methods. G. PC-3M cells were transiently transfected with STAT3-siRNA or the non-_targeting scrambled siRNA for 48 hours and the interference effect of STAT3 was detected by Western blotting. Cells were treated with Lycorine (0, 10 and 25 μM) for another 48 hours and cell viability was tested by MTS assay.
Figure 6
Figure 6. Effect of Lycorine on JAK/STAT3 pathway in PCa cells
A. PC-3M cells were treated with 10ng/ml EGF and 10 μM Lycorine for 24 hours, then prepared for Western blotting of the expression of EGFR, JAK1/2, STAT3 and their phosphorylation. B. PC-3M cells were treated with increasing concentrations of Lycorine (from 0 μM to 25 μM) for 24 hours before being applied to ChIP assay to test the STAT3-dependent transcriptional activity of Bcl-xL, cyclin D1, and Twist. Beta-actin was used as internal controls. C. Schematic model of the hypothesized mechanism by which Lycorine inhibits PCa growth and metastasis.
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