Lycorine exerts antitumor activity against osteosarcoma cells in vitro and in vivo xenograft model through the JAK2/STAT3 pathway

Abstract

Background: Lycorine, a natural alkaloid, has been indicated to have various physiological effects, including a potential effect against cancer. However, the anticancer effect of lycorine on osteosarcoma (OS) and the detailed molecular mechanisms involved remain unclear. Purpose: The purpose of this study was to examine the effect of lycorine on human OS and elucidated it underlying mechanisms Materials and methods: In vitro assays, OS cells were treated with lycorine at various concentrations. Then the cell proliferation, colony formation, cell cycle distribution, apoptosis, migration and invasion were assayed to detect the anticancer effect of lycorine on OS cell lines. Western blotting analysis was used to verify the expression of related proteins. In addition, the mouse xenograft model was performed to evaluate lycorine's therapeutic potential on OS in vivo. Results: The in vitro results demonstrated that lycorine induced apoptosis and cell cycle arrest and suppressed the migration and invasion by suppressing constitutive signal transducers and activators of transcription 3 (STAT3) activation through enhancing the expression of SH2 domain-containing phosphatase 1 (SHP-1) and downregulating the expression of STAT3 target proteins. Moreover, our mouse xenograft model revealed that lycorine inhibited the tumor growth in vivo. Conclusion: These results demonstrated that the anti-OS effects of lycorine were at least partly due to the suppression of the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2)/STAT3 pathway. Taken together, these results indicate that lycorine possesses the potential to be a promising candidate in clinical therapy for human OS in the future.

Keywords: JAK2/STAT3; SHP-1; anticancer; lycorine; osteosarcoma.

Figure 1

Effect of lycorine on the proliferation and colony formation of OS cells. Notes: (A) The anti-proliferative effect of lycorine on OS cell lines was determined by CCK8 assay. Cells were treated with different concentrations of lycorine for 24, 48, and 72 h. (B) Comparison of the effect of lycorine on human bone marrow stromal cells with that on OS cells for 24 h. (C) EdU assay detecting cell proliferation. EdU assays were performed 48 h after treatment with lycorine at various concentrations. (D) EdU-positive cells were caculated with the following formula: (EdU add-in cells/Hoechst stained cells) ×100%. (E) Representative images of the colony formation assays of MNNG/HOS and U2OS cells are shown. (F, G) The colonies that consisted of at least 50 cells were counted. Data are presented as the mean ± SD of three independent experiments. **P<0.001 vs control. Abbreviations: OS, osteosarcoma; EdU, 5-ethynyl-2-deoxyuridine.

Figure 2

Lycorine induces apoptosis and cell cycle arrest in MNNG/HOS and U2OS cells. Notes: (A) MNNG/HOS and U2OS cells treated with lycorine were stained with Annexin V-PE/7-AAD and analysed by flow cytometry. (B) The proportion of apoptotic MNNG/HOS and U2OS cells. (C-E) MNNG/HOS and U2OS cells were treated with Lycorine for 48 h. The expressions expression levels of cell apoptosis-related proteins were measured by Western blot. And the Bcl-2 protein expression levels in both cells were statistically analysed. (F, G) Flow cytometric analysis of the percentage of cells treated with Lycorine in various phases of the cell cycle. (H) The expressions of cell cycle-regulated proteins were measured by Western blot. Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs control, **P<0.001 vs control. Abbreviation: Annexin V-PE/7-AAD, Annexin V-phycoerythrin/7-amino actinomycin.

Figure 3

Effects of lycorine on the migration and invasion of MNNG/HOS and U2OS cells. Notes: (A, B) Micrographs of wound healing assays for MNNG/HOS and U2OS cells treated with Lycorine. Images were obtained at 0 and 24 h (×100 magnification). The percentage of wound closure (original width –width at 24 h after scraping/original width) was calculated. (E, F) A Transwell assay was used to detect the migration of MNNG/HOS and U2OS cells. The number of migratory cells was observed and counted by using a light microscope (×100 magnification). (G, H) A Transwell assay was used to detect the invasion of MNNG/HOS and U2OS cells. The number of the invasive cells was observed and counted by using a light microscope (×100 magnification). (I) The tumour metastasis-related protein levels were detected by Western blotting. Data are presented as the mean ± SD of three independent experiments **P<0.001 vs control.

Figure 4

Lycorine inhibits the JAK2/STAT3 signaling pathway by enhancing SHP-1 expression. Notes: (A) MNNG/HOS and U2OS cells were treated with various concentrations of lycorine for 48 h and then the cell lysates were extracted for Western blot analysis by using antibodies specific to JAK2, p-JAK2, STAT3, p-STAT3, and SHP-1. (B, C) SHP-1 activity was measured in the cells 24 h after treatment with or without lycorine. (D) A schematic illustration of the hypothesized mechanism for the anti-OS activity of lycorine. Data are presented as the mean ± SD of three independent experiments. **P<0.001 vs control. Abbreviations: JAK2, Janus kinase 2; STAT3, signal transducers and activators of transcription 3; SHP-1, SH2 domain-containing phosphatase 1.

Figure 5

Lycorine inhibits OS xenograft growth in vivo. Notes: MNNG/HOS cells were injected into the right axilla of nude mice. Four days after tumour inoculation, the mice were randomly divided into four groups and then treated with lycorine (10 mg/kg every 2 days or 20 mg/kg every 2 days), DMSO (negative control) or cisplatin (positive control). (A, B) An image of the nude mice xenograft tumours was taken after the lycorine treatment for 10 days. (C) Tumour weights were measured after ten days of lycorine administration. (D, E) Tumour volumes and nude mice weights were measured and calculated every 2 days. (F) H&E staining indicated that no major organ(heart, liver, spleen, lung, and kidney) toxicities were observed (×200 magnification). (G) Immunohistochemical analysis of Ki-67 to indicate cell proliferation in tumour tissues (×500 magnification). Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs control, **P<0.001 vs control.