Effect of Spicatoside a on Anti-Osteosarcoma MG63 Cells through Reactive Oxygen Species Generation and the Inhibition of the PI3K-AKT-mTOR Pathway

Abstract

Osteosarcoma is a primary malignant tumor found in the bones of children and adolescents. Unfortunately, many patients do not respond well to treatment and succumb to the illness. Therefore, it is necessary to discover novel bioactive compounds to overcome therapeutic limitations. Liriope platyphylla Wang et Tang is a well-known herb used in oriental medicine. Studies have shown that metabolic diseases can be clinically treated using the roots of L. platyphylla. Recent studies have demonstrated the anticarcinoma potential of root extracts; however, the exact mechanism remains unclear. The aim of this study was to examine the anti-osteosarcoma activity of a single compound extracted from the dried roots of L. platyphylla. We purified Spicatoside A (SpiA) from the dried roots of L. platyphylla. SpiA significantly inhibited the proliferation of human osteosarcoma MG63 cells in a dose- and time-dependent manner. SpiA also regulated the expression of various downstream proteins that mediate apoptosis (PARP, Bcl-2, and Bax), cell growth (cyclin D1, Cdk4, and Cdk6), angiogenesis (VEGF), and metastasis (MMP13). The Proteome Profiler Human Phospho-Kinase Array Kit showed that the AKT signaling protein was a target of SpiA in osteosarcoma cells. We also found that SpiA suppressed the constitutive activation of the PI3K-AKT-mTOR-p70S6K1 signaling pathway. We further validated the effects of SpiA on the AKT signaling pathway. SpiA induced autophagosome formation and suppressed necroptosis (a form of programmed cell death). SpiA increased the generation of reactive oxygen species (ROS) and led to the loss of mitochondrial membrane potential. N-acetylcysteine (NAC)-induced inhibition of ROS generation reduced SpiA-induced AKT inhibition, apoptotic cell death, and anti-metastatic effects by suppressing cell migration and invasion. Overall, these results highlight the anti-osteosarcoma effect of SpiA by inhibiting the AKT signaling pathway through ROS generation, suggesting that SpiA may be a promising compound for the treatment of human osteosarcoma.

Keywords: AKT; ROS; Spicatoside A; apoptosis; autophagy; necroptosis; osteosarcoma.

Figure 1

Isolation and characterization of Spicatoside A (SpiA) from Liriope platyphylla roots. (A) The strategy for extracting SpiA. (B,C) ¹H NMR (500 MHz, Pyridine-d₅) (B) and ¹³C-NMR (125 MHz, Pyridine-d₅) (C) spectra of SpiA. (D) HPLC evaluation and chemical structure of isolated SpiA.

Figure 1

Isolation and characterization of Spicatoside A (SpiA) from Liriope platyphylla roots. (A) The strategy for extracting SpiA. (B,C) ¹H NMR (500 MHz, Pyridine-d₅) (B) and ¹³C-NMR (125 MHz, Pyridine-d₅) (C) spectra of SpiA. (D) HPLC evaluation and chemical structure of isolated SpiA.

Figure 2

Anti-tumor effects of SpiA on cell proliferation and apoptosis in human MG63 cells. (A) MTT assay after SpiA or Vincristine treatment with the indicated doses for 3 days. The red dotted line indicates the boundary marks for 0, 24, 48, and 72 h. (B) BrdU cell proliferation assay after SpiA with the indicated doses for 3 days in cells. (C–F). Western blotting was performed after SpiA treatment with the indicated doses for 24 h. Western blotting of PARP, cleaved PARP (C), Bcl-2, Bax (D), cyclin D1, Cdk4/6 (E), VEGF, and MMP13 (F). The total levels of β-actin were used as a loading control for the samples. * indicates statistical significance at p < 0.05. Representative results of three independent experiments.

Figure 3

Anti-tumor effects of SpiA on AKT signaling in human MG63 cells. (A) Kinases in the Proteome Profiler Human Phospho-Kinase Array are shown in the table. (B) The phosphorylation profiles of 37 different kinases were analyzed after SpiA treatment for 24 h. Red rectangles indicate double spots with large differences. The density is illustrated in a bar graph. (C) Western blotting was performed after SpiA treatment with the indicated doses for 24 h. (D) Immunofluorescence assay to monitor phosphorylation levels of p70S6K (red) after SpiA treatment with the indicated doses for 24 h. DAPI staining (blue) indicates nuclei. Scale bar: 50 μm. Representative results of three independent experiments.

Figure 4

Anti-tumor effects of SpiA on autophagic and necroptotic processes in human MG63 cells. (A) After SpiA treatment with the indicated doses for 24 h, Western blotting was performed to assess autophagy. The relative level (%) normalized to β-actin is illustrated in the bar graph. The red dotted line indicates the boundary marks. (B,C) Immunofluorescence assay to monitor DAPGreen-positive autophagosomes (green) after SpiA treatment with the indicated doses for 24 h (SpiA) or control untreated cells (Con). The arrows indicate representative cells. (B). The DAPGreen (fold) is illustrated in a bar graph (C). (D) After SpiA treatment for 24 h, Western blotting was performed to assess necroptotic signaling. The total levels of β-actin were used as a loading control for the samples. The relative level (%) normalized to β-actin is illustrated in the bar graph. The red dotted line indicates the boundary marks. * indicates statistical significance at p < 0.05. Representative results of three independent experiments.

Figure 5

Anti-tumor effects of SpiA on ROS generation and mitochondria potential in human MG63 cells. (A,B) After treatment with SpiA for 24 h (SpiA) or no treatment (Con), the cells were incubated with CellROX™ Green reagent (A) and MitoTracker™ Red CMXRos (B) and analyzed under a fluorescence microscope. DAPI staining (blue) indicates nuclei. The arrows indicate representative cells. The relative fold is illustrated in a bar graph. Scale bar: 20 μm. (C) Mitochondrial membrane potential was detected using Rhodamine123 (Rh123) under a fluorescence microscope. The arrows indicate representative cells. The Rh123 (fold) is illustrated in a bar graph. Scale bar: 20 μm. (D) Cells were treated with SpiA for 24 h in the absence or presence of 5 mM NAC and Western blotting was performed. The total levels of β-actin were used as a loading control for the samples. The relative level (%) normalized to β-actin is illustrated in the bar graph. * and # indicate statistical significance at p < 0.05. Representative results of three independent experiments.

Figure 6

Anti-tumor effects of SpiA-induced ROS on cell death, migration, and invasion in human MG63 cells. (A,B) Cells were treated with SpiA in the absence or presence of 5 mM NAC for 24 h, and then apoptotic cell death was detected using the TUNEL assay. The relative level (%) is illustrated in the bar graph (B). Scale bar: 50 μm. (C,D) After SpiA treatment for 24 h in the absence or presence of 5 mM NAC, cell migration using the wound assay was monitored under a light microscope. The white dots indicate the movement of cells in the wounded area. (C). The migration rate (%) is illustrated in the bar graph (D). Scale bar: 100 μm (E,F). Cell invasion using the Boyden chamber assay was monitored under a light microscope (E). The invasion rate (%) is illustrated in the bar graph (F). Scale bar: 50 μm. *, and # indicate statistical significance at p < 0.05. Representative results of three independent experiments.