Differential Effects of Wedelia chinensis on Human Glioblastoma Multiforme Cells

Abstract

Introduction: Glioblastoma multiforme (GBM) is the most aggressive glioma, and its diffuse nature makes resection of it difficult. Moreover, even with the administration of postoperative radiotherapy and chemotherapy, prolonged remission is often not achieved. Hence, innovative or alternative treatments for GBM are urgently required. Traditional Chinese herbs and their functional components have long been used in the treatment of various cancers, including GBM. The current study investigated the antitumor activity of Wedelia chinensis and its major functional components, luteolin and apigenin, on GBM.

Materials and methods: MTT assay, Transwell migration assay, and flow cytometry analysis were adopted to assess the cell viability, invasive capability, and cell cycle. Immunofluorescence staining and Western blotting were used to detect the expressions of apoptotic and autophagy-related signaling molecules.

Results: The W. chinensis extract (WCE) significantly inhibited the proliferation and invasive ability of both GBM8401 and U-87MG cells in a dose-dependent manner. Moreover, differential effects of WCE on GBM8401 and U-87MG cells were observed: WCE induced apoptosis in GBM8401 cells and autophagy in U-87MG cells. Notably, WCE had significant effects in reducing the cell survival and invasive capability of both GBM8401 and U-87MG cells than the combination of luteolin and apigenin.

Conclusions: Taken together, these findings indicate the potential of using WCE and the combination of luteolin and apigenin for GBM treatment. However, further investigations are warranted before considering recommending the clinical use of WCE or the combination of luteolin and apigenin as the standard for GBM treatment.

Keywords: Wedelia chinensis extract; apoptosis; autophagy; glioblastoma multiforme; traditional Chinese medicine.

Figure 1.

Effects of Wedelia chinensis extract (WCE) on the viability of GBM8401, U-87MG, and human brain astrocyte (HBA) cells. Relative cell survival of GBM8401, U-87MG, and HBA cells after treatment with various concentrations of WCE for (A) 24 and (B) 48 hours. Similar results were observed in 3 repeated experiments. The numbers 1, 2, and 3 indicate a significant difference (P < .05) compared with control (0 μg/mL), HBA, and GBM8401 cells, respectively.

Figure 2.

Effects of Wedelia chinensis extract (WCE) on the invasive ability of GBM8401 and U-87MG cells. The representative photos of invaded (A) GBM8401 and (B) U-87MG cells treated with different concentrations of WCE for 24 hours. Quantified results of invaded percentage for both cells were shown in the lower panel, respectively. Similar results were observed in 3 repeated experiments. The symbol * indicates significant differences (P < .05) compared with control.

Figure 3.

Representative results of flow cytometry in GBM8401 and U-87MG cells in the presence of various concentrations of WCE for 24 hours. The arrow indicates the sub-G1 proportion. Similar results were observed in 3 repeated experiments.

Figure 4.

Expression of apoptotic proteins. (A) The expression of Bcl-2, Bax, cytochrome c (Cyt-C), Apaf-1, cleaved caspase-9, and cleaved caspase-3 proteins in GBM8401 cells treated with various concentrations of WCE for 24 hours. (B) Bars represent protein quantification relative to β-actin. Similar results were observed in 3 repeated experiments. The symbol * indicates P < .05 compared with control (0 μg/mL).

Figure 5.

Detection of LC3B puncta in U-87MG cells. Representative images of immunofluorescence staining with specific antibodies against LC3B proteins in U-87MG cells in the presence of different concentrations of WCE for 24 hours. Middle panel shows the images U-87MG cells stained with DAPI and right panel presents the merged images of DAPI and LC3B. Arrows indicate the expression of LC3B puncta. Similar results were observed in 3-repeated experiments. Scale bars = 15 μm.

Figure 6.

Expression of autophagy-related proteins. (A) The expression of LC3, Atg-7, Atg-5, Beclin-1, and P62 proteins in U-87MG cells treated with different concentrations of WCE for 24 hours. Bars represent the ratio of (B) LC3-II/LC3-I and the protein quantification of (C) Atg-7, (D) Atg-5, (E) Beclin-1, (F) P62 relative to β-actin. Similar results were observed in 3 repeated experiments. *Indicates P < .05 compared with control (0 μg/mL).

Figure 7.

Involvement of autophagy in the response of U-87MG cells on WCE. U-87MG cells were pre-treated (1 hour) with 25 µM CQ (autophagy inhibitor) before WCE treatment (3.72 μg/mL). (A) Cell lysates were collected after 24 hours and expressions of P62 and LC3-II were detected by western blot. Bars represent protein quantification of (B) P62 and (C) LC3-II relative to β-actin. Similar results were observed in 3 repeated experiments. The numbers 1, 2, and 3 indicate a significant difference (P < .05) compared with control (0 μg/mL), CQ (25 µM), and WCE (3.72 μg/mL), respectively.

Figure 8.

Effects of WCE, luteolin, apigenin, and combination of luteolin and apigenin on the viability of GBM8401 and U-87MG cells. Relative cell survival of (A) GBM8401 and (B) U-87MG in the presence of various concentrations of luteolin, apigenin, and WCE for 24 hours. (C) Relative cell survival of GBM8401 and U-87MG in the presence of luteolin, apigenin, the combination of luteolin and apigenin, and WCE for 24 hours. Similar results were obtained in 3-repeated experiments. The numbers 1, 2, 3, and 4 indicate a significant difference (P < .05) compared with control (0 μg/mL), luteolin, apigenin, and the combination of luteolin and apigenin, respectively.

Figure 9.

Effects of WCE, luteolin, apigenin, and combination of luteolin and apigenin on the invasive ability of GBM8401 and U-87MG cells. The representative photos of invaded (A) GBM8401 and (B) U-87MG cells treated with different concentrations of WCE for 24 hours. Quantified results of invaded percentage for both cells are shown in the lower panel, respectively. Similar results were observed in 3-repeated experiments. The numbers 1, 2, 3, and 4 indicate a significant difference (P < .05) compared with control (0 μg/mL), luteolin, apigenin, and the combination of luteolin and apigenin, respectively.