Cordyceps militaris Fraction induces apoptosis and G2/M Arrest via c-Jun N-Terminal kinase signaling pathway in oral squamous carcinoma KB Cells

Abstract

Background: Cordyceps militaris fraction (CMF) has been shown to possess in vitro antitumor activity against human chronic myeloid leukemia K562 cells in our previous research.

Materials and methods: The in vitro inhibitory activities of CMF on the growth of KB cells were evaluated by viability assay. The apoptotic and cell cycle influences of CMF were detected by 4',6-diamidino-2-phenylindole staining and flow cytometry assay. The expression of different apoptosis-associated proteins and cell cycle regulatory proteins was examined by Western blot assay. The nuclear localization of c-Jun was observed by fluorescence staining.

Objective: The objective of this study was to investigate the antiproliferative effect of CMF as well as the mechanism underlying the apoptosis and cell cycle arrest it induces in KB cells.

Results: CMF suppressed KB cells' proliferation in a dose- and time-dependent manner. Flow cytometric analysis indicated that CMF induced G2/M cell cycle arrest and apoptosis. Western blot analysis revealed that CMF induced caspase-3, caspase-9, and PARP cleavages, and increased the Bax/Bcl-2 ratio. CMF also led to increased expression of p21, decreased expression of cyclin B1, mitotic phosphatase cdc25c, and mitotic kinase cdc2, as well as unchanged expression of p53. In addition, CMF stimulated c-Jun N-terminal kinases (JNK) protein phosphorylations, resulting in upregulated expression of c-Jun and nuclear localization of c-Jun. Pretreatment with JNK inhibitor SP600125 suppressed CMF-induced apoptosis and G2/M arrest.

Conclusions: CMF is capable of modulating c-Jun caspase and Bcl-2 family proteins through JNK-dependent apoptosis, which results in G2/M phase arrest in KB cells. CMF could be developed as a promising candidate for the new antitumor agents.

Summary: CMF exhibited strong anticancer activity against oral squamous carcinoma KB cellsCMF inhibited KB cells' proliferation via induction of apoptosis and G2/M cell cycle arrestCMF activated JNK signaling pathway and promoted the nuclear localization of c-JunCMF regulated the apoptosis- and cell cycle-related proteins in a manner dependent on JNK/c-Jun pathway. Abbreviations used: CMF: Cordyceps militaris fraction; OSCC: Oral squamous cell carcinoma; JNK: c-Jun N-terminal kinase.

Keywords: Apoptosis; Cordyceps militaris fraction; KB cells; c-Jun N-terminal kinases/c-Jun; cell cycle arrest.

Figure 1

Cordyceps militaris fraction inhibits proliferation of KB cells. Cells were seeded at a density of 3.5 × 10³ cells/well in 96-well plates overnight and then treated with the different concentrations of Cordyceps militaris fraction for 24, 48, or 72 h. Cell viability was assessed by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Each value represents the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01, compared to the control

Figure 2

Cordyceps militaris fraction induces apoptosis and activates the intrinsic apoptotic pathway in KB cells. (a) Apoptotic morphological changes in KB cells. Apoptotic nuclei manifested condensed or fragmented DNA brightly stained by 4′,6-diamidino-2-phenylindole (48 h) (magnification, ×200). Arrows pointed to morphological changes in the cells. (b) Determination of apoptosis of KB cells by flow cytometry. After treatment of cells with Cordyceps militaris fraction, the percentage of apoptotic cells was detected by flow cytometry using Annexin V/propidium iodide staining. (c and d) Effect of Cordyceps militaris fraction on the expression of apoptosis-related proteins in KB cells. Cells were treated with the indicated concentrations of Cordyceps militaris fraction for 24 h, and cell lysates were analyzed by Western blot analysis

Figure 3

Cordyceps militaris fraction induces G2/M phase arrest and regulates the expression of cell cycle-related mitotic proteins in KB cells. (a) Effect of Cordyceps militaris fraction on cell cycle distribution of KB cells. After treatment of cells with Cordyceps militaris fraction, the cell cycle proportions were detected by flow cytometry using propidium iodide staining. (b) Histograms of cell cycle distribution in nontreated and treated KB cells. Each value represents the mean ± standard deviation of three independent experiments. *P < 0.05 and **P < 0.01, compared to the control group. (c and d) Effect of Cordyceps militaris fraction on the expression of cell cycle-related mitotic proteins in KB cells. Cells were treated with the indicated concentrations of Cordyceps militaris fraction for 8 h, and cell lysates were analyzed by Western blot analysis

Figure 4

Cordyceps militaris fraction induces activation of c-Jun N-terminal kinase and c-Jun in KB cells. (a and b) Effect of Cordyceps militaris fraction on the expression of phosphor mitogen-activated protein kinases and p-c-Jun in KB cells. Cells were treated with 50 μg/ml of Cordyceps militaris fraction for indicated hours (a) or with indicated concentrations of Cordyceps militaris fraction for 8 h (b), and cell lysates were analyzed by Western blot analysis. (c) Effect of p-38 inhibitor SB203580 on Cordyceps militaris fraction-regulated expression of p-p38 in KB cells. Cells were treated with 20 μM SB203580 for 1 h prior to exposure to 50 μg/ml of Cordyceps militaris fraction for 6 h, and cell lysates were analyzed by Western blot analysis. (d) Effect of c-Jun N-terminal kinase inhibitor SP600125 on Cordyceps militaris fraction-regulated expression of p-c-Jun N-terminal kinase in KB cells. Cells were treated with 20 μM SP600125 for 1 h prior to exposure to 50 μg/ml of Cordyceps militaris fraction for 6 h, and cell lysates were analyzed by Western blot analysis. (e) Effects of c-Jun N-terminal kinase inhibitor SP600125 and p-38 inhibitor SB203580 on Cordyceps militaris fraction-regulated expression of p-c-Jun in KB cells. Cells were treated with 20 μM SP600125 or 20 μM SB203580 for 1 h prior to exposure to 50 μg/ml of Cordyceps militaris fraction for 6 h, and cell lysates were analyzed by Western blot analysis

Figure 5

Cordyceps militaris fraction increases the nuclear localization of c-Jun. Exposure to Cordyceps militaris fraction causes nuclear accumulation of phospho-c-Jun in KB cells. Cells were treated with Cordyceps militaris fraction for 24 h, and then fixed and immunostained with phospho-c-Jun antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Localization was visualized using laser scanning confocal microscope. Images were taken under × 40 objective

Figure 6

Cordyceps militaris fraction induces apoptosis and cell cycle arrest in a manner dependent on c-Jun N-terminal kinase/c-Jun signaling pathway. (a) Effect of c-Jun N-terminal kinase inhibitor SP600125 on Cordyceps militaris fraction-induced apoptosis in KB cells. Cells were treated with 20 μg/ml of Cordyceps militaris fraction alone or in combination with 20 μM SP600125 for 48 h, and then Annexin V-FITC/PI double-staining analysis was performed. (b) Effect of c-Jun N-terminal kinase inhibitor SP600125 on Cordyceps militaris fraction-induced G2/M arrest in KB cells. Cells were treated with 20 μg/ml of Cordyceps militaris fraction alone or in combination with 20 μM SP600125 for 24 h, and then PI staining analysis was performed. (c) Effect of c-Jun N-terminal kinase inhibitor SP600125 on Cordyceps militaris fraction-regulated expression of caspase-9 and PARP in KB cells. Cells were cotreated with 20 μg/ml of Cordyceps militaris fraction and 20 μM SP600125, and cell lysates were analyzed by Western blot analysis. (d) Effect of c-Jun N-terminal kinase inhibitor SP600125 on Cordyceps militaris fraction-regulated expression of mitotic cyclin/kinase/phosphatase-related proteins in KB cells. Cells were cotreated with 20 μg/ml of Cordyceps militaris fraction and 20 μM SP600125, and cell lysates were analyzed by Western blot analysis