Cordycepin Induces Apoptosis and G2/M Phase Arrest through the ERK Pathways in Esophageal Cancer Cells

Abstract

Esophageal cancer is one of the most aggressive and lethal gastrointestinal tract malignancies, with a poor overall five-year survival rate. Cordycepin, a major compound of Cordyceps sinensis, has been shown to have anticancer potential. This study focuses on the anticancer properties of cordycepin that target esophageal cancer and reveals molecular aspects underlying these effects. In our CCK-8 assays and colony formation assays, cordycepin significantly suppressed esophageal cancer cell proliferation. Moreover, cordycepin induced chromatin condensation in esophageal cancer cells and significantly increased the number of apoptotic cells through activation of caspase cascades, apoptotic signaling, and the regulation of Bcl-2 family members. Cell cycle assays showed that cordycepin altered cyclin-dependent kinase1 and cyclinB1 expression, which resulted in a G2/M phase blockade. Mechanistically, ERK pathway inactivation was involved in the anti-tumor functions of cordycepin. The same results were also observed in vivo. Taken together, these findings reveal that cordycepin induces pro-apoptosis and anti-proliferation mechanisms in cancer cells, and may represent a novel therapeutic agent.

Keywords: ERK; apoptosis; cell cycle; cordycepin; esophageal cancer.

Figure 1

Cordycepin suppresses ESCC cell proliferation. (A,B) ECA109 and TE-1 cells were treated with cordycepin (0, 20,40, 60, 80 and 100μg/ml) for 24, 48, and 72 h. Cell viability was assessed using a CCK-8 assay. (C) 293T cell viability was evaluated by CCK-8 analysis after treatment with cordycepin. (D,E) ECA109 and TE-1 cells were exposed to cordycepin and were allowed to form colonies for 14 days. Colony numbers were counted and recorded. Results are presented as mean ± SD (n= 3 independent experiments). *p<0.05, **p<0.01, and ***p<0.001.

Figure 2

Cordycepin induces apoptosis in ECA109 and TE-1 cells. (A) ECA109 and TE-1 cells were treated with cordycepin (0,40, 60, 80μg/ml) for 48h. Apoptosis was assessed by flow cytometry with annexin V-FITC/propidium iodide (PI) staining. The x-axis represents annexinV-FITC, and the y-axis represents propidium iodide (PI) staining. (B,C) The Q3 quadrant (annexin V/PI), Q4 quadrant (annexin V+/PI), and Q2 quadrant (Annexin V+/PI+) indicate the percentages of normal cells, early apoptotic and late apoptotic cells, respectively. (D,E) Nuclear morphological changes associated with apoptosis were evaluated by Hoechst 33342 staining. Results are presented as mean ± SD (n = 3 independent experiments). *p<0.05, **p<0.01, and ***p<0.001.

Figure 3

Cordycepin induces cell cycle arrest at G2/M phase and regulates the expression of cell cycle-related proteins. (A) ECA109 and TE-1 cells were treated with cordycepin (0,40, 60, 80μg/ml) for 48h.The cell cycle phases of treated cells were evaluated by flow cytometry. (B,C) The percentage of cells in the G1, S , and G2/M phases of the cell cycle are shown. (D) The protein levels of cell cycle regulators, such as CDK1 and cyclinB1, were examined by western blotting analysis and GAPDH was used as a loading control.

Figure 4

Cordycepin alters the expression of apoptosis-related proteins and MEK/ERK signaling proteins. (A) ECA109 and TE-1 cells were treated with cordycepin (0,40, 60, 80μg/ml) for 48h. Apoptosis-related proteins, PARP,cleaved PARP, cleaved caspase-3, cleaved caspase-9, Bax and Bcl-2 were analysed by Western blot analysis. GAPDH was used as a loading control. (B,C) The Bcl-2/Bax ratio was evaluated by the band density compared with the control. (D) After pretreatment with 10mM Z‑VAD‑FMK for 30min, ECA109 and TE-1 cells were incubated with 60 μg/ml cordycepin for 24h, and cellular viability was determined. (E) Western blotting analysis of MAPK/ERK signalling-related proteins in ECA109 and TE-1 cell lines. GAPDH was used as a loading control. (F) ECA109 and TE-1 cells treated with or without the ERK inhibitor SCH772984 were analysed by CCK-8 assay. Results are presented as mean ± SD (n = 3 independent experiments). *p<0.05, **p<0.01, and ***p<0.001.

Figure 5

Cordycepin suppressed tumor growth in vivo. (A-C) Mice were treated with different concentrations (0, 5 mg/kg and 10 mg/kg) of cordycepin for 24 days. Images of 5 representative mice from each group are shown. Tumor sizes and tumor weights were measured. (D) PARP,cleaved PARP, cleaved-caspase3, cleaved-caspase9, Bax and Bcl-2, P-MEK, P-ERK expression levels in xenograft tumors were analyzed using western blot analysis. (E) The expression of cleaved caspase-3, Ki67, P-MEK and P-ERK were evaluated by IHC and HE staning in xenograft tumor. Results are presented as mean ± SD (n = 3 independent experiments). *p<0.05, **p<0.01, and ***p<0.001.