Anti-cancer effect of Cordyceps militaris in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis

Abstract

Background: Cordyceps militaris has been used as a traditional medicine in Asian countries for a long time. Different types of Cordyceps extract were reported to have various pharmacological activities including an anti-cancer effect. We investigated the inhibitory effect of Cordyceps militaris ethanol extract on a human colorectal cancer-derived cell line, RKO.

Methods: RKO cells were treated with various concentrations of nucleosides-enriched ethanol extract of Cordyceps militaris for 48 h and cytotoxicity was measured using a CCK-8 assay. Then, xenograft Balb/c nude mice were injected with RKO cells and subsequently orally administered with ethanol extract of Cordyceps militaris every day for 3 weeks to examine the inhibitory effect on tumor growth. Lastly, the effect of Cordyceps militaris on cell cycle as well as apoptosis was measured using flow cytometry. Also, the expression of p53, caspase 9, cleaved caspase-3, cleaved PARP, Bim, Bax, Bak, and Bad were detected using western blot assay.

Results: RKO cells were highly susceptible to the ethanol extract of Cordyceps militaris (CME) and the growth of RKO cells-derived tumor was significantly delayed by the treatment of Cordyceps militaris. Cordyceps militaris induced cell cycle arrest in G2/M phase (untreated; 20.5 %, CME 100 μg/ml; 61.67 %, CME 300 μg/ml; 66.33 %) and increased early apoptosis (untreated; 1.01 %, CME 100 μg/ml; 8.48 %, CME 300 μg/ml; 18.07 %). The expression of p53, cleaved caspase 9, cleaved caspase-3, cleaved PARP, Bim, Bak, and Bad were upregulated by the treatment of Cordyceps militaris.

Conclusion: Ethanol extract of Cordyceps militaris was highly cytotoxic to human colorectal carcinoma RKO cells and inhibited the growth of tumor in xenograft model. The anti-tumor effect of Cordyceps militaris was associated with an induction of cell cycle arrest and mitochondrial-mediated apoptosis.

Fig. 1

Cell cytotoxicity of Cordyceps militaris in human colorectal carcinoma RKO cells. a RKO cells were treated with various concentrations (0–1000 μg/mL) of ethanol extract of Cordyceps militaris (CME) for 48 h, and cell viability was determined by CCK-8 assay. The results are presented as mean ± standard deviation (SD) for five independent experiments. b Cells were treated with or without ethanol extract of Cordyceps militaris (CME) for 24 h or 48 h. Cell morphology was observed by light microscopy (×200)

Fig. 2

Anti-cancer effect of Cordyceps militaris in a xenograft mouse bearing RKO cell–derived human colorectal cancer. Mice were injected with human colorectal carcinoma RKO cells (1 × 10⁶ cells per mouse) subcutaneously into the back next to the right hind leg. Mice were sorted into 2 groups (n = 10/group) and orally administered ethanol extract of Cordyceps militaris (CME) (100 mg/kg) or drinking water. 14 days later, tumors were identified and measured every two days until the experimental endpoint. a Study design for animal experiment. b Photograph of xenograft mice bearing RKO cell-derived human colorectal cancer in right hind leg. The pictures of untreated and treated groups were taken at 13 days since the tumor volume was measured. c Inhibitory effect of Cordyceps militaris (CME) on RKO cell-derived tumor growth. Compared between drinking water group and CME group *P < 0.05. d Correlation of survival rate of xenograft mice bearing human colorectal cancer

Fig. 3

Induction of cell cycle arrest by Cordyceps militaris in RKO cells. Cells were treated with or without ethanol extract of Cordyceps militaris (CME) (100 and 300 μg/ml) for 24 h. Then, cells were harvested for PI staining, which was measured by flow cytometry. a Representative PI staining for cell cycle progress in RKO cells. b The percentage of cell cycle distribution is presented as mean ± standard deviation (SD) for five independent experiments. Compared with other groups *P =0.006, **p = 0.0002

Fig. 4

Induction of cell apoptosis by Cordyceps militaris in RKO cells. Cells were treated with or without ethanol extract of Cordyceps militaris (CME) (100 and 300 μg/mL) for 48 h and then stained with Annexin V and propidium iodide (PI). Apoptosis of RKO cells was analyzed by flow cytometry. a Representative Annexin V and PI staining for cell apoptosis in RKO cells. b The percentage of early apoptosis is presented as mean ± standard deviation (SD) for five independent experiments. Compared with other groups *P = 0.003

Fig. 5

Induction of mitochondrial-mediated apoptosis by Cordyceps militaris in a p53-dependent manner. Cells were treated with or without ethanol extract of Cordyceps militaris (CME) (0, 100, and 300 μg/ml) for 48 h and then western blot analysis for various apoptotic related molecules were performed. β-actin served as the loading control. a Expression of p53 and Bcl-2 family proteins. b Expression of caspases 9, 7, and 3, and PARP proteins. c The relative intensity of each band compared to the loading control