Cordycepin Enhanced Therapeutic Potential of Gemcitabine against Cholangiocarcinoma via Downregulating Cancer Stem-Like Properties

Abstract

Cordycepin, a valuable bioactive component isolated from Cordyceps militaris, has been reported to possess anti-cancer potential and the property to enhance the effects of chemotherapeutic agents in various types of cancers. However, the ability of cordycepin to chemosensitize cholangiocarcinoma (CCA) cells to gemcitabine has not yet been evaluated. The current study was performed to evaluate the above, and the mechanisms associated with it. The study analyzed the effects of cordycepin in combination with gemcitabine on the cancer stem-like properties of the CCA SNU478 cell line, including its anti-apoptotic, migratory, and antioxidant effects. In addition, the combination of cordycepin and gemcitabine was evaluated in the CCA xenograft model. The cordycepin treatment significantly decreased SNU478 cell viability and, in combination with gemcitabine, additively reduced cell viability. The cordycepin and gemcitabine co-treatment significantly increased the Annexin V+ population and downregulated B-cell lymphoma 2 (Bcl-2) expression, suggesting that the decreased cell viability in the cordycepin+gemcitabine group may result from an increase in apoptotic death. In addition, the cordycepin and gemcitabine co-treatment significantly reduced the migratory ability of SNU478 cells in the wound healing and trans-well migration assays. It was observed that the cordycepin and gemcitabine cotreatment reduced the CD44^highCD133^high population in SNU478 cells and the expression level of sex determining region Y-box 2 (Sox-2), indicating the downregulation of the cancer stem-like population. Cordycepin also enhanced oxidative damage mediated by gemcitabine in MitoSOX staining associated with the upregulated Kelch like ECH Associated Protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) expression ratio. In the SNU478 xenograft model, co-administration of cordycepin and gemcitabine additively delayed tumor growth. These results indicate that cordycepin potentiates the chemotherapeutic property of gemcitabine against CCA, which results from the downregulation of its cancer-stem-like properties. Hence, the combination therapy of cordycepin and gemcitabine may be a promising therapeutic strategy in the treatment of CCA.

Keywords: Cancer stem cell; Chemoresistance; Cholangiocarcinoma; Cordycepin; Gemcitabine.

Fig. 1

Inhibitory effects of cordycepin on the growth of SNU478 cells. (A) The chemical structure of cordycepin. Cell viability in SNU478 cells (B) treated with cordycepin with different concentrations for 72 h and (C) treatment of cordycepin and/or gemcitabine with different incubation times (24, 48, and 72 h) assessed using WST assay. (D) Representative images of colony formation in SNU478 cells and (E) quantitative analysis of colonies. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. **p<0.01 vs. control group; ^##p<0.01 vs. cordycepin group; ^@@p<0.01 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine; IC₅₀, half-maximal inhibitory concentration.

Fig. 2

Effects of cordycepin on gemcitabine-mediated apoptotic changes in SNU478 cells. The cells stained with Annexin V/PI were assessed after treatment of cordycepin and/or gemcitabine by FACS analysis and expression levels of apoptosis-related proteins were analyzed by Western blot. (A) Representative flow cytometry plot of SNU478 cells for Annexin V/PI staining and (B) percentage of apoptotic cells. (C) Representative band images of proteins and (D) the relative intensity ratio of Bcl-2/β-actin were presented. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. *p<0.05 and **p<0.01 vs. control group; ^#p<0.05 and ^##p<0.01 vs. cordycepin group; ^@@p<0.01 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine.

Fig. 3

Effects of cordycepin on migratory ability in SNU478 cells. Cell migration after treatment of cordycepin and/or gemcitabine was assessed by wound healing assay and trans-well migration assay. (A) Representative images of wound healing were captured with different incubation times (at 0, 24, and 48 h). (B) Wound areas of SNU478 cells were measured by image J and the percentage of wound closure was calculated. (C) Representative images of migratory cells and (D) the number of migrated cells were assessed. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. *p<0.05 and **p<0.01 vs. control group; ^#p<0.05 vs. cordycepin group; ^@@p<0.01 vs. gemcitabine group (Dunnet’s test). Bar=500 μm. Cordy, cordycepin; Gem, gemcitabine.

Fig. 4

Effects of cordycepin treatment on cancer stem-like cell population in SNU478 cells. SNU478 cells were treated with cordycepin and/or gemcitabine, and expression of CD44/CD133 was detected by flow cytometry. (A) Representative flow cytometry plots of SNU478 cells for CD44 and CD133 and (B) percentage of CD44^highCD133^high cells in total cells were presented. Protein expression levels of p-Akt, Akt, and Sox-2 in SNU478 after treatment of cordycepin and/or gemcitabine were confirmed by Western blot. (C) Representative band images of each protein and the relative intensity ratio of (D) p-Akt/Akt and (E) Sox-2/β-actin were presented. The control group received no treatment at all. Data are expressed as means ± SD. from at least three independent experiments. *p<0.05 and **p<0.01 vs. control group; ^##p<0.01 vs. cordycepin group; ^@@p<0.01 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine.

Fig. 5

Effects of co-treatment cordycepin and gemcitabine on mitochondrial ROS generation in SNU478 cells. The content of mitochondrial ROS was determined by MitoSOX staining and related protein levels were confirmed by Western blot. (A) Representative images of SNU478 cells stained with MitoSOX (red, mitochondria peroxide) and Hoechst 33342 (blue, nuclei) are shown. (B) MitoSOX fluorescence intensity was quantified. (C) Representative band images of proteins and (D) the relative intensity ratio of Keap1/Nrf2 were presented. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. **p<0.01 vs. control group; ^@@p<0.01 vs. gemcitabine group (Dunnet’s test). Bar=200 μm. Cordy, cordycepin; Gem, gemcitabine.

Fig. 6

Effects of cordycepin in combination with gemcitabine on tumor growth in SNU478 xenograft mouse model. (A) Scheme of in vivo experiment. (B) The tumor volumes and (C) body weights in SNU478 xenografted mice were measured thrice a week after treatment, and (D) the tumor weights were measured at study termination (day 26). Saline (0.9% NaCl) was used as a vehicle for administration. Data are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. vehicle group; ^#p<0.05 and ^##p<0.01 vs. cordycepin group; ^@p<0.05 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine.

Fig. 7

Effects of cordycepin and/or gemcitabine treatment on PCNA and Ki-67 expression in xenografted SNU478 tumors. The expression levels of PCNA and Ki-67 in xenografted SNU478 tumors were analyzed by IHC staining. (A) Representative IHC images against PCNA and Ki-67 in tumors and the relative DAB intensity of (B) PCNA and (C) Ki-67 were analyzed. Data are expressed as means ± SEM. *p<0.05 vs. vehicle group; ^@@p<0.01 vs. gemcitabine group (Dunnet’s test). Bar=50 μm. Cordy, cordycepin; Gem, gemcitabine.