Fermented So-Cheong-Ryong-Tang (FCY) induces apoptosis via the activation of caspases and the regulation of MAPK signaling pathways in cancer cells

Abstract

Background: So-Cheong-Ryong-Tang (CY), a traditional herbal formula, mainly has been shown to possess allergic rhinitis and asthma for hundreds of years in Asian countries. Although this medicine has been attracted Asian scientists with investigating mechanisms of action against inflammatory-related diseases, there is a little available information on the anti-cancer effect of CY, especially on the fermented form (FCY). In this study, we explored the chemopreventive/chemotherapeutic efficacy of FCY against cancer cells and proved the efficacy of FCY through performing in vivo xenograft assay.

Methods: CY was fermented with bacteria and lyophilized. For analysis of the constituents of CY and FCY, high performance liquid chromatography (HPLC)-DAD system was performed. To detect the anti-cancer effect of FCY, cell viability assay, caspase activity assay, cell cycle analysis, and Western blot analysis were performed in AGS human gastric cancer cells. The inhibitory effects of tumor growth by CY and FCY were evaluated in athymic nude mice inoculated with HCT116 human colon cancer cells.

Results: As a result of analyzing the 11components present in CY and FCY, the contents of ephedrine HCl, glycyrrhizin, gingerol, schisandrin, and gomisin A were respectively increased by fermentation in FCY. The treatment of CY or FCY inhibited the viability of AGS cells, interestingly, the inhibition of cancer cell growth was enhanced by fermentation of CY. FCY induced the apoptosis through activating the caspase-3, -8, and -9. Additionally, FCY regulated the activation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). In vivo xenografts, administration of FCY significantly inhibited the tumor formation, and improved the anti-tumor effect compared to that of CY in athymic nude mice.

Conclusions: FCY indicated significant anti-cancer effects, and its efficacy against tumor formation was improved than that of CY, therefore, FCY might be used for applications of traditional medicine against cancer in modern complementary and alternative therapeutics. Graphical Abstract ᅟ.

Graphical Abstract

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Fig. 1

HPLC-DAD analysis of CY and FCY. a Determination of eleven standard components in CY and FCY. (a) Mixed standards, (b) CY, and (c) FCY. b Identification of changed components by bacterial fermentation in FCY compared to CY

Fig. 2

Inhibition of human gastric cancer cell viability by CY or FCY. AGS and NUGC cells were treated with 500 and 1000 μg/mL for 48 h. Cell viability was determined by MTT assay and the results are expressed as the percentages of viable cells compared to untreated cells. The data are shown as the means ± SD of three independent experiments

Fig. 3

Induction of apoptosis by FCY in AGS cells. a Effects of FCY on the expressions of caspase-3, −8, and −9, and PARP cleavage. The cells were exposed to 500 and 1000 μg/mL FCY for 24 and 48 h and protein levels were determined by Western blot analyses. The band intensity was calculated and compared to untreated cells using ImageJ after normalization relative to GAPDH expression. b The relative luminescence indicated the dose-dependent activations of caspase-3/7, −8, and −9 that were induced by 48 h of treatment with the indicated dose of FCY. The data are shown as the means ± SD of three independent experiments. ^* P < 0.05 and ^** P < 0.01 versus untreated control cells

Fig. 4

Effects of FCY on cell cycle progression in AGS cells. a The cells were treated with 500 or 1000 μg/mL of FCY for 48 h, fixed with pre-chilled 70 % ethanol, stained with propidium iodide solution and then subjected to flow cytometry for determination of cell cycle distribution. b The levels of cell cycle regulatory proteins in cells treated with FCY for 24 h were examined by Western blot analyses. c After incubation with 500 or 1000 μg/mL FCY, apoptosis was assessed using YO-PRO-1 staining by flow cytometry. The percentage of the M2 population depicts apoptosis, which increased in conjunction with the dose. The data are representative of three independent experiments

Fig. 5

Identification of the relationship between MAPK activation and the anti-proliferative effects of FCY on AGS cells. a Cells were prepared after treatment with 500 μg/mL FCY for 0.5, 1, 3, and 6 h, and then subjected to Western blot analyses to determine the levels of MAPK proteins, including ERK, p38, and JNK (or AKT) and their phosphorylated forms. b Investigation of the anti-proliferative effects of FCY for 48 h using the MAPK cascade inhibitors PD98059 (10 μM), SB203580 (5 μM), and SP600125 (10 μM), and the PI3K inhibitor LY294005 (10 μM). Cell viability was determined by MTT assay. The results show the means ± SD of three independent experiments. ^* P < 0.05 and ^** P < 0.01 versus untreated control cells and ^## P < 0.01 versus cells treated with FCY only

Fig. 6

Inhibitory effects of FCY on in vivo tumor growth in a xenograft model. (a) The cells were injected into athymic nude mice and, 5 days after tumor implantation, the mice were treated daily with saline (vehicle), CY (157.5 mg/kg or 315 mg/kg), or FCY (157.5 mg/kg) for 14 days. Treatment was terminated after 15 days. (b) Changes in body weight during the administration of CY or FCY and tumor growth inhibition by (c) CY or (d) FCY. Comparison of anti-tumor activity between vehicle and herbal formulations (CY and FCY), according to representative tumor images and tumor weights. Data are shown as means ± SD, ^* P < 0.05 versus vehicle