Network pharmacology analysis of the anti-cancer pharmacological mechanisms of Ganoderma lucidum extract with experimental support using Hepa1-6-bearing C57 BL/6 mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (GL) is an oriental medical fungus, which was used to prevent and treat many diseases. Previously, the effective compounds of Ganoderma lucidum extract (GLE) were extracted from two kinds of GL, [Ganoderma lucidum (Leyss. Ex Fr.) Karst.] and [Ganoderma sinense Zhao, Xu et Zhang], which have been used for adjuvant anti-cancer clinical therapy for more than 20 years. However, its concrete active compounds and its regulation mechanisms on tumor are unclear. AIM OF THE STUDY: In this study, we aimed to identify the main active compounds from GLE and to investigate its anti-cancer mechanisms via drug-target biological network construction and prediction. MATERIALS AND METHODS: The main active compounds of GLE were identified by HPLC, EI-MS and NMR, and the compounds related targets were predicted using docking program. To investigate the functions of GL holistically, the active compounds of GL and related targets were predicted based on four public databases. Subsequently, the Identified-Compound-Target network and Predicted-Compound-Target network were constructed respectively, and they were overlapped to detect the hub potential targets in both networks. Furthermore, the qRT-PCR and western-blot assays were used to validate the expression levels of target genes in GLE treated Hepa1-6-bearing C57 BL/6 mice. RESULTS: In our work, 12 active compounds of GLE were identified, including Ganoderic acid A, Ganoderenic acid A, Ganoderic acid B, Ganoderic acid H, Ganoderic acid C2, Ganoderenic acid D, Ganoderic acid D, Ganoderenic acid G, Ganoderic acid Y, Kaemferol, Genistein and Ergosterol. Using the docking program, 20 targets were mapped to 12 compounds of GLE. Furthermore, 122 effective active compounds of GL and 116 targets were holistically predicted using public databases. Compare with the Identified-Compound-Target network and Predicted-Compound-Target network, 6 hub targets were screened, including AR, CHRM2, ESR1, NR3C1, NR3C2 and PGR, which was considered as potential markers and might play important roles in the process of GLE treatment. GLE effectively inhibited tumor growth in Hepa1-6-bearing C57 BL/6 mice. Finally, consistent with the results of qRT-PCR data, the results of western-blot assay demonstrated the expression levels of PGR and ESR1 were up-regulated, as well as the expression levels of NR3C2 and AR were down-regulated, while the change of NR3C1 and CHRM2 had no statistical significance. CONCLUSIONS: The results indicated that these 4 hub target genes, including NR3C2, AR, ESR1 and PGR, might act as potential markers to evaluate the curative effect of GLE treatment in tumor. And, the combined data provide preliminary study of the pharmacological mechanisms of GLE, which may be a promising potential therapeutic and chemopreventative candidate for anti-cancer.

[Study on synergistic effect of sodium cantharidinate combined with chemotherapeutic drugs on hepatic carcinoma and its effective mechanism].

OBJECTIVE: To study the synergistic effect on hepatoma cell(SMMC-7721) and the reduction killing effect on normal liver cells(LO-2) treated with sodium cantharidinate (SCA) in combination with fluorouracil(5-FU) or cisplatin(DDP) as well as the related mechanism. METHODS: MTT assay was used to select the best ratio of SCA with 5-FU or SCA with DDP which had less toxicity on LO-2 cell line and had synergistic effect on SMMC-7721 cell line; Flow cytometry assay was used to analyze the apoptosis-induction of the different ratio of drugs on both cell lines; Hoechst-33258 fluorescent staining assay was used to observe the nuclear morphological changes of cells; Immunoblotting assay was used to analyze the Ras/Raf/ERK1/2 signaling pathway and the apoptosis related signaling pathway in both cell lines. RESULTS: MTI assay indicated that the proliferation inhibition of SCA,5-FU and DDP on SMMC-7721 cell line was in a time-and dose-dependent manner respectively. Among them, SCA had a more significant inhibition on SMMC-7721 cell line than on LO-2 after 12 h or 24 h treatment (P <0. 01). Moreover, after a treatment of 48 h,the ratio of 2. 5 µg/mL SCA and 2 µg/mL DDP showed a more significant inhibition on SMMC-7721 cell line than on LO-2 cell line,which was then be considered as the optimal concentration ratio for the following experiment. Co-treatment of SCA (2. 5 µg/mL) with DDP (2 µg/mL) induced a more significant apoptosis on SMMC-7721 cell line compared with single treatment with SCA (2. 5 µg/mL) or DDP (2 µg/mL) respectively (P < 0. 01). After a 48 h treatment of the optimal ratio of drugs, the significant morphological apoptotic characteristics were observed both under inverted microscope and by Hoechst-33258 fluorescent staining assay in both cell lines. The results of Western blot assay showed that this ratio of drugs could significantly increase the protein expression of Bax,P53 and P21 and decreased the expression of BCL-2, Casepase-3, p-Erk, p-Ras and p-c-Raf in SMMC-7721 cells. Meanwhile,the effect on the proteins mentioned above was lesser in LO-2 cells. CONCLUSION: These results indicates that 2. 5 µg/mL SCA + 2 µg/mL DDP showed a higher inhibition on the hepatic carcinoma cells and a relatively lower cytotoxicity on normal liver cells. The major anti-cancer mechanism is related with the inhibition on Erk signaling pathway and the induction of apoptosis through the mitochondrial pathway.