Baicalin induces cellular senescence in human colon cancer cells via upregulation of DEPP and the activation of Ras/Raf/MEK/ERK signaling

Abstract

Baicalin is a natural flavonoid glycoside which has potent anti-tumor and antioxidant activity in cancer cells. In the present study, we found that baicalin treatment significantly induced senescence in colon cancer cells. Furthermore, baicalin upregulated the expression of decidual protein induced by progesterone (DEPP) in HCT116 colon cancer cells, which accompanied with the activation of Ras/Raf/MEK/ERK and p16^INK4A/Rb signaling pathways. Meanwhile, these phenomena also appeared under the anti-oxidation effect exerted by baicalin. In addition, ectopic expression of DEPP in HCT116 cells significantly induced the activity of senescence-associated β-galactosidase (SA-β-Gal) in tumor cells regulated by Ras/Raf/MEK/ERK signaling pathway. Knockdown of DEPP by RNA interference efficiently counteracted the baicalin-mediated growth inhibition, senescence and cell cycle arrest in cancer cells. Importantly, in a xenograft mouse model of human colon cancer, we further confirmed that baicalin treatment dramatically inhibited tumor growth, which was due to the induction of tumor cellular senescence via the upregulation of DEPP and the activation of Ras/Raf/MEK/ERK signaling in vivo. In addition to baicalin treatment, we found that the hypoxia-response protein DEPP functions as a positive regulator involving the regulations of Ras/Raf/MEK/ERK signaling pathway and inhibition of human colon cancer by other anti-oxidative drugs, such as curcumin and sulforaphane, resulting in tumor cellular senescence. These results collectively suggest that baicalin upregulates the expression of DEPP and activates its downstream Ras/Raf/MEK/ERK and p16^INK4A/Rb pathways by acting as an antioxidant, leading to senescence in colon cancer cells.

Fig. 1. Baicalin-induced senescence in colon cancer cells.

a HCT116 and SW480 were incubated with the indicated doses of baicalin for 24 h. The mean value of OD450 is shown as mean ± SD of six wells. Shown are representative of 5 independent repeated experiments. b SA-β-Gal staining was performed in HCT116 and SW480 cells treated with baicalin. Shown are representative of four experiments. The quantifications of SA-β-Gal staining are means ± SD. c Flow cytometric analysis of DNA content was performed in HCT116 and SW480 cells treated with baicalin. The cell cycle distribution is represented as the percentage of cells in each cycling phase (subG1, G0/G1, S, G2/M), the data are representative of three experiments. d EdU staining was performed in HCT116 and SW480 cells treated with baicalin. Shown are representative of three experiments. The quantifications of integrated optical density are means ± SD. e Colony formation assay was implemented in HCT116 cells treated with baicalin. Shown are representative of 3 experiments. The quantifications of colonies formed are means ± SD. f, g Flow cytometric analysis of Annexin V/PI double staining was performed in HCT116 cells treated with baicalin. The percentage of apoptotic cells are representative of three experiments. (*P ≤ 0.05, *P ≤ 0.01 and ***P ≤ 0.001 versus the control group)

Fig. 2. Effects of baicalin on ROS level and SOD activity in colon cancer cells.

a Cells were incubated with the indicated doses of baicalin and H₂O₂ for indicated times. The represented results were obtained 4 h after treatment. The cells were stained with DCFH-DA and analyzed by flow cytometry to determine ROS level. Shown are representative of 4 experiments. b The mean fluoresce intensity of DCF is shown as mean values ± SD of four independent experiments. c The data for SOD activity in HCT116 cells treated with 40 μM baicalin were normalized to controls and are shown as the means ± SD (n = 4). d–f The same protocols for testing ROS level and SOD activity were conducted in SW480 cell line. g Double staining for the DNA damage response marker γH2AX (red) and DAPI (blue) in HCT116 cells treated with vehicle and indicated concentrations of baicalin. Shown are representative of four experiments. h Cells treated with vehicle or baicalin were analyzed by flow cytometry to determine the levels of p-ATM. Shown are graphical mean fluoresce intensity of 4. (**P ≤ 0.01, ***P ≤ 0.001 versus the control group)

Fig. 3. DEPP was upregulated by baicalin in cancer cells in vitro.

a HCT116 cells treated with vehicle or baicalin were subjected to whole-genome microarray analysis. The histogram depicts a compilation of hypoxia-response genes. b The expression of DEPP mRNA in the vehicle-treated or baicalin-treated cells was verified by qRT–PCR. The data shown are means ± SD from three independent experiments. c HCT116 cells were exposed to hypoxic condition for indicated times, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16^INK4A, pRb, Rb and β-actin were analyzed by western blotting. d The expression of DEPP mRNA in HCT116 cells exposed to hypoxic condition was verified by qRT–PCR. The data shown are means ± SD from three independent experiments. e HCT116 cells were treated with vehicle or baicalin at indicated concentrations, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK and β-actin were analyzed by Western blotting. f HCT116 cells were treated with vehicle or varying concentrations of baicalin (10, 20 and 40 μM) for 24 h, and the protein levels of p16^INK4A, pRb, Rb, p21, p27, cleaved-caspase 3 and caspase 3 were analyzed by western blotting. g, h A549 and Panc-1 cells were treated with vehicle or baicalin at indicated concentrations, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16^INK4A, pRb, Rb and β-actin were analyzed by Western blotting. (***P ≤ 0.001 versus the control group)

Fig. 4. Baicalin upregulated DEPP expression and induced senescence in colon cancer cells in vivo.

a, b Effects of baicalin on tumor volume and body weight. The data are representative of results obtained from 5 mice. c Tumor sections were subjected to hematoxylin and eosin staining, SA-β-Gal staining and IHC for Ki67, p16^INK4A and cleaved-caspase 3. Original magnification was 400×. Every single shown picture is representative of five sections from every group. Different sections represent different tumors. The representative images and the calculation of the percentage of positive cells are shown in d–g; the data are the means ± SD (***P ≤ 0.001). h Tumors were collected for western blot analysis of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16^INK4A, pRb, Rb and β-actin. Every single lane represents one tumor from an individual mouse. i Tumor sections were subjected to cleaved-caspase 3. Original magnification was 400×

Fig. 5. Knockdown of DEPP attenuated baicalin-induced senescence in colon cancer cells.

a The validation of two different siRNA targeting DEPP was analyzed by western blot of DEPP and β-actin. b SA-β-Gal staining was performed in HCT116 cells transfected with siDEPP during baicalin-induced senescence. Shown are representative of four experiments. The quantifications of SA-β-Gal staining are shown in e; the data are means ± SD. c Flow cytometric analysis of DNA content was performed in HCT116 cells transfected with siDEPP during baicalin-induced cell cycle arrest. Data are representative of three experiments. The cell cycle distribution is represented as the percentage of cells in each cycling phase (G0/G1, S, G2/M) in f. d Colony formation assay was implemented in HCT116 cells transfected with siDEPP during baicalin-induced proliferation inhibition. Shown are representative of three experiments. The quantifications of colonies formed are shown in g; the data are means ± SD. (***P ≤ 0.001 versus the control group)

Fig. 6. DEPP controlled baicalin-induced senescence and the activation of Ras/Raf/MEK/ERK and p16^INK4A/Rb signaling pathways in cancer cells.

HCT116 cells transfected with either control siRNA or siDEPP (no. 1 and 2) were exposed to baicalin (40 μM), and 24 h later, immunoblotting analysis a was performed. Panc-1 and A549 cells transfected with either control siRNA or siDEPP (no. 2) were exposed to baicalin (40 µM), and 24 h later, immunoblotting analysis b, c were performed. d, e SA-β-Gal staining was performed in Panc-1 and A549 cells treated with baicalin. Shown are representative of three experiments. The quantifications of SA-β-Gal staining are means ± SD. f EdU staining was performed in Panc-1 and A549 cells treated with baicalin. Shown are representative of three experiments. The quantifications of integrated optical density are means ± SD. g Colony formation assay was implemented in Panc-1 and A549 cells treated with baicalin. Shown are representative of 3 experiments. The quantifications of colonies formed are means ± SD. (***P ≤ 0.001 versus the control group)

Fig. 7. Ras/Raf/MEK/ERK signaling pathway was involved in DEPP-mediated tumor cellular senescence in colon cancer cells.

HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with siRNA targeting Raf1 and ERK, then immunoblotting analysis a and SA-β-Gal staining c were carried out. Shown are representative of 4 experiments. HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with Sorafenib or U0126 for 24 h, then immunoblotting analysis b, SA-β-Gal staining d, EdU staining e and colony formation assay f were carried out. Shown are representative of 4 experiments. The quantification of SA-β-Gal and crystal violet staining represents the means ± SD. The integrated optical density of EdU staining represents the means ± SD. (***P ≤ 0.001 versus the control group). g Immunoblotting of Flag-DEPP immunoprecipitates from HCT116 cells with or without the treatment with baicalin or siDEPP. The binding of DEPP to Ras in cells is presented relative to that in the untreated cells. Shown are representative of three experiments

Fig. 8. Curcumin and sulforaphane upregulated DEPP expression and induced senescence in HCT116 cells. SA-β-Gal staining, colony formation assay and EdU staining was performed in HCT116 cells treated with curcumin or sulforaphane in.

The images are representative of 4 experiments. The representative images and quantifications of SA-β-Gal staining are shown in a, e. The cell cycle distribution is represented as the percentage of cells in each cycling phase (G0/G1, S, G2/M) in b, f. The fluorescence intensity of EdU is represents as the images of immunofluorescence staining and the integrated optical density. The data are the means ± SD (**P ≤ 0.01, ***P ≤ 0.001). Cells treated with curcumin or sulforaphane were collected for western blot analysis of DEPP and associated signaling proteins. d Curcumin. h Sulforaphane