Baicalein and baicalin inhibit colon cancer using two distinct fashions of apoptosis and senescence

Abstract

Baicalein and baicalin are active components of the Scutellaria baicalensis Georgi and both have broad anti-tumor activity. However, how and whether baicalein and baicalin inhibit colon cancer is unclear. Here we demonstrate that baicalein and baicalin can significantly inhibit human colon cancer cell growth and proliferation. Furthermore, both can induce cell cycle arrest, and suppress cancer cell colony formation and migration. The suppressive effects are mechanistically due to the induction of colon cancer cell apoptosis and senescence mediated by baicalein and baicalin, respectively. Furthermore, we revealed that baicalin-induced senescence in tumor cells is due to its inhibition of telomerase reverse transcriptase expression in tumor cells, and that MAPK ERK and p38 signaling pathways are causatively involved in the regulation of colon cancer cell apoptosis and senescence mediated by baicalein and baicalin. In addition, our in vivo studies using human colon cancer cells in humanized mouse xenograft models, further demonstrated that baicalein and baicalin can induce tumor cell apoptosis and senescence, resulting in inhibition of tumorigenesis and growth of colon cancer in vivo. These data clearly suggest that baicalein and baicalin have potent anti-cancer effects against human colon cancer and could be potential novel and effective target drugs for cancer therapy.

Keywords: Scutellaria baicalensis georgi; apoptosis; baicalein; baicalin; colon cancer.

Figure 1. Baicalein and baicalin inhibit colon cancer cell growth and proliferation

Three colon cancer cell lines (HCT116, SW480 and HT29) were cultured at a started number of 2 × 10⁵/well in 6-well plates, or 5 × 10³/well in 96-well plates, and treated with the indicated concentrations of baicalein or baicalin. The cell growth was evaluated at different time points using cell number counting (in A), and cell proliferation was determined using [³H]-thymidine assays (in B). Human foreskin fibroblast HFF cells treated with baicalein or baicalin for 72 h were included as a normal cell control (in A). Data shown in (A) and (B) are mean ± SD from three independent experiments with similar results. ^*p < 0.05 and ^**p < 0.01 compared with the medium control group.

Figure 2. Baicalein and baicalin significantly promote colon cancer cell cycle arrest in S phase and decrease in G0/G1 phase

(A) and (B) HCT116 and SW480 colon cancer cells were cultured for 72 h in the presence of the indicated concentrations of baicalein or baicalin. Cell cycle distribution in tumor cells was analyzed after incubation with 10 μg/ml propidium iodide and 100 μg/ml RNase A. Data are representative of three independent experiments with similar results.

Figure 3. Baicalein and baicalin inhibit colon cancer cell colony formation and migration

(A) and (B) Baicalein and baicalin treatments dramatically decreased the numbers and sizes of tumor colonies in HCT116 and SW480 cells after 2 weeks of culture. Two hundred to five hundred per well of colon cancer cells pre-treated with baicalein or baicalin, were seeded in 6-well plates for culture, and cell colonies counted after 10-14 days of culture. Results shown in the histogram (in B) are summaries of mean ± SD from three independent experiments. ^*p < 0.05 and ^**p < 0.01 compared with the medium control group. (C) Baicalein and baicalin treatments in HCT116 and SW480 colon cancer cells significantly inhibited the migration of tumor cells compared with the medium control group in the wound closure assays. Data shown are representative from three independent experiments with similar results.

Figure 4. Baicalein- and baicalin-mediated inhibition of colon cancer cell growth and proliferation is due to induction of cell apoptosis and senescence, respectively

(A) and (B) Significantly increased apoptotic cell populations were induced in HCT116 and SW480 cells after treatment with baicalein but not baicalin. (C) and (D) Treatment with baicalin but not with baicalein in HCT116 and SW480 cancer cells markedly induced SA-β-Gal positive cell populations in tumor cells. Tumor cells were cultured in the presence of indicated concentrations of baicalein and baicalin for 72 h or 120 h. Apoptosis in treated tumor cells was analyzed after staining with PE-labeled Annexin V and 7-AAD (in A). Senescent cells were analyzed using the SA-β-Gal activity assay and the SA-β-Gal positive cells were identified with dark blue granules as indicated by the arrows (in C). Data in (B) and (D) are mean ± SD from three independent experiments with similar results. ^*p < 0.05 and ^**p < 0.01 compared with the medium control group.

Figure 5. Baicalin, but not baicalein, significantly suppresses hTERT expression in colon cancer cells

(A) Both baicalin and baicalein did not induce phosphorylated activation of ATM and its associated molecule CHK2 in colon cancer cells. HCT116 cells and SW480 cells were treated with baicalein or baicalin at indicated concentration for 72 h. The p-ATM and p-CHK2 expression in treated colon cancer cells were analyzed by the flow cytometry. Results shown are a representative of three experiments with similar results. (B) HCT116 cells were treated with baicalein or baicalin at indicated concentration for 24 h. mRNA in cells was purified for quantitative PCR analysis of hTERT expression. mRNA levels in each group were normalized to the relative quantity of GAPDH expression and then adjusted to hTERT levels in medium group (set as 1). Results shown in the histogram are mean ± SD from three independent experiments. ^*p < 0.05 and ^**p < 0.01 compared with the medium control group.

Figure 6. MAPK p38 and ERK1/2 signaling pathways involve colon cancer cell apoptosis and senescence induced by baicalein and baicalin

(A) and (B) Both baicalein and baicalin treatment induced phosphorylation of ERK and p38 in HCT116 and SW480 tumor cells. Colon cancer cells were cultured in the presence of 50 μM baicalein and baicalin for the indicated times and cell lysates were prepared for western blot analyses.

Figure 7. Baicalein and baicalin inhibit tumor growth and development in vivo in a colon cancer xenograft model

(A) Experimental scheme for tumorigenesis studies with a colon cancer xenograft model. HCT116 cells (5 × 10⁶/mouse) were subcutaneously injected into NSG mice. After 8 days post tumor injection, the tumor-bearing mice were administrated with baicalein (50 mg/kg) or baicalin (50 mg/kg), and solvent control through intraperitoneal injection, respectively, at every other day for 2 weeks. (B) Both baicalein and baicalin dramatically inhibited HCT116 tumor growth in NSG immunodeficient mice. Tumor volumes were measured and presented as mean ± SD (n = 4 mice per group). P values were determined by the one-way analysis of variance (ANOVA). Similar results were obtained in three repeated experiments. (C) Representative image of the xenograft tumors obtained from the indicated groups at the endpoint of the experiments (day 29). (D) Treatments with both baicalein and baicalin had much lower tumor weight compared with that of solvent control group. Results shown are mean ± SD of the xenograft tumor weights from the indicated groups in the model at the endpoint of the experiments (day 29) (n= 4 mice per group). ^**p < 0.01, compared with the solvent control group using unpaired t-test. (E) and (F) Increased apoptotic cells were observed in tumor tissues from the treatment group of baicalein but not baicalin in NSG mice. Cell apoptosis in the paraffin-embedded tissues was analyzed by the TUNEL and PI staining at the endpoint of experiment. Panel in (E) is photomicrographs of the TUNEL and PI staining in sections of tumor tissues from different treatment groups. Panel in (F) is the mean ± SD of percentages of apoptotic cells (TUNEL⁺PI⁺ cells) in the total PI⁺ cells in the tumor tissues from 4 mice of each group. ^**p < 0.01, compared with the control treatment mice using unpaired t-test. (G) and (H) Large amounts of senescent tumor cells were observed in tumor tissues from both treatments of baicalein and baicalin in NSG mice. SA-β-Gal expression was determined in the tumor frozen tissues from different groups at the endpoint of experiment. Panel in (G) is photomicrographs of SA-β-Gal expression in tumor tissues from different groups. Panel in (H) is the mean ± SD of percentages of SA-β-Gal⁺ cells per high microscope field (× 400) in the tumor tissues from 4 mice of each group. ^**p < 0.01, compared with the control treatment mice using unpaired t-test.