Inhibitory effects of petasin on human colon carcinoma cells mediated by inactivation of Akt/mTOR pathway

Abstract

Background: Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored.

Methods: Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests.

Results: Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues.

Conclusions: Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.

Figure 1

Effects of petasin on the proliferation of human colon carcinoma cell lines as measured using the MTT assay. Changes in the viability of SW-620 (A), Caco-2 (B), Lovo (C), and HT-29 cells (D) after 24, 48, and 72 h of treatment with 1, 5, and 25 μmol/L of petasin, n = 3. One-way analysis of variance was used for intergroup comparisons and multiple comparisons. Post hoc tests between groups were evaluated with Student's t tests. ^∗P< 0.05, ^†P< 0.01, ^‡P< 0.001 vs. control cells. MTT: 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide.

Figure 2

Petasin induced apoptosis in SW-620 cells. SW-620 cells were treated with 25 μmol/L petasin for 48 h. (A) Apoptosis was detected with Annexin-V-fluorescein isothiocyanate/propidium iodide staining and flow cytometry. (B) Morphological changes in apoptotic cells were evaluated by Hoechst 33258 staining, original magnification ×200. Differences between the two groups were assessed by analysis of independent sample t tests. n = 3, ^†P < 0.01 vs. control cells.

Figure 3

Petasin inhibited the migration and invasion abilities of SW-620 cells. SW-620 cells were treated with 25 μmol/L petasin for 24 h. (A) Cell migration was assessed using a wound-healing assay. (B) Cell invasion was assessed using Transwell chambers. Invasive cells were stained with 0.2% crystal violet, original magnification ×200. Differences between the two groups were assessed by analysis of independent sample t tests. n = 3, ^†P < 0.01 vs. control cells.

Figure 4

Petasin inactivated the Akt/mTOR signaling pathway, upregulated apoptotic proteins, and decreased MMP expression in SW-620 cells. SW-620 cells were treated with 25 μmol/L petasin for 24 h. Western blotting analysis was performed to determine the expression of Akt/mTOR/P70S6K, caspase-3, caspase-9, Bcl-2, MMP-3, and MMP-9. (A) Representative images of immunoblots. (B) Quantification of protein expression levels in experimental groups. GAPDH was used as the loading control. Differences between two groups were assessed by analysis of independent sample t tests. n = 3, ^∗P < 0.05, ^†P < 0.01 vs. control cells. mTOR: Mammalian target of rapamycin; MMP: Matrix metalloproteinase; p-mTOR: Phosphorylation of mTOR; P70S6K: P70S6 kinase; p-P70S6K: Phosphorylation of P70S6K; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Petasin inhibited tumor growth and induces apoptosis in SW-620 cells from xenograft in vivo. (A) Tumor volume was measured in every 7 days. (B) TUNEL assay was performed to detect DNA fragmentation, original magnification ×200. SW-620 cells were injected intradermally into the Balb/c nude mice and 10 mg/kg petasin was orally administered twice a day for 28 days. One-way analysis of variance was used for intergroup comparisons and multiple comparisons. Post hoc tests between groups were evaluated with Student's t tests. n = 6, ^∗P < 0.05, ^†P < 0.01 vs. control group. TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.