Herbal formula YGJDSJ inhibits anchorage-independent growth and induces anoikis in hepatocellular carcinoma Bel-7402 cells

Abstract

Background: Based on clinical medications and related studies, we established a Yang-Gan Jie-Du Sang-Jie (YGJDSJ) herbal formula for hepatocarcinoma treatment. In present study, we evaluated the anti-cancer potential of YGJDSJ on suspension-grown human hepatocellular carcinoma Bel-7402 cells.

Methods: Bel-7402 cells were cultured in poly(2-hydroxyethyl methacrylate) (poly-HEMA) coated plates and treated with YGJDSJ. Anchorage-independent cell growth was detected by cell Counting Kit-8 (CCK-8) assay and soft agar colony formation assay. Anoikis was detected by ethdium homodimer-1 (EthD-1) staining and flow cytometry analysis. Caspases activities were detected by the cleavage of chromogenic substrate. Reactive oxygen species (ROS) was detected by 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining. Protein expression and phosphorylation was identified by western blot. Protein expression was knocked-down by siRNA.

Results: YGJDSJ inhibited the proliferation of Bel-7402 cells in poly-HEMA coated plates and anchorage-independent growth of Bel-7402 cells in soft agar. YGJDSJ also induced anoikis in Bel-7402 cells as indicated by EthD-1 staining and flow cytometry analysis. YGJDSJ activated caspase-3, - 8, and - 9 in suspension-grown Bel-7402 cells. The pan-caspase inhibitor Z-VAD-FMK significantly abrogated the effects of YGJDSJ on anoikis in suspension-grown Bel-7402 cells. In addition, YGJDSJ increased ROS in suspension-grown Bel-7402 cells. The ROS scavenger N-acetyl-L-cysteine (NAC) partially attenuated YGJDSJ-induced activation of caspase-3, - 8 and - 9 and anoikis in suspension-grown Bel-7402 cells. Furthermore, YGJDSJ inhibited expression and phosphorylation of protein tyrosine kinase 2 (PTK2) in suspension-grown Bel-7402 cells. Over-expression of PTK2 significantly abrogated YGJDSJ induced anoikis.

Conclusions: YGJDSJ inhibits anchorage-independent growth and induce caspase-mediated anoikis in Bel-7402 cells, and may relate to ROS generation and PTK2 downregulation.

Keywords: Anoikis; Caspases; Chinese herb; Hepatocellular carcinoma; Protein tyrosine kinase 2; Reactive oxygen species.

Fig. 1

YGJDSJ inhibited anchorage-independent growth of Bel-7402 cells. a Bel-7402 cells were cultured in poly-HEMA coated 96-well plate and treated with different concentrations of YGJDSJ for 24 h, cell viability was evaluated by CCK-8 assay. b Bel-7402 cells were treated with different dose of YGJDSJ twice a week for 2 weeks in soft-agar colony formation assay. Data shown are representative of three independent experiments. *P < 0.01, versus control group

Fig. 2

YGJDSJ induced anoikis in Bel-7402 cells. Suspension-grown Bel-7402 cells were treated with different dose of YGJDSJ for 24 h, stained with EthD-1, and observed under fluorescence microscope (× 200) (a), and quantitated with a fluorescence microplate reader (b). YGJDSJ treated or untreated Bel-7402 cells were stained with Annexin V-FITC/PI, analyzed in FACScalibour flow cytometer (c), and expressed as mean ± SD (d). Data illustrated are from three separate experiments. *P < 0.01, versus control group

Fig. 3

YGJDSJ activated caspases in Bel-7402 cells. After 24 h YGJDSJ (100–400 μg/ml) treatment, caspase-3 (a), caspase-3 (b) and caspase-9 (c) activities in suspension-cultured Bel-7402 cells were detected as described in Materials and Methods. Caspases activities were expressed as fold activation over control. d suspension-cultured Bel-7402 cells were pretreated with Z-VAD-FMK (50 μmol/L) for 2 h before treatment with YGJDSJ for 24 h, stained with Annexin V-FITC/PI and analyzed by flow cytometry. Data presented are from three separate experiments. *P < 0.01, versus control group; ^#P < 0.01, versus corresponding dose of YGJDSJ treated Z-VAD-FMK (−) group

Fig. 4

YGJDSJ increased ROS level in Bel-7402 cells. After 24 h YGJDSJ (100–400 μg/ml) treatment, intracellular ROS production in suspension-cultured Bel-7402 cells was stained with DCFH-DA, observed under fluorescence microscope (× 200) (a), quantitated with a fluorescence microplate reader and expressed as fold of control (b) Suspension-cultured Bel-7402 cells were pretreated with NAC (50 mmol/L for 2 h) for ROS inhibition, followed by YGJDSJ (100–400 μg/ml) treatment for 24 h, and subjected to caspase-3 (c), caspase-8 (d) and caspase-9 (e) activities and anoikis detection (f). Caspases activities were expressed as fold activation over control. Data shown are representative of three independent experiments. *P < 0.01, versus control group; ^#P < 0.01, versus corresponding dose of YGJDSJ treated NAC (−) group.

Fig. 5

YGJDSJ inhibited PTK2 expression and phosphorylation in Bel-7402 cells. Suspension-cultured Bel-7402 cells were collected after YGJDSJ treatment, subjected to western blots using indicated antibodies (a), and the expression of proteins were expressed as fold of GAPDH (b). Bel-7402 cells were transfected with recombinant human PTK2 and empty vector, and subjected to suspension-culture, YGJDSJ (200 μg/ml) treatment for 24 h, western blots using indicated antibodies (c) and anoikis detection (d). *P < 0.01, versus control group; ^▲P > 0.05, versus control YGJDSJ group; ^#P < 0.01, versus YGJDSJ treated vector group