Molecular mechanisms of Bombyx batryticatus ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Abstract

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.

Keywords: Apoptosis; Bax; Bcl-2; Bombyx batryticatus; P21; SGC-790 cells.

Fig. 1

Inhibition of proliferation by BBE. SGC-7901 cells (a) and BGC-823 cells (b). Cells were treated with 1.0–10.0 mg/mL BBE for 24, 36, 48 and 60 h. Cell viability was determined by MTT assay. Results are expressed as percentages of proliferation compared to the untreated control (mean ± SE, n = 3)

Fig. 2

Apoptosis of SGC-7901 cells treated with BBE. 1 Morphologic changes include rupture, aggregation, swelling, of the membrane of cells observed under an inverted microscope (×25). SGC-7901 cells were treated with different concentrations of BBE for 24 h. a Control, b 2 mg/mL, c 4 mg/mL, d 6 mg/mL. 2 Morphology of apoptotic cells observed by laser confocal fluorescence microscopy. SGC-7901 cells were treated with 6 mg/mL BBE for 3, 6, 12 h. Cells were observed by confocal fluorescence microscopy. a Early apoptosis, b late apoptosis, c late apoptosis, more distortion in both cytomembrane and nucleus. 3 Fluorescence-activated cell sorter analysis for Annexin-V and propidium iodide (PI) staining of SGC-7901 cells incubated with BBE at different concentrations for 24 h. a Control, b 2 mg/mL, c 4 mg/mL, d 6 mg/mL. Upper right necrotic cells and late apoptotic cells. Lower left fully viable cells. Lower right early apoptotic cells. 4 DNA fragmentation influenced by BBE in SGC-7901 cells for 24 h. Lanes 1–4 stand for 0 mg/mL (control), 2, 4 and 6 mg/mL, respectively

Fig. 3

Possible pathway of apoptosis in SGC-7901 cells treated with BBE. a Relative expression levels of Bcl-2, Bax and P21 in SGC-7901 cells. Cells were treated with 0 mg/mL (control), 2, 4, 6, 8 mg/mL BBE for 36 h. Results were expressed as average of three replicates ± SD. β-actin was used as an internal reference gene. The relative expression was calculated based on the value of the lowest expression (expression levels of Bcl-2 at 8 mg/mL), which was ascribed an arbitrary value of 1. b Effects of BBE on β-actin, Bcl-2, Bax and P21 protein expression in SGC-7901 cells. Cells were treated with 0 mg/mL (control), 2, 4, 6, 8 mg/mL BBE for 36 h. The protein levels were monitored by Western blot analysis. The signal was normalized using β-actin as an internal standard

Fig. 4

Cell cycle analysis (b, c) of SGC 7901 cells treated with 4 and 6 mg/ml BBE for 24h. Cells distribution was increased in G1 and G2 phase, when it was decreased in S phase. The difference in G1, G2 and S phase summarized in (d), while (a) is the control treatment