Silk fibroin peptide suppresses proliferation and induces apoptosis and cell cycle arrest in human lung cancer cells

Abstract

Silkworm cocoon was recorded to cure carbuncle in the Compendium of Materia Medica. Previous studies have demonstrated that the supplemental silk protein sericin exhibits anticancer activity. In the present study, we investigated the effects of silk fibroin peptide (SFP) extracted from silkworm cocoons against human lung cancer cells in vitro and in vivo and its possible anticancer mechanisms. SFP that we prepared had high content of glycine (~ 30%) and showed a molecular weight of ~ 10 kDa. Intragastric administration of SFP (30 g/kg/d) for 14 days did not affect the weights, vital signs, routine blood indices, and blood biochemical parameters in mice. MTT assay showed that SFP dose-dependently inhibited the growth of human lung cancer A549 and H460 cells in vitro with IC₅₀ values of 9.921 and 9.083 mg/mL, respectively. SFP also dose-dependently suppressed the clonogenic activity of the two cell lines. In lung cancer H460 xenograft mice, intraperitoneal injection of SFP (200 or 500 mg/kg/d) for 40 days significantly suppressed the tumor growth, but did not induce significant changes in the body weight. We further examined the effects of SFP on cell cycle and apoptosis in H460 cells using flow cytometry, which revealed that SFP-induced cell cycle arrest at the S phase, and then promoted cell apoptosis. We demonstrated that SFP (20-50 mg/mL) dose-dependently downregulates Bcl-2 protein expression and upregulates Bax protein in H460 cells during cell apoptosis. The results suggest that SFP should be studied further as a novel therapeutic agent for the treatment of lung cancer.

Fig. 1

Molecular weight and amino-acid composition of SFP. a Mass spectrogram of the SFP solution before dialysis. b Mass spectrogram result of the SFP solution after dialysis with a molecular weight cutoff of 8 kDa

Fig. 2

Antiproliferative effects of SFP on lung cancer cells and normal lung epithelial cells in vitro. a A549, H460, and BEAS-2b cells treated with varying concentrations (0–120 mg/mL) of SFP for 72 h. Cell viability was analyzed via MTT assay. b, c A549 and H460 cells treated with varying concentrations (0–120 mg/mL) of SFP for different incubation periods. d Colony formation assays of the A549 and H460 cells treated with SFP at the indicated concentrations. Mean ± SD. n = 3 **P < 0.01, *P < 0.05 vs control

Fig. 3

a, b Tumor-bearing mice were treated with vehicle, SFP (200 and 500 mg/kg), or cisplatin for 21 days. Vehicle was administered via intragastrically in the control group. SFP was administered via intragastrically in the SFP group. Cisplatin was administered via intraperitoneal injection. The body weights a and tumor growth rates b were measured as described in the Materials and Methods section. c, d Tumor-bearing mice were intraperitoneally injected with vehicle, SFP (200 and 500 mg/kg), and cisplatin for 40 days. Body weights c and tumor growth rates d were measured as described in the Materials and Methods. e The anticancer ratio of the two routes of administration. f Antitumor effect of SFP in vivo. BALB/c (nude) mice were administered normal saline, DDP, low-dose SFP (200 mg/kg), and high-dose SFP (500 mg/kg). Photos of tumors were taken after the end of drug administration. Mean ± SD. n = 5 **P < 0.01, *P < 0.05 vs control

Fig. 4

SFP triggers cell cycle arrest at the S phase and regulates apoptosis in H460 cells. a, b H460 cells were treated with various concentrations of SFP for 48 h. Cell cycle distribution a and cell apoptosis b were analyzed using Verity’s Modfit LT 3. 0 software. Mean ± SD. n = 3 **P < 0.01, *P < 0.05 vs control

Fig. 5

H460 cells were incubated with various concentrations of SFP for 48 h. Bax and Bcl-2 protein expression levels were detected via western blotting using β-actin as internal control. **P < 0.01, *P < 0.05 vs control