Diallyl trisulfide inhibits osteosarcoma 143B cell migration, invasion and EMT by inducing autophagy

Abstract

Background: Diallyl trisulfide (DATS), a compound derived from garlic, has been demonstrated its anti-cancer properties. While it has been shown to inhibit the expression of epidermal growth factor receptor (EGFR) in various cancers, its effects on osteosarcoma (OS) cells remain unclear. This study aimed to investigate the impacts of DATS on OS cells growth, migration, invasion, epithelial-mesenchymal transition (EMT) and autophagy, as well as its underlying mechanisms which was involving in the EGFR/PI3K/AKT/mTOR pathway.

Methods: In this study, human osteosarcoma cells (143B) were treated with different concentrations of DATS (10, 50, 100 and 200 μM) for 24 and 48 h, respectively. Cell viability was measured using CCK8, the half lethal concentration was selected for the following experiments. Wound healing and transwell assays were performed to evaluate migration and invasion abilities, while flow cytometry was used to measure apoptosis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and confocal imaging were employed to analyze the related mRNA and protein expression levels of epithelial-mesenchymal transition (EMT), EGFR/Phosphoinositide 3 kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) signaling pathway and autophagy-related markers.

Results: DATS significantly inhibited proliferation, migration and EMT in osteosarcoma cells. Additionally, DATS promoted cell apoptosis and induced autophagy, which could be rescued by the autophagy inhibitor 3-methyladenine (3-MA). Moreover, DATS treatment led to the inactivation of the EGFR/PI3K/AKT/mTOR pathway in osteosarcoma cells.

Conclusions: This study demonstrated that DATS inhibited osteosarcoma cell growth, migration and EMT, but inducing apoptosis and autophagy. These effects were mediated by the inactivation of the EGFR/PI3K/AKT/mTOR signaling pathway. These findings suggested that DATS could serve as a potential therapeutic agent for osteosarcoma treatment.

Keywords: Autophagy; Diallyl trisulfide; EGFR/PI3K/AKT/mTOR; Osteosarcoma.

Fig. 1

DATS inhibited OS cells growth, migration, invasion and EMT. (A) OS cells were treated with DATS (0, 10, 50, 100, 200 μM) for 24 and 48 h, CCK8 assay was conducted to test cell viability of OS cells. (B–C) Wound healing and transwell were used to assess the potential of DATS on migrative and invasive abilities. Scale bar: 1 mm for B and 500 μm for C. (D) Western blot was employed to measure the expression levels of E-cad, N-cad, MMP-2, Snail and Vimentin in OS cells. *p < 0.05 (compared with NC). #p < 0.05 (compared with NC). Data are represented as means ± SD; n = 3 per group.

Fig. 2

DATS induced autophagy and apoptosis. (A) Western blot was employed to measure the expression of LC3BI/II, p62 and Beclin-1 in OS cells after DATS treatment. (B) OS cells were transfected with a pEGFP-C3-MAP1LC3B reporter (green) and stained with DAPI (blue), then analyzed with confocal microscopy, scale bar = 10 μm. (C) The apoptosis rates of OS cells were examined by flow cytometry after the treatment of DATS for 24 h. *p < 0.05 (compared with NC). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The overview of RNA-seq. (A) Pearson correlation map between each sample of RNA-seq. (B) Volcano map of differential expressed genes after DATS treatment. (C) Heatmap of up-regulating expressed genes after DATS treatment. (D) Heatmap of down-regulating expressed genes after DATS treatment.

Fig. 4

Bioinformatic analysis of RNA-seq data. (A) The histogram image of top 20 altered GO terms after DATS treatment in GO enrichment results. (B) The scatterplot image of KEGG pathways after DATS treatment in KEGG enrichment results. (C) The EGFR/PI3K/AKT/mTOR signaling pathway in PD-L1 and PD-1 pathway. (D) PPI network for EGFR and PI3K/AKT/mTOR signaling pathway.

Fig. 5

DATS regulated EGFR/AKT/mTOR signaling pathway. (A) Western blot was employed to measure the relative expressions of EGFR, p-ERK/ERK, PI3K, p-AKT/AKT, mTOR and p-mTOR in OS cells. (B) qPCR was used to measure the expression of PI3K, mTOR, EGFR and AKT in OS cells. *p < 0.05 (with NC). Data are represented as means ± SD; n = 3 per group.

Fig. 6

Autophagy can be reversed by 3-MA. (A) Western blot was employed to measure the expression of LC3 I/II, p62 and Beclin-1, OS cells were transfected with pEGFP-C3-MAP1LC3B reporter and analyzed with confocal microscopy images. Scale bar: 10 μm. (B–C) Wound healing and transwell were used to assess the potential of DATS and 3-MA on migrative and invasive abilities. Scale bar: 1 mm for B and 500 μm for C. (D) The expression levels of E-cad, N-cad, MMP-2, Snail and Vimentin in OS cells was measured by Western blot. (E) Flow cytometric assays showed the apoptosis rate of OS treated with DATS and 3-MA. *p < 0.05 (with NC). #p < 0.05 (with DATS). **p < 0.05 (with NC and DATS). Data are shown as means ± SD; n = 3 per group.

Fig. 7

DATS inactivated EGFR/PI3K/AKT/mTOR pathway. (A) Western blot was used to detect the EGFR, ERK, p-ERK, PI3K, AKT, p-AKT, mTOR and p-mTOR expression. (B) qRT-PCR was used to examine the expression of PI3K, mTOR, EGFR and AKT in OS cells after DATS and/or 3-MA treatment. *p < 0.05 (with NC). Data are shown as means ± SD; n = 3 per group.