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: Diallyl trisulfide, Osteosarcoma, Autophagy,
   EGFR/PI3K/AKT/mTOR

1. Introduction

   Osteosarcoma (OS) is the most prevalent bone malignancy characterized
   by locally aggressive growth and early metastatic potential in children
   and adolescents. The incidence rate of OS is 2 cases per million, with
   a 5-year survival rate of less than 68% under 14 years old in the
   United States [[37]1]. Despite significant advances in the treatment of
   OS, including surgical resection and the use of chemotherapy drugs such
   as cisplatin, methotrexate, and doxorubicin [[38][2], [39][3],
   [40][4]], the subsequent side effects, for example myelosuppression,
   nephrotoxicity, cardiac issues, and neurotoxicity, profoundly reduces
   patients’ quality of life [[41]5,[42]6]. Therefore, the novel effective
   and non-toxic agents are urgently needed to improve the therapeutic
   efficacy in osteosarcoma.

   Autophagy plays a crucial role in assessing the efficacy of anticancer
   drugs, as it facilitates the transport of cytoplasmic substances to
   lysosomes for degradation through vesicles. It is the primary mechanism
   by which eukaryotes degrade their own organelles or proteins to
   regulate cell proliferation and functions [[43]7,[44]8]. Growing
   evidence indicated that autophagy has a significant role in tumor
   progression. By modulating various autophagy-specific markers, like
   MAP1LC3 (LC3) and Beclin-1, autophagy can control tumor growth [[45]9].
   The phosphatidylinositol 3-kinase (PI3K) family of enzymes are involved
   in several cellular functions, including cell growth, proliferation,
   movement, differentiation and intracellular transport. Protein kinase B
   (AKT) and mammalian rapamycin target (mTOR) are crucial regulators of
   autophagy in tumors. Recent studies have demonstrated that the
   PI3K/AKT/mTOR pathway is implicated in autophagy arrest in osteosarcoma
   cells [[46]10]. Moreover, the epidermal growth factor receptor (EGFR)
   has emerged as an autophagy marker in OS [[47]11]. Also the increased
   EGFR can rapid the malignant process of osteosarcoma [[48]12].
   Consequently, EGFR/AKT/mTOR inhibitors have attracted considerable
   attention in cancer research.

   Diallyl trisulfide (DATS), also known as allicin, is the principal
   bioactive component found in garlic. It has been extensively studied
   and proven to possess anti-inflammatory, antiseptic, cardiovascular
   protective, and immune-enhancing properties [[49]13]. Importantly,
   numerous studies have reported the inhibitory effects of DATS in
   various cancers, including breast cancer, gastric cancer, glioblastoma,
   hepatocellular carcinoma, esophageal cancer, and bladder cancer
   [[50][14], [51][15], [52][16], [53][17], [54][18]]. Furthermore, it has
   been reported that DATS has the ability to reduce the risk of
   gastrointestinal cancers [[55]19], reverse chemotherapy resistance
   [[56]20,[57]21], and enhance anti-cancer activity when combined with
   biomaterials [[58]22]. These findings highlight that DATS has potential
   to fight against cancer due to its multifaceted and multi-targeted
   feature, as well as its apparent lack of toxicity [[59]23]. In recent
   years, although some progress has been made in exploring the molecular
   functions of DATS in OS [[60][24], [61][25], [62][26]], the underlying
   mechanism remains unclear.

   Baesd on above, we aim to found out how DATS regulates osteosarcoma
   cell malignant processes. Our investigation revealed that DATS could
   inhibit proliferation, migration and EMT, but induce autophagy and
   apoptosis in OS cells by downregulating EGFR, and its downstream
   PI3K/AKT/mTOR signaling pathway. These results confirmed that DATS
   might be a useful candidates for OS therapy.

2. Results

2.1. DATS inhibited OS cells growth, migration and EMT

   First, OS cells viability was determined by CCK8 assay. As shown in
   [63]Fig. 1A, the cell viabilities were significantly reduced in
   response to 24 and 48 h DATS treatment in a dose-dependent manner. DATS
   induced approximately 50% growth inhibition in OS cells after 24 h
   incubation. Therefore, 100 μM of DATS was used as working condition in
   the following experiments. To assess the potential of DATS in migration
   and invasion, we conducted wound healing and transwell experiments.
   After 48 h, the scratch gap area in the DATS group decreased slowly
   compared with the normal control (NC) group, as shown in [64]Fig. 1B.
   The transwell results showed a significant decrease in the number of
   invasive cells, upon DATS treatment for 24 h ([65]Fig. 1C), indicating
   that DATS blocked the migratory and invasive abilities of OS cells. To
   further investigate the effect of DATS on epithelial-mesenchymal
   transition (EMT), five EMT-related proteins—E-cadherin (E-cad),
   N-cadherin (N-cad), matrix metalloproteinase-2 (MMP-2), vimentin, and
   the E-cad blocker snail—were examined using Western blot analysis. The
   results showed that DATS suppressed the protein levels of N-cad, Snail,
   MMP-2 and Vimentin, but increased the level of E-cad in OS cells
   ([66]Fig. 1D). These findings indicated that DATS inhibited cell
   growth, migration, invasion and EMT in OS.

Fig. 1.

   [67]Fig. 1
   [68]Open in a new tab

   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.

2.2. DATS promoted OS cells apoptosis and autophagy

   To estimate the effect of DATS on autophagy, Western blot was carried
   out to examine the autophagic markers p62, LC3B I/II, and Beclin-1.
   LC3B II is an indicator of autophagic induction, formed by the
   conversion of cytoplasmic LC3B I [[69]27]. Thus, in [70]Fig. 2A, we
   observed an significantly increase in the conversion rate from LC3B–I
   to LC3B-II in DATS group, along with the upregulation of Beclin-1, an
   autophagy promoter. The expression of autophagy substrate p62 was
   notably reduced in DATS group compared to NC group, suggesting that
   DATS could induce autophagy in OS cells. Furthermore, OS cells were
   transfected with a pEGFP-C3-MAP1LC3B reporter, the autophagosomes was
   examined by confocal imaging. The results revealed that the content of
   autophagy vesicles in DATS group was higher than that in NC group,
   where autophagosomes were indicated by green signal. ([71]Fig. 2B).
   This founding was consistent with the alternation of Western blot
   analysis. Additionally, flow cytometric assay was performed to
   investigate the effect of DATS on cell apoptosis. As we can see in
   [72]Fig. 2C, the apoptosis rate in the blank group was only 1.46%,
   whereas it increased to 16.20% after DATS intervention.

Fig. 2.

   [73]Fig. 2
   [74]Open in a new tab

   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