Hui Ling

Erk is involved in the differentiation induced by diallyl disulfide in the human gastric cancer cell line MGC803.

Diallyl disulfide (DADS) is a major constituent of garlic. Previously, we found that DADS both inhibited proliferation in human gastric cancer cells in vitro and in vivo, and induced G2/M arrest. In this study, we investigated whether this differentiation effect was induced by DADS in human gastric cancer MGC803 cells, and whether it was related to an alteration in ERK activity. The results showed that the growth of MGC803 cells was inhibited by DADS. Cells treated with DADS displayed a lower nucleocytoplasmic ratio and tended to form gland and intercellular conjunction structures. The ConA-mediated cell agglutination ratio and cells’ ALP specific activity decreased. In MGC803 cells, dye transfer was limited to a few cells neighbouring the dye-injected cell and to a depth of 1–2 layers beneath the scrape site. However, after treatment with DADS, the LY (Lucifer Yellow) was transferred to several cells immediately neighbouring the microinjected cell and to a depth of 2–4 cell layers from the scrape site. This indicated that DADS induced differentiation in MGC803 cells. Western blot analysis revealed that although DADS did not influence the quantity of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls. At 30 mg·L−1, DADS inhibited the activation of ERK1/2 in 15–30 min. These results suggested that the DADS-induced differentiation of MGC803 cells involved an alteration of the ERK1/2 signaling pathway.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

Inhibition of ERK and activation of p38 are involved in diallyl disulfide induced apoptosis of leukemia HL-60 cells

We investigated the effects of diallyl disulfide (DADS) on the induction of apoptosis in human Leukemia cell line HL-60 and explored the roles of mitogen-activated protein kinase (ERK and p38 MAPK) pathways in the growth inhibition and apoptosis induced by DADS. MTT assay was used to determine the DADS induced cell growth inhibition in HL-60 cells. Flow cytometry and DNA fragmentation were used to examine the roles of apoptosis in DADS-mediated cell death. Western blot analysis of the expression of phospho-MAPKs (ERK and p38) was employed to elucidate the possible mechanisms of DADS induced apoptosis. We found that growth inhibition of HL-60 cells treated with DADS exhibited a dose-dependent response (P<0.05) and DADS induced significant apoptosis. DADS at the concentration of 10 mg/L persistently activated p38 and simultaneously reduced ERK activity. PD98059, an inhibitor of ERK upstream activators MAPK kinase MKK1 and MKK2, promoted cytotoxicity and apoptosis in HL-60 cells treated with DADS. In contrast, SB203580, an inhibitor of p38, decreased cytotoxicity and apoptosis induced by DADS. Therefore, DADS can effectively inhibit the proliferation and induce apoptosis of human leukemia cell line HL-60. Inhibition of ERK signaling pathways and activation of p38 signaling pathways are likely involved in DADS induced apoptosis in HL-60 cells.

Diallyl disulfide suppresses epithelial-mesenchymal transition, invasion and proliferation by downregulation of LIMK1 in gastric cancer

Diallyl disulfide (DADS) has been shown to have multi-targeted antitumor activities. We have previously discovered that it has a repressive effect on LIM kinase-1 (LIMK1) expression in gastric cancer MGC803 cells. This suggests that DADS may inhibit epithelial-mesenchymal transition (EMT) by downregulating LIMK1, resulting in the inhibition of invasion and growth in gastric cancer. In this study, we reveal that LIMK1 expression is correlated with tumor differentiation, invasion depth, clinical stage, lymph node metastasis, and poor prognosis. DADS downregulated the Rac1-Pak1/Rock1-LIMK1 pathway in MGC803 cells, as shown by decreased p-LIMK1 and p-cofilin1 levels, and suppressed cell migration and invasion. Knockdown and overexpression experiments performed in vitro demonstrated that downregulating LIMK1 with DADS resulted in restrained EMT that was coupled with decreased matrix metalloproteinase-9 (MMP-9) and increased tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. In in vitro and in vivo experiments, the DADS-induced suppression of cell proliferation was enhanced and antagonized by the knockdown and overexpression of LIMK1, respectively. Similar results were observed for DADS-induced changes in the expression of vimentin, CD34, Ki-67, and E-cadherin in xenografted tumors. These results indicate that downregulation of LIMK1 by DADS could explain the inhibition of EMT, invasion and proliferation in gastric cancer cells.

Identification of potential targets for diallyl disulfide in human gastric cancer MGC-803 cells using proteomics approaches.

Diallyl disulfide (DADS) is characterized as an effective agent for the prevention and therapy of cancer, however, mechanisms regarding its anticancer effects are not fully clarified. In the present study, we compared the protein expression profile of gastric cancer MGC-803 cells subjected to DADS treatment with that of untreated control cells to explore potential molecules regulated by DADS. Using proteomic approaches, we identified 23 proteins showing statistically significant differences in expression, including 9 upregulated and 14 downregulated proteins. RT-PCR and western blot analysis confirmed that retinoid-related orphan nuclear receptor α (RORα) and nM23 were increased by DADS, whereas LIM kinase-1 (LIMK1), urokinase-type plasminogen activator receptor (uPAR) and cyclin-dependent kinase-1 (CDK1) were decreased. DADS treatment and knockdown of uPAR caused suppression of ERK/Fra-1 pathway, downregulation of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9) and vimentin, and upregulation of tissue inhibitor of metalloproteinase-3 (TIMP-3) and E-cadherin, concomitant with inhibition of cell migration and invasion. Moreover, knockdown of uPAR potentiated the effects of DADS on MGC-803 cells. These data demonstrate that downregulation of uPAR may partially be responsible for DADS-induced inhibition of ERK/Fra-1 pathway, as well as cell migration and invasion. Thus, the discovery of DADS-induced differential expression proteins is conducive to reveal unknown mechanisms of DADS anti-gastric cancer.

Diallyl disulfide inhibits TGF‑β1‑induced upregulation of Rac1 and β‑catenin in epithelial‑mesenchymal transition and tumor growth of gastric cancer

Transforming growth factor‑β1 (TGF‑β1) has been demonstrated to promote epithelial‑mesenchymal transition (EMT), invasion and proliferation in tumors via the activation of Rac1 and β‑catenin signaling pathways. The present study investigated the effects of diallyl disulfide (DADS) on TGF‑β1‑induced EMT, invasion and growth of gastric cancer cells. TGF‑β1 treatment augmented EMT and invasion, concomitantly with increased expression of TGF‑β1, Rac1 and β‑catenin in gastric cancer cells. DADS downregulated the expression levels of TGF‑β1, Rac1 and β‑catenin. DADS, TGF‑β1 receptor inhibitor as well as Rac1 inhibitor antagonized the upregulation of the TGF‑β1‑induced expression of these genes, abolishing the enhanced effects of TGF‑β1 on EMT and invasion. Blocking the TGF‑β1 receptor through inhibition resulted in the decreased expression of Rac1 and β‑catenin. Rac1 inhibitor reduced the TGF‑β1‑induced β‑catenin expression. In addition, DADS and the aforementioned inhibitors attenuated the TGF‑β1‑induced tumor growth and the expression changes of E‑cadherin, vimentin, Ki‑67 and CD34 in nude mice. These data indicated that the blockage of TGF‑β1/Rac1 signaling by DADS may be responsible for the suppression of EMT, invasion and tumor growth in gastric cancer.