In 2002, Fingrut et al. first reported the jasmonates-mediated suppression of cellular proliferation and induction of cell death in various human and mouse cancer cell lines, including breast cancer, prostate cancer, melanoma, lymphoblastic leukemia, and lymphoma
. In the past decade, several groups have demonstrated that members of jasmonate family and their synthetic derivatives exhibit anti-cancer activity on other kinds of tumor cells, including lung cancer
, colon cancer
, cervical cancer
[29, 30], neuroblastoma
[12, 13], and myeloid leukemia
[31, 32]. To date, several mechanisms have been proposed to explain the anti-cancer effects of jasmonates, including induction of severe ATP depletion via mitochondrial perturbation
, induction of re-differentiation via mitogen-activated protein kinase activity
, induction of a significant decrease in survivin levels via the β-catenin/T-cell factor pathway
, and induction of apoptosis via pro-apoptotic proteins of the Bcl-2 family
, opening the mitochondrial permeability transition pore complex
 and activation of extrinsic apoptotic pathway
. However, the anti-cancer activity of sub-cytotoxic jasmonates and underlying mechanisms still warrant further investigation.
Recent evidence shows that MJ can inhibit melanoma cell migration and suppress the development of melanoma growth in mouse lungs
, suggesting the potential anti-metastatic activities of MJ. In the current study, we demonstrated that in addition to the cytotoxic properties of MJ in cancer therapy, sub-cytotoxic MJ attenuated the migration and invasion of human gastric cancer SGC-7901 and MKN-45 cells. The SGC-7901 cell line was first established from the metastatic lymph node of a 56-year-old female patient suffering from gastric adenocarcinoma
, while the MKN-45 cell line was derived from a metastatic liver tumor of a 62-year-old female with gastric cancer
. It is well known that the extracellular matrix (ECM) is a barrier to prevent tumor cells from invasion and metastasis
. Specific enzymes produced by cancer cells and activated by certain signals, such as matrix metalloproteinases (MMPs), have been reported to degrade ECM, and are associated with the progression of gastric cancer
[23, 39]. MMP-14, also named as membrane type-1 matrix metalloproteinase, functions as a pericellular collagenase and plays an important role in tumor invasion and metastasis by facilitating the cancer cells to remodel and penetrate ECM
[40–42]. Clinical evidence has shown the linkage between high MMP-14 expression and cancer progression, such as lymph node metastases, invasion, poor clinical stage, larger tumor size, and increasing tumor stage
. In this study, we found that sub-cytotoxic MJ selectively down-regulated the expression of MMP-14, but not of MMP-7 and MMP-9, in gastric cancer cells. In addition, restoration of MMP-14 rescued the sub-cytotoxic MJ-induced inhibition on the migration and invasion of cancer cells, suggesting the role of MMP-14 down-regulation in the anti-metastatic activities of sub-cytotoxic MJ.
Since MMP-14-mediated degradation of ECM occurs throughout the angiogenic process and contributes to vascular regression
, we further demonstrated that sub-cytotoxic MJ attenuated the angiogenic capabilities of gastric cancer cells. In addition, sub-cytotoxic MJ did not induce the cell death of human umbilical vein endothelial cells, ruling out the influence of direct cytotoxicity on angiogenesis. In a previous study, MJ was noted to consistently impair the vascular growth in the Chorioallantoic model of angiogenesis
, while the underlying mechanisms remain largely unknown. VEGF, a dimeric and heparin-binding glycoprotein that functions as a potent mitogen of vascular endothelial cells, is a major inducer of angiogenesis that can promote the growth and metastasis of tumors
. In this study, for the first time, we demonstrate that VEGF functions as a downstream gene of MMP-14 in gastric cancer cells. Since our findings indicated that sub-cytotoxic MJ abolished the VEGF expression in gastric cancer cells, combining the evidence that restoration of VEGF expression through MMP-14 over-expression rescued the decrease in tube formation of endothelial cells, we believe that the decreased expression of MMP-14 and downstream VEGF, at least in part, contributes to the anti-angiogenic function of sub-cytotoxic MJ in gastric cancer cells.
To elucidate the mechanisms underlying the down-regulation of MMP-14 by sub-cytotoxic MJ, we further demonstrated that the MMP-14 transcript levels were abolished in MJ-treated gastric cancer cells. Because of the lack of typical TATA box and the presence of a GC-rich sequence immediately upstream of the transcriptional start sites within the promoter, the transcriptional control of MMP-14 is unique compared to other MMP family members
. The GC-rich region within human MMP-14 promoter serves as the putative binding site for transcription factor Sp1 in fibrosarcoma and prostate cancer cells
[20, 44]. In addition, constitutive Sp1 binding to the MMP-14 promoter was also noted in rat glomerular mesangial cells
. In this study, we demonstrated the over-expressed Sp1 and its positive correlation with MMP-14 transcription in gastric cancer tissues. Then, we sought to determine whether Sp1 played a role in regulating MMP-14 in gastric cancer cells. Electrophoretic mobility shift assay is a traditional method to investigate the transcription factor-DNA interaction via studying the binding of protein to known DNA oligonucleotide probes
. However, this method is an in vitro assay and relatively difficult to be quantified
. In recent years, ChIP assay is an alternative method to monitor transcriptional regulation through transcription factor-DNA binding interaction in living cells, which is quantitative when coupled with qPCR analysis
. Here, we showed that Sp1 bound to the MMP-14 promoter in gastric cancer cells via ChIP assay. Additionally, administration of sub-cytotoxic MJ attenuated the Sp1 expression and binding to MMP-14 promoter, implying the important role of Sp1 in sub-cytotoxic MJ-induced down-regulation of MMP-14 in gastric cancer cells.