Downregulation of Tie2 gene by a novel antitumor sulfolipid, 3'-sulfoquinovosyl-1'-monoacylglycerol, targeting angiogenesis

Abstract

We previously reported that 3'-sulfoquinovosyl-1'-monoacylglycerol (SQMG) was effective in suppressing the growth of solid tumors due to hemorrhagic necrosis in vivo. In the present study, we investigated the antiangiogenic effect of SQMG. In vivo assessment of antitumor assays showed that some tumor cell lines, but not others, were sensitive to SQMG. Microscopic study suggested that in SQMG-sensitive tumors, but not SQMG-resistant tumors, angiogenesis was reduced. We next investigated gene expression relating to angiogenesis in tumor tissues by quantitative real-time polymerase chain reaction. Consequently, although vascular endothelial growth factor gene expression was not detected with significant differences among the cases, significant downregulation of Tie2 gene expression was observed in all SQMG-sensitive tumors as compared with controls, but not in SQMG-resistant tumors. These data suggested that the antitumor effects of SQMG could be attributed to antiangiogenic effects, possibly via the downregulation of Tie2 gene expression in SQMG-sensitive tumors.

Figure 1

Structure of 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG). SQMG contains a single fatty acid, R = C18:1.

Figure 2

In vivo study of the antitumor effects of 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG). Human tumor cells (10⁶) of the cell lines (a) MDA‐MB‐231, (b) A549, (c) WiDr, (d) SAS, (e) PC‐3, (f) TE‐8, and (g) LU65 were injected subcutaneously into mice, and when tumors grew to 30–40 mm³, mice were injected with saline (control), 5 mg/kg (SQMG 5), or 20 mg/kg (SQMG 20) every day for 14 days. The means ± SE of tumor volumes from each group (n = 4/group) are shown. *P < 0.01.

Figure 3

Antiangiogenesis assessment by immunohistochemical analysis. Cryosections of MDA‐MB‐231 treated (a,b) without and (c,d) with 3′‐sulfoquinovosyl‐1′‐monoacylglycerol were stained with antimouse CD31 monoclonal antibody and antirat IgG conjugated with AlexaFlour 488, and nuclei were counterstained with propidium iodide. Arrows indicate CD31‐positive blood vessels. Insets in (a) and (c) are magnified and shown in (b) and (d), respectively. Scale bar = 100 µm.

Figure 4

Influence of 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG) on cell proliferation and apoptosis. (a) Human umbilical vein endothelial cells (HUVEC) and (c) NIH3T3 were cultured in the presence or absence of SQMG at the indicated concentrations. Cell proliferation was examined by MTT assay. Results represent means ± SE of triplicate wells on one of three independent experiments. (b) HUVEC were cultured in the presence or absence of SQMG at the concentrations indicated for 48 h, harvested, and double‐stained with annexin‐V–fluorescein and propidium iodide (PI). The percentage of apoptotic (annexin V and PI double positive) cells was determined by flow cytometric analysis. Results represent means ± SE of three independent experiments.

Figure 5

Effect of 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG) on angiogenesis in vitro. (a–c) Human umbilical vein endothelial cells (HUVEC) grown on a fibroblast sheet on Matrigel were cultured (a) without and (b,c) with SQMG at the indicated concentrations. After cultivation, the cells were fixed and stained with an antihuman CD31 antibody for 1 h. CD31 molecules were detected using the alkaline phosphate method. (d) The tube formation of HUVEC was quantitated using Image ++ software. Results represent means ± SE of five independent areas on one of three independent experiments. *P < 0.01.

Figure 6

Angiogenic receptor gene expression on capillary‐formed human umbilical vein endothelial cells (HUVEC) in vitro. Total RNA of capillary‐formed HUVEC was assessed as to the amount of mRNA copy number of human (a) Flt‐1, (b) KDR, and (c) Tie2 by quantitative real‐time reverse transcription–polymerase chain reaction analysis. Results represent means ± SE of triplicate wells on one of three independent experiments.