Docosahexaenoic acid suppresses breast cancer cell metastasis by targeting matrix-metalloproteinases

Abstract

Breast cancer is one of the most prevalent cancers in women, and nearly half of breast cancer patients develop distant metastatic disease after therapy. Despite the significant advances that have been achieved in understanding breast cancer metastasis in the past decades, metastatic cancer is still hard to cure. Here, we demonstrated an anti-cancer mechanism of docosahexaenoic acid (DHA) that suppressed lung metastasis in breast cancer. DHA could inhibit proliferation and invasion of breast cancer cells in vitro, and this was mainly through blocking Cox-2-PGE2-NF-κB-MMPs cascades. DHA treatment significantly decreased Cox-2 and NF-κB expression as well as nuclear translocation of NF-κB in MDA-MB-231 cells. In addition, DHA also reduced NF-κB binding to DNA which may lead to inactivation of MMPs. Moreover, in vivo studies using Fat-1 transgenic mice showed remarkable decrease of tumor growth and metastasis to EO771 cells to lung in DHA-rich environment. In conclusion, DHA attenuated breast cancer progression and lung metastasis in part through suppressing MMPs, and these findings suggest chemoprevention and potential therapeutic strategy to overcome malignant breast cancer.

Keywords: DHA; MMP; breast cancer; omega-3 PUFA.

Figure 1. DHA suppresses various oncogenic signaling pathways

A. MDA-MB-231 cells were treated with various concentration of DHA for 24 hrs and the expression of β-catenin, cyclin D1, Cox-2, PGDH, p65, pIκB, and VEGF were analyzed by Western blot analysis. The densitometric ratio to actin band intensity for each sample was normalized to the control and shown below the blot. B. MDA-MB-231 cells were transfected with luciferase reporter containing Cox-2 and VEGF promoter and treated with DHA for 24 hrs. Then, the cells were lysed and the luciferase activity was measured with the dual luciferase assay. *, p <0.01 compared with control, Student's t test, significantly different from the control. C. MDA-MB-231 cells were transfected with luciferase reporter containing TCF/LEF or NF-κB binding site and treated with DHA for 24 hrs. D. MDA-MB-231 cells were treated with DHA (10 μM) or AA (10 μM) for 24 hrs, and cells were immunostained with anti-β-catenin antibody.

Figure 2. DHA treatment reduces in vitro cell invasion and motility by modulating MMP expression and activity

A. MDA-MB-231 cells loaded onto Matrigel-coated upper chamber transwell were treated with DHA (5, 10 μM) or AA (10 μM) 24 hrs. Then the numbers of invasive cells were counted in 10 random fields and normalized by Con cells as 1. B. MDA-MB-231 cells were loaded onto upper chamber transwell without Matrigel, and treated with DHA or AA for 24 hrs. The filtrated cells were stained and quantified. C. MDA-MB-231 cells transfected with MMP-2 or MMP-9 promoter were treated with increased dose of DHA (0, 5, 10 μM) for 24 hrs. Then, the cells were lysed and the luciferase activity was measured with the dual luciferase assay. D. MDA-MB-231 cells were treated with increased dose of DHA (0-10 μM) for 24 hrs and MMP-2, MMP-9 and TIMPs mRNA levels were analyzed by RT-PCR. E. MDA-MB-231 cells were treated with increased dose of DHA (0-25 μM) for 24 hrs, and condition media were collected and MMP activity was analyzed using gelatin zymography for secreted enzymatic activity.

Figure 3. DHA antagonizes the effect of PGE₂

A. MDA-MB-231 cells transfected with MMP-2 or MMP-9 promoter were treated with 10 μM AA for 24 hrs. Then, the cells were lysed and the luciferase activity was measured with the dual luciferase assay. B. MDA-MB-231 cells were treated with increased dose of AA (0, 5, 10 μM) in serum-free media for 24 hrs. Then the condition media were prepared and the activity of MMP-2 and MMP-9 were analyzed using gelatin zymography. C. MDA-MB-231 cells were transfected with MMP-2 and MMP-9 promoter, and then treated with PGE₂ (0, 5, 10 μM) for 12 hrs and luciferase activity was measured with the dual luciferase assay. D-E. MDA-MB-231 cells transfected with MMP-2 or MMP-9 promoter were pretreated with DHA (10 μM) for 2 hrs, and then PGE₂ (10 μM) (D) or AA (10 μM) (E) were added, respectively. After incubation for 12 hrs, the luciferase activity was measured with dual luciferase assay. F. MDA-MB-231 cells were treated with PGE₂ (10 μM) in presence or absence of DHA (10 μM), and the activity of MMP-2 and MMP-9 promoter were analyzed by gelatin zymography.

Figure 4. DHA blocks NF-κB signaling which can bind to MMP promoter

A. MDA-MB-231 cells transfected with reporter construct containing NF-κB binding site were pretreated with DHA (10 μM) for 2 hrs, and then PGE₂ (10 μM) were added. After 12 hrs, the luciferase activity was measured with dual luciferase assay. B. Cells were treated with increased dose of DHA (0-25 μM) for 24 hrs, and whole cell lysates were immunoprecipitated with anti-IKKγ. Then immune complex kinase assay was performed with [γ-³²P]ATP and GST-IκB-α as an exogenous substrate. GST-IκB-α phosphorylation was assessed by SDS-PAGE and autoradiography. The densitometry ratio to IKKγ band intensity for each sample was normalized to the control and shown below the blot. C. MDA-MB-231 cells were treated with DHA (10 μM) or AA (10 μM) for 24 hrs, and cells were immunostained with p65 antibody. D. MDA-MB-231 cells were treated with DHA for 24 hrs. To induce binding, TPA was pretreated for 4 hrs and then DHA was added. And the nuclear extracts were incubated with the radiolabeled oligonucleotide containing NF-κB and EMSA was performed. F, blank without nuclear extract; FD, free DNA probe.

Figure 5. DHA-rich environment retards tumor growth in vitro

A-B. Fat-1 transgenic and C57BL/6 wild-type mice were implanted with murine breast carcinoma cell line EO771 cells. After inoculation, animals were closely monitored for the development of subcutaneous tumor. After two weeks, the animals were sacrificed and xenograft tissues were collected for further experiment. Day of the implantation of the tumor cells was designated day 0. WT, representative xenograft tumor from wild type mouse; Fat-1, two representative xenograft tumors from fat-1 transgenic mice. C. The tumor size was measured at indicated time with a caliper. Tumor volume=0.5×(width)²×length. D. The animals were sacrificed at two weeks after implanted, tumor tissues were fixed with formalin and TUNEL stain was performed. E. The nuclear extracts were prepared from mice xenograft tissues. Then the nuclear extracts were incubated with the radiolabeled oligonucleotide containing NF-κB and EMSA was performed. F, blank without nuclear extract; Comp., competitor without isotope labeling; FD, free DNA probe.

Figure 6. DHA-rich environment prevents lung metastasis

A. Murine breast carcinoma cell line EO771 cells were injected into tail vein of Fat-1 transgenic and wild-type mice. After 4 weeks, the animals were sacrificed and dissected to check the metastasis to lung. B. The xenograft tissue in lung were fixed and stained with endothelial marker CD31 antibody. C. The xenograft tissues in lung were fixed with formalin and TUNEL stain was performed. D. The mechanism of DHA on suppressing metastasis was illustrated.