Fig 1: MMP3 is SIRT1-dependently secreted from fibroblasts and stimulates cancer cell growthA. Conditioned media were collected from MEF-1 cells transfected with SIRT1 (2 μg) or siSIRT1 (80 nM). The media were applied to ARY015 mouse antibody arrays. Representative array images are shown in the upper three panels. Three SIRT1-dependently secreted factors are listed in the bottom panel. B. MEFs and B16F10 cells were transfected as indicated and then co-cultured. C. MEF-1 and B16F10 cells were transfected with 2 μg of empty vector or SIRT1 plasmid, or 80 nM siRNA targeting none or SIRT1. After incubated for 48 h, cells were lysed to immunoblot the indicated proteins. D. SKOV3, SNU840, and CCD18Lu cells were transfected and co-cultured as indicated. Cell growth (mean ± s.d.; n=4) was analyzed by MTT staining. *, p < 0.05. E, MMP3 levels in B16F10 tumors (see Figure 1) were analyzed by immunohistochemistry. Representative images were captured under a microscope at 400× magnification (scale bar, 100 μm).
Fig 2: MMP3 acts as a growth factor for cancer cellsA. Cancer cells (B16F10, SKOV3, or SNU840) cells were co-cultured with fibroblasts (MEF-1 or CCD18Lu) which were transfected with SIRT1. MMP3 inhibitor at the indicated concentrations was administered into the co-culture media. B. Conditioned media were collected from fibroblasts transfected with si-SIRT1 (80 nM). Cancer cells were cultured in a 1:1 (v/v) mixture of the conditioned medium and fresh medium, and treated with recombinant human MMP3 (rhMMP3) at the indicated concentrations. Cancer cell growth was estimated using MTT. Data (mean ± s.d.; n=4-6) are plotted as a function of incubation time. *, p < 0.05.
Fig 3: Fibroblast-derived MMP3 promotes tumor growthA. MEF cells obtained from WT mice were infected with sh-LTviral-Control or -SIRT1 (0.9 × 108 TU/mL) for 72 hour. The MEF cells (1×105) and B16F10 cells (1×106) were mixed with Matrigel and injected into the flanks of nude mice. Tumor volumes are expressed at the means ± s.e.m. (n, 10; *, p < 0.05) in the left panel. Tumors (a, b) were excised and weighed on the final day. Each tumor weight is plotted in the right panel and pictures of tumors are shown below the plot. B. MEFs obtained from WT and SIRT1-TG mice were transfected with the indicated siRNAs (80 nM). MEFs (1×105) and B16F10 cells (1×106) were mixed with Matrigel and the cell mixtures were injected into the flanks of nude mice. Tumor volumes were measured from day 7 after implantation. Results are expressed as the means ± s.e.m. (n, 6-8; *, p < 0.05) in the left panel. Each tumor weight is plotted in the right panel and pictures of tumors are shown below the plot.
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