Mullerian inhibiting substance inhibits invasion and migration of epithelial cancer cell lines

Abstract

Objective: Given the fact that Mullerian Inhibiting Substance (MIS) causes complex remodeling of the urogenital ridge and regression of the Mullerian ducts during male embryonic development, we examined whether MIS could affect similar cell properties such as migration and invasion that could contribute ultimately to micro-metastasis of cancers arising from Mullerian tissues. MIS receptor expressing cell lines found to be invasive and migratory in vivo are examined in an in vivo assay that is cost-effective.

Methods: We designed in vitro and in vivo experiments to determine if MIS inhibited the movement of cancer lines IGROV-1, HEp3, MDA-MB-231, and HT1080 in cell culture invasion/migration chamber assays and in chick embryo metastasis assays.

Results: MIS, at concentrations below those that inhibit cell proliferation, blocked in vitro invasion and in vivo migration of epithelial cancer cells that express the MIS receptor.

Conclusions: While our laboratory has previously established MIS as an inhibitor of cancer cell proliferation using in vitro assays and in vivo xenografts, we now show that MIS can also inhibit in vivo tumor migration.

Fig. 1

The presence of the MISRII is detected by quantitative PCR. (A) The relative amount of mRNA transcript (Ct) for MISRII expression for each cell line is shown. (B) Agarose gel of the PCR products confirms the appropriate length bands for both GAPDH and MISRII.

Fig. 2

MIS dose responses for inhibition of cell proliferation (% Cell Growth) at concentrations between 0 and 60 ug/ml for MDA-MB-231, HEp3, IGROV-1, and HT1080 cells at either (A) 3 days or (B) 7 days. Paclitaxel cell proliferation dose responses between 0 and 8 nM are also shown for the same cell lines at 3 (C) and 7 (D) days of incubation.

Fig. 3

50nM MIS inhibits the invasion in vitro (Cell invasion count) of the epithelial cell lines IGROV-1 (A, 60.50 ± 8.99, n = 8 vs. control 371.1 ± 70.99, n = 7, p < 0.0006), MDA-MB-231 (B, 134.8 ± 33.51, n = 8 vs. control 640.7 ± 117.4, n = 9, p < 0.0015), and HEp3 (C, 114.4 ± 18.24, n = 8 vs. control 406.4 ± 44.64, n = 10, p < 0.0001), but not the fibrosarcoma cell line HT 1080 (D, 1282 ± 91.67 n = 6 vs. control 1171 ± 140.1, n = 9, p = 0.57). Longer bars indicate more invasion. 1nM paclitaxel did not inhibit invasion. Error bars show the standard error of the mean. Asterisks indicate treatment outcomes significantly different from controls (p < 0.05).

Fig. 4

Chick chorioallantoic membrane (CAM) assay for cell migration (A). Pretreatment with 7 ug/ml (50 nM) MIS of IGROV-1 (B, C) and HEp3 cells (D, E) significantly reduces relative migration to the posterior CAM (B, D) and lung (C, E) when compared to buffer controls ([B] 11.44 ± 1.43, n = 5 vs. 23.32 ± 2.19, n = 6, p < 0.002; [C] 8.87 ± 1.58, n = 4 vs. 15.04 ± 1.15, n = 6, p < 0.02; [D] 10.62 ± 1.18, n = 8 vs. 17.76 ± 0.97, n = 5, p < 0.002; [E] 8.04 ± 0.52, n = 9 vs. 11.49 ± 1.37, n = 11, p < 0.045). Relative migration levels were not statically different from the non-metastatic negative control cell line WI-38. Longer bars indicate more migration. Error bars represent the standard error of the mean. Asterisks indicate p < 0.05. Ns = not significant.