Normal Basal Epithelial Cells Stimulate the Migration and Invasion of Prostate Cancer Cell RM-1 by TGF-β1/STAT3 Axis in vitro

Abstract

Aim: Basal epithelial cells are absent in distant prostate cancer. This study aimed to investigate whether basal epithelial cells could suppress migration and invasion of prostate cancer cells to become a new treatment strategy for prostate cancer.

Main methods: Basal epithelial cells were identified by immunofluorescence with anti-p63. Wound healing assays or transwell assays were used to explore the effects of basal epithelial cells, TGF-β1, SB431542 (inhibitor of TGF-β type I receptor) or stattic (inhibitor of phosphorylated STAT3) on migration or invasion of mouse prostate cancer cell (RM-1). Concentration of TGF-β1 was measured by ELISA assay. HE staining was used to investigate cell morphology. Immunocytochemistry with anti-p63 was used to identify basal epithelial cells. Levels of STAT3, p-STAT3 (Ser727) and proteins associated with EMT were measured with Western blot assay. Cell proliferation was measured with MTT or CCK8 assay.

Results: Normal basal epithelial cells acquired from mouse prostate were specific to anti-p63 and more than 90%. Basal epithelial cells and RM-1 could both secrete TGF-β1. Basal epithelial cells and TGF-β1 promoted the migration and invasion of RM-1 through changing the cell morphology and up-regulating expression of ZEB1, N-cadherin, vimentin, snail and p-STAT3 (Ser727), at the same time down-regulating E-cadherin of RM-1. SB431542 strongly suppressed migration, invasion as well as the expressions of EMT relevant proteins and p-STAT3 (Ser727) of co-cultured RM-1. In addition, stattic suppressed proliferation, migration and invasion of non-treated RM-1 and co-cultured RM-1.

Conclusion: Our study suggests that normal basal epithelial cells might stimulate the migration and invasion of RM-1 by TGF-β1/STAT3 axis which could be suppressed by inhibitor of TGF-β receptor and inhibitor of p-STAT3. So, basal epithelial cells might not become a treatment strategy for prostate cancer, but our results could provide some researching references for other diseases which include basal epithelial cells such as prostatic intraepithelial neoplasia, prostatic hyperplasia, cervical cancer, or urinary bladder cancer.

Keywords: RM-1; STAT3; TGF-β1; invasion; migration; normal basal epithelial cells.

Figure 1

Simple map of cell co-cultured system.

Figure 2

Identification of basal epithelial cells acquired from mouse prostate with anti-p63 in immunofluorescence assay (Objective 10X). Basal epithelial cells were specific to anti-p63 and more than 90%.

Figure 3

Normal basal epithelial cells could stimulate the migration of RM-1 directly or indirectly. (A) Effect of basal epithelial cells on cells wound healing of RM-1 was determined after being mixed-cultured (1:1) for 0 h and 24 h. (B) Quantification of migrated width of wound healing assay. (C) Effect of basal epithelial cells on migration of RM-1 was determined after being co-cultured for 20 h. (D) Quantification of migrated cells in (C). (E) Effect of 10% conditional medium of basal epithelial cells on migration of RM-1 was estimated after being cultured for 20 h. (F) Quantification of migrated cells in (E). Values are represented by mean±SD from at least three independent experiments. “*” represents “p<0.05”, “***” represents “p<0.001” vs control group.

Figure 4

Normal basal epithelial cell promoted the invasion of RM-1 directly or indirectly. (A) Effect of basal epithelial cells on invasion of RM-1 was determined after being co-cultured for 20 h. (B) Quantification of invasive cells in (A). (C) Effect of 10% conditional medium of basal epithelial cells on invasion of RM-1 was estimated after being cultured for 20 h. (D) Quantification of invasive cells in (C). Values are represented by mean±SD from at least three independent experiments. “**” represents “p<0.01”, “***” represents “p<0.001” vs control group.

Figure 5

Basal epithelial cells secreted TGF-β1 and additional TGF-β1 stimulated the migration and invasion of RM-1. (A) Quantification of TGF-β1 levels measurement in ELISA assay. Effect of 5 ng/mL TGF-β1 on migration (B) and invasion (C) of RM-1 in transwell chamber with or without matrigel after being cultured for 20 h. (D) Quantification of invasive cells in (C). Values are represented by mean±SD from at least three independent experiments. “***” represents “p<0.001” vs control group.

Figure 6

Basal epithelial cells secreted TGF-β1 to promote the migration and invasion of RM-1. (A) Basal epithelial cells and TGF-β1 (5 ng/mL) changed the morphology of RM-1 from cuboid to spindle-shape. (B) HE staining and immunocytochemistry with anti-63 in mixed-cultured group. Arrowhead indicates basal epithelial cell of which p63 is positive. (C) Basal epithelial cells and TGF-β1 regulated the proteins associated with EMT. (D) Quantification of relative proteins expression in (C). (E) Effect of different concentration of SB431542 on RM-1 death was measured by CCK8 assay. SB431542 (2000 nM, 4000 nM) inhibited the migration (F and G) and invasion (H and I) of co-cultured RM-1. (J) Expressions of EMT relevant proteins were down-regulated when SB431542 (2000 nM or 4000 nM) was added into medium of co-cultured cells. (K) Quantification of relative expressions of proteins in (J). Values are represented by mean±SD from at least three independent experiments. “*” represents “p<0.05”. “**” represents “p<0.01”. “***” represents “p<0.001”.

Figure 7

TGF-β1 which was secreted by basal epithelial cells could promote migration and invasion of RM-1 depending on the phosphorylation of STAT3. (A and B) p-STAT3 was up-regulated by basal epithelial cells, conditional medium of basal epithelial cells and TGF-β1. (C and D) Expression of p-STAT3 in co-cultured group was down-regulated when SB431542 was added. (E) Stattic down-regulated the expression of p-STAT3 and STAT3. Stattic inhibited the proliferation (F), migration (G) and invasion (H) of untreated RM-1 and co-cultured RM-1. Values are represented by mean±SD from at least three independent experiments. “**” represents “p<0.01”. “***” represents “p<0.001”.