Research Paper

Molecular Characterization of Epithelial to Mesenchymal Transition in Human Prostatic Epithelial Cells

[+] Author and Article Information
Victor K. Lin1

Department of Urology,University of Texas Southwestern Medical Centervictor.lin@utsouthwestern.edu

Shih-Ya Wang, Claus G. Roehrborn

Department of Urology,University of Texas Southwestern Medical Center

Lanxiao Wu

Department of Bioengineering,University of Texas at Arlington

Smitha M. Rao, J. C. Chiao

Department of Electrical Engineering,University of Texas at Arlington


Corresponding author.

J. Nanotechnol. Eng. Med 1(2), 021011 (May 14, 2010) (6 pages) doi:10.1115/1.4001542 History: Received March 16, 2010; Revised March 31, 2010; Published May 14, 2010; Online May 14, 2010

Epithelial to mesenchymal transition (EMT) has been believed to play a critical role in cancer metastasis. TGFβ has been described as an inducer of EMT in normal mammary epithelial cells by signaling through receptor serine/threonine kinase pathways to regulate epithelial cell plasticity and invasion. In this study, we investigated the EMT cellular responses, including morphologic changes, phenotype switches, invasiveness enhancement, and cellular contraction alteration, in TGFβ stimulated human prostate normal epithelial cells (PZ-HPV-7). Migration of TGFβ treated PZ-HPV-7 cells across matrigel was measured in invasion chambers (8μm pore size). The cells were treated with or without TGFβ (2 ng/ml) in PrEGM media for 3 days. Immunoblot assay was conducted and it was demonstrated that the induction of vimentin when stimulated by TGFβ was accompanied by a downregulation of E-cadherin, though p-cadherin level was not altered. It was also observed that there was a decrease in cytokaretin 5/6 expression associated with the downregulation of E-cadherin during the induction of EMT. In order to study the cell contraction, three-dimensional collage lattice assay was performed. It was demonstrated that TGFβ-stimulated PZ-HPV-7 cells gained contractility. Our results showed that TGFβ stimulation induced PZ-HPV-7 cells to undergo epithelial to mesenchymal transition. EMT characteristics such as acquisition of mesenchymal markers and loss of epithelial markers were evident in the induction of vimentin and downregulation of E-cadherin and cytokeratins, as well as phenotypic alterations including increased contraction and enhanced mobility were detected.

Copyright © 2010 by American Society of Mechanical Engineers
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Figure 1

PZ-HPV-7 morphologic change during TGFβ stimulated EMT. PZ-HPV-7 cells were seeded at 1×104 cell/cm2 24 h before the onset of TGFβ stimulation. Panel A: cells without TGFβ. Panel B: cells with 1ng/ml TGFβ. Panel C: cells with 5 ng/ml TGFβ. The arrows indicate the cells with elongated fibroblastoid mesenchymal-like morphology, a characteristic of EMT.

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Figure 2

Immunoblot of vimentin induction in PZ-HPV-7 cells upon TGFβ stimulation. Immunoblot shows that HPV-7 treated with TGFβ at 1 ng/ml and 5 ng/ml media for indicated times exhibited the induction of vimentin expression. HPS 307, a primary culture of human prostate stromal cells, expressed vimentin endogenously and the expression was not affected by TGFβ stimulation. β-actin expression on the same blot is shown as a protein loading control.

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Figure 3

E-cadherin and cytokeratin expression on TGFβ treated PZ-HPV-7 cells. (a) Immunoblot results demonstrated that the induction of vimentin upon TGFβ stimulation was accompanied with a downregulation of E-cadherin, though p-cadherin level appeared not altered. (b) Decreasing cytokaretin 5/6 expression was also seen associated with the downregulation of E-cadherin during the induction of EMT. HPS 307 (stromal cells) expressed no cytokeratin 5/6 at all as part of the control.

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Figure 4

FACS analysis of adherent molecule expression. PZ-HPV-7 cells were treated with 2 ng/ml TGFβ for 0, 3, or 6 days, incubated with antibodies and subjected for FACS analysis. The results indicated that both E-cadherin and ZO-1 positive cells had decreased responses to TGFβ stimulation in a time dependent manner.

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Figure 5

TGFβ-stimulated PZ-HPV-7 cells gained contractility as a marker of mesenchymal functionality. HPV-7 cells were pretreated without or with TGFβ (2 ng/ml) for 3 days and embedded (2×105 cells/200 μl) in the collagen lattice for additional incubation as indicated without or with TGFβ. (a) Column A is the lattices with cells that were TGFβ pretreated (2 ng/ml) for 3 days and then continued the TGFβ treatment with indicated times in three-dimensional lattices. Column B: cells with TGFβ pretreatment for 3 days and then without TGFβ for the indicated time in the 3D lattice. Column C: cells without TGFβ pretreatment for 3 days and then treated with TGFβ for indicated time in the 3D lattice. Column D: cells without TGFβ at all as neither the pretreatment nor in the 3D lattice as the control. The reduction in lattice in diameter was measured at indicated times in the rows as in an indicator for cellular contractility. (b) The reduction rate of the lattice size for different treatments.

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Figure 6

Inhibition of MEK-1/2 abolished the induction of vimentin and downregulated E-cadherin upon TGFβ-induced EMT in PZ-HPV-7 cells. Cells were treated with pathway inhibitors for 2 days (d2) or 4 days (d4), with (2 ng/ml) or without TGFβ. The cells were assessed for vimentin and E-cadherin expressions. Immunoblot results indicated that the MAPK signaling including p38MAPK and MEK pathways may play critical roles in the TGFβ induction of vimentin expression in PZ-HPV-7 cells, while PKC and PI3 kinase pathways have no apparent effect. (SB: SB203580, p38MAPK inhibitor; PD: PD98059, MEK inhibitor; UO: U0126, MEK-1/2 inhibitor; LY: LY294002, PI3K inhibitor; and SC: SC-791, COX-2 inhibitor; NI: no inhibitor added).

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Figure 7

Blocking of the MEK-1/2 pathway by U0126 also abolished the increased contractivity in the TGFβ-stimulated PZ-HPV-7 cells. Cells were treated with ( 2ng/ml) or without TGFβ in combination with or without U0126 (10 μM) in 3D lattices for 1.5 h or 48 h as described in Fig. 5. The reduction in the lattice size indicated that blocking of MEK pathway by U0126 also blocked the increased contraction by TGFβ while U0126 alone gave no apparent effect on cellular contraction.

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Figure 8

TGFβ-Induced EMT in HPV-7 cells was associated with an enhanced cellular invasiveness. HPV-7 cells were pretreated with (+) or without (−) TGFβ (2 ng/ml) in PrEGM media for 3 days before they were seeded to invasion assay chambers with or without continuous TGFβ treatment for 3 days. (a) The stained membranes of cell invasion clearly indicated that TGFβ stimulated HPV-7 cells exhibited a higher invasiveness. The rows are labeled as (−/−) no pretreatment and no continuous treatment of TGFβ. (−/+) no pretreatment with continuous treatment in the invasion chamber. (+/−) with pretreatment of TGFβ but no continuous treatment in the invasion chamber. (+/+)TGFβ treated in the whole 6 day period. Each experiments were repeated three times (N=3) in the columns. (b) Quantification of the stained cells indicated that there is an almost a twofold increase in invasiveness for the treated cells.



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