Background: Extra-cellular matrix (ECM) proteins adhesion, and invasion of the basement membrane are mechanisms which might favor or counteract pancreatic cancer (PC) progression. We ascertained whether TGF-b1 stimulation modifies: 1. cell growth, 2. ECM adhesion and 3. Matrigel invasion of two PC cell lines, one primary (MIA PaCa 2) and one metastatic (CAPAN-1). Materials and Methods: The cells were cultured with 10% FCS for 3 days. Then they were cultured with FCS 1% in the absence (PBS) or presence of TGF- 1 (1 ng/mL), for another three days, followed by invasion and adhesion experiments. Adhesion: 96 wells plates coated with fibronectin (FN, 20 ug/mL), laminin (LM, 10 ug/mL) or type I collagen (COL I, 10 ug/mL) were used. Adherent cells were evidenced using crystal violet (Abs 570 nm). Invasion: coculture plates with inserts covered or not with Matrigel (300 ug) were used. After 48 h at 37ºC, the cells passing into the lower chamber were considered invasive and counted. Results: TGF- 1 reduced MIA PaCa 2 (87 12%, mean SD with respect to control cells) and CAPAN-1 (66.4 4) cell growth. PBS stimulated MIA PaCa 2 cells were significantly more invasive than CAPAN-1 cells, considering both inserts with (37.9 5.1 vs 13.4 1.5, t 5.28, p 0.001) or without Matrigel (55.5 8.4 vs 30.2 1.7, t 3.7, p 0.01). In the presence of Matrigel, TGF- 1 significantly enhanced the number of invasive CAPAN-1 (t 2.47, p 0.05), while significantly reduced the number of invasive MIA PaCa 2 cells (t 3.16, p 0.01). TGF- 1 significantly enhanced FN and LM adhesion of CAPAN-1 (t 7.75, p 0.001 and t 9.58, p 0.001), but not of MIA PaCa 2 cells (t 1.53, p:ns and t 0.97, p:ns). Conclusion: TGF- 1 reduced MIA PaCa 2 and enhanced CAPAN-1 invasion pattern in vitro, and this might be consequent to an increased adhesion to the ECM proteins FN and LM. These differences may depend on DPC4 alterations: this gene is wild in MIA PaCa 2 while presents a 343 stop codon in CAPAN-1 and is involved in TGF 1 intracellular signaling.
Transforming growth factor-beta 1 (TGF-B1) stimulation of pancreatic cancer cells evokes a different invasion pattern in vitro
GRECO, ELIANA;BASSO, DANIELA;FOGAR, PAOLA;ZAMBON, CARLO-FEDERICO;PLEBANI, MARIO;PEDRAZZOLI, SERGIO
2002
Abstract
Background: Extra-cellular matrix (ECM) proteins adhesion, and invasion of the basement membrane are mechanisms which might favor or counteract pancreatic cancer (PC) progression. We ascertained whether TGF-b1 stimulation modifies: 1. cell growth, 2. ECM adhesion and 3. Matrigel invasion of two PC cell lines, one primary (MIA PaCa 2) and one metastatic (CAPAN-1). Materials and Methods: The cells were cultured with 10% FCS for 3 days. Then they were cultured with FCS 1% in the absence (PBS) or presence of TGF- 1 (1 ng/mL), for another three days, followed by invasion and adhesion experiments. Adhesion: 96 wells plates coated with fibronectin (FN, 20 ug/mL), laminin (LM, 10 ug/mL) or type I collagen (COL I, 10 ug/mL) were used. Adherent cells were evidenced using crystal violet (Abs 570 nm). Invasion: coculture plates with inserts covered or not with Matrigel (300 ug) were used. After 48 h at 37ºC, the cells passing into the lower chamber were considered invasive and counted. Results: TGF- 1 reduced MIA PaCa 2 (87 12%, mean SD with respect to control cells) and CAPAN-1 (66.4 4) cell growth. PBS stimulated MIA PaCa 2 cells were significantly more invasive than CAPAN-1 cells, considering both inserts with (37.9 5.1 vs 13.4 1.5, t 5.28, p 0.001) or without Matrigel (55.5 8.4 vs 30.2 1.7, t 3.7, p 0.01). In the presence of Matrigel, TGF- 1 significantly enhanced the number of invasive CAPAN-1 (t 2.47, p 0.05), while significantly reduced the number of invasive MIA PaCa 2 cells (t 3.16, p 0.01). TGF- 1 significantly enhanced FN and LM adhesion of CAPAN-1 (t 7.75, p 0.001 and t 9.58, p 0.001), but not of MIA PaCa 2 cells (t 1.53, p:ns and t 0.97, p:ns). Conclusion: TGF- 1 reduced MIA PaCa 2 and enhanced CAPAN-1 invasion pattern in vitro, and this might be consequent to an increased adhesion to the ECM proteins FN and LM. These differences may depend on DPC4 alterations: this gene is wild in MIA PaCa 2 while presents a 343 stop codon in CAPAN-1 and is involved in TGF 1 intracellular signaling.
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