Extracellular pressure stimulates colon cancer cell proliferation via a mechanism requiring PKC and tyrosine kinase signals

Abstract

Pressure in colonic tumours may increase during constipation, obstruction or peri-operatively. Pressure enhances colonocyte adhesion by a c-Src- and actin-cytoskeleton-dependent PKC-independent pathway. We hypothesized that pressure activates mitogenic signals.

Methods: Malignant colonocytes on a collagen I matrix were subjected to 15 mmHg pressure. ERK, p38, c-Src and Akt phosphorylation and PKCalpha redistribution were assessed by western blot after 30 min and PKC activation by ELISA. Cells were counted after 24 h and after inhibition of each signal, tyrosine phosphorylation or actin depolymerization.

Results: Pressure time-dependently increased SW620 and HCT-116 cell counts on collagen or fibronectin (P < 0.01). Pressure increased the SW620 S-phase fraction from 28 +/- 1 to 47 +/- 1% (P = 0.0002). Pressure activated p38, ERK, and c-Src (P < 0.05 each) but not Akt/PKB. Pressure decreased cytosolic PKC activity, and translocated PKCalpha to a membrane fraction. Blockade of p38, ERK, c-Src or PI-3-K or actin depolymerization did not inhibit pressure-stimulated proliferation. However, global tyrosine kinase blockade (genistein) and PKC blockade (calphostin C) negated pressure-induced proliferation.

Conclusions: Extracellular pressure stimulates cell proliferation and activates several signals. However, the mitogenic effect of pressure requires only tyrosine kinase and PKCalpha activation. Pressure may modulate colon cancer growth and implantation by two distinct pathways, one stimulating proliferation and the other promoting adhesion.

Figure 1

Pressure stimulates time‐dependent SW620 cell proliferation. (a) 15 mmHg pressure application for 24 h results in a 51 ± 7% increase in cell number compared with the ambient pressure control at 24 h (n = 14, P = 0.0001). (b) Time course of the pressure effect. Cells were exposed to pressure for 0, 3, 4.5, 6, 12 or 24 h and then maintained at ambient pressure for the balance of the 24‐h period. Cell numbers are significantly (P < 0.05) increased over control values after 6 h of exposure (data shown are two‐point normalized between t = 0 and 24 h of exposure to pressure) and remain elevated for 24 h. (c) Flow cytometry in cells rendered quiescent by serum deprivation. The ratio of cells in S‐phase after 24 h of pressure was 46.6 ± 0.8% compared with 27.8 ± 0.1% in cells maintained at ambient pressure (P = 0.0002, n = 6). All data, mean ± SEM; t = 0, initial cell count; control, ambient pressure; pressure, 15 mmHg.

Figure 2

Pressure‐stimulated colon cancer cell proliferation is independent of matrix and cell line. (a) In SW620 cells on a fibronectin matrix, 24 h of exposure to 15 mmHg increased pressure increased cell number by 29 ± 6% over ambient pressure controls at 24 h (P = 0.0013, n = 6). (b) Similarly, in HCT116 cells plated on collagen I, pressure enhanced cell number by 27 ± 8% (P = 0.009, n = 6). All data, mean ± SEM normalized to cell counts at t = 0.

Figure 3

Pressure induces early (30 min) signal activation. (a) Western blot assays of ERK, p38, c‐Src and Akt/PKB phosphorylation presented as the ratio of phosphorylated to total forms. Bars depict the results of densitometric analysis (mean ± SEM, n = 3–7, P < 0.05). Representative western blots are shown above the bars. ERK, p38, and c‐Src but not Akt/PKB, were activated by 30 min of pressure. (b) Cytosolic PKC activity, expressed as percentage of control, is significantly decreased by pressure (P = 0.026, n = 3). Activation by 1 µg/ml PMA was used as a positive control. DMSO‐treated cells were assayed as a vehicle control for the PMA. (c) Western blot analysis of PKCα subcellular distribution in response to pressure. Bars represent densitometric ratios of PKCα to GAPDH for the cytosolic fraction (Cyto) and to Na/K ATPase for the membrane fraction (Memb) (*P = 0.05 membrane pressure vs. membrane control, n = 3). A representative western blot is shown above the bars. Control, ambient; pressure, 15 mmHg.

Figure 4

Tyrosine kinase and PKC inhibition block pressure‐induced proliferation. (a) Pharmacological blockade of PI‐3‐kinase with 20 µm LY294002 (LY), actin depolymerization with 10 µm phalloidin (Ph), c‐Src blockade with 20 µm PP2 (PP2), p38 inhibition with 20 µm SB203580 (SB) and MEK inhibition with 20 µm PD98059 (PD) all failed to negate the pressure effect on proliferation. *P < 0.05 vs. respective control (ambient). Appropriate vehicle controls yielded similar pressure mediated increases in cell number. (b) Genistein (10 µm) and calphostin C (100 nm) abrogate pressure‐stimulated cell proliferation, limiting increases in cell number to 11 ± 9% with genistein and 5 ± 3% with calphostin C (n = 5, each).