The growth of malignant keratinocytes depends on signaling through the PGE(2) receptor EP1

Abstract

Recent discoveries shed light on the importance of prostaglandin (PG) production in the development of skin cancer. Work by Fischer et al. demonstrates that skin tumor promotion caused by ultraviolet B radiation can be decreased by up to 89% by blocking cyclooxygenase-2 (COX-2) with the drug Celecoxib. A similar study showed that Celecoxib can decrease new tumor formation by 44% in mice that already have tumors. These studies demonstrate the importance of COX-2 and PGs in the development of squamous cell carcinoma. We have explored growth signaling in a model of skin tumor progression. Because changes in PG production have been implicated in skin carcinogenesis, we examined this pathway. We found that malignant cell lines secrete more prostaglandin E(2) (PGE(2)) than the parental cells. We observed increased expression of COX-1 and -2. We also found that these cells express the PGE(2) receptors EP1 and EP4. When the cells are grown in the presence of indomethacin, the growth rate of the malignant cells is decreased. This effect can be reversed by addition of PGE(2) or an EP1 agonist to the medium. Thus, we have shown that skin tumor cells depend in part on PGE(2) signaling through the EP1 prostanoid receptor for their in vitro growth.

Figure 1

The cell line 6RI is invasive. A total of 5x10⁶ cells were injected subcutaneously into athymic nude mice. Tumors were harvested and H&E-stained. A representative margin is shown. The bars in the upper left corners represent 500 µm. (A) Tumor formed by the cell line 6R90. The arrow shows a regular margin that does not invade the underlying tissue. (B) Tumor formed by the cell line 6RI. The arrow shows the tumor invading the underlying muscle.

Figure 2

The malignant cell lines have increased COX expression. This is a Western blot analysis of COX-1 and -2 in 308, 6R90, and 6RI cells. Forty micrograms of protein lysate was fractionated by SDS-PAGE. After transferring the protein to PVDF membrane, the blots were probed with antibody specific to either COX-1 or -2 as indicated.

Figure 3

PGE₂ production. EIA was performed to measure PGE₂ production. The cells were grown under normal culture conditions in 96-well plates and lysed in the plate. A sample was then taken for analysis by EIA. The bars indicate 95% confidence intervals. The asterisk indicates a significant difference compared to 308 cells with a P value <.01 by analysis of variance.

Figure 4

Immunohistochemical staining for EP receptors. Cells were grown on cover slips under normal culture conditions and stained for the EP receptors. The counterstain was DAPI staining of the nucleus. (A) 308 Cells incubated with secondary antibody. (B) 6R90 Cells incubated with secondary antibody. (C) 6RI Cells incubated with secondary antibody. (D) 308 Cells stained for EP1. (E) 6R90 Cells stained for EP1. (F) 6RI Cells stained for EP1. (G) 308 Cells stained for EP4. (H) 6R90 Cells stained for EP4. (I) 6RI Cells stained for EP4.

Figure 5

Quantification of EP receptors. A total of 10⁶ cells were stained for the EP1 and EP4 receptors. Data are expressed as fold of 308 staining. The bars indicate the standard deviation from five experiments.

Figure 6

A total of 10⁴ cells were plated and counted every 12 hours for 48 hours. Cells were treated with vehicle control (ethanol, EtOH) or 10 µM indomethacin (indo.) or 10 µM indomethacin and PGE₂ (indo., PGE₂). The bars indicate the standard deviation from three experiments. (A) Growth curve of 308 cells. (B) Growth curve of 6R90 cells. (C) Growth curve of 6RI cells.

Figure 7

A total of 10⁴ cells were plated and counted every 12 hours for 48 hours. Cells were treated with vehicle control (ethanol, EtOH) or 10 µM indomethacin (indo.) or 10 µM indomethacin and 17-phenyl trinor PGE₂ (an EP1 and EP3 agonist) (indo., EP1 ag). The bars indicate the standard deviation from three experiments. (A) Growth curve of 308 cells. (B) Growth curve of 6R90 cells. (C) Growth curve of 6RI cells.