Prostanoid receptor expression in colorectal cancer related to tumor stage, differentiation and progression

Figures & data

Table I.  The primers used in LightCycler® realtime PCR from 5′ to 3′.

	Primer	Annealingtemp. (C°)	Amplicon size (bp)	Primer conc. (µM)	MgCl2 conc. (mM)
PGD2 receptor DP1	f TGATGACCGTGCTCTTCACT r CCAAGGGTCCACAATTGAAA	55	158	0.5	PLUS
PGD2 receptor DP2	f CCTCTGTGCCCAGAGCCCCACGATGTCGGC r CACGGCCAAGAAGTAGGTGAAGAAG	68	301	0.5	5
PGF2α receptor FP	Hs_PTGFR_1_SG (Qiagen)	55	95	*	PLUS
PGI2 receptor IP	f TGCTCCCTGCCTCTCACGAT r TGGCTTCTGCTTTGGACGAC	65	387	0.5	5
TXA2 receptor TP	Hs_TBXA2R_1_SG (Qiagen)	60	99	*	2

(f = forward, r = reverse). ref Hawcroft et al.* 2 µl primer were added according to the manufactures protocol PLUS MgCl₂ included in the master mix and not added separately

Figure 1.  Prostanoid receptor expression in tumor tissue (n = 62) compared to normal colon tissue (n = 43). * p < 0.05 vs. normal colon tissue.

Table II.  Transcript expression of PG receptors in normal colon tissue and colorectal cancers grouped according to Dukes A-D stage.

			Tumor Tissue
PG	Receptor	Normal colon tissue (43)	Dukes A (10)	Dukes B (26)	Dukes C (16)	Dukes D (10)	Statistics K-W among all p <	K-W within Dukes p <
PGD2	DP1	5.73±1.09	0.29±0.23	0.98±0.29	0.58±0.23	3.86±3.04	<0.0001	ns
PGD2	DP2	4.26±0.59	3.93±1.75	1.30±0.24	7.01±5.29	3.10±1.88	<0.0001	0.07
PGF2α	FP	2.88±0.53	1.26±0.67	2.37±0.99	1.76±0.73	0.73±0.27	<0.003	ns
PGI2	IP	2.63±0.26	1.32±0.40	1.67±0.61	2.03±0.95	0.94±0.24	<0.0001	ns
TXA2	TP	2.84±0.55	9.72±7.94	3.45±1.46	3.49±1.54	6.44±3.57	<0.02	0.07

Kruskal-Wallis test (K-W)

Mean±SEM

Results are units per units GAPDH

Table III.  Transcript expression of prostanoid receptors in tumor tissue during progression of colorectal carcinoma.

Receptor	Dukes A + B (36)	Dukes C + D (26)	Mann-Whitney p <
DP1	0.87±0.22	1.88±1.18	Ns
DP2	2.03±0.54	5.51±3.31	Ns
FP	2.06±0.74	1.36±0.47	Ns
IP	1.58±0.45	1.61±0.59	Ns
TP	5.19±2.42	4.62±1.65	Ns

Mean±SEM

Units per units GAPDH

Events of tumor progression are reflected by grouping tumors to Dukes A + B

versus Dukes C + D.

Table IV.  Transcript expression of prostanoids receptors in tumor tissue from colorectal cancers of various differentiation.

PG	Receptor	High (5)	Medium (42)	Low (10)	Kruskal-Wallis p <
PGD2	DP1	0.54±0.13	1.62±0.75	0.48±0.12	ns
PGD2	DP2	1.10±0.23	4.43±2.09	1.91±0.81	ns
PGF2α	FP	1.16±0.31	2.08±0.68	0.73±0.27	ns
PGI2	IP	1.85±0.93	1.69±0.51	1.26±0.45	ns
TXA2	TP	1.30±0.28	6.60±2.25	1.83±0.42	ns

Mean±SEM

Units per units GAPDH

Table V.  Estimates of prostanoid receptor expression in two established colorectal cancer cell lines with high (HCA) and low (HT-29) PGE₂ production. Results are provided relative to expression levels in colon cancer tissue and as raw measures obtained by Light Cycler and Taqman determinations as indicated.

Receptors:	HCA-7^a		HT-29^b
	Relative to colon cancer	Direct measurement	Relative to colon cancer	Direct measurement
DP1 receptor	0	0^c	0	0^c
DP2 receptor	4%	0.34^c	7%	1.3^c
FP receptor	1%	0.04^c	1%	0.1^c
IP receptor	0	0^c	0	0^c
TP receptor	4%	0.45^c	3%	0.9^c
EP1 receptor	2%	0.1^d	433%	519^e
EP2 receptor	720%	31^d	0	0^e
EP3 receptor	14%	0.2^d	0	0^e
EP4 receptor	742%	4^d	86%	85^e

a: PGE₂ concentration in cell medium at confluence 120–130 pg/ml

b: PGE₂ concentration in cell medium at confluence < 2.5 pg/ml

c: Light Cycler units/units GAPDH

d: Taqman Relative gene expression versus a Dukes c colon cancer

e: mol/mol GAPDH