There are four subtypes of EP receptor with the majority localised to the plasma membrane. The binding of prostaglandins to cell surface receptors triggers selleck chem changes in second messen gers. PGE2 modulates processes fundamental to tumour cell survival such as enhanced proliferation and resistance to apoptosis, however, the precise molecular mechanisms remain unclear. There is therefore a strong rationale to seek a more profound understanding of the downstream targets of COX 2 activity. Selective COX 2 inhibitors have shown promise as chemo preventive agents, but their adverse cardiovascular effects have undermined their suitability for long term use. Renewed attention must now therefore focus on the altered signalling occurring downstream of COX 2 in can cer as a source for new refined therapeutic targets.
Methods Cell culture HT 29 cells were purchased from the ATCC and maintained in McCoys 5 A medium containing 1. 5 mM L glutamine, 10% FBS, penicillin 100 Uml and streptomycin 100 gml. HCA7 cells were kindly donated by Susan Kirkland and were cultured in DMEM with 10% FBS, supplemented with 1 mM sodium pyruvate and 100 gml kanamycin. SC236, a selective COX 2 inhibitor was a Inhibitors,Modulators,Libraries gift from Dr. Peter Isakson. PGE2 was purchased from Cayman. L 161982, a selective antagonist of the EP4 receptor was a kind gift of Merck Frosst, Canada. PD153035 was purchased from Calbiochem. Wortmannin was purchased from Sigma Aldrich. Quantitative RT PCR Total RNA was isolated from cells and tissue following homogenisation in RNA lysis buffer Inhibitors,Modulators,Libraries supplemented with 1% ? mercaptoethanol.
Extraction was performed using RNeasy Mini Kits. Total RNA was reverse transcribed using Moloney Murine Leukaemia Virus reverse transcriptase Inhibitors,Modulators,Libraries according to the manufacturers instructions. Gene expres sion was quantified by RT PCR using SYBR Green Univer Inhibitors,Modulators,Libraries sal Master Mix. Reactions were carried out in a 96 well format in the ABI 7700 Sequence Detector. Results were then normalized to 18S rRNA amplified from the same cDNA mix. Sequences of the primer pairs used are listed below. Immunohistochemical staining for COX 2 and amphiregulin Samples of formalin fixed, paraffin embedded tissue were deparaffinised and rehydrated in Xylene and Methanol. Detection of COX 2. Endogenous peroxidase activity was quenched with 0. 3% H2O2 in Methanol. Specimens were blocked in 1.
5% normal serum and then incubated with antibody to COX 2 diluted 1200, Inhibitors,Modulators,Libraries followed by sec ondary antibody and ABC complex from Vectastain Elite kit. Sections were exposed to diaminobenzidine and counterstained with hematoxylin and mounted using DPX. Detection of Amphiregulin. Antigen retrieval was performed by heating slides in 10 kinase inhibitor EPZ-5676 mM citrate buffer for 4 minutes in a pressure cooker. Blocking was performed with goat serum for 30 minutes.