Suppression of N-methyl-N-nitrosourea/testosterone-induced rat prostate cancer growth by celecoxib: effects on cyclooxygenase-2, cell cycle regulation, and apoptosis mechanism(s).
OBJECTIVE
This study was aimed at examining the mechanisms underlying the chemopreventive effect of celecoxib against prostate cancer. We focused our attention on events at the cellular level to show the ability of celecoxib to inhibit prostate cancer growth, by inducing cell cycle arrest and apoptosis. Moreover, we attempted to demonstrate the expression of genes involved in the downstream events related to cyclooxygenase-2 (COX-2) regulation and apoptosis.


METHODS
To determine the level of COX-2 expression, we used paraffin-embedded tumor tissue sections and cancer cells (I-26) derived from N-methyl-N-nitroso-urea/testosterone-induced rat dorsolateral prostate, and we used immunofluorescence detection and Western blot analyses with anti-COX-2 monoclonal antibodies. We conducted clonogenic cell survival assays to demonstrate cell growth inhibition at very low doses of celecoxib. Flow cytometric analysis demonstrated the effects on the cell cycle. Reverse transcription-PCR and Western blot analyses were performed to show the effect of celecoxib on the downstream events of COX-2 and apoptosis-related targets.


RESULTS
The summary of our findings indicates that (a). these cells from chemically induced rat prostate tumors express COX-2 at both the mRNA and the protein level; (b). celecoxib significantly reduces COX-2 expression in these cancer cells; and (c). celecoxib induces cell cycle arrest at the G(1)-S phase transition point and modifies cell cycle regulatory proteins such as cyclin D1, retinoblastoma (Rb), and phosphorylated Rb, cyclin E, p27(KIP1), and p21(WAF1/CIP1). Furthermore, celecoxib inhibits DNA synthesis and induces apoptosis. Most importantly, celecoxib-induced apoptosis was associated with down-regulation of COX-2, nuclear factor kappaBp65, and with activation of peroxisome proliferator-activated receptor gamma, apoptosis activating factor-1, and caspase-3.


CONCLUSIONS
Results from the present study clearly indicate that celecoxib exerts its anticancer effect partly through COX-2-independent mechanisms in addition to the known primary function of COX-2 inhibition.
Narayanan
Bhagavathi A
BA
Division of Nutritional Carcinogenesis, American Health Foundation, Valhalla, New York 10595, USA. bnarayan@ifcp.us
Condon
Mark S
MS
Bosland
Maarten C
MC
Narayanan
Narayanan K
NK
Reddy
Bandaru S
BS
D000477:Alkylating Agents; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D017209:Apoptosis; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002273:Carcinogens; D000068579:Celecoxib; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D056741:Cyclin D; D019927:Cyclin E / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D051546:Cyclooxygenase 2; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005434:Flow Cytometry; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000096:biosynthesis*; D008297:Male; D008770:Methylnitrosourea; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D011471:Prostatic Neoplasms / Q000139:chemically induced* / Q000188:drug therapy*; D011720:Pyrazoles; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D016160:Retinoblastoma Protein / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000627:therapeutic use*; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D000477:Alkylating Agents; D000970:Antineoplastic Agents; D002273:Carcinogens; C493958:Cdkn1a protein, rat; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D004396:Coloring Agents; D056741:Cyclin D; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D007527:Isoenzymes; D011720:Pyrazoles; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D013449:Sulfonamides; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008770:Methylnitrosourea; D004247:DNA; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D001973:Bromodeoxyuridine; D000068579:Celecoxib