Study of molecular mechanisms of pro-apoptotic activity of NCX 4040, a novel nitric oxide-releasing aspirin, in colon cancer cell lines.
BACKGROUND
Despite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, in in vitro human colon cancer models.


METHODS
The effect on tumor growth was evaluated in four human colon cancer cell lines (LoVo, LRWZ, WiDr and LoVo Dx) by sulforhodamine B assay, oxidative stress by immunohistochemistry, apoptosis by laddering assay, mitochondrial membrane potential (DeltaPsim) by flow cytometry, and apoptosis- and chemoresistance-related markers by western-blot and real-time method, respectively. Prostaglandin E2 levels were determined by ELISA.


RESULTS
NCX 4040 produced a higher cytotoxic effect in all the cell lines than that produced by other NO donors tested. In particular, in LoVo and LRWZ cells, NCX 4040 induced a cytocidal effect and apoptosis through p53 and NAG-1 expression, an early DeltaPsim collapse, and a sequential release of cytoplasmatic cytochrome c and caspase -9 and -3 active forms. 8-hydroxyguanine lesions, indicative of oxidative stress, were also observed. Conversely, in WiDr line, the drug caused a cytocidal effect, albeit not through apoptosis, and a concomitant increase in COX-2 activity. In LoVo Dx line, characterized by high levels drug resistance and DNA repair-related markers, only a cytostatic effect was observed, again in concomitance with the increase in COX-2 enzyme activity.


CONCLUSIONS
This study highlights the multiplicity of mechanisms involved in sensitivity or resistance to NCX 4040 and could provide useful indications for tailored therapy by identifying potentially drug-responsive tumors.
Tesei
Anna
A
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Italy. anna.tesei@ausl.fo.it
Rosetti
Marco
M
Ulivi
Paola
P
Fabbri
Francesco
F
Medri
Laura
L
Vannini
Ivan
I
Bolla
Manlio
M
Amadori
Dino
D
Zoli
Wainer
W
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001241:Aspirin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000473:pathology*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D008968:Molecular Conformation; D009569:Nitric Oxide / Q000378:metabolism; D009574:Nitro Compounds / Q000737:chemistry / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; C498045:NCX 4040; D009574:Nitro Compounds; D009599:Nitroprusside; D009569:Nitric Oxide; D001241:Aspirin