Modulation of p38MAPK and PI3K/Akt/NκFB pathways is involved in the COX-2-independent antitumor effects of celecoxib on two murine mammary adenocarcinoma cell lines

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Nonsteroidal anti-inflammatory drug (NSAID) treatment exhibited antitumor properties on several tumor models, including mammary cancers. The cyclooxygenases (COXs) are the canonical targets of NSAIDs, being the inducible COX-2 largely implicated in tumor progression and overexpressed in many tumor types. It is now becoming evident that other targets besides COX-2 should explain the antitumor effects of NSAIDs like celecoxib. LM3 and LMM3 are two murine mammary tumor cell lines originally related but with different COX-2 expression. Whereas LMM3 has low COX-2 protein levels, it is barely detected in LM3. When syngeneic BALB/c mice were treated with celecoxib at 500, 1000 and 1500ppm, a significantly reduced sc tumor growth was observed (p<0.001). The number of spontaneous metastasis as well as those established after i.v. LM3 and LMM3 cell delivery was also reduced (p<0.001 and 0.01 respectively). Celecoxib and its derivative, dimethylcelecoxib (DMC), which lacks COX-2 inhibitory properties, decreased LM3 and LMM3 cell viability at 10uM after 72h of treatment, effect that could not be reverted by exogenously added PGE2 (at 5 and 10 μM). Under the same conditions celecoxib and DMC rendered an increased number of apoptotic cells evaluated by staining with ethidium bromide/acridine orange and confirmed by flow cytometry of propidium iodide stained cells. The cell cycle profile after the treatments did not differ as well as no change in p21 and p27 levels was observed, suggesting that the antitumor effects of celecoxib are due to a decreased survival or increased apoptosis. We observed diminished phospho-Akt levels in cell lysates and tumor homogenates and increased phospho-p38 after treatment with celecoxib and DMC. The transfection of both cell lines with a dominant negative p38 or a constitutively active subunit of PI3K partially but significantly abolished the cytotoxicity of celecoxib and DMC, effect that was even increased by the cotransfection. These results indicate that both a downregulation of the PI3K/Akt and upregulation of p38 signals are modulated by celecoxib. We also observed a decreased phospho-JNK level in LM3 and LMM3 cell lines after celecoxib and DMC treatments which parallels a lower activity of AP1 evaluated in a gene reporter assay (55.2 and 56.3% of control respectively for LM3 cells). Given that celecoxib and DMC reduced the levels of p-Akt, we next studied the activation of NFBκ, a target of the Akt signaling network. Both celecoxib and DMC reduced NFκB activity in a gene reporter assay by 65 and 67 % respectively. Taken together, these data indicate that the independent antitumor effect of cxb may involve the modulation of PI3K/Akt/NFkB, p38MAPK and JNK/AP1 signaling pathways.