Objective:The present study sought to determine the potential role of p38 and PI3K signaling pathways in mediating phenotypic switching between angiogenic and angiostatic elements among squamous cell carcinoma (SCC) cell lines. Methods: We investigated the effects of gaseous hypoxia and those of cobalt chloride, which mimics the hypoxic response including the production of reactive oxygen species on such phenotypic shifts. SCC-4, -9, -15, and –25 cell lines all have been shown to possess mutations, insertions or deletions in p53. Consequently, both hypoxia and treatment with cobalt chloride were utilized to provide a model by which to compare phenotypic switching independent of the complexities of apoptosis as determined by Annexin V staining. Real-time PCR was used to determine the relative expression and Western blot analyses were used to determine production of collagen XVIII, CBP2/Hsp47, and HIF-1alpha and to provide a measure of an angiostatic phenotype while VEGF expression was used to appraise proangiogenic states. Results: These studies revealed that Hypoxia and treatment with cobalt chloride produced transient up regulation of collagen XVIII and CBP2/Hsp47 expression that was modulated with time and by increasing HIF-1alpha production. These changes were reversed following inhibition with the PI3K inhibitor, Wortmannin. Conversely, VEGF expression was increased following hypoxia and cobalt chloride treatment and inhibited both by the respective p38 and PI3K inhibitors SB203580 and Wortmannin. Conclusion:These studies show that phenotypic switching between angiostatic elements, such as collagen XVIII, and proangiogenic elements, determined by VEGF expression, are controlled by cross-talk between p38 and PI3K signaling pathways, thus, lending further evidence that a balance of angiogenic stimulators and inhibitors may regulate tumor progression in changing tumor microenvironments. This work was supported in part by PHS grants DE12606 and DE13118.

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