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Hypoxia and signaling via hypoxia-inducible factor-1 (HIF-1) is a key feature of solid tumors and is related to tumor progression as well as treatment failure. Although it is generally accepted that HIF-1 provokes tumor cell survival and induces chemoresistance under hypoxia, HIF-1-independent mechanisms operate as well. We present evidence that conditioned medium obtained from A549 cells, incubated for 24 h under hypoxia, protected naive A549 cells from etoposide-induced cell death. Lipid extracts generated from hypoxiaconditioned medium still rescued cells from apoptosis induced by etoposide. Specifically, the bioactive lipid sphingosine-1-phosphate (S1P) not only was essential for cell viability of A549 cells but also protected cells from apoptosis. We noticed an increase in sphingosine kinase 2 (SphK2) protein level and enzymatic activity under hypoxia, which correlated with the release of S1P into the medium. Knockdown of SphK2 using specific small interfering RNA relieved chemoresistance of A549 cells under hypoxia and conditioned medium obtained from SphK2 knockdown cells was only partially protective. Coincubations of conditioned medium with VPC23019, a S1P 1 /S1P 3 antagonist, reduced protection of conditioned medium, with the further notion that p42/ 44 mitogen-activated protein kinase transmits autocrine or paracrine survival signaling downstream of S1P 1 /S1P 3 receptors. Our data suggest that hypoxia activates SphK2 to promote the synthesis and release of S1P, which in turn binds to S1P 1 /S1P 3 receptors, thus activating p42/44 mitogen-activated protein kinase to convey autocrine or paracrine protection of A549 cells.

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Hypoxia inducible factor 1 is regulated by the appearance of the HIF-1a subunit. HIF-1a is subjected to proteasomal destruction or enhanced protein translation, which requires the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. We investigated how PI3K/Akt and glycogen synthase kinase 3b (GSK3b) affect HIF-1a in human RKO cells under prolonged periods of severe hypoxia/anoxia. 16-to 32-h lasting incubations attenuated Akt activity and decreased HIF-1a protein. This was reproduced by blocking PI3K with LY294002. GSK3b inhibition by indirubins circumvented the effect of hypoxia/anoxia or LY294002 on HIF-1a. Ruling stability regulation of HIF-1a protein and/or enhanced transcription of HIF-1a mRNA via GSK3b inhibition out is suggestive for translational modulation of HIF-1a under the influence of GSK3b.