The p53 tumour suppressor is involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 stabilization occurs under hypoxic and DNA damage conditions. However, only in the latter scenario is stabilized p53 capable of inducing the expression of its pro-apoptotic targets. Here we present evidence that under hypoxia-mimicking conditions p53 acetylation is reduced to a greater extent at K320 site targeted by P300/ CBP-associated factor (PCAF) than at K382 site targeted by p300/CBP. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the p21 WAF-1/CIP-1 gene, which appears to be unaffected by hypoxia, but are not recruited to the BID promoter and hence p53 is incapable of upregulating pro-apoptotic BID in hypoxic conditions. As the K320 p53 acetylation is the site predominantly affected in hypoxia, the PCAF histone acetyltransferase activity is the key regulator of the cellular fate modulated by p53 under these conditions. In addition, we provide evidence that PCAF acetylates hypoxia-inducible factor-1a (HIF-1a) in hypoxic conditions and that the acetylated HIF-1a is recruited to a particular subset of its targets. In conclusion, PCAF regulates the balance between cellcycle arrest and apoptosis in hypoxia by modulating the activity and protein stability of both p53 and HIF-1a.
The sesquiterpene lactone dihydroartemisinin (DHA), a semisynthetic derivative of the herbal antimalaria drug artemisinin, is cytotoxic to human tumor cells. Treatment of Jurkat T-lymphoma cells with DHA induced a breakdown of the mitochondrial transmembrane potential, release of cytochrome c, activation of caspases, and DNA fragmentation indicative of apoptosis induction. Although the absence of FADD or caspase-8 did not alter apoptosis rates in Jurkat cells, overexpression of dominant-negative caspase-9 or of antiapoptotic Bcl-xL or Bcl-2 largely decreased the cytotoxicity of DHA, demonstrating a role of the intrinsic death pathway. The proapoptotic Bcl-2 effector protein Bak and the Bcl-2 homology domain 3-only protein NOXA turned out to be important mediators of DHA-induced apoptosis in Jurkat cells. DHA treatment triggered the expression of NOXA and the activation of Bak. Furthermore, DHA-induced apoptosis was completely abrogated by loss of Bak and largely reduced in cells with siRNA-mediated downregulation of Bak or NOXA. Proapoptotic signaling of DHA also involved the formation of reactive oxygen species and membrane oxidation. Pretreatment with the lipophilic radical scavenger vitamin E or the hydrophilic radical scavengers glutathione and N-acetylcysteine reduced DHA-induced membrane oxidation and apoptosis, respectively. Oxidative changes also occurred in cells with disruption of the mitochondrial death pathway, suggesting a role of reactive oxygen species and oxidative membrane changes in death signaling upstream of the mitochondria. Interestingly, DHA increased the cytotoxic action of ionizing radiation and of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand in Jurkat cells, suggesting a potential benefit of DHA in combined treatment strategies. Mol Cancer Ther; 9(9); 2497-510. ©2010 AACR.
Tumor hypoxia is one main biological factor that drives resistance to chemotherapy and radiotherapy. To develop a novel strategy for overcoming hypoxia-induced therapy resistance, we examined the anti-neoplastic activity of the reactive oxygen donor dihydroartemisinin (DHA) in human colon cancer cell lines in normoxia and severe hypoxia. In addition, we analyzed the involvement of the intrinsic apoptosis pathway for DHA-mediated cytotoxicity in HCT116 cells in short-term and long-term in vitro assays. When applied at lower concentrations (≤25 μM), DHA induced apoptosis in Colo205, HCT15, and HCT116 cells, whereas necrotic cell death was increased when cells were treated with higher DHA concentrations (50 μM). However, no preference for DHA-induced apoptosis or necrosis could be detected between the treatment under normoxic or hypoxic conditions. Moreover, DHA potently reduced clonogenic survival of HCT116 cells in normoxia and hypoxia. Treatment of HCT116 cells with 25 μM DHA resulted in activation of Bax under normoxic and hypoxic conditions. Interestingly, cytochrome c release from the mitochondria and caspase-activation were observed only under normoxic conditions, whereas, under hypoxic conditions DHA induced a caspase-independent apoptosis-like cell death. However, under both conditions, generation of reactive oxygen species was an important mediator of DHA-induced toxicity. Further molecular analysis suggests that DHA-mediated cell death involves different sets of pro-apoptotic Bcl-2 family members. The pronounced cytotoxic activity of DHA in severe hypoxia as well as normoxia offers new perspectives for targeting the hypoxic tumor cell fraction to improve treatment outcome for cancer patients.
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