Limits...
Paradoxical regulation of hypoxia inducible factor-1α (HIF-1α) by histone deacetylase inhibitor in diffuse large B-cell lymphoma.

Bhalla S, Evens AM, Prachand S, Schumacker PT, Gordon LI - PLoS ONE (2013)

Bottom Line: We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway.We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival.We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Hematology/Oncology Northwestern University Feinberg School of Medicine and the Robert H Lurie Comprehensive Cancer Center, Chicago, Illinois, United States of America.

ABSTRACT
Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.

Show MeSH

Related in: MedlinePlus

PCI-24781 induced Apoptosis is mediated by PI3K/Akt pathway.(A) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. (B) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. (C) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. (D) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. (E) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842257&req=5

pone-0081333-g004: PCI-24781 induced Apoptosis is mediated by PI3K/Akt pathway.(A) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. (B) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. (C) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. (D) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. (E) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).

Mentions: We next investigated the factors that might regulate HIF-1α in lymphoma cells under normoxia. As MEK/ERK [38] and PI3K/AKT pathways [39] have been implicated as upstream activator of HIF-1α, it is conceivable that the HIF-1α suppressive effects of PCI-24781 in DLBCL cells are attributable to an interaction of the drug with an upstream kinase. Therefore we examined the effects of PCI-24781 on expression of ERK (pERK and total ERK) and AKT (pAKT and total AKT) protein by Western blotting in DLBCL cell lines. Treatment of cells with PCI-24781 resulted in a significant decrease in phosphorylation of AKT (Ser 473) and ERK proteins (Figure 4A). An increase in the proapoptotic protein Bim was also observed suggesting induction of apoptosis in association with the decrease in Akt or ERK activation.


Paradoxical regulation of hypoxia inducible factor-1α (HIF-1α) by histone deacetylase inhibitor in diffuse large B-cell lymphoma.

Bhalla S, Evens AM, Prachand S, Schumacker PT, Gordon LI - PLoS ONE (2013)

PCI-24781 induced Apoptosis is mediated by PI3K/Akt pathway.(A) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. (B) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. (C) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. (D) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. (E) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842257&req=5

pone-0081333-g004: PCI-24781 induced Apoptosis is mediated by PI3K/Akt pathway.(A) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. (B) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. (C) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. (D) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. (E) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).
Mentions: We next investigated the factors that might regulate HIF-1α in lymphoma cells under normoxia. As MEK/ERK [38] and PI3K/AKT pathways [39] have been implicated as upstream activator of HIF-1α, it is conceivable that the HIF-1α suppressive effects of PCI-24781 in DLBCL cells are attributable to an interaction of the drug with an upstream kinase. Therefore we examined the effects of PCI-24781 on expression of ERK (pERK and total ERK) and AKT (pAKT and total AKT) protein by Western blotting in DLBCL cell lines. Treatment of cells with PCI-24781 resulted in a significant decrease in phosphorylation of AKT (Ser 473) and ERK proteins (Figure 4A). An increase in the proapoptotic protein Bim was also observed suggesting induction of apoptosis in association with the decrease in Akt or ERK activation.

Bottom Line: We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway.We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival.We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Hematology/Oncology Northwestern University Feinberg School of Medicine and the Robert H Lurie Comprehensive Cancer Center, Chicago, Illinois, United States of America.

ABSTRACT
Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.

Show MeSH
Related in: MedlinePlus