Korean J Hematol.  2010 Sep;45(3):158-163. 10.5045/kjh.2010.45.3.158.

Induction of hypoxia-inducible factor-1alpha inhibits drug-induced apoptosis in the human leukemic cell line HL-60

• Yook YJ
• Seo YJ
• Kang HJ
• Ko SH
• Shin HY
• Lee JJ
• Jeong G
• Ahn HS

• KMID: 2252055
• DOI: http://doi.org/10.5045/kjh.2010.45.3.158

Abstract

BACKGROUND
Leukemic cells originate from hypoxic bone marrow, which protects them from anti-cancer drugs. Although many factors that cause drug resistance in leukemic cells have been studied, the effect of hypoxia on drug-induced apoptosis is still poorly understood.
METHODS
In this study, we examined the effect of hypoxia on anti-leukemic drug resistance in leukemic cell lines treated with cobalt chloride (CoCl2), a hypoxia-mimetic agent. Cellular proliferation was evaluated using the methyl thiazolyl tetrazolium (MTT) assay. Flow cytometry analysis and western blots were performed to investigate apoptosis-related proteins.
RESULTS
Unlike its previously known apoptotic effect, the expression of HIF-1alpha increased the survival rate of human promyelocytic leukemia HL-60 cells when these cells were exposed to anti-leukemic drugs; these effects were mediated by heat-shock protein HSP70 and the pro-apoptotic protein Bax.
CONCLUSION
These findings may provide new insights for understanding the mechanisms underlying hypoxia and for designing new therapeutic strategies for acute myeloid leukemia.

Keyword

Hypoxia; Arsenic trioxide; HIF-1alpha; Cobalt chloride; Bax; HSP70

MeSH Terms

Anoxia
Apoptosis
Arsenicals
Blotting, Western
Bone Marrow
Cell Line
Cell Proliferation
Cobalt
Drug Resistance
Flow Cytometry
Heat-Shock Proteins
HL-60 Cells
Humans
Leukemia
Leukemia, Myeloid, Acute
Oxides
Proteins
Survival Rate
Arsenicals
Cobalt
Heat-Shock Proteins
Oxides
Proteins

Figure

• 

  Fig. 1 CoCl2 increases the resistance of HL-60 to Actinomycin-D (Act-D). (A) HL-60, U937, K562, and CCRF-CEM cells were cultured for 8 hrs with or without CoCl2 (100 µM) and for additional 15 hrs in the presence of Act-D (1 µg/mL). Cells were then analyzed for PI negative population by flow cytometry. CTR, control. (B) HL-60 cells were pre-treated with various doses of CoCl2 for 8 hrs and then cultured for 15 hrs with indicated doses of Act-D. Cell viability was evaluated by the MTT assay. The relative viable cell number was calculated as the ratio of each optical density (OD) to the control OD. Results are expressed as relative viability±SEM. An asterisk (*) represents statistical significance when the P-value is lower than 0.05.

• 

  Fig. 2 CoCl2 increases the resistance of HL-60 to anti-leukemic drugs. HL-60, U937, K562, and CCRF-CEM cells were cultured for 15 hrs in the presence or absence of CoCl2. Cells were then cultured for another 15 hrs with or without (A) ATO (2.5 µM), (B) 17-AAG (1 µM), (C) valproic acid (5 mM), or (D) cytarabine (1 µM). Cell viability was analyzed by the MTT assay. The relative viable cell number was calculated as the ratio of each OD to the OD of cells treated with drug alone (set as 1.0). Results are expressed as relative viability±SEM. Asterisk (*) indicates statistical significance when the P-value is lower than 0.05.

• 

  Fig. 3 CoCl2 decreases the apoptotic rate of HL-60 cells through the HSP70/Bax pathway. (A) HL-60 or K562 cells were cultured for 24 hrs in the presence or absence of CoCl2. Expression levels of HSP90, HSP70, and Bax were analyzed by western blotting. An α-tubulin antibody was used as an internal control. (B) HL-60 cells were cultured with indicated dose of CoCl2 for 24 hrs. The expression levels of Bax, Bcl-2, ERK, phospho-ERK, HSP70, HSP90, p38, and phospho-p38 were analyzed by western blotting. (C) HL-60 cells were cultured with or without CoCl2 in the presence of ATO for 24 hrs. The amount of cleaved caspase-3 was assessed by flow cytometry. (D) HL-60 cells were cultured with or without ATO in the presence or absence of CoCl2 for 24 hrs. Cells were stained with annexin V/PI and then analyzed by flow cytometry.

• 

  Fig. 4 The expression of HIF-1α increases the resistance of HL-60 cells to ATO. (A) HL-60 cells were cultured in the presence of the indicated doses of CoCl2 for 24 hrs. Expression of HIF-1α was then analyzed by western blotting. A β-actin antibody was used as an internal control. (B) HL-60 cells were cultured in the presence or absence of CdCl2 and/or CoCl2 for 12 hrs and with ATO for additional 48 hrs. Cell viability was analyzed by the MTT assay. The relative viable cell number was calculated as the ratio of each OD to the control OD. Results are expressed as relative viability±SEM. Two asterisks (*) represents statistical significance when the P-value is lower than 0.01.

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