Journal List > Korean J Physiol Pharmacol > v.14(5) > 1025699

Lee: CaMKII Inhibitor KN-62 Blunts Tumor Response to Hypoxia by Inhibiting HIF-1α in Hepatoma Cells

Abstract

In rapidly growing tumors, hypoxia commonly develops due to the imbalance between O2 consumption and supply. Hypoxia Inducible Factor (HIF)-1α is a transcription factor responsible for tumor growth and angiogenesis in the hypoxic microenvironment; thus, its inhibition is regarded as a promising strategy for cancer therapy. Given that CamKII or PARP inhibitors are emerging anticancer agents, we investigated if they have the potential to be developed as new HIF-1α-targeting drugs. When treating various cancer cells with the inhibitors, we found that a CamKII inhibitor, KN-62, effectively suppressed HIF-1α specifically in hepatoma cells. To examine the effect of KN-62 on HIF-1α-driven gene expression, we analyzed the EPO-enhancer reporter activity and mRNA levels of HIF-1α downstream genes, such as EPO, LOX and CA9. Both the reporter activity and the mRNA expression were repressed by KN-62. We also found that KN-62 suppressed HIF-1α by impairing synthesis of HIF-1α protein. Based on these results, we propose that KN-62 is a candidate as a HIF-1α-targeting anticancer agent.

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Fig. 1.
HIF-1α protein level is regulated by CamKII inhibitor KN-62 in Hepatoma cell lines. (A) Hep3B and HepG2 cells were pre-treated with 0, 1, 2, or 5 μM KN-62 and incubated with 1% O2 hypoxia for 8 hr. Western blot analysis was done with anti-HIF-1α and anti-β-Tubulin antibodies. (B) Western blot analysis of HIF-1α and β-Tubulin in Hep3B cell line before (N) and after (H) 8 hr incubation in 1% O2 in the presence of 2 μM and 5 μM KN-62, 100 μM and 500 μM 3-AB, or 10 μM and 30 μM DPQ. (C) MCF7 and SK-N-MC cells were incubated in 1% O2 for 8 hr in the presence of 0, 5, 10, or 15 μM KN-62 and western blot analysis was performed with anti-HIF-1α and anti-β-Tubulin antibodies.
kjpp-14-331f1.tif
Fig. 2.
KN-62 repressed HIF-1α activity in Hep3B cell line. Hep3B cells were transiently transfected with an Epo-Luc construct. The cells were treated with increasing concentrations of KN-62 (A), 3AB (B) and DPQ (C) in 21% (Normoxia) or 1% (Hypoxia) O2 for 16 h. Luciferase activity is shown as the fold change from the value with 21% O2 and no drug. Denotes p<0.01 between indicated groups. n.s., not significant.
kjpp-14-331f2.tif
Fig. 3.
The mRNA level of HIF-1α downstream gene was decreased in the presence of KN-62 under hypoxia. Quantitative real-time measurement of Epo (A), LOX (B) and CA9 (C) mRNA level was done using total RNA from Hep3B cells treated with 16-hr hypoxia with or without KN-62. 18S signal was used for normalization of signals and values were shown as relative value to normoxia sample. Denotes p<0.05 between indicated groups.
kjpp-14-331f3.tif
Fig. 4.
The protein level of HIF-1α was regulated by synthesis rate. (A) Top: Hep3B cells were treated with 1% O2 for 8 hr with or without KN-62 and re-oxygenated for the indicated time. Western blot signals of HIF-1α and β-tubulin are detected. Bottom: The HIF-1α protein band densities were quantified using the ImageJ program and plotted as a function of time. (B) Top: Western blot signals of HIF-1α and β-tubulin are detected with Hep3B cell extract treated with MG132 with or without KN-62 for the indicated time. Bottom: The protein band densities were quantified using ImageJ and plotted as a function of time.
kjpp-14-331f4.tif
Fig. 5.
HIF-1α protein translation was decreased by KN-62 treatment via inhibition of Akt signaling. (A) The reporter activity of Tk/HIF-1α-5′-UTR Luciferase in Hep3B cells treated with Normoxia, Hypoxia or Hypoxia with KN-62 was analyzed. Values are shown as the relative value to normoxia sample. (B) The reporter activity of CMV/GFP-5'UTR-Luciferase in Hep3B cells treated with Normoxia, Hypoxia or Hypoxia with KN-62 was analyzed. Values are shown as relative value to normoxia sample. (C) KN-62 inhibits phosphorylation of AKT. After 8-hr incubation under hypoxic conditions with 0, 1, 2, 5, or 10 μM KN-62, Hep3B cells were lysed and subjected to Western blotting with anti-phospho S473 Akt antibody, anti-total Akt antibody and anti-β tubulin antibody. Denotes p<0.05 between indicated groups.
kjpp-14-331f5.tif
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