kjpp The Korean Journal of Physiology & Pharmacology Korean J Physiol Pharmacol 1226-4512 2093-3827 Korean J Physiol Pharmacol 10.4196/kjpp.2010.14.5.331 kjpp-14-331 Original Article CaMKII Inhibitor KN-62 Blunts Tumor Response to Hypoxia by Inhibiting HIF-1α in Hepatoma Cells LeeKyoung-Hwa Department of Physiology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799, Korea Corresponding to: Kyoung-Hwa Lee, Department of Physiology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 28, Yongon-dong, Chongno-gu, Seoul 110-799, Korea. (Tel) 82-2-3668-7632, (Fax) 82-2-745-7996, (E-mail) lee12042@snu.ac.kr 022010 18022010 14 5 331 336 27092010 08102010 18102010 Copyright © 2010 Korean J Physiol Pharmacol 2010 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 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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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.

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.

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.

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.

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.