Doxorubicin‐induced Activation of Intracellular Matrix Metalloproteinase‐2 by Oxidative Stress

Abstract

Matrix metalloproteinase‐2 (MMP‐2) activity is involved in several heart pathologies resulting from increased oxidative stress including ischemia‐reperfusion injury. In addition to its roles in extracellular matrix remodeling, MMP‐2 is also an intracellular protease with specific intracellular substrates. MMP‐2 mRNA and protein levels are elevated in cancer patients treated with doxorubicin (DXR). Though effective in chemotherapy, DXR's dose‐limiting cardiotoxicity may cause heart failure by activating intracellular MMP‐2. Human fibrosarcoma cells (HT1080 cells) and neonatal rat ventricular myocytes (NRVM) were treated with 0.01‐1 μM DXR for 0.5 and 2 h at 37°C. DXR treatment had no effect on cell viability. MMP‐2 activity and protein levels were measured by gelatin zymography and immunoblotting, respectively. Oxidative stress was measured by changes in mitochondrial aconitase activity. To visualize changes in intracellular MMP‐2 activity in real time, we will use a genetically encoded, fluorescence resonance energy transfer (FRET)‐based biosensor. In HT1080 cells 0.1‐1 μM DXR stimulated a 1.6 fold increase in intracellular MMP‐2 activity after 2 h without changing its protein level. DXR had no effect on MMP‐2 activity in NRVM. The FRET‐based MMP‐2 biosensor detected MMP but not other intracellular proteases, such as calpains and caspases, in vitro. In human cancer cells, acute treatment with DXR activates intracellular MMP‐2. The biosensors are now being expressed in cells to directly study intracellular MMP‐2 activity. The FRET‐based biosensor will allow us to better understand how oxidative stress stimuli affects intracellular MMP‐2 activity and its specific substrates.