Fluorescence polarization based detection of hyaluronidase activity as a biomarker for skin cancer.




Chib, Rahul
Shah, Sunil
Mummert, Mark
Raut, Sangram
Bora, Ilkay
Pedro, Bobby
Gryczynski, Ignacy
Gryczynski, Zygmunt
Fudala, Rafal


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Purpose: Malignant melanoma (MM) is a type of skin cancer that has a high potential to metastasize to distant organs and cause death. MM is the third most common skin cancer in the United States and has an incidence of 18 new cases per 100,000. It is more lethal compared to other type of skin cancers due to its higher rate of metastasis and has a 5 years survival rate of 7-18%. In MM, the levels of hyaluronidase are overexpressed. Hyaluronidase is an endoglycosidase that degrades glycosaminoglycan and the hyaluronic acid (HA). Therefore, monitoring the hyaluronidase activity can be used as a contrasting mechanism for its detection. A novel fluorescence polarization based detection of enzyme activity using a fluorophore with a long fluorescence lifetime can enable a simple wide field molecular analysis of the cancer activities at the cellular and tissue level. Hyaluronic acid is a large biopolymer (over 1MDa) that is cleaved by the hyaluronidase enzyme to smaller elements. Monitoring kinetics of HA degradation by fluorescence polarization will be a simple and precise tool reflecting hyaluronidase activity and can be used for the detection, diagnosis and monitoring of the malignant melanoma. Methods: A long lifetime fluorophore (ADOTA) with a fluorescence lifetime of ̴ 20 ns was used to label hyaluronic acid. The hyaluronidase activity was measured as a function of change in the steady state fluorescence intensity and fluorescence polarization. Results: An intact HA will rotate slowly and the fluorescence polarization will be high, on the other hand, the cleaved HA ( ̴300KDa) will depolarize within the fluorescence lifetime of ADOTA yielding low polarization. The change in the polarization directly reflects hyaluronidase activity. We found an increase in the fluorescence intensity with increasing time. This is due to release of HOMO-FRET. We also observed a decrease in the fluorescence correlation time as cleaved hyaluronic acid fragments needs smaller time to rotate. Conclusions: Thus, preliminary results show the ability of HA-ADOTA probe to efficiently detect hyaluronidase activity. This study will develop a new technology for the non-invasive detection of the molecular activity of tumor in situ. In future, this technology can be used to construct a devise which can be used in the primary care setting for the detection of melanoma and monitoring the therapy progress.


Research Appreciation Day Award Winner - 2015 Quest Award
Research Appreciation Day Award Winner - 2015 Graduate Student Association Awards - 3rd Place Poster