Highlighting Our Magic Red Kits!
Stay tuned for a new series of posts on our popular Magic Red products! We will highlight several publications that have used these kits in their research. First, read below for a little background on how we developed our Magic Red substrate:
Back in 2004, ICT released what was then a novel fluorogenic substrate format for the detection of intracellular caspase and cathepsin enzyme activity. These kit products formed the basis for the Magic Red intracellular protease detection product concept. These products employ the use of the red fluorogenic fluorophore, Cresyl violet. When linked to various peptide target sequences, Cresyl violet dye fluorescence is quenched. Red fluorescence potential is enabled upon proteolytic cleavage of both peptide targeting sequences by the target enzymes (it excites at 590 nm and emits at 628 nm). Thus, its first use was described for measuring dipeptidyl peptidase IV (CD26) activity when (Ala-Pro)2-cresyl violet was used as the fluorogenic substrate (1). Utilizing this same principle, apoptotic cells were identified using (z-DEVD)2-cresyl violet as a fluorogenic substrate for caspases 3 and 7 (2). ICT offers a Caspase-3/7 kit based on this chemistry.
Cathepsins are house-keeping proteases primarily localized and active within lysosomes. They are defined by their substrate specificities (3). Elevated cathepsin enzyme activity levels within serum or extracellular matrix regions is often indicative of a variety of clinically significant pathologies. Cathepsin-mediated diseases include: Alzheimer’s disease, numerous types of cancer, and autoimmune related diseases such as arthritis, and osteoporosis. Osteoporosis is usually associated with the accelerated breakdown of bone structure (4). ICT offers three different preferred-specificity, Magic Red kits. Each preferred-specificity cathepsin B, K, and L detection kit contains the preferred dipeptide targeting sequences for the respective B, K, or L cathepsin enzymes.
Cathepsin B is thought to play an important role in intracellular proteolysis. Overexpression of cathepsin B is often correlated with invasive and metastatic cancers (5). It plays a specific role in pathologies of the central nervous system particularly in relation to Alzheimer’s disease (6). Other disease pathologies known to involve some level of cathepsin B participation include ischemia (7) and various neuronal dysfunction (8).
Cathepsin K is essential for osteoclast-mediated bone resorption. The enzyme’s ability to catabolize elastin, collagen, and gelatin is instrumental in the breakdown of bone and cartilage. This catabolic activity is also partially responsible for the loss of lung elasticity and recoil in emphysema. Cathepsin K inhibitors show great potential in the treatment of osteoporosis. Cathepsin K enzyme expression is reported in prostate (9), breast cancer (10), and is linked to further increased in metastasis of breast cancer to the bone (11). Cathepsin K inhibitors are promising therapeutic-candidates for treating breast cancer metastasis (12)
Cathepsin L is a lysosomal endopeptidase involved in antigen processing, bone resorption, tumor invasion and metastasis, and protein turnover during growth regulation (13). Cathepsin L can also be secreted, allowing for its involvement in the degradation of extracellular proteins such as collagen, elastin, and other basement membrane proteins (14). This degradation of the basement membrane and interstitial matrix may promote tumor cell invasion and metastasis by allowing cancer cells to breach the basement membrane barrier and invade local and distant tissue sites (15). Increases in the production of cathepsin L were found to correlate with elevated migration and invasiveness in human glioma U251 cells undergoing X-ray treatment compared to control populations (16). Activation of cathepsin L contributes to the irreversible depolarization induced by oxygen and glucose deprivation in rat hippocampal CA1 neurons (17).
Magic Red Cathepsin-B Assay Kit (Catalog 937, 938)
Magic Red Cathepsin K Assay Kit (Catalog 939, 940)
Magic Red Cathepsin L Assay Kit (Catalog 941, 942)
fluorogenic substrate for the analysis of kinetic parameters of dipeptidyl peptidase IV (CD26) in individual living rat hepatocytes. Anal Biochem 252, 71-77, doi:10.1006/abio.1997.2312 (1997).
2. Lee, B. W. et al. DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. Biotechniques 35, 1080-1085 (2003).
3. Conus, S. & Simon, H. U. Cathepsins: key modulators of cell death and inflammatory responses. Biochem Pharmacol 76, 1374-1382, doi:10.1016/j.bcp.2008.07.041 (2008).
4. Buhling, F. et al. Review: novel cysteine proteases of the papain family. Adv Exp Med Biol 477, 241-254, doi:10.1007/0-306-46826-3_26 (2000).
5. Ruan, J. et al. Over-expression of cathepsin B in hepatocellular carcinomas predicts poor prognosis of HCC patients. Mol Cancer 15, 17, doi:10.1186/s12943-016-0503-9 (2016).
6. Hook, G., Yu, J., Toneff, T., Kindy, M. & Hook, V. Brain pyroglutamate amyloid-beta is produced by cathepsin B and is reduced by the cysteine protease inhibitor E64d, representing a potential Alzheimer’s disease therapeutic. J Alzheimers Dis 41, 129-149, doi:10.3233/JAD-131370 (2014).
7. Yoshida, M. et al. Primate neurons show different vulnerability to transient ischemia and response to cathepsin inhibition. Acta Neuropathol 104, 267-272, doi:10.1007/s00401-002-0554-4 (2002).
8. Hook, G. R. et al. The cysteine protease cathepsin B is a key drug target and cysteine protease inhibitors are potential therapeutics for traumatic brain injury. J Neurotrauma 31, 515-529, doi:10.1089/neu.2013.2944 (2014).
9. Brubaker, K. D., Vessella, R. L., True, L. D., Thomas, R. & Corey, E. Cathepsin K mRNA and protein expression in prostate cancer progression. J Bone Miner Res 18, 222-230, doi:10.1359/jbmr.2003.18.2.222 (2003).
10. Littlewood-Evans, A. J. et al. The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma. Cancer Res 57, 5386-5390 (1997).
11. Le Gall, C. et al. A cathepsin K inhibitor reduces breast cancer induced osteolysis and skeletal tumor burden. Cancer Res 67, 9894-9902, doi:10.1158/0008-5472.CAN-06-3940 (2007).
12. Duong, L. T., Wesolowski, G. A., Leung, P., Oballa, R. & Pickarski, M. Efficacy of a cathepsin K inhibitor in a preclinical model for prevention and treatment of breast cancer bone metastasis. Mol Cancer Ther 13, 2898-2909, doi:10.1158/1535-7163.MCT-14-0253 (2014).
13. Kane, S. E. & Gottesman, M. M. The role of cathepsin L in malignant transformation. Semin Cancer Biol 1, 127-136 (1990).
14. Korenc, M., Lenarcic, B. & Novinec, M. Human cathepsin L, a papain-like collagenase without proline specificity. FEBS J 282, 4328-4340, doi:10.1111/febs.13499 (2015).
15. Lankelma, J. M. et al. Cathepsin L, target in cancer treatment? Life Sci 86, 225-233, doi:10.1016/j.lfs.2009.11.016 (2010).
16. Xiong, Y. et al. Cathepsin L is involved in X-ray-induced invasion and migration of human glioma U251 cells. Cell Signal 29, 181-191, doi:10.1016/j.cellsig.2016.10.012 (2017).
17. Kikuta, S., Murai, Y. & Tanaka, E. Activation of cathepsin L contributes to the irreversible depolarization induced by oxygen and glucose deprivation in rat hippocampal CA1 neurons. Neurosci Lett 636, 120-126, doi:10.1016/j.neulet.2016.11.006 (2017).