Caspase 3/7 Activation in response to p97 inhibition
Assay Purpose: To evaluate the ability of specific p97 inhibitors to induce apoptosis in cells by monitoring activation of caspase 3/7 with Caspase 3/7 Glo kit from Promega after a 7 hour incubation. This assay will classify p97 inhibitors into either caspase-activating compounds, which maybe useful for developing anti-cancer agents, or non-activating compounds, which may have other therapeutic potentials. For a compound to be considered as a caspase-activating agent, it should activate caspase 3/7 more than 5 fold at a concentration less than 20 microM after 7 h treatment. ..more
BioActive Compounds: 5
Assay Provider: Raymond Deshaies, California Institute of Technology
Assay Performer: Tsui-Fen Chou, California Institute of Technology
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH085687-01
Grant Proposal PI: Raymond Deshaies, California Institute of Technology
Assay Purpose: To evaluate the ability of specific p97 inhibitors to induce apoptosis in cells by monitoring activation of caspase 3/7 with Caspase 3/7 Glo kit from Promega after a 7 hour incubation. This assay will classify p97 inhibitors into either caspase-activating compounds, which maybe useful for developing anti-cancer agents, or non-activating compounds, which may have other therapeutic potentials. For a compound to be considered as a caspase-activating agent, it should activate caspase 3/7 more than 5 fold at a concentration less than 20 microM after 7 h treatment.
Description: Misfolded proteins accumulate in the endoplasmic reticulum (ER) in response to environmental stress (1). To reduce the burden these proteins place on the secretory pathway, eukaryotic cells have evolved a process, known as ER-associated degradation (ERAD), to recognize and eliminate these proteins (1, 2). The highly conserved p97 ATPase functions in ERAD by hydrolyzing ATP needed to export ubiquitinated substrates to the cytosol for degradation by the proteasome (2, 3). The discovery of p97 missense mutations in a genetic form of human dementia (5-7), the localization of p97 in ubiquitylated inclusions in affected neurons of amyotrophic lateral sclerosis (ALS) and Parkinson's disease (8), and the overproduction of p97 in multiple cancers (10-14), suggests that p97 has diverse and essential cellular roles. Thus, the identification of probes that selectively target p97 activity may provide insights into the biological roles of p97. Given that the existing p97 inhibitor, DBeQ (CID676352), rapidly induced caspase 3/7 (15) in cells, specific p97 inhibitor as determined by not inhibiting ODD-Luc degradation were assayed to measure their ability to activate caspase 3/7.
1. Raasi S, Wolf DH. Ubiquitin receptors and ERAD: a network of pathways to the proteasome. Semin Cell Dev Biol. 2007 Dec;18(6):780-91. PMID: 17942349.
2. Halawani D, Latterich M. p97: The cell's molecular purgatory? Mol Cell. 2006 (22)6: 713-717. PMID: 16793541.
3. Wang Q, Li L, Ye Y. Inhibition of p97-dependent protein degradation by Eeyarestatin I. J Biol Chem. 2008 Mar 21;283(12):7445-54. PMID: 18199748.
4. Ye Y., Meyer H., Rapoport, T. A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER to the cytosol. Nature. 2001. 414(6864): p. 52-656. PMID: 11740563.
5. Watts, G.D., J. Wymer, M.J. Kovach, S.G. Mehta, S. Mumm, D. Darvish, A. Pestronk, M.P. Whyte, and V.E. Kimonis, Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet. 2004. 36(4): p. 377-81. PMID: 15034582.
6. Weihl, C.C., Dalal, S., Pestronk, A., and Hanson, P. I., Inclusion body myopathy-associated mutations in p97/VCP impair endoplasmic reticulum-associated degradation. Hum. Mol. Genet. 2006. 15(2): p. 189- 199. PMID: 16321991.
7. Mizuno, Y., Hori, S., Kakizuka, A. and Okamoto, K. (2003) Vacuole-creating protein in neurodegenerative diseases in humans. Neurosci. Lett. 343, 77-80. PMID: 12759168.
8. Ishigaki S, Hishikawa N, Niwa J, Iemura S, Natsume T, Hori S, Kakizuka A, Tanaka K, Sobue G. (2004) Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders. J Biol Chem. 2004 Dec 3;279(49):51376-85. PMID: 15456787.
9. Hirabayashi, M., Inoue, K., Tanaka, K., Nakadate, K., Ohsawa, Y., Kamei, Y., Popiel, A.H., Sinohara, A., Iwamatsu, A., Kimura, Y. Uchiyama, Y.,Hori, S., Kakizuka, A. VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration. Cell Death
Differ. 2001, 8, 977-984. PMID: 11598795.
10. Mitas, M., Mikhitarian, K., Walters, C., Baron, P. L., Elliott, B. M., Brothers, T. E., Robison, J. G., Metcalf, J. S., Palesch, Y. Y., Zhang, Z., Gillanders, W. E., and Cole, D. J., Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel. Int. J. Cancer. 2001. 93(2): p. 162-171. PMID: 11410861.
11. Marchetti, A., Buttitta, F., Bertacca, G., Zavaglia, K., Bevilacqua, G., Angelucci, D., Viacava, P., Naccarato, A., Bonadio, A., Barassi, F., Felicioni, L., Salvatore, S., Mucilli, F., mRNA markers of breast cancer nodal metastases: comparison between mammaglobin and carcinoembryonic antigen in 248 patients. J. Pathol. 2001. 195(2): p. 186-190. PMID: 11592097.
12. Smith, L.M., Nesterova, A., Alley, S. C., Torgov, M. Y., Carter, P. J., Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97. Mol. Cancer Ther, 2006. 5(6): p.1474-1482. PMID: 16818506.
13. Yamamoto, S., Tomita, Y., Uruno, T., Hoshida, Y., Qiu, Y., Iizuka, N., Nakamichi, I., Miyauchi, A., and Aozasa, K., Increased expression of valosin-containing protein (p97) is correlated with disease recurrence in follicular thyroid cancer. Ann. Surg. Oncol., 2005. 12(11): p. 925-934. PMID: 16189643.
14. Yamamoto, S., Tomita, Y., Uruno, T., Hoshida, Y., Qiu, Y., Iizuka, N., Nakamichi, I., Miyauchi, A., and Aozasa, K., Expression level of valosin-containing protein (p97) is associated with prognosis of esophageal carcinoma. Clin. Cancer Res., 2004. 10(16): p. 5558-5565. PMID: 15328197."
15. Chou T.-F., Brown, S. J., Minond, D., Nordin, B.E., Li, K., Jones, A.C., Chase, P., Porubsky, P. R., Stoltz, B.M., Schoenen, F. J., Patricelli, M.P., Hodder, P., Rosen, H., and Deshaies, R. J. (2011) Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proc Natl Acad Sci USA 108:4834-4839.
SW403 cells were seeded onto a 384 well white clear bottom plate (3000 cells per well). Cells were treated with compounds for 7 h and caspase-3/7 Glo (Promega) was added into each well and the contents of the well were mixed by shaking at 500 rpm for 1 min. Luminescence signal was determined after incubation at room temperature for 1 h with 0.1 ms integration time on the Synergy HT Microplate Reader (BioTek). Normalized Caspase3/7 activity is calculated as luminescence of test compound/luminescence of DMSO (1 %).
Score = maximal fold activation of each compound / maximal fold activation observed for all compounds x 100
For a compound to be considered as caspase activating agent, its score should be more than 15. This corresponds to activity scores greater than 50. Activity scores were calculated by normalizing the highest fold activation to 100 and the lowest fold activation to 0.
p97, AAA ATPase, valosin-containing protein, VCP, cancer, neurodegenerative disease, inclusion body myopathy associated with Paget disease of the bone and frontotemporal
dementia (IBMPFD), dose response, inhibitor, luciferase
Data Table (Concise)