The loss or decreased expression of beta 2m-microglobulin (beta2m) causes cellular resistance to chemotherapy. Moreover, exogenous beta 2m protein causes caspase-dependent apoptosis, but independently of caspases-3, -8, and -9, in drug sensitive and resistant variants. In this application, we propose to explore the molecular mechanism(s) by which beta2m triggers apoptosis and how it increases the efficacy of anticancer agents. Our long-term objectives are to unravel the molecular mechanism(s) of beta2m-induced apoptosis, and to apply this knowledge to identify and/or develop new therapeutic regimens.
The Specific Aims are to (1) restore beta2 expression in drug resistant cells and assess its influence on the cytotoxic effects of chemotherapeutic drugs, (2) identify specific caspases or non-caspase proteins involved in the execution of beta 2m-induced apoptosis, and (3) investigate the role of the tumor necrosis factor-alpha (TNF-alpha) receptor family and increased reactive oxygen species (ROS) in beta 2m-induced apoptosis.
Specific Aim 1 will (a) determine whether the increase in endogenous J32m in MCF-7 and MCF-7/Adr-5 cells increases the efficacy of DOX, VCR, Taxol and VP-16, (b) determine whether exogenous beta 2m synergistically enhances apoptosis induced by these anticancer agents in these cells, (c) evaluate whether [beta 2m alone or in combination with these drugs exerts similar apoptotic effects on the estrogen receptor-negative breast cancer cell line MDA-MB-231 and its DOX-resistant derivative MDA-MB-231/Adr, and (d) examine whether [beta 2m modulates the effects of anticancer drugs on caspases during apoptosis and affects the cell cycle in these cells, and ascertain biochemically how apoptosis-triggering by [beta 2m and anticancer agents differs.
Specific Aim 2 will (a) explore by Western blot analysis whether caspases-4, -7, -10, -11, -12 and -13 are activated during beta 2m-triggered apoptosis, (b) assess beta 2m-induced caspase activation by detecting cleavage of the caspase substrate Z-VAD-afc, (c) identify the proteins that bind specifically to the biotin-VAD-fmk and biotin-YVAD-cmk affinity probes and 125I-labeled YVAD-cmk in the cytosols from beta 2m-treated and untreated control cells, and (d) determine the identity of the proteins specifically labeled with these affinity probes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and N-terminal sequencing.
Specific Aim 3 will (a) explore the mechanisms by which beta 2m induces apoptosis through death receptors, (b) determine whether [beta 2m induces increased ROS through the mitochondria, and (c) investigate whether cellular redox state plays a role in cytochrome c release by [beta 2m. These studies will aid in understanding the molecular mechanism(s) of beta 2m-induced apoptosis, and will be useful for the development of more effective chemotherapeutic or potential gene therapy strategies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA080734-05A1
Application #
6618606
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Muszynski, Karen
Project Start
1999-03-03
Project End
2006-02-28
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
5
Fiscal Year
2003
Total Cost
$282,940
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Safa, Ahmad R (2016) Resistance to Cell Death and Its Modulation in Cancer Stem Cells. Crit Rev Oncog 21:203-219
Safa, Ahmad R (2013) Roles of c-FLIP in Apoptosis, Necroptosis, and Autophagy. J Carcinog Mutagen Suppl 6:
Safa, A R (2012) c-FLIP, a master anti-apoptotic regulator. Exp Oncol 34:176-84
Safa, Ahmad R; Pollok, Karen E (2011) Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy. Cancers (Basel) 3:1639-71
Park, Soo-Jung; Bijangi-Vishehsaraei, Khadijeh; Safa, Ahmad R (2010) Selective TRAIL-triggered apoptosis due to overexpression of TRAIL death receptor 5 (DR5) in P-glycoprotein-bearing multidrug resistant CEM/VBL1000 human leukemia cells. Int J Biochem Mol Biol 1:90-100
Choi, Mi-Ran; Najafi, Farhad; Safa, Ahmad R et al. (2008) Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI. Biochem Pharmacol 75:2276-88
Safa, Ahmad R; Day, Travis W; Wu, Ching-Huang (2008) Cellular FLICE-like inhibitory protein (C-FLIP): a novel target for cancer therapy. Curr Cancer Drug Targets 8:37-46
Zhong, Xiaoling; Safa, Ahmad R (2007) Phosphorylation of RNA helicase A by DNA-dependent protein kinase is indispensable for expression of the MDR1 gene product P-glycoprotein in multidrug-resistant human leukemia cells. Biochemistry 46:5766-75
Park, Soo-Jung; Wu, Ching-Huang; Choi, Mi-Ran et al. (2006) P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5. Biochem Pharmacol 72:293-307
Day, Travis W; Najafi, Farhad; Wu, Ching-Huang et al. (2006) Cellular FLICE-like inhibitory protein (c-FLIP): a novel target for Taxol-induced apoptosis. Biochem Pharmacol 71:1551-61

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