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PRO- AND ANTI-APOPTOTIC FUNCTIONS OF ETOPOSIDE AND THE MECHANISM OF CARDIOLIPIN OXIDATION BY CYTOCHROME C.

Kini, Vidisha (2005) PRO- AND ANTI-APOPTOTIC FUNCTIONS OF ETOPOSIDE AND THE MECHANISM OF CARDIOLIPIN OXIDATION BY CYTOCHROME C. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Cytochrome c (cyt c) binds with high affinity with anionic phospholipids such as phosphatidylserine (PS) and cardiolipin (CL) to form a complex with peroxidase activity capable of oxidizing polyunsaturated phospholipids, including CL. Release of cyt c from mitochondria plays a pivotal role in cytosolic triggering of apoptotic caspase cascades whereby CL oxidation is involved in mitochondrial membrane permeabilization. Thus control of CL oxidation is critical to regulation of early stages of apoptosis. Given the lipid antioxidant potency of etoposide as well as its ability to induce apoptosis, we hypothesized that cyt c catalyzed CL oxidation during apoptosis can be sensitive to etoposide, hence affect execution of the apoptotic program. We analyzed how the apparent inability of etoposide to prevent apoptosis is related to the mechanism of mitochondrial cardiolipin oxidation by hydrogen peroxide catalyzed by cyt c molecules which has public health relevance since etoposide is a commonly used anti-tumor drug. In a model biochemical system, we showed that 160 pmol lipid hydroperoxides / nmol CL were generated when 100µM liposomes containing a mixture tetralinoleoyl-CL (TLCL) with dioleoyl phosphatidylcholine (DOPC) [1:1] were incubated with 4µM cyt c and 100µM H2O2 or 250µM AMVN. Etoposide inhibited CL hydroperoxide production in a concentration dependent manner but with different sensitivity to the different oxidizing systems. Etoposide was more effective in AMVN system (I50=3µM) compared to cyt c/H2O2 system (I50=15µM), suggesting CL is not oxidized randomly but via a definite pathway. Next we tried to outline the pathway of cardiolipin oxidation using EPR techniques and PAGE studies. Characterizing the protein derived (tyrosyl) radical and etoposide-phenoxyl radical we noted that cardiolipin oxidation occurs via the heme of the cyt c peroxidase and also partly by the protein derived (tyrosyl) radical generated as result of cyt c peroxidase reaction. Etoposide, in the model system inhibits cardiolipin oxidation by preventing formation of protein derived (tyrosyl) radical. However, in HL-60 cells, etoposide enhanced CL oxidation while suppressing AMVN-induced oxidation of other phospholipids. Thus etoposide-dependent inhibition of CL oxidation is not likely to interfere with the execution of apoptotic program via prevention of mitochondrial membrane permeabilization.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Kini, Vidishak_vidisha@yahoo.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKagan, Valeriankagan@pitt.eduKAGAN
Committee CoChairFabisiak, Jamesfabs@pitt.eduFABS
Committee MemberYalowich, Jackyalowich@pitt.eduYALOWICH
Committee MemberEpperly, Michaelepperly@pitt.eduEPPERLY
Committee MemberEvans, Rhobertrwe2@pitt.eduRWE2
Date: 8 July 2005
Date Type: Completion
Defense Date: 6 June 2005
Approval Date: 8 July 2005
Submission Date: 7 June 2005
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Environmental and Occupational Health
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Cardiolipin; Cytochrome c; Etoposide
Other ID: http://etd.library.pitt.edu/ETD/available/etd-06072005-134536/, etd-06072005-134536
Date Deposited: 10 Nov 2011 19:46
Last Modified: 15 Nov 2016 13:44
URI: http://d-scholarship.pitt.edu/id/eprint/8027

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