Date of Award

2012

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Ana-Liisa Nieminen

Second Advisor

John J. Lemasters

Third Advisor

Craig C. Beeson

Fourth Advisor

Scott T. Eblen

Fifth Advisor

Christina Voelkel Johnson

Abstract

Photodynamic therapy (PDT) is an FDA-approved, minimally invasive treatment modality that utilizes light in the presence of oxygen to activate photosensitizing agents to produce cell death. Phthalocyanine 4 (Pc 4), a second generation photosensitizer has shown efficacy in vitro, in vivo and in a phase I clinical trial. Pc 4 localizes primarily to mitochondria and endoplasmic reticulum, where it causes apoptotic cell death during PDT. Previously, our laboratory showed that photo sensitizers that localize to lysosomes are more effective in killing cancer cells than ones directed to mitochondria after PDT. Here, we investigated the interactions between lysosomes and mitochondria in promoting the efficiency of PDT cell killing efficiency. Three head and neck cancer cell lines (UMSCC1, UMSCC14A and UMSCC22A) were exposed to Pc 4-PDT. The 3 cell lines responded differently: UMSCC1 and UMSCC14A cells were more resistant, whereas UMSCC22A cells were more sensitive to Pc 4-PDT. In non-erythroid cells, the mitochondrial iron transporter mitoferrin2 (Mfrn2) localizes on the mitochondrial inner membrane and transports iron from the cytosol into the mitochondria. PDT-sensitive cells expressed higher Mfrn2 mRNA and protein levels compared to PDT-resistant cells. High Mfrn2 expressing cells showed higher rates of mitochondrial Fe2+ uptake compared to low Mfrn2 expressing cells. Bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes that releases lysosomal iron to the cytosol, enhanced PDT-induced cell killing of both resistant and sensitive cells. Inhibition of the divalent metal transporter 1 (DMT1) on lysosomal membranes by ferristatin markedly protected high Mfrn2 expressing cells against bafilomycin-enhanced PDT toxicity, suggesting that iron release after bafilomycin occurs via DMT1. Iron chelators and the inhibitor of the mitochondrial Ca2+ (and Fe2+) uniporter, Ru360, protected against PDT plus bafilomycin toxicity. Knockdown of Mfrn2 in UMSCC22A cells decreased the rate of mitochondrial Fe2+ uptake and delayed PDT plus bafilomycin-induced mitochondrial depolarization and cell killing. Conversely, increased expression of Mfrn2 in low Mfrn2 expressing UMSCC1 cells increased PDT plus bafilomycin-induced killing. Chloroquine, which also releases iron from lysosomes, significantly delayed tumor regrowth in high Mfm2 expressing tumors after PDT. Taken together, the data suggest that lysosomal iron release and mitochondrial iron uptake through Mfrn2 act synergistically to induce PDT-mediated and iron-dependent mitochondrial dysfunction and subsequent cell killing. Furthermore, Mfm2 expression levels in tumors might be utilized as a biomarker predicting response to PDT in head and neck cancers.

Rights

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