Proteasome Inhibition Potentiates Antitumor Effects of Photodynamic Therapy in Mice through Induction of Endoplasmic Reticulum Stress and Unfolded Protein Response

Angelika Szokalska ¹, Marcin Makowski ¹, Dominika Nowis ¹, Grzegorz M. Wilczyski ^{2, 3}, Marek Kujawa ², Cezary Wójcik ⁴, Izabela Mynarczuk-Biay ², Pawel Salwa ¹, Jacek Bil ¹, Sylwia Janowska ¹, Patrizia Agostinis ⁵, Tom Verfaillie ⁵, Marek Bugajski ¹, Jan Gietka ¹, Tadeusz Issat ¹, Eliza Godkowska ¹, Piotr Mrówka ¹, Tomasz Stoklosa ¹, Michael R. Hamblin ⁶, Pawe Mróz ⁶, Marek Jakóbisiak ¹, and Jakub Golab ^1*

Departments of ¹Immunology and ²Histology and Embryology, Center of Biostructure Research, Medical University of Warsaw; ³Laboratory of Molecular and Systemic Neuromorphology, M. Nencki Institute of Experimental Biology, Warsaw, Poland; ⁴Department of Anatomy and Cell Biology, Indiana University School of Medicine-Evansville, Evansville, Indiana; ⁵Department of Molecular and Cell Biology, Catholic University of Leuven, Campus Gasthuisberg, Leuven, Belgium; and ⁶Department of Dermatology, Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts

^* To whom correspondence should be addressed. E-mail: jakub.golab{at}wum.edu.pl.

	Abstract

Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors. [Cancer Res 2009;69(10):4235–43]

Key Words: photodynamic therapy, Photofrin, proteasome, bortezomib, cancer