Apoptosis and autophagy after mitochondrial or endoplasmic reticulum photodamage

Abstract

Photodynamic therapy (PDT) can cause lethal photodamage by both direct and indirect mechanisms. Direct modes of cell death relate to nonspecific necrosis and the initiation of signaling pathways that elicit apoptosis, autophagy or both. In this report, effects of low-dose and high-dose PDT are explored, comparing sensitizers that localize in the endoplasmic reticulum (the porphycene termed CPO) or mitochondria (mesochlorin). To explore the role of autophagy, two cell lines were examined--the murine L1210 leukemia and an Atg7 knockdown derivative of L1210. The Atg7 gene is central to the process of autophagy. High-dose PDT with either sensitizer resulted in a substantial loss of the Bcl-2 protein. As Bcl-2 regulates both apoptosis and autophagy, loss of this protein can lead to initiation of either or both processes. Low-dose PDT with either sensitizer resulted in the initiation of apoptosis in the L1210/Atg7- cell line and a 20% loss of viability. In contrast, the same PDT dose led to the rapid appearance of autophagic cells in the L1210 line, less apoptosis and only a 5% loss of viability. These results are consistent with autophagy serving as a pro-survival response via the recycling of damaged organelles. At a higher PDT dose more apoptosis was again seen in the L1210/Atg7- line, but both cell lines exhibited comparable cytotoxicity in colony formation assays. We conclude that autophagy offers protection from the phototoxic effects of low-dose PDT, but can serve as an alternate death mode when the PDT dose is increased.

Figure 1

Fluorescence localization patterns of MC (a) and MTG (b) in L1210 cells.

Figure 2

Western blot analyses of Atg7 expression in L1210 cells before (a) and directly after an LD₉₀ PDT dose with CPO (b) or MC (c). Lane (d) depicts Atg7 expression in the L1210/Atg7⁻ cell line. In this and all other Western blots, the total protein loading was 40 µg per well.

Figure 3

(a) Phase-contrast images (1000×) of control L1210 and L1210/Atg7⁻ cells. (b) Phase-contrast and HO342 stained images of L1210 and L1210/Atg7⁻ cells after low-dose or high-dose PDT (light doses are defined in Table 1). MC-sensitized cells were photographed 30 min after irradiation.

Figure 4

Electron microscopic examination of vacuoles in L1210 cells 30 min after exposure to low-dose PDT using MC as the photosensitizing agent.

Figure 5

Western blots showing processing of the LC3-I protein to the faster-migrating LC3-II species in L1210 vs L1210/Atg7⁻ cells. Analyses are of control cultures (top), and cultures harvested 30 min or 24 h after administration of low- or high-dose PDT with MC (left) or CPO (right).

Figure 6

Western blot analyses of Bcl-2 expressions in L1210 and L1210/Atg7⁻ cells. Cultures were sensitized with MC or CPO and irradiated at low vs high PDT doses (see Table 1 for details). Cultures were harvested immediately after irradiation for analyses of Bcl-2. a = controls, b = MC low dose, c = CPO low dose, d = MC high dose, e = CPO high dose.