Signaling events in apoptotic photokilling of 5-aminolevulinic acid-treated tumor cells: inhibitory effects of nitric oxide

Abstract

Antitumor photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that can destroy tumor and tumor vasculature cells. NO produced by these cells could be procarcinogenic by inhibiting apoptosis and promoting angiogenesis and tumor growth. We recently showed that NO from a chemical donor or activated macrophages makes COH-BR1 breast tumor cells more resistant to photokilling sensitized by 5-aminolevulinic acid (ALA)-generated protoporphyrin IX (PpIX). Signaling events associated with this hyperresistance have now been examined. ALA-treated COH-BR1 cells containing mitochondria-localized PpIX died mainly by apoptosis after being irradiated. Underlying redox signaling associated with MAP kinase (ERK1/2, p38, JUN) phosphorylation-activation, and heme oxygenase-1 (HO-1) upregulation was studied using immunoprecipitation and Western blot methodology. ALA/light treatment resulted in activation of proapoptotic JNK and p38 alpha, and deactivation of prosurvival p38 beta and ERK1/2. Involvement of both JNK and p38 in apoptosis was established by using a specific inhibitor for each. Spermine NONOate-derived NO, introduced immediately before irradiation, provided substantial protection against apoptosis. This was accompanied by greater HO-1 induction and a strong inhibition of each MAP kinase effect seen in the absence of NO. Downstream of JNK and p38 alpha activation, a marked upregulation/activation of proapoptotic Bax and Bid was observed along with down-regulation of antiapoptotic Bcl-xL, each response being reversed by NO. These findings provide new insights into signaling activity associated with the intrinsic apoptotic pathway in ALA-PDT and how this activity can be modulated by NO.

Figures

Figure 1

Subcellular localization of PpIX in ALA-treated cells: susceptibility to PpIX-sensitized photokilling in the absence vs. presence of SPNO. COH-BR1 cells at ~60% confluency in serum-free DME/F12 medium were incubated with 1.0 mM ALA for 45 min, washed, and then incubated with Rh123 (1 μg/ml) for 10 min, all in the dark at 37 °C. (A) Confocal images of cells showing sites of major PpIX fluorescence, Rh123 fluorescence, and overlaying of the two. Each bar represents 75 μm. (B) Apoptotic vs. necrotic photokilling of cells preincubated with 1.0 mM ALA for 45 min. Twenty hours after exposure to a light fluence of 2 J/cm2 (Irrad), cells were stained with Ho and PI, and examined for numbers of apoptotic and necrotic nuclei by fluorescence microscopy. ALA-treated cells kept in the dark were analyzed alongside as a control (Dark). Approximately 100 cells in 4 viewing fields were scored for each sample. Plot shows extent of apoptosis (black bars) and necrosis (gray bars) after cell exposure to the following fluences: 0, 2, 4, and 6 J/cm2. (C) and (D) Effect of active vs. decomposed SPNO on photokilling. ALA-treated cells were irradiated in the presence of active donor (SPNO) or decomposed donor (dSPNO); 20 h later they were stained with Ho and PI and scored for apoptosis and necrosis. Plots in (B), (C), and (D) show means ± SD of values from three separate experiments.

Figure 2

MAPK status in photodynamically challenged cells: increasing light fluence/single post-fluence time format. COH-BR1 cells were dark-incubated with 1.0 mM ALA for 45 min and then exposed to the indicated fluences (J/cm2), either directly (ALA/hν) or after addition of 0.2 mM SPNO (ALA/SPNO/hν). A control not treated with ALA or SPNO was irradiated alongside (hν). Twenty hours after each irradiation, the cells were recovered by trypsinization, washed, and lysed in preparation for Western blot analyses. Samples of equivalent total cellular protein (120 μg per lane) were subjected to SDS-PAGE using 10% acrylamide/bis-acrylamide, followed by transfer to a PVDF membrane. After blocking, the membrane was treated sequentially with primary antibodies against p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, and JNK, followed in each case by reporter secondary antibody and chemiluminescence measurement, with antibody stripping between each of the six determinations. β-Actin was also monitored as a loading reference. For p38 and ERK1/2 (but not JNK because p-JNK was undetectable), densitometrically-determined intensity of phosphorylated enzyme relative to overall intensity (e.g. p-p38/p38) was as follows: p38: hν [1.0±0.1 (2), 1.1±0.0 (4), 1.0±0.2 (6)]; ALA/hν [1.1±0.1 (2), 1.0±0.2 (4), 1.1±0.0 (6)]; ALA/SPNO/hν [0.9±0.1 (2), 0.8±0.1 (4), 0.9±0.0 (6)]. ERK1/2: hν [0.9±0.0 (2), 0.8±0.1 (4), 0.8±0.1 (6)]; ALA/hν [0.4±0.0 (2), 0.2±0.0 (4), 0.3±0.0 (6)]; ALA/SPNO/hν [1.2±0.1 (2), 0.8±0.1 (4), 0.9±0.0 (6)]. Numbers in brackets are means ± deviation of values from two separate experiments for each reaction condition, light fluences being indicated in parentheses. The immunoblots shown are from one of these experiments. Values are relative to the dark control (0-fluence) in each case.

Figure 3

MAPK status in photodynamically challenged cells: single light fluence/increasing post-fluence time format. COH-BR1 cells sensitized with ALA-generated PpIX as described in Figs. 1 and 2, were exposed to a 4 J/cm2 light fluence in the absence (ALA/hν) or presence (ALA/SPNO/hν) of 0.2 mM SPNO, then trypsinized and recovered, either immediately (0 min) or after increasing periods of dark incubation ranging from 15 min to 4 h. For each condition, a non-irradiated dark control (DC) was run alongside and cells were recovered at the 15 min time point. Samples with the same amount of total cell protein (120 μg per lane) were subjected to SDS-PAGE and Western blotting, using primary antibodies against p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, and JNK. For each MAPK (except JNK in ALA/SPNO/hν system), densitometrically-measured intensity of phosphorylated enzyme relative to overall intensity was as follows: JNK: ALA/hν [2.4±0.2 (0.25), 1.7±0.2 (0.5), 1.3±0.1 (1), 1.1±0.1 (2), 1.0±0.1 (4)]. p38: ALA/hν [3.2±0.2 (0.25), 3.4±0.3 (0.5), 1.5±0.1 (1), 1.0±0.1 (2), 0.9±0.1(4)]; ALA/SPNO/hν [1.0±0.1 (0), 1.1±0.1 (0.25), 1.0±0.0 (0.5), 0.9±0.2 (2), 0.9±0.1 (4)]. ERK1/2: ALA/hν [0.9±0.1 (0.25), 1.0±0.1 (0.5), 0.9±0.1 (1), 0.9±0.1 (2), 0.9±0.1 (4)]; ALA/SPNO/hν [1.4±0.1 (0), 4.5±0.1 (0.25), 4.3±0.0 (0.5), 3.8±0.1 (2), 4.8±0.1 (4)]. Numbers in brackets are means ± SD of values from three separate experiments for each reaction condition, cells being sampled at the post-irradiation times indicated in parentheses (hours). The immunoblots shown are from one of these experiments. Values are relative to the dark control (0-fluence) in each case.

Figure 4

Phosphorylation state of p38α and p38β in photochallenged cells. ALA-treated cells were (A) exposed to a light fluence of 4 J/cm2 and examined after 15 min in the dark, or (B) exposed to fluences of 2, 4, and 6 J/cm2 and examined after 20 h in the dark. ALA-treated cells were also irradiated in the presence of 0.2 mM SPNO and examined 20 h later (C). Non-irradiated ALA-treated cells are also represented in each case (0 J/cm2). The p38α and p38β isoforms were immunoprecipitated using monoclonal antibodies specific for each and then analyzed by immunoblotting, using p-p38 antibody to probe for extent of phosphorylation and the individual antibodies to probe for total amount of each p38 isoform. The latter values were used for standardizing sample loads and normalizing the amounts of corresponding p-p38 in the different sample lanes. Graphs beside the immunoblots show integrated levels of each p-p38 isoform: in (A) relative to p-p38α at 0 J/cm2; in (B) and (C), each relative to its own level at 0 J/cm2. Means ± deviation of values from two separate experiments are plotted in (B).

Figure 5

Effects of MAPK inhibitors on extent of photosensitized MAPK phosphorylation and apoptotic cell killing. (A) Cell sensitization with ALA-derived PpIX was as described in Fig. 1. Where indicated, cells were pre-incubated with p38 or JNK inhibitors for 1 h (ALA being included during the last 45 min), after which irradiation was carried out (4 J/cm2) and apoptosis assessed 20 h later. DC, dark control without inhibitor; IC, irradiated control without inhibitor; SB, 20 μM SB202190 before irradiation; SP, 25 μM SP600125 before irradiation. Plotted data are means ± SD of values from three separate experiments; *significantly less than corresponding IC value, p < 0.005. (B) Phosphorylation status of p38 and JNK after irradiation of sensitized cells that had been treated with 0, 10, or 20 μM SB202190; *significantly lower than the value at 0 μM inhibitor, p < 0.005. (C) Phosphorylation status of JNK and p38 after irradiation of sensitized cells that had been treated with 0, 10, or 25 μM SP600125; *significantly lower than the value at 0 μM inhibitor, p < 0.001. Western analyses in (B) and (C) were carried out 15 min after 4 J/cm2 of light exposure. Plotted data in each case are means ± SD of values from three separate experiments, one of which is represented in the immunoblots (inset).

Figure 6

HO-1 expression in relation to apoptosis in ALA/light-treated cells: effects of NO and hemin. ALA-treated cells were either kept in the dark (0 J/cm2) or exposed to light fluences of 2, 4, and 6 J/cm2, either directly (ALA/hν) or after addition of 0.2 mM SPNO (ALA/SPNO/hν) or 5 μM hemin (ALA/hemin/hν). A control not treated with ALA or the other agents was irradiated alongside (hν). Twently hours after irradiation, samples were recovered and analyzed for degree of HO-1 expression and apoptosis. (A) HO-1 protein level, as determined by Western analysis. Band intensities normalized to β-actin and relative to 0-fluence were as follows: ALA/hν [1.7±0.0 (2), 1.8±0.2 (4), 2.0±0.0 (6)]; ALA/SPNO/hν [3.4±0.2 (2), 2.7±0.2 (4), 3.9±0.3 (6)]; ALA/hemin/hν [0.9±0.1 (2), 1.0±0.2 (4), 1.3±0.1 (6)]. Numbers in brackets are means ± deviation of values from two independent experiments at fluence levels indicated in parentheses; the blots shown are from one of these experiments. (B) apoptosis, as determined by fluorescence microscopy of Ho and PI-stained cells. IC, irradiated control without SPNO or hemin. Plotted data are means ± SD of values from three separate experiments; *significantly less than corresponding IC value, p <0.001.

Figure 7

Changes in Bcl-2 family protein levels in ALA/light- and ALA/SPNO/light-treated cells. Cells sensitized with ALA-induced PpIX were exposed to light fluences of 2 and 4/cm2 in the absence (A) or presence (B) of SPNO (0.2 mM). A non-irradiated control (0 J/cm2) was run alongside. Twenty hours after each irradiation, samples were analyzed for Bax and Bcl-xL by Western blotting, β-actin serving as a loading standard. Plots in (A) and (B) show changes in Bax (●) and Bcl-xL (○) levels relative to the non-irradiated control for the respective blots. Samples were also analyzed for Bid by Western blotting (C); plots show changes in Bid level after irradiation in the absence (△) or presence (▲) of SPNO. Means ± deviation of immunoblot data from two separate experiments for each reaction condition are plotted in (A), (B), and (C). The blots shown are from one of these experiments.