Site-directed photoproteolysis of 8-oxoguanine DNA glycosylase 1 (OGG1) by specific porphyrin-protein probe conjugates: a strategy to improve the effectiveness of photodynamic therapy for cancer

Abstract

The specific light-induced, non-enzymatic photolysis of mOGG1 by porphyrin-conjugated or rose bengal-conjugated streptavidin and porphyrin-conjugated or rose bengal-conjugated first specific or secondary anti-IgG antibodies is reported. The porphyrin chlorin e6 and rose bengal were conjugated to either streptavidin, rabbit anti-mOGG1 primary specific antibody fractions or goat anti-rabbit IgG secondary antibody fractions. Under our experimental conditions, visible light of wavelengths greater than 600 nm induced the non-enzymatic degradation of mOGG1 when this DNA repair enzyme either directly formed a complex with chlorin e6-conjugated anti-mOGG1 primary specific antibodies or indirectly formed complexes with either streptavidin-chlorin e6 conjugates and biotinylated first specific anti-mOGG1 antibodies or first specific anti-mOGG1 antibodies and chlorin e6-conjugated anti-rabbit IgG secondary antibodies. Similar results were obtained when rose bengal was used as photosensitizer instead of chlorin e6. The rate of the photochemical reaction of mOGG1 site-directed by all three chlorin e6 antibody complexes was not affected by the presence of the singlet oxygen scavenger sodium azide. Site-directed photoactivatable probes having the capacity to generate reactive oxygen species (ROS) while destroying the DNA repair system in malignant cells and tumors may represent a powerful strategy to boost selectivity, penetration and efficacy of current photodynamic (PDT) therapy methodologies.

Figure 1. Photolysis of mOGG1 by first specific porphyrin-conjugated anti-mOGG1 antiserum (direct method)

Recombinant purified mOGG1 was reacted with either chlorin e6-conjugated anti-mOGG1 antibodies or pyropheophorbide-a-conjugated anti-mOGG1 antibodies in the presence of visible light as described in Materials and Methods. All irradiations were conducted at 25°C for 30 min. Each lane of a 7.5–15% (w/v) polyacrylamide gel was loaded with 10 μl of reaction mixture. Gels were stained overnight with Coomassie blue. Lane 1, mOGG1 photolyzed with chlorin e6-conjugated anti-mOGG1 antibodies diluted 1:20. Lane 2, mOGG1 photolyzed with chlorin e6-conjugated anti-mOGG1 antibodies diluted 1:50. Lane 3, mOGG1 photolyzed with pyropheophorbide-a-conjugated anti-mOGG1 antibodies diluted 1:20. Lane 4, mOGG1 photolyzed with pyropheophorbide-a-conjugated anti-mOGG1 antibodies diluted 1:50. Lane 5, unphotolyzed mOGG1 (Molecular mass/Gel migration standard).

Figure 2. Photolysis of mOGG1 by anti-mOGG1 first specific rabbit IgG fractions either conjugated to porphyrin or rose bengal (direct method)

MOGG1 was reacted with chlorin e6, pyropheophorbide-a or rose bengal-conjugated anti-mOGG1 rabbit IgG fractions in the presence of visible light as described in Materials and Methods. All irradiations were conducted at 25°C for 30 min. Each lane of a 7.5–15% (w/v) polyacrylamide gel was loaded with 10 μl of reaction mixture. Gels were stained overnight with Coomassie blue. (A) Lane 1, mOGG1 photolyzed with chlorin e6-conjugated anti-mOGG1 antibodies diluted 1:100. Lane 2, mOGG1 photolyzed with chlorin e6-conjugated anti-mOGG1 antibodies diluted 1:1000. Lane 3, mOGG1 photolyzed with rose bengal-conjugated anti-mOGG1 antibodies diluted 1:100. Lane 4, mOGG1 photolyzed with rose bengal-conjugated anti-mOGG1 antibodies diluted 1:1,000. (B) Lane 1, mOGG1 photolyzed with pyropheophorbide-a-conjugated anti-mOGG1 antibodies diluted 1:10. Lane 2, mOGG1 photolyzed with pyropheophorbide-a-conjugated anti-mOGG1 antibodies diluted 1:100. Lane 3, mOGG1 photolyzed with pyropheophorbide-a-conjugated anti-mOGG1 antibodies diluted 1:1,000. Lane 4, mOGG1 photolyzed with pyropheophorbide-a-conjugated anti-mOGG1 antibodies diluted 1:10,000. (A, B) Lane 5, unphotolyzed mOGG1 (Molecular mass/Gel migration standard).

Figure 3. Photolysis of mOGG1 by either first specific anti-mOGG1 rabbit IgG fractions or anti-rabbit IgG secondary antibodies conjugated to porphyrins

MOGG1 was incubated with chlorin e6 or pyropheophorbide-a-conjugated anti-mOGG1 rabbit IgG fractions (direct method) or with a mixture containing unconjugated anti-mOGG1 rabbit IgG fractions and either chlorin e6 or pyropheophorbide-a-conjugated goat anti-rabbit IgG secondary antibodies (indirect method) in the presence or absence of visible light as described in Materials and Methods. All irradiations were conducted at 25°C for 30 min. Each lane of a 7.5–15% (w/v) polyacrylamide gel was loaded with 10 μl of reaction mixture. Gels were stained overnight with Coomassie blue. (A) Lane 1, mOGG1 photolyzed with 1 μl chlorin e6-conjugated anti-mOGG1 antibodies. Lane 2, mOGG1 photolyzed with 1 μl unconjugated anti-mOGG1 antibodies diluted 1:50 and 1 μl chlorin e6-conjugated secondary antibodies. Lane 3, mOGG1 photolyzed with 1 μl pyropheophorbide-a-conjugated anti-mOGG1 antibodies. Lane 4, mOGG1 photolyzed with 1 μl unconjugated anti-mOGG1 antibodies diluted 1:50 and 1 μl pyropheophorbide-a-conjugated secondary antibodies. (B) Lanes 1–4 are as in A, unirradiated (dark controls). (A,B) Lane 5, unphotolyzed mOGG1 (Molecular mass/Gel migration standard).

Figure 4. Photolysis of mOGG1 by chlorin e6-conjugated secondary antibodies (indirect method) in the presence and absence of sodium azide

MOGG1 was incubated with a mixture containing unconjugated anti-mOGG1 rabbit IgG fractions and chlorin e6-conjugated goat anti-rabbit IgG secondary antibodies in the presence or absence of sodium azide in the presence of visible light as described in Materials and Methods. All irradiations were conducted at 25°C for 30 min. Each lane of a 7.5–15% (w/v) polyacrylamide gel was loaded with 10 μl of reaction mixture. Gels were stained overnight with Coomassie blue. First specific anti-mOGG1 rabbit IgG fractions were diluted 1:500 and anti-rabbit IgG secondary antibodies were diluted 1:10. Lane 1, mOGG1 photolyzed with unconjugated anti-mOGG1 antibodies and chlorin e6-conjugated secondary antibodies. Lane 2, mOGG1 photolyzed with unconjugated anti-mOGG1 antibodies and chlorin e6-conjugated secondary antibodies in the presence of 0.5 mM sodium azide. Lane 3, mOGG1 photolyzed with unconjugated anti-mOGG1 antibodies and chlorin e6-conjugated secondary antibodies in the presence of 2 mM sodium azide. Lane 4, unphotolyzed mOGG1 (Molecular mass/Gel migration standard).

Figure 5. Photolysis of mOGG1 by biotinylated anti-mOGG1 antibodies-rose bengal-conjugated streptavidin complexes

MOGG1 was incubated with biotinylated anti-mOGG1 rabbit IgG fractions and either unconjugated streptavidin or rose bengal-conjugated streptavidin in the presence of visible light as described in Materials and Methods. All irradiations were conducted at 25°C for 30 min. Each lane of a 7.5–15% (w/v) polyacrylamide gel was loaded with 10 μl of reaction mixture. Gels were stained overnight with Coomassie blue. (A) Lane 1, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies and 2 μg of unconjugated streptavidin. Lane 2, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies and 0.5 μg of rose bengal conjugated-streptavidin. Lane 3, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies and 1 μg of rose bengal conjugated-streptavidin. Lane 4, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies and 2.0 μg of rose bengal conjugated-streptavidin. Lanes 1–4, biotinylated anti-mOGG1 antibodies diluted 1:50. (B) Lane 1, mOGG1 photolyzed with 1 μl biotinylated anti-mOGG1 antibodies and rose bengal-conjugated streptavidin. Lane 2, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies diluted 1:2 and rose bengal-conjugated streptavidin. Lane 3, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies diluted 1:5 and rose bengal-conjugated streptavidin. Lane 4, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies diluted 1:10 and rose bengal-conjugated streptavidin. (B) Lanes 1–4 contain 1 μg of rose bengal-conjugated streptavidin. Lane 5, unphotolyzed mOGG1 (Molecular mass/Gel migration standard).

Figure 6. Photoproteolysis of mOGG1 by biotinylated anti-mOGG1 antibodies-chlorin e6-conjugated streptavidin complexes

MOGG1 was incubated with 1 μl biotinylated anti-mOGG1 rabbit IgG fractions and either unconjugated streptavidin or chlorin e6-conjugated streptavidin in the presence or absence of visible light as described in Materials and Methods. All irradiations were conducted at 25°C for 30 min. Each lane of a 7.5–15% (w/v) polyacrylamide gel was loaded with 10 μl of reaction mixture. Gels were stained overnight with Coomassie blue. Lane 1, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies and 1 μg unconjugated streptavidin. Lane 2, mOGG1 photolyzed with biotinylated anti-mOGG1 antibodies and 1 μg chlorin e6-conjugated streptavidin. Lane 3, mOGG1 incubated in the dark with biotinylated anti-mOGG1 antibodies and 1 μg chlorin e6-conjugated streptavidin. Lane 4, unphotolyzed mOGG1 (Molecular mass/Gel migration standard).