5þ ; AA, ascorbic acid.

Transcription

1 A B C SUPPLEMENTAL FIG. S1. Synergistic effect of MnTMPyP, AA, and GSH on PC-3 cell proliferation. PC-3 cells were incubated with different concentrations of AA (A) and GSH (B) with MnTMPyP for various times of treatment. (C) Combined treatment of MnTMPyP (1 mm) and GSH (0.8 mm) with different concentrations of AA for 1 and 2 hours. Cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at 3 days after the treatment. Data are expressed as the mean (of triplicates) standard error of the mean. MnTMPyP, Mn(III) tetrakis(n-methylpyridinium-2-yl)porphyrin 5þ ; AA, ascorbic acid.

2 SUPPLEMENTAL FIG. S2. Morphological changes of PC-3 cells treated with MnTMPyP and/or AA. The cells were treated for 1 hour with AA (0.5 and 1 mm) and/or MnTMPyP (1 mm), and photographs were taken at different time points thereafter. The panel consists of representative micrographs with magnifications of 80(top panel, 2 hours), 160(top panel insets), and 40(middle [24 hours] and lower [48 hours] panels). MnTMPyP, Mn(III) tetrakis(n-methylpyridinium-2-yl)porphyrin 5þ ; AA, ascorbic acid.

3 SUPPLEMENTAL FIG. S3. In vitro (cell-free) reduction of Mn(III)TMPyP to Mn(II)TMPyP, measured using spectrophotometry. The solution of M alone [Mn(III)TMPyP, 5 mm], MA (M [5 mm] and A [ascorbic acid, 1 mm]), and MG (M [5 mm] and G [GSH, 1 mm]) were prepared in PBS and incubated in anoxia chamber for 6 hours. The UV absorbance spectrum ( nm) was measured with a sealable cuvette in a spectrophotometer. As shown by the arrows, the absorbance of Mn(II)TMPyP is at 441 nm (Harriman A, Porter G. Photochemistry of manganese porphyrins. Part 1. Characterisation of some water soluble complexes. J Chem Soc Faraday Transact 2 Mol Chem Phys 1979;75:1532).

4 SUPPLEMENTAL FIG. S4. H 2 O 2 generation by AA and MnTMPyP in PBS and DMEM/10% FBS. AA and MnTMPyP were mixed in concentrations as indicated, and the generation of H 2 O 2 was measured using Amplex-Red methods as described in the Materials and Methods section. The data expressed are the mean of duplicates SEM. H 2 O 2, hydrogen peroxide; AA, ascorbic acid; MnTMPyP, Mn(III) tetrakis(n-methylpyridinium-2-yl)porphyrin 5þ ;DMEM,Dulbecco s modified Eagle s medium, FBS, fetal bovine serum; RFU, relative fluorescence unit. SUPPLEMENTAL FIG. S6. Change of cellular ROS level after treatment with MnTMPyP and/or AA in PBS. PC-3 cells were incubated with CM-H 2 DCFDA (10 mm, 30 minutes) and then placed in PBS with different concentrations of MnTMPyP, in the presence or absence of AA (1 mm). Cellular ROS was determined as the DRFU over 1 hour of treatment and expressed as the mean (of triplicates) SEM. ROS, reactive oxygen species; MnTMPyP, Mn(III) tetrakis(nmethylpyridinium-2-yl)porphyrin 5þ ; AA, ascorbic acid, RFU, relative fluorescence unit. SUPPLEMENTAL FIG. S5. Generation of H 2 O 2 by AA (1 mm) and/or MnTMPyP (1 mm) in purified deionized and distilled H 2 O, PBS, and DMEM/10% FBS. H 2 O 2 was measured using Amplex-Red methods as described in the Materials and Methods section. The data expressed are the mean of duplicates SEM. Blank, without Amplex-Red; Cont, control; M, MnTMPyP; A, AA; MnTMPyP, Mn(III) tetrakis(n-methylpyridinium-2-yl)porphyrin 5þ ; AA, ascorbic acid; H 2 O 2, hydrogen peroxide; RFU, relative fluorescence unit. DRFU1000. SUPPLEMENTAL FIG. S7. In vitro (cell-free) time course of the Fenton reaction. HO generation by Fe (5 mm) and hydrogen peroxide (250 mm) in PBS was measured using the terephthalic acid assay as described in the Materials and Methods section, over a time course of 24 hours. RFU, relative fluorescence unit.

5 SUPPLEMENTAL FIG. S8. The effect of chelators on in vitro (cell-free) O 2 and H 2 O 2 generation. (A) O 2 was generated by MnTMPyP (M, 5 mm) and/or AA (A, 1 mm) in PBS, in the presence or absence of DP (8 mm), DFO (5 mm), and EDTA (20 mm). O 2 was measured using the nitro blue tetrozolium assay as described in the Materials and Methods section. (B) H 2 O 2 was generated by MnTMPyP (M, 5 mm) and/or AA (A, 1 mm) in PBS, in the presence or absence of DP (8 mm), DFO (5 mm), and EDTA (20 mm). H 2 O 2 was measured using AMPLEX-Red as described in the Materials and Methods section. Cont, control; H 2 O 2, hydrogen peroxide; MnTMPyP, Mn(III) tetrakis(n-methylpyridinium-2-yl)porphyrin 5þ ; AA, ascorbic acid; DFO, desferrioxamine; DP, dipyridyl. RFU, relative fluorescence unit; mod, milli optical density.

6 SUPPLEMENTAL FIG. S9. Hypoxia did not change the reactive oxygen species and HO generation by MnTMPyP, but decreased the MnTMPyP/AA cytotoxicity. (A) PC-3 cells were plated at a density of 1000 cells/well on a 96-well plate. After overnight incubation, cells were placed in hypoxia chambers with different O 2 concentrations for 2 hours at 378C, then treated (1 hour) with AA (1 mm) and/or MnTMPyP (1 mm), and returned to fresh medium under normoxic condition. Cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at 3 days after treatment. Data are expressed as the mean OD (of triplicates) SEM. (B) PC-3 cells were plated at a density of 25,000 cells/well on a 96-well plate. After overnight incubation, the cells were placed in different O 2 concentrations for 2 hours at 378C, then incubated with CM-H 2 DCFDA for 30 minutes, and treated with different concentrations of MnTMPyP dissolved in PBS. The fluorescence (RFU) was read at the end (1 hour) of the treatment by using a microplate reader. (C) In vitro (cell-free) generation of HO was measured using terephthalic acid in the reactions of Fe (FeSO 4 7H 2 O, 5 mm), AA (1 mm), and different concentrations of MnTMPyP under 0.5% O 2 concentration. RFU was measured at 1 hour after incubation at 378C. MnTMPyP, Mn(III) tetrakis(n-methylpyridinium-2-yl)porphyrin 5þ ; AA, ascorbic acid.