Photochemistry and Photobiology Val. 55, No. 3, pp. 431-435. 1992 Printed in Great Britain. All rights reserved
0031-8655192 $05.00+O .OO Copyright 0 1992 Pergamon Press plc
PHOTODESTRUCTION OF TUMOR CELLS BY INDUCTION OF ENDOGENOUS ACCUMULATION OF PROTOPORPHYRIN IX: ENHANCEMENT BY 1,lO-PHENANTHROLINE NATALIE REBEIZ’,CAROLE C. REBEIZ~, SEANARKINS’,KEITHW. KELLEY’and CONSTANTIN A. R E B E I Z ~ * ‘Laboratory of Immunophysiology, Department of Animal Sciences and *Laboratory of Plant Pigment Biochemistry and Photobiology, University of Illinois, 1301 W. Gregory, -Urbana. IL 61801, USA (Received 9 July 1991; accepted 13 September 1991)
Abstract-Rapidly proliferating transformed mammalian cells can be photodesroyed in vitro upon inducing the accumulation of endogenous protoporphyrin IX (Proto). Proto biosynthesis and accumulation were triggered by manipulation of the porphyrin-heme biosynthetic pathway. Proto accumulation in cultured cells was induced by treatment with 1.0 mM 6-aminolevulinic acid (ALA), a naturally occurring 5-carbon amino acid, for 3.5 h. In darkness, significant Proto accumulation became evident within 3.5 h of incubation. In the light, the accumulated tetrapyrroles triggered destruction of treated cells within the first 30 min of illumination, probably via the rapid oxidation of cellular constituents by singlet oxygen. Protoporphyrin IX accumulation and specific cell lysis increased significantly by inclusion of 0.75 mM 1,lO-phenanthroline (Oph), a tetrapyrrole biosynthesis modulator. Slower growing untransformed cells did not accumulate significant amounts of Proto following ALA and Oph treatment unless stimulated to proliferate with the mitogenic lectin Concanavalin A.
(Oph), a porphyrin biosynthesis modulator, to ALA would increase the amount of Proto accumulation Photodynamic herbicides and porphyric insecticides and cell lysis. Malik (1989) recently demonstrated are two novel technologies that manipulate the that treatment of transformed cells in virro with photosensitizing capability of endogenous porphyALA alone induces Proto accumulation. However, rins. These two novel technologies destroy undesiraccumulation of Proto was slow, non-selective, and able plants and insects following co-treatment with the amount of light energy needed to destroy the 6-aminolevulinic acid (ALA)?, a naturally occurring treated cells was relatively high. In this report it is 5-carbon amino acid, and one of a number of tetrashown that treatment of rapidly multiplying immorpyrole biosynthesis modulators (Rebeiz et d., talized cells, with ALA and Oph causes the cells to 1988a, b, 1990). The amino acid and the modulator accumulate several-hundred-fold greater amounts of act in concert. The amino acid serves as a building proto than untreated cells. This endogenous Proto block of intracellular tetrapyrrole accumulation, accumulation causes rapid cell death in the light. while the modulator amplifies the accumulation of Slower growing cells respond to such treatments by harmful tetrapyrroles. In the light, it is believed that accumulating much lower levels of Proto. the accumulated tetrapyrroles photosensitize the formation of singlet oxygen which kills treated MATERIALS AND METHODS plants or insects by oxidation of their cellular memCell lines and cell culture. The gibbon monkey lymbranes. Protoporphyrin IX (Proto) is the immediate pre- phoma cell line MLA 144 and the human myelogenous leukemia cell line K562 were obtained from the American cursor of protoheme in plant and animal cells. Pro- Type Culture Collection, Rockville, MD. The murine toheme in turn is the prosthetic group of hemoglo- methyl-cholanthrene induced sarcoma cells (Meth-A) were bin, cytochromes, catalases and peroxidases. Since provided by Dr. Robert M. Lorence, RUSH Presbyterian plant and animal cells share the same tetrapyrrole St. Lukes Hospital, Chicago, IL. The murine fibrosarcoma biosynthetic pathway from ALA to Proto, we won- cell line WEHI 164 clone 13 was kindly provided by Dr. Terje Espevik, Institute of Cancer Research, Frondheirn, dered whether the addition of 1,lo-phenanthroline Norway. All cells were cultured in RPMI 1640 (GIBCO, INTRODUCTION
* To whom correspondence should be addressed.
t Abbreviarions: ALA, G’aminolevulinic acid; Con
A,
Concanavalin A; DM, dimethyl; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; LSD, least significant difference; Oph, 1,lo-phenanthroline; PDT, exogenous porphyrin photodynamic therapy; Proto, protoporphyrin IX.
Grand Island, NY) with 5% FBS (ICNFLOW, Costa Mesa, CA) at 3TC, 7% C 0 2 and 95% relative humidity. Cells were maintained in logarithmic phase of growth by passage 24 h prior to assay. Chemicals. 6-Aminolevulinic acid was purchased from Biosynth International, Chicago, IL, and Oph was obtained from Aldrich, St. Louis, MO. Protoporphyrin IX dimethyl ester (DM) and Mg-Proto were obtained from Porphyrin Products, Logan. UT. Unless otherwise indi-
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437
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cated, all chemicals. except Proto DM and Mg-Proto, were dissolved in RPMI 1640 tissue culture medium (GIBCO. Grand Island. NY) supplemented with 25 mM Hepes, and the pH adjusted to 7.2 immediately before use. Murine splenoryres. Single cell suspensions of splenocytes from BALB!c mice were prepared as described by Davila er a/. (1987). Erythrocytes were lysed by resuspending the pelleted cells in 1.0 mL of 0.83% NH,CI in 0.1% KHCO,: 0.01 mM EDTA at 4°C for 2 min. Splenocytes were washed three times with RPMI 1640, and resuspended at 3 x l(P cellsimL in RPMI 1640 supplemented with lW0 FBS. Splenocytes were then incubated in the absence or presence of the mitogenic lectin concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO) at an optimal concentration for proliferation of 1.25 p g h L for 40 h. Dererrniriarion o j cellular cyroroxicify. Target cells were incubated at 37°C with 100 pCi of Na5' Cr (Amersham, Arlington Heights, IL; specific activity. 1 pCi/pL) per 1 x lo7cells in 1 mL of RPMI 1640 medium supplemented with 5% FBS. After 7 h the cells were washed thoroughly three times with RPMI 1640 and resuspended in RPMI 1640 + 25 mM Hepes without serum. Washed cells (100 pL) were placed in microtiter plates (3.0 x 10' cells1well) and were treated with the indicated concentrations of ALA and Oph (in 100 pL) in darkness. Controls were treated with 100 p L RPMI containing 25 mM Hepes. After 3 h of dark incubation some of the $'Cr loaded cells were exposed to 30 min of white light (1.11 mW cm-?. sodium halide lamp) while other cells were kept for a similar period of time in darkness. 'I Cr release from the cells exposed to 30 min of white light or darkness was determined by removing 100 pL of supernatant, mixing with 3 mL liquid scintillation cocktail and counting in a Beckman LS5801 scintillation counter (Beckman Instruments, Fullerton, CA). One hundred percent cell lysis was determined from the 51Crrelease by control cells treated with 0.02% Triton X-100. The "Cr release from control cells treated with medium only did not exceed 18%. "Cr release from ALA and Oph treated cells was used to determine the specific '/o cell lysis (Davila er a / . . 1987) as follows: (CpmtrcatcJce1i
0031-8655192 $05.00+O .OO Copyright 0 1992 Pergamon Press plc
PHOTODESTRUCTION OF TUMOR CELLS BY INDUCTION OF ENDOGENOUS ACCUMULATION OF PROTOPORPHYRIN IX: ENHANCEMENT BY 1,lO-PHENANTHROLINE NATALIE REBEIZ’,CAROLE C. REBEIZ~, SEANARKINS’,KEITHW. KELLEY’and CONSTANTIN A. R E B E I Z ~ * ‘Laboratory of Immunophysiology, Department of Animal Sciences and *Laboratory of Plant Pigment Biochemistry and Photobiology, University of Illinois, 1301 W. Gregory, -Urbana. IL 61801, USA (Received 9 July 1991; accepted 13 September 1991)
Abstract-Rapidly proliferating transformed mammalian cells can be photodesroyed in vitro upon inducing the accumulation of endogenous protoporphyrin IX (Proto). Proto biosynthesis and accumulation were triggered by manipulation of the porphyrin-heme biosynthetic pathway. Proto accumulation in cultured cells was induced by treatment with 1.0 mM 6-aminolevulinic acid (ALA), a naturally occurring 5-carbon amino acid, for 3.5 h. In darkness, significant Proto accumulation became evident within 3.5 h of incubation. In the light, the accumulated tetrapyrroles triggered destruction of treated cells within the first 30 min of illumination, probably via the rapid oxidation of cellular constituents by singlet oxygen. Protoporphyrin IX accumulation and specific cell lysis increased significantly by inclusion of 0.75 mM 1,lO-phenanthroline (Oph), a tetrapyrrole biosynthesis modulator. Slower growing untransformed cells did not accumulate significant amounts of Proto following ALA and Oph treatment unless stimulated to proliferate with the mitogenic lectin Concanavalin A.
(Oph), a porphyrin biosynthesis modulator, to ALA would increase the amount of Proto accumulation Photodynamic herbicides and porphyric insecticides and cell lysis. Malik (1989) recently demonstrated are two novel technologies that manipulate the that treatment of transformed cells in virro with photosensitizing capability of endogenous porphyALA alone induces Proto accumulation. However, rins. These two novel technologies destroy undesiraccumulation of Proto was slow, non-selective, and able plants and insects following co-treatment with the amount of light energy needed to destroy the 6-aminolevulinic acid (ALA)?, a naturally occurring treated cells was relatively high. In this report it is 5-carbon amino acid, and one of a number of tetrashown that treatment of rapidly multiplying immorpyrole biosynthesis modulators (Rebeiz et d., talized cells, with ALA and Oph causes the cells to 1988a, b, 1990). The amino acid and the modulator accumulate several-hundred-fold greater amounts of act in concert. The amino acid serves as a building proto than untreated cells. This endogenous Proto block of intracellular tetrapyrrole accumulation, accumulation causes rapid cell death in the light. while the modulator amplifies the accumulation of Slower growing cells respond to such treatments by harmful tetrapyrroles. In the light, it is believed that accumulating much lower levels of Proto. the accumulated tetrapyrroles photosensitize the formation of singlet oxygen which kills treated MATERIALS AND METHODS plants or insects by oxidation of their cellular memCell lines and cell culture. The gibbon monkey lymbranes. Protoporphyrin IX (Proto) is the immediate pre- phoma cell line MLA 144 and the human myelogenous leukemia cell line K562 were obtained from the American cursor of protoheme in plant and animal cells. Pro- Type Culture Collection, Rockville, MD. The murine toheme in turn is the prosthetic group of hemoglo- methyl-cholanthrene induced sarcoma cells (Meth-A) were bin, cytochromes, catalases and peroxidases. Since provided by Dr. Robert M. Lorence, RUSH Presbyterian plant and animal cells share the same tetrapyrrole St. Lukes Hospital, Chicago, IL. The murine fibrosarcoma biosynthetic pathway from ALA to Proto, we won- cell line WEHI 164 clone 13 was kindly provided by Dr. Terje Espevik, Institute of Cancer Research, Frondheirn, dered whether the addition of 1,lo-phenanthroline Norway. All cells were cultured in RPMI 1640 (GIBCO, INTRODUCTION
* To whom correspondence should be addressed.
t Abbreviarions: ALA, G’aminolevulinic acid; Con
A,
Concanavalin A; DM, dimethyl; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; LSD, least significant difference; Oph, 1,lo-phenanthroline; PDT, exogenous porphyrin photodynamic therapy; Proto, protoporphyrin IX.
Grand Island, NY) with 5% FBS (ICNFLOW, Costa Mesa, CA) at 3TC, 7% C 0 2 and 95% relative humidity. Cells were maintained in logarithmic phase of growth by passage 24 h prior to assay. Chemicals. 6-Aminolevulinic acid was purchased from Biosynth International, Chicago, IL, and Oph was obtained from Aldrich, St. Louis, MO. Protoporphyrin IX dimethyl ester (DM) and Mg-Proto were obtained from Porphyrin Products, Logan. UT. Unless otherwise indi-
431
437
NATALIEREBEIZel a / .
cated, all chemicals. except Proto DM and Mg-Proto, were dissolved in RPMI 1640 tissue culture medium (GIBCO. Grand Island. NY) supplemented with 25 mM Hepes, and the pH adjusted to 7.2 immediately before use. Murine splenoryres. Single cell suspensions of splenocytes from BALB!c mice were prepared as described by Davila er a/. (1987). Erythrocytes were lysed by resuspending the pelleted cells in 1.0 mL of 0.83% NH,CI in 0.1% KHCO,: 0.01 mM EDTA at 4°C for 2 min. Splenocytes were washed three times with RPMI 1640, and resuspended at 3 x l(P cellsimL in RPMI 1640 supplemented with lW0 FBS. Splenocytes were then incubated in the absence or presence of the mitogenic lectin concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO) at an optimal concentration for proliferation of 1.25 p g h L for 40 h. Dererrniriarion o j cellular cyroroxicify. Target cells were incubated at 37°C with 100 pCi of Na5' Cr (Amersham, Arlington Heights, IL; specific activity. 1 pCi/pL) per 1 x lo7cells in 1 mL of RPMI 1640 medium supplemented with 5% FBS. After 7 h the cells were washed thoroughly three times with RPMI 1640 and resuspended in RPMI 1640 + 25 mM Hepes without serum. Washed cells (100 pL) were placed in microtiter plates (3.0 x 10' cells1well) and were treated with the indicated concentrations of ALA and Oph (in 100 pL) in darkness. Controls were treated with 100 p L RPMI containing 25 mM Hepes. After 3 h of dark incubation some of the $'Cr loaded cells were exposed to 30 min of white light (1.11 mW cm-?. sodium halide lamp) while other cells were kept for a similar period of time in darkness. 'I Cr release from the cells exposed to 30 min of white light or darkness was determined by removing 100 pL of supernatant, mixing with 3 mL liquid scintillation cocktail and counting in a Beckman LS5801 scintillation counter (Beckman Instruments, Fullerton, CA). One hundred percent cell lysis was determined from the 51Crrelease by control cells treated with 0.02% Triton X-100. The "Cr release from control cells treated with medium only did not exceed 18%. "Cr release from ALA and Oph treated cells was used to determine the specific '/o cell lysis (Davila er a / . . 1987) as follows: (CpmtrcatcJce1i
