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Combination therapy: Photochemotherapy; electric current; and ionizing radiation. Different combinations studied in a WiDr human colon adenocarcinoma cell line
J. Photochem. Photobiol. B: Biol., 21 (1993) 149-154
149
therapy: photochemotherapy; Combination . 1. lomzmg raalation. Different combinations colon adenocarcinoma cell line
electric current; and studied in a WiDr human
Liwei Ma, Vladimir Iani and Johan Moan+ Deparhent (Received
of Biophysics, Institute for Cancer Research, Montebello 0310, Oslo (Norway) March
16, 1993; accepted
August
16, 1993)
Abstract The interactions of pairs of the modalities Photofrin II-sensitized photochemotherapy (PCT), ionizing radiation and an electric current were investigated by the colony formation assay in WiDr cells, a human colon adenocarcinoma cell line. When the cells were treated simultaneously with PCT and an electric current, a slightly synergistic effect was observed at low exposures (surviving fraction -0.1) while a seemingly antagonistic effect was found at higher exposures. The same was found to be true for the combinations of PCT plus ionizing radiation and ionizing radiation plus electric current.
Key words: Photochemotherapy;
Ionizing
radiation;
Electricity;
1. Introduction Several investigators have proposed that photochemotherapy (PCT) of tumors with porphyrins as sensitizers mainly acts by type II photosensitization [l, 21. However, this is not generally proven, and type I process may also play a role [3]. If so, free radicals may be produced during the therapy. Some of these radicals may be ions formed by electron transfer [4]. A substantial fraction of such charge pairs may undergo geminate recombination similar to what occurs during radiolysis of water [5]. An electric current may interfere with this recombination process, both by the action of the electric field on ion pairs with a low but finite probability of geminate recombination and by the interference of ions flowing through the medium surrounding the cells. Even though cell membranes act as insulators preventing easy passage of charged species, lethal reactions may take place in or at the plasma membrane (which is charged). Electricity has been shown to produce lesions in cell membranes [6]. The plasma membrane is known to be a sensitive target also to PCI [7, S]. Changes in membrane permeability have been documented to occur after PCT with-porphyrins m +Author to whom correspondence
loll-1344/93/$6.00
should be addressed.
Photofrin
II
Previous studies have shown that an electric current increased the photodynamic inactivation of Herpes simplex virus [9] and Pseudorabies virus [lo] with methylene blue and light. The purpose of the present study is to investigate if PCT with Photofrin II, the most widely used sensitizer in clinical trials, ionizing radiation and an electric current can be combined in a way that is favourable for tumor therapy.
2. Materials
and methods
2.1. Cell cultures Cells of the established line WiDr, derived from a primary adenocarcinoma of the rectosigmoid colon [ll] were used. The cells were grown in monolayers at 37 “C in a humidified incubator with 5% CO2 and were subcultured twice a week in medium RPM1 1640 supplemented with 10% fetal calf serum (FCS) 2.2. Photosensitizer Photofrin II was provided by Lederle Parenterals (Carolina, Puerto Rico) as an aqueous solution at a concentration of 2.5 pg ml-’ and stored at -20 “C.
0 1993 - Elsevier
Sequoia.
All rights reserved
1.50
L. Ma et al. / Combination therapy of a human colon adenocarcinoma cell line
2.3. Irradiation sources The light source consisted of a bank of four fluorescent tubes (model 3026. Applied Photophysics London) emitting light in the region of 375-450 nm with the highest fluence rate around 405 nm [12]. The fluence rate of the light reaching the cells was 36 W m-‘. The source of ionizing radiation was a Siemens Stabilipan operated at 200 kV, 20 mA and with 0.5 mm Cu filtration. The dose rate to the cells was 5.3 Gy min-‘. 2.4. Current source The timed d.c. current source was a 3-volt constant current power unit built in our laboratory. The trans-sample current was adjusted to 1.0 mA. Preliminary experiments were performed to determine a relevant current level. A current of 1.0 mA produces a slightly lethal effect (up to 8% cell death) on the cells at an exposure time relevant for PCT as well as for ionizing radiation at the exposure rates used in the present work. A specially designed lid on the plastic culture dish (Falcon) was made from Stanyl Polyamide. Two glass supporters with platinum electrodes were fitted to the inside top of the lid, and the electrodes were contacted close to the cells as shown in Fig. 1. The polyamide lid with electrodes was sterilized by autoclaving prior to use. 2.5. Labelling with PI4 irradiation and electrical treatment single numbers of cells Appropriate (1.54.0x 103) were plated in 35 mm diameter plastic culture dishes and incubated at 37 “C for 8 h for proper attachment to the substratum. Subsequently, in one series of experiments, the cells were washed with PBS and incubated with 0.1 pg ml-’ of PI1 in PBS for 30 min at 37 “C, and then exposed to graded fluences of light as shown on the figures with or without concomitant Dish
application of a d.c. electric current of 1.0 mA (3.0 V) in the presence of sensitizer. In a second series, after the culture medium was replaced by PBS, the cells were irradiated with 5.3 Gy min-’ ionizing radiation for varying time lengths alone or treated simultaneously with electricity. In a third series, the cells containing PI1 were irradiated with graded doses of the light alone or simultaneously with ionizing radiation. After treatments, the cells were washed twice and incubated in fresh medium with 10% FCS for 11 days for colony formation. The procedure to determine the ability of the cells to form colonies was the same as that described earlier [13].
3. Results and discussion 3.1. The combined effect of PCT and electricity Figure 2 shows a plot of the survival of the cells given PCT and electricity separately or the two treatments in combination simultaneously. When these data are analysed by an isobologram (Fig. 3) it seems that the combined treatment is of a synergistic nature at low exposures (surviving fraction (SF) -0.1) while at higher exposures (SF -0.01) it is of a slightly antagonistic nature, i.e. the effect of the combined treatment is lower
0.1
lid with electrodes
0 Cells
Platinum
on the bottom
electrodes
Fig. 1. A specially designed cell culture dish used to expose cells to combination therapy of electricity and another treatment.
20
40
Exposure
60
time
60
100
(set)
Fig. 2. Survival curves for WiDr cells exposed to: 0, PCT alone (cells incubated with 0.1 pg ml-’ PI1 in PBS for 30 min before irradiation); 0, electricity alone (cells treated with a d.c. electric current of 1.0 mA for different times); X, PCT plus electricity (PII-loaded cells given light and electricity simultaneously). Bars, s.d. All the data are from three independent experiments. When bar is not shown the s.d. is smaller than the symbol.
L. Ma et al, / Combination therapy of a human colon adenocarcinoma cell line
0
20
Exposure
40
times
60
80
of PCT (s)
Fig. 3. Isobologram for the combined effects of PCI and electricity on WiDr cells. Lines of additivity were calculated as described in ref. 25. The point at the left of the envelope of additivity (x) indicates a synergistic interaction of PCT and electricity. The point at the right of the envelope (0) represents an antagonistic effect between these two treatments. Interaction points were obtained at surviving fraction of 0.1 (X) and 0.01 (e) in the survival curve shown in Fig. 2.
than one might expect from the effects of the separate treatments. The mechanisms of the combined effect of the two treatments are not clear, and will require further study. Earlier studies have indicated that membrane damage to cells may be mainly responsible for the phototoxicity of PCT [7, 8, 141. Such damage is produced by ‘02 but may certainly also be produced via a type I process involving charged species. Interactions between such charged species and the electric field might be an origin of synergism or antagonism. A cell membrane function as a dielectric layer protects the internal components of the cell from external influence [15]. The integrity of a membrane can be damaged by the application of an electric current [6]. Hence, PI1 presented outside the cells may get increased access to the interior of the cells as a result of the effect of the current. Membrane damage caused by small doses of PCT has been shown to result in increased cellular uptake of sensitizer [16]. Superoxide anion (O,-), an oxygen radical with a long lifetime and high oxidative effect, can also be produced electrolytically at the platinum cathode in buffered aqueous solutions following application of an electrical current [17]. 02- produced in this way can probably cause DNA single-strand breaks [6] and presumably may also damage membranes. 02- as a charged particle produced outside
1.51
the cells may have easier access to the DNA through a membrane being damaged by PCT. PCT itself can also produce DNA single-strand breaks in cells although probably not at a level that is lethal [13, 181. A possible origin of antagonism at high exposures could be a destructive interaction between some of the products formed by the two treatment modalities. For instance, one might speculate that the electric current may induce a product of low toxicity which is able to quench either ‘02 or other toxic products induced by PCT. Survival curves of the cells exposed to PBS solution given an electric current of 1.0 mA for different times are shown in Fig. 4. Firstly, pure PBS was given an electric current for 20, 60 or 100 s, and then immediately the solution of PBS treated by the electric current was added to the cells for 1, 5, or 10 min. After the treatments, PBS was replaced with fresh medium and the cells were incubated in an incubator for colony formation. Figure 4 shows that the survival fraction of the cells was decreased by 25% when PBS treated by the electric current with a longer exposure time (100 s) was given to the cells for 10 min. One possible contributing factor for this phenomenon might be the production of peroxides in PBS by the applied electric current. In fact, as mentioned above, the work of For-man er al. has demonstrated that after the treatment of an electric current, superoxide anion (OJ can be found in buffered aqueous solutions [ 171. Therefore, this result implies that survival of the cells may be
d
0
.d
’ 0.9 ‘_._ . .._
;.:I, , , , , , 0
20
40
Exposure
60
60
time
(set)
100
Fig. 4. Survival curves for WiDr cells exposed to PBS solution given an electric current of 1.0 mA for 20, 60 or 100 s. 0, After the electric current was given to pure PBS for the different times, the cells were exposed immediately to PBS treated by the electric current for 1 min; 0, after the electric current was provided to PBS only, the cells were exposed to PBS treated by the electric current for 5 min; 0, after the electric current was given to PBS alone, the cells were exposed to PBS treated by the electric current for 10 min.
152
L. Ma et al. I Combination theram of a human colon adenocarcinoma cell line
affected by oxidizing species produced by the electric current in the medium surrounding the cells. This experiment indicates that such toxic products have a relatively long lifetime such that their concentration will be built up during the exposure. 3.2. 7Yhe effect of ionizing radiation combined with electricity The effect on cell survival of separately administered electricity and ionizing radiation or of the combined treatment is shown in Fig. 5. When also analysed by an isobologram, these two treatment modalities appeared to act slightly synergistically at low exposures (SF-0.1) and antagonistically at large exposures (SF-0.01) (Fig. 6). The reports of Capella et al. [6, 191 show a synergistic effect of electricity combined with ionizing radiation or PCT with methylene blue on Escherichia coli bacteria. As with PCT plus electricity in the present work, the reason for the possible synergism at low exposures can only be speculated upon. Capella et al. [6] proposed that an increased yield of single-strand breaks in DNA may be a main reason for the synergism between the two treatments. The reason for the antagonism at the largest exposures in the present work are unclear. Since ionizing radiation also acts, to a large extent,
01 0
I
I
/
I
20
40
60
80
Exposure times of ionizing radiation (s) Fig. 6. Isobologram for the combined effects of electricity and ionizing radiation on WiDr cells. Interaction points were obtained at surviving fraction of 0.1 (X) and 0.01 (0) in the survival curve shown in Fig. 5. The symbols represent the same meaning as described in Fig. 3.
1
0.1
0.1
0.01
0.001
1
0
’
20
’ 40
Exposure
0.001
I
0
20
40
Exposure
60
80
time
(set)
100
Fig. 5. Survival curves for WiDr cells exposed to: 0, ionizing radiation alone (cells treated with ionizing radiation of 5.3 Gy min-‘); 0, electricity alone under the same experimental conditions as described in Fig. 2; X ionizing radiation plus electricity (cells treated with ionizing radiation and electricity simultaneously). Bars, s.d. All the data are from three independent experiments. When bar is not shown the s.d. is smaller than the symbol.
,
w
’
’ 60
time
’
’ 60
’
’ 100
(set)
Fig. 7. Survival curves for WiDr cells exposed to: 0, ionizing radiation alone under the same experimental conditions as described in Fig. 4; 0, PCT alone under the same experimental conditions as described in Fig. 2; X, PCT plus ionizing radiation (P&loaded cells given light and ionizing radiation of 5.3 Gy/ min simultaneously). Bars, s.d. All the data are from three independent experiments. When bar is not shown the s.d. is smaller than the symbol.
via production of oxidizing species (OH) a similar explanation as proposed above for PCT plus electricity may be possible.
L. Ma et al. / Combination therapy of a human colon adenocarcinoma cell line
153
three survival curves for the combined treatments show reduced shoulders. This is in agreement with the slightly synergistic effects seen at low doses. If the shoulders of the survival curves are related to repair processes, the present data indicate that the treatment modalities studied here interact destructively with each other’s repair mechanisms.
60
40
Acknowledgments 20
0 0
40
20
Exposure
60
80
times of PCT (s)
Fig. 8. Isobologram for the combined effects of PCT and ionizing radiation on WiDr cells. Interaction points were obtained at surviving fraction of 0.1 ( x ) and 0.01 (0) in the survival curve shown in Fig. 7. The symbols represent the same meaning as described in Fig. 3.
The present work was supported by The Association for International Cancer Research in the UK. The authors appreciate the critical reading of the manuscript by Professor Claude Rimington, FRS, and the supply of PI1 from Lederle Parenterals Company.
References 1 D. Kessel,
3.3. The effect of PCT combined with ionizing radiation Figure 7 shows the survival of cells treated separately with PCT and ionizing radiation or treated with the two modalities in combination. The combined treatment curve shows mainly an additive effect of the individual treatments at low exposures ( < 20 s). This would be in agreement with the results from other work [20-221, which demonstrated that ionizing radiation combined with PCT resulted in no more than an additive effect in vitro. Thus, in these systems ionizing radiation and PCT seem to act independently. In other systems, however, PCT was reported to act synergistically with ionizing radiation [23, 241. In the present study, the isobologram shows that the interaction of these two treatments on exposure, resulted in the surviving fraction of 0.1, was slightly synergistic. Whereas, at large exposures (SF - 0.01) these two modalities seem to act antagonistically (Fig. 8). Again an explanation as proposed in Section 3.1. might be possible.
4. Conclusion In conclusion, the present results show that combinations of two of the modalities PCT, an electric current and ionizing radiation acted mainly additively or synergistically in the present system. Large exposures to all three modalities resulted in seemingly antagonistic effects. Furthermore, all
2 3
4 5
6
7
8
9
10
11
12
Hematoporphyrin and HPD: photophysics, photochemistry and phototherapy, Photochem. Photobiol., 39 (1984) 851-859. T.J. Dougherty, Photodynamic therapy, Clin. Chest. Med., 6 (1985) 219-236. G. Jori, Photosensitizing properties of porphyrins and photodynamic therapy, Photobiochem. Photobiophys., Suppl. (1987) 373-384. B.W. Henderson and T.J. Dougherty, How does photodynamic therapy work? Photochem. PhotobioZ., 55 (1992) 145-157. M. Tubiana, J. Dutreix and A. Wambersie, Radiation chemistry, in M. Tubiana, J. Dutreix and A. Wambersie (eds.), Introduction to radiobiology, Taylor & Francis, London, 1990, pp. 22-33. M.A.M. Capella, M.E.F. Fonseca and S. Menezes, Synergism between electricity and ionizing radiation, Z. Photochem. Photobiol. B: BioL, 8 (1991) 371-383. J. Moan, T. Christensen and J.B. Mcghie, Porphyrin-sensitized photoinactivation of human cells in vitro, Am. Z. Pathol., 109 (1982) 184-192. D. Kessel, Effects of photoactivated porphyrins at the cell surface of leukemia L1210 cells, Biochemistry, 16 (1977) 3444-3449. M.R. Swartz, L.E. Schnipper, A.A. Lewin and C.S. Crumpacker, Inactivation of Herpes Simplex Virus with methylene blue, light and electricity, Proc. Sot. Ep. Biol. Med., 161 (1979) 204-209. J.A. Badylak, G. Scherba and P.D. Gustafson, Photodynamic inactivation of Pseudorabies Virus with methylene blue dye, light and electricity, J. Clin. Microbial., 17 (1983) 374-376. P. Noguchi, R. Wallace, J. Johnson, E.M. Earley, S. O’Brien, S. Ferrone, M.A. Pellegrino, J. Milstein, C. Needy, W. Browne and J. Petricciani, Characterization of WiDr: A human colon carcinoma cell line, In vitro, 15 (1979) 401408. K. Berg, E. Hovig and J. Moan, Sister chromatid exchanges induced by photodynamic treatment of cells in the presence of Photofrin II, aluminium phthalocyanine tetrasulfonate and tetra (3-hydroxyphenyl) porphyrin, in R.H. Douglas, J. Moan and F. Dall’Acqua (eds.), Light in Biology and Medicine, Vol. 1, Plenum, New York, 1988, pp. 95-103.
L. Ma et al. / Combination therapy of a human colon adenocarcinoma cell line
154
13 J. Moan, E.O. Pettersen and T. Christensen; The mechanism of photodynamic inactivation of human cells in vitro in the presence of haematoporphyrin, Br. J. Cancer, 39 (1979) 389-407. 14 D. Kessel and T.-H. Chou, Tumor-localizing components of the porphyrin preparation hematoporphyrin derivative, Cancer Rex, 43 (1983) 1994-1999. fusion and related 15 U. Zimmerman, Electric field-mediated electrical phenomena, Biochim. Biophys. Acta, 694 (1982) 227-277. 16 J. Moan and T. Christensen, Cellular uptake and photodynamic effect of hematoporphyrin, Photobiochem. Photobiophys., 2 (1981) 291-299. 17 H.J. Forman and I. Fridovich, Electrolytic univalent reduction of oxygen in aqueous solution demonstrated with superoxide dismutase, Science, 175 (1972) 339. effect on DNA and 18 J. Moan and E. Boye, Photodynamic cell survival of human cells sensitized by hematoporphyrin, Photobiochem. Photobiophys., 2 (1981) 301-307. 19 M.A.M. Capella and S. Menezes, Synergism between electrolysis and methylene blue photodynamic action inEschenchia cob, Int. .I. Radiat. Biol., 62 (1992) 321-326.
/’
20 J. Winther, J. Overgaard and N. Ehlers, The effect of photodynamic therapy alone and in combination with misonidazole or X-rays for management of a retinoblastoma-like tumour, Photochem. Photobiol., 47 (1988) 419-423. 21 D.A. Bellnier and T.J. Dougherty, Haematoporphyrin derivative photosensitization and y-radiation damage interaction in Chinese hamster ovary fibroblasts, ht. J. Radiat. Biol., 50 (1986) 659-664. 22 N. Ramakrishnan, M.E. Clay, L.R. Friedman, A.R. Antunez and N.L. Oleinick, Post-treatment interactions of photodynamic and radiation-induced cytotoxic lesions, Photochem. Photobiol., 52 (1990) 555-559. 23 G. Graschew and M. Shopova, Photodynamic therapy and y-irradiation of tumors: effect of tumor-cell reoxygenation, Lasers Med. Sci., 1 (1986) 193-195. 24 F.-Y. Zhao, K.-H. Zhang, H.-N. Huang, K.-H. Sun, Q.-B. Ling and B. Xu, Use of haematoporphyrin derivative as a sensitizer for radiotherapy of oral and maxillofacial tumors: a preliminary report, Lasers Med. Sci., I (1986) 253-256. 25 L. W. Ma, J. Moan, K. Berg, Q. Peng and H.B. Steen, Potentiation of photodynamic therapy by mitomycin c in cultured human colon adenocarcinoma cells, Radiat. Rex, 134 (1993) 22-28.
149
therapy: photochemotherapy; Combination . 1. lomzmg raalation. Different combinations colon adenocarcinoma cell line
electric current; and studied in a WiDr human
Liwei Ma, Vladimir Iani and Johan Moan+ Deparhent (Received
of Biophysics, Institute for Cancer Research, Montebello 0310, Oslo (Norway) March
16, 1993; accepted
August
16, 1993)
Abstract The interactions of pairs of the modalities Photofrin II-sensitized photochemotherapy (PCT), ionizing radiation and an electric current were investigated by the colony formation assay in WiDr cells, a human colon adenocarcinoma cell line. When the cells were treated simultaneously with PCT and an electric current, a slightly synergistic effect was observed at low exposures (surviving fraction -0.1) while a seemingly antagonistic effect was found at higher exposures. The same was found to be true for the combinations of PCT plus ionizing radiation and ionizing radiation plus electric current.
Key words: Photochemotherapy;
Ionizing
radiation;
Electricity;
1. Introduction Several investigators have proposed that photochemotherapy (PCT) of tumors with porphyrins as sensitizers mainly acts by type II photosensitization [l, 21. However, this is not generally proven, and type I process may also play a role [3]. If so, free radicals may be produced during the therapy. Some of these radicals may be ions formed by electron transfer [4]. A substantial fraction of such charge pairs may undergo geminate recombination similar to what occurs during radiolysis of water [5]. An electric current may interfere with this recombination process, both by the action of the electric field on ion pairs with a low but finite probability of geminate recombination and by the interference of ions flowing through the medium surrounding the cells. Even though cell membranes act as insulators preventing easy passage of charged species, lethal reactions may take place in or at the plasma membrane (which is charged). Electricity has been shown to produce lesions in cell membranes [6]. The plasma membrane is known to be a sensitive target also to PCI [7, S]. Changes in membrane permeability have been documented to occur after PCT with-porphyrins m +Author to whom correspondence
loll-1344/93/$6.00
should be addressed.
Photofrin
II
Previous studies have shown that an electric current increased the photodynamic inactivation of Herpes simplex virus [9] and Pseudorabies virus [lo] with methylene blue and light. The purpose of the present study is to investigate if PCT with Photofrin II, the most widely used sensitizer in clinical trials, ionizing radiation and an electric current can be combined in a way that is favourable for tumor therapy.
2. Materials
and methods
2.1. Cell cultures Cells of the established line WiDr, derived from a primary adenocarcinoma of the rectosigmoid colon [ll] were used. The cells were grown in monolayers at 37 “C in a humidified incubator with 5% CO2 and were subcultured twice a week in medium RPM1 1640 supplemented with 10% fetal calf serum (FCS) 2.2. Photosensitizer Photofrin II was provided by Lederle Parenterals (Carolina, Puerto Rico) as an aqueous solution at a concentration of 2.5 pg ml-’ and stored at -20 “C.
0 1993 - Elsevier
Sequoia.
All rights reserved
1.50
L. Ma et al. / Combination therapy of a human colon adenocarcinoma cell line
2.3. Irradiation sources The light source consisted of a bank of four fluorescent tubes (model 3026. Applied Photophysics London) emitting light in the region of 375-450 nm with the highest fluence rate around 405 nm [12]. The fluence rate of the light reaching the cells was 36 W m-‘. The source of ionizing radiation was a Siemens Stabilipan operated at 200 kV, 20 mA and with 0.5 mm Cu filtration. The dose rate to the cells was 5.3 Gy min-‘. 2.4. Current source The timed d.c. current source was a 3-volt constant current power unit built in our laboratory. The trans-sample current was adjusted to 1.0 mA. Preliminary experiments were performed to determine a relevant current level. A current of 1.0 mA produces a slightly lethal effect (up to 8% cell death) on the cells at an exposure time relevant for PCT as well as for ionizing radiation at the exposure rates used in the present work. A specially designed lid on the plastic culture dish (Falcon) was made from Stanyl Polyamide. Two glass supporters with platinum electrodes were fitted to the inside top of the lid, and the electrodes were contacted close to the cells as shown in Fig. 1. The polyamide lid with electrodes was sterilized by autoclaving prior to use. 2.5. Labelling with PI4 irradiation and electrical treatment single numbers of cells Appropriate (1.54.0x 103) were plated in 35 mm diameter plastic culture dishes and incubated at 37 “C for 8 h for proper attachment to the substratum. Subsequently, in one series of experiments, the cells were washed with PBS and incubated with 0.1 pg ml-’ of PI1 in PBS for 30 min at 37 “C, and then exposed to graded fluences of light as shown on the figures with or without concomitant Dish
application of a d.c. electric current of 1.0 mA (3.0 V) in the presence of sensitizer. In a second series, after the culture medium was replaced by PBS, the cells were irradiated with 5.3 Gy min-’ ionizing radiation for varying time lengths alone or treated simultaneously with electricity. In a third series, the cells containing PI1 were irradiated with graded doses of the light alone or simultaneously with ionizing radiation. After treatments, the cells were washed twice and incubated in fresh medium with 10% FCS for 11 days for colony formation. The procedure to determine the ability of the cells to form colonies was the same as that described earlier [13].
3. Results and discussion 3.1. The combined effect of PCT and electricity Figure 2 shows a plot of the survival of the cells given PCT and electricity separately or the two treatments in combination simultaneously. When these data are analysed by an isobologram (Fig. 3) it seems that the combined treatment is of a synergistic nature at low exposures (surviving fraction (SF) -0.1) while at higher exposures (SF -0.01) it is of a slightly antagonistic nature, i.e. the effect of the combined treatment is lower
0.1
lid with electrodes
0 Cells
Platinum
on the bottom
electrodes
Fig. 1. A specially designed cell culture dish used to expose cells to combination therapy of electricity and another treatment.
20
40
Exposure
60
time
60
100
(set)
Fig. 2. Survival curves for WiDr cells exposed to: 0, PCT alone (cells incubated with 0.1 pg ml-’ PI1 in PBS for 30 min before irradiation); 0, electricity alone (cells treated with a d.c. electric current of 1.0 mA for different times); X, PCT plus electricity (PII-loaded cells given light and electricity simultaneously). Bars, s.d. All the data are from three independent experiments. When bar is not shown the s.d. is smaller than the symbol.
L. Ma et al, / Combination therapy of a human colon adenocarcinoma cell line
0
20
Exposure
40
times
60
80
of PCT (s)
Fig. 3. Isobologram for the combined effects of PCI and electricity on WiDr cells. Lines of additivity were calculated as described in ref. 25. The point at the left of the envelope of additivity (x) indicates a synergistic interaction of PCT and electricity. The point at the right of the envelope (0) represents an antagonistic effect between these two treatments. Interaction points were obtained at surviving fraction of 0.1 (X) and 0.01 (e) in the survival curve shown in Fig. 2.
than one might expect from the effects of the separate treatments. The mechanisms of the combined effect of the two treatments are not clear, and will require further study. Earlier studies have indicated that membrane damage to cells may be mainly responsible for the phototoxicity of PCT [7, 8, 141. Such damage is produced by ‘02 but may certainly also be produced via a type I process involving charged species. Interactions between such charged species and the electric field might be an origin of synergism or antagonism. A cell membrane function as a dielectric layer protects the internal components of the cell from external influence [15]. The integrity of a membrane can be damaged by the application of an electric current [6]. Hence, PI1 presented outside the cells may get increased access to the interior of the cells as a result of the effect of the current. Membrane damage caused by small doses of PCT has been shown to result in increased cellular uptake of sensitizer [16]. Superoxide anion (O,-), an oxygen radical with a long lifetime and high oxidative effect, can also be produced electrolytically at the platinum cathode in buffered aqueous solutions following application of an electrical current [17]. 02- produced in this way can probably cause DNA single-strand breaks [6] and presumably may also damage membranes. 02- as a charged particle produced outside
1.51
the cells may have easier access to the DNA through a membrane being damaged by PCT. PCT itself can also produce DNA single-strand breaks in cells although probably not at a level that is lethal [13, 181. A possible origin of antagonism at high exposures could be a destructive interaction between some of the products formed by the two treatment modalities. For instance, one might speculate that the electric current may induce a product of low toxicity which is able to quench either ‘02 or other toxic products induced by PCT. Survival curves of the cells exposed to PBS solution given an electric current of 1.0 mA for different times are shown in Fig. 4. Firstly, pure PBS was given an electric current for 20, 60 or 100 s, and then immediately the solution of PBS treated by the electric current was added to the cells for 1, 5, or 10 min. After the treatments, PBS was replaced with fresh medium and the cells were incubated in an incubator for colony formation. Figure 4 shows that the survival fraction of the cells was decreased by 25% when PBS treated by the electric current with a longer exposure time (100 s) was given to the cells for 10 min. One possible contributing factor for this phenomenon might be the production of peroxides in PBS by the applied electric current. In fact, as mentioned above, the work of For-man er al. has demonstrated that after the treatment of an electric current, superoxide anion (OJ can be found in buffered aqueous solutions [ 171. Therefore, this result implies that survival of the cells may be
d
0
.d
’ 0.9 ‘_._ . .._
;.:I, , , , , , 0
20
40
Exposure
60
60
time
(set)
100
Fig. 4. Survival curves for WiDr cells exposed to PBS solution given an electric current of 1.0 mA for 20, 60 or 100 s. 0, After the electric current was given to pure PBS for the different times, the cells were exposed immediately to PBS treated by the electric current for 1 min; 0, after the electric current was provided to PBS only, the cells were exposed to PBS treated by the electric current for 5 min; 0, after the electric current was given to PBS alone, the cells were exposed to PBS treated by the electric current for 10 min.
152
L. Ma et al. I Combination theram of a human colon adenocarcinoma cell line
affected by oxidizing species produced by the electric current in the medium surrounding the cells. This experiment indicates that such toxic products have a relatively long lifetime such that their concentration will be built up during the exposure. 3.2. 7Yhe effect of ionizing radiation combined with electricity The effect on cell survival of separately administered electricity and ionizing radiation or of the combined treatment is shown in Fig. 5. When also analysed by an isobologram, these two treatment modalities appeared to act slightly synergistically at low exposures (SF-0.1) and antagonistically at large exposures (SF-0.01) (Fig. 6). The reports of Capella et al. [6, 191 show a synergistic effect of electricity combined with ionizing radiation or PCT with methylene blue on Escherichia coli bacteria. As with PCT plus electricity in the present work, the reason for the possible synergism at low exposures can only be speculated upon. Capella et al. [6] proposed that an increased yield of single-strand breaks in DNA may be a main reason for the synergism between the two treatments. The reason for the antagonism at the largest exposures in the present work are unclear. Since ionizing radiation also acts, to a large extent,
01 0
I
I
/
I
20
40
60
80
Exposure times of ionizing radiation (s) Fig. 6. Isobologram for the combined effects of electricity and ionizing radiation on WiDr cells. Interaction points were obtained at surviving fraction of 0.1 (X) and 0.01 (0) in the survival curve shown in Fig. 5. The symbols represent the same meaning as described in Fig. 3.
1
0.1
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I
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Fig. 5. Survival curves for WiDr cells exposed to: 0, ionizing radiation alone (cells treated with ionizing radiation of 5.3 Gy min-‘); 0, electricity alone under the same experimental conditions as described in Fig. 2; X ionizing radiation plus electricity (cells treated with ionizing radiation and electricity simultaneously). Bars, s.d. All the data are from three independent experiments. When bar is not shown the s.d. is smaller than the symbol.
,
w
’
’ 60
time
’
’ 60
’
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Fig. 7. Survival curves for WiDr cells exposed to: 0, ionizing radiation alone under the same experimental conditions as described in Fig. 4; 0, PCT alone under the same experimental conditions as described in Fig. 2; X, PCT plus ionizing radiation (P&loaded cells given light and ionizing radiation of 5.3 Gy/ min simultaneously). Bars, s.d. All the data are from three independent experiments. When bar is not shown the s.d. is smaller than the symbol.
via production of oxidizing species (OH) a similar explanation as proposed above for PCT plus electricity may be possible.
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three survival curves for the combined treatments show reduced shoulders. This is in agreement with the slightly synergistic effects seen at low doses. If the shoulders of the survival curves are related to repair processes, the present data indicate that the treatment modalities studied here interact destructively with each other’s repair mechanisms.
60
40
Acknowledgments 20
0 0
40
20
Exposure
60
80
times of PCT (s)
Fig. 8. Isobologram for the combined effects of PCT and ionizing radiation on WiDr cells. Interaction points were obtained at surviving fraction of 0.1 ( x ) and 0.01 (0) in the survival curve shown in Fig. 7. The symbols represent the same meaning as described in Fig. 3.
The present work was supported by The Association for International Cancer Research in the UK. The authors appreciate the critical reading of the manuscript by Professor Claude Rimington, FRS, and the supply of PI1 from Lederle Parenterals Company.
References 1 D. Kessel,
3.3. The effect of PCT combined with ionizing radiation Figure 7 shows the survival of cells treated separately with PCT and ionizing radiation or treated with the two modalities in combination. The combined treatment curve shows mainly an additive effect of the individual treatments at low exposures ( < 20 s). This would be in agreement with the results from other work [20-221, which demonstrated that ionizing radiation combined with PCT resulted in no more than an additive effect in vitro. Thus, in these systems ionizing radiation and PCT seem to act independently. In other systems, however, PCT was reported to act synergistically with ionizing radiation [23, 241. In the present study, the isobologram shows that the interaction of these two treatments on exposure, resulted in the surviving fraction of 0.1, was slightly synergistic. Whereas, at large exposures (SF - 0.01) these two modalities seem to act antagonistically (Fig. 8). Again an explanation as proposed in Section 3.1. might be possible.
4. Conclusion In conclusion, the present results show that combinations of two of the modalities PCT, an electric current and ionizing radiation acted mainly additively or synergistically in the present system. Large exposures to all three modalities resulted in seemingly antagonistic effects. Furthermore, all
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