Dye-Sensitized Photoinactivation of Tumor Cells in Vitro

Dye-sensitized Photoinactivation of Tumor Cells in Vitro

JUmTH S. BELLIN,* STEVEN C. MOHOS, A~D GERALD OSTER

(Department of Pathology, State University of New York, Do~zmslale Medical Center, and Department of Chemistry, Polytechnic Institute of Brooklyn, Brooklyn, N.Y.)

SUMMARY The in vitro photodynamic inactivation of a wide variety of tumor cells was studied with various dyes used as sensitizcrs. The capacity of dyes to act as photosensitizers in this system parallels their capacity to sensitize the photoinactivation of many other substrates and correlates with their capacity to undergo photoreduction. Some dyes are able to inactivate tumor cells in the abscnce of light. :Photodynamic inactivation of the tumor cells dcstroys their tumor-producing capacity but docs not change their dyc- binding capacity.

It has long been known that some microorgan- Menke (16) has shown that both normal and neo- isms, when stained with certain dyes, can be in- plastic cells grown in tissue culture (lie when sub- activated with visible light. This process, com- jected to the action of visible light in the presence monly referred to as photodynamic action, in- of a xanthene dye. The criterion of death in this volves the participation of oxygen and consists of author's publication was a subjective one, and, a dye-sensitized photoautoxidation of some es- since relevant controls were omitted, his results sential elements of the substrate involved. Many could be interpreted as being due to indirect ef- diverse substrates are affected by photodynamic fects, resulting from the photodynamic destruc- action. These include enzymes (~3), transfornfing tion of nutrients in the culture medium. principle (1), viruses (11), bacteria (10), and The present paper is concerned with the in vitro erythrocytes (8). The autoxidation of many simple photodynamic inactivation of tumor cells as organic substrates is enormously accelerated by judged by their inability to elicit tumor growth. light in the presence of certain sensitizing dyes. A In conformity with previous results on the photo- mechanism for the photosensitized autoxidation dynamic inactivation of transforming principle of para-toluenediamine and criteria for the capac- and para-toluenediamine, we have found that ity of dyes to act as sensitizers in such a system only certain classes of dyes can act as photo- were established (18). It was found that those dyes sensitizers in this system. which can photosensitize the autoxidation of aromatic amines can also act as sensitizers for the MATERIALS AND METHODS photodynamic inactivation of transforming prin- Tv,~ons A:ND DYES E.M1)LOYED ciple (1). The tumors used were: the Gardner lymphoma There is an analogous relationship between (6C3HED) grown in ~0-gram C3H/Hej mice (both photodynamie action and the effect of x-rays in the tumor and mice were obtained from the Jack- that both phenomena involve autoxidations. For son Memorial Laboratory); Sarcoma 180 and example, ferrous sulfate is autoxidized by the Ehrlich ascites tumor, both obtained from Dr. H. action of x-rays as well as by dyes and visible T. Sugiura at the Sloan-Kettering Institute and light; viruses can be inactivated by the action of both grown in ~0-gram Swiss mice from the both agents when oxTgen is present. Since some pathogen-free colony at the State University of tumor cells are susceptible to the action of ioniz- New York Downstate Medical Center; mammary ing radiation when oxygen is present, they might adenocarcinoma, dbrB, grown in DBA/1 mice, likewise be inactivated by photodynamic action. both obtained from the Jackson Laboratory. * Public Health Service Research Fellow of tile National Dyes (histological grade) were obtained from Cancer Institute. Eastman Kodak Company. The dye concentra- Received for publication April 10, 1961. tions employed were of the order of 10-5 M, so that 1365

the small amounts of added salts which these com- containing 100 units penicillin/ml, as detailed mercial samples contained should not influence above. the results obtained. DETERMINATION OF CELL VIABILITY: PROCEDURES FOR PHOTODYNAMIC TUMOR-PRODUCING CAPACITY INACTIVATION The samples of 6C3HED and dbrB tumor cells In the case of solid mouse tumors (6C3HED and were tested for their tumor-producing capacity by dbrB).--Growths were removed with aseptic pre- subcutaneous injection, 0.5 cc. of each suspension cautions, and a suspension of tumor cells was pre- being injected into each of four mice in the right pared by pressing the tumor through a 40-mesh or left axilla or groin. The animals were examined Monel metal sieve into physiological saline ad- daily for signs of tumor growth, and the earliest justed to pH 7.4. The suspension was diluted day such growth became palpable was noted. The with the same medium to give suspensions con- increase in tumor size as measured by palpation taining 106 cells per ml. as determined by hemo- was charted until the mice died. On autopsy tu- cytometer counts. All cell suspensions were mor-bearing mice showed huge localized tumor routinely tested for bacterial contamination by masses at the sites of injection. Previous experi- culture in trypticase soy broth at 37 ~ C. Aliquots ments in our laboratory have shown by methods of the counted suspension were pipetted into four of serial dilution that, under the experimental sterile test tubes, to two of which were added ali- conditions outlined above, Gardner lymphoma quots of a ]0-ZM sterile stock solution in saline of cannot be successfully transplanted if fewer than the dye under study. The final dye concentration 100 viable cells are injected. Since we implanted was 10 -~ M, except in those experiments in which it 500,000 cells, the fact that in "inactivated" was desired to investigate the effect of various samples no tumor growth was noted in more than dye concentrations. A dye concentration of 80 days means that in such samples fewer than 10 -5 M was routinely employed, because this con- 0.0~ per cent of the cells could have survived centration gives an optical density of about one photodynamic action. The suspensions of Sarcoma for most dyes at their respective absorption 180 and of Ehrlich ascites tumor cells were tested maxima. Unit optical density gives nearly optimal by the intraperitoneal injection of 0.5 cc. (500,- photometric efficiency. Of the resulting cell sus- 000 cells) into each of four mice. The first signs of pensions two (with and without dye) were kept in tumor growth, determined by the appearance of the dark at room temperature as controls, and ascites, were usually noted in 4-5 days, and death two with and without dye were illuminated in test followed about 10 days later. tubes at a distance of 15 cm. from the front surface of a 500-watt TDC brand slide projector. 1 BINDING OF DYES TO TUMOR CELLS This projector has a built-in heat filter and was Fresh suspensions of 6C3HED cells, obtained equipped with a Corning Number 3070 filter to as noted above, were washed with pH 7.4 saline eliminate radiation below 400 mtt. There was no and resuspended to a concentration of 107 cells/ml detectable rise in temperature of the illuminated of suspension; 0.0~ ml. of 10-3M dyes were added samples. The samples were illuminated for 30 to duplicate ~-ml. aliquots of this suspension. minutes, except where otherwise noted. One sample was illuminated for 30 minutes, and The suspensions were then centrifuged in a one was kept as a dark control at room tempera- size 1, model CM International Centrifuge, with ture. Two other samples of the suspension, to a #s head, at 800 r.p.m, for 10 minutes, after which no dye had been added, served as illumi- which the ~sediments were resuspended in their nated and dark controls. After illumination all original volume of sterile saline containing 100 samples were centrifuged at 800 r.p.m, for 10 units penicillin/ml. minutes; the resulting supernatants (which were In the case of ascites tumors (Sarcoma 180, Ehr- lich ascites).--Tumor cell suspensions were har- not turbid) are referred to as S1. The sedimented vested 5 days after inoculation as milky-white sus- cells were washed with pH 7.4 saline, and the re- pensions, which were tested as above for bacterial sulting supernatant was labeled S~. By means of contamination. To diluted suspensions, containing a Zeiss spectrophotometer, the optical density at 106 cells per ml. of pH 7.4 saline, dye was added to the absorption maximum of each dye in question a concentration of 10 -5 M, and aliquots were illumi- was then determined in (a) the solution containing nated, centrifuged, and resuspended in saline only 10 -5 M dye in saline, (b) supernatant S1, and (c) supernatant S~. From the difference in optical 1 Manufactured by Bell and Howell Company, Chicago, Illinois. density of the appropriate solutions and the molar

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. BELLIN et al.--Photoinactivation of Tumor Cells 1367 extinction coefficient of the dye in question, the experiments are summarized in Table 3. With amount of dye bound to the cells was determined. 10 -5 M methylene blue or proflavine, 5 minutes' illumination gave only partial inhibition (100 per RESULTS cent takes, but after an increased latent period); Photodynamic inactivation of tumor ceUs.--In with 10 minutes of illumination at the same dye Table 1 are summarized experiments demonstrat- concentration complete tumor inhibition was ob- ing the photodynamic effect on 6C3HED tumor served. Rose bengal, as previously noted, had a cells of thirteen dyes which were chosen as repre- "dark effect," and was able to inactivate the tumor sentative members of the various families of dyes; cells even in the absence of light. Methylene blue all were used in 10 -5 M concentration. The values had a similar, but less pronounced effect: if, in- listed represent averages for eight to twelve de- stead of being washed after exposure, in order terminations. The standard error of these determi- that they be free of excess dye, the cells were in- nations varied from zero to +0.4. The first six jected in a suspension containing 10 -5 M methylene dyes listed were able to photosensitize the in- blue, tumor inhibition was noted. activation of tumor cells. It is evident that the participation of both dye and light is required for TABLE 1 the process of inactivation to occur: neither light PItOTODYNAMIC INACTIVATION OF TUMOR itself, nor the presence of dye per se, was able to CELLS in Vitro reduce the tumor-producing capacity of the cells. (Gardner Lymphosarcoma) In another experiment (data not illustrated) it was shown that irradiation of a solution of proflavine TUMOR VIABILITY in saline did not subsequently inactivate the cells. (Number of days before tumor Tumor cells which have been photoinactivated becomes palpable) differ from viable cells in much the same way as do cells which have been subjected to x-ray ir- DYE radiation (15): frequently the nuclei appear Without dye With dye shrunken and the cytoplasm swollen, with a con- siderable amount of "blebbing" of cytoplasmic I No Light lNht I_ ight light material. The next five dyes listed in Table 1 inactivated the tumor cells even in the absence of Eosin Y 11 12 -- 15 >30 Methylene blue l0 11 11 >30 light ("dark effect"), thus precluding the obser- Thionine 11 1~2 13 >30 vation of a possible photodynamic action; methyl Proflavine 9 10 10 >30 green produced some retardation in the absence of Acridine orange 11 12 1'2 >30 Neutral red 11 1~2 14 >30 light; and congo red was totally without effect. Rose bengal 11 11 >30 >30 The observed slight retardation in tumor growth Crystal violet II 12 >30 >80 produced in the absence of light by eosin Y, Thioflavine TG II 12 >30 >30 Janus green B ll ll >30 >30 thionine, and neutral red could not be quantita- 2,6-dichloroindophenol II I~ >30 >30 tively related to the percentage of tumor cells Methyl green II l! 18 >30 killed with our methods. Congo red ll II 1~2 1~2 In Table ~ are listed analogous experiments with Sarcoma 180, Ehrlich ascites, and dbrB Dyes used in 10 -~ M concentration; 500,000 tumor cells implanted. tumor cells. It can be seen that the dyes tested exhibited a photodynamic effect on this wide To test whether the presence of oxygen is variety of tumor cells just as they do in the case of necessary for the photodynamic inactivation of Gardner lymphoma: the photodynamic action, the tumor cells, attempts were made to eliminate therefore, is not confined to specific tumor cells. oxygen from the solution. Bubbling with nitrogen In contrast to observations on Gardner lymphoma denatures the cells, as shown by their inability to cells (cf. Table 1), rose bengal did not affect Ehr- cause tumor growth on transplantation. Therefore, lich ascites cells or Sarcoma 180 cells in the absence lymphoma cell suspensions in a Warburg ap- of light; proflavine had some "dark action" on paratus were flushed across the surface of the Ehrlich ascites cells. suspension with oxygen-free nitrogen for 45 In a series of experimenls with methylene blue, minutes prior to and during 15 minutes of subse- proflavine, and rose bengal used as sensitizers, it quent illumination. A photodynamic effect was was established that, in suspensions containing still observed, and control suspensions did not 10 6 cell per ml. and 10 -5 .~ dye, all the tumor cells show any reduced viability. This was presumably were inactivated in 10 minutes of exposure. These due to the fact that there was still enough oxygen

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. 1368 Cancer Research Vol. ~1, November 1961 available to permit photodynamic action to occur supernatant solution ($1) had an optical density (a concentration of only 5 )< 10 -6 M oxygen is suf- of 0.6~, from which we subtract 0.044, the optical ficient to allow for photodynamic action [18]). density of the supernatant of a cell suspension The capacity of the Gardner lymphoma cells to equilibrated with saline in the absence of dye. The bind dyes was investigated as detailed above. In optical density of the dye in the supernatant, the case of eosin Y, for instance, the optical therefore, was 0.578, representing a concentration density of a 10-SM solution was 0.840 at 516 m#. of 6.88 X 10 -s M dye. The amount of (lye absorbed After 80 minutes' equilibration in the dark the by the cells was 3.1~ X 10 -9 moles/ml (31.~ per

TABLE 2 PHOTODYNAMIC INACTIVATION OF TUMOR CELLS in Vitro (Other Tumor Types)

TL'MOR VIABILITY

(Av. survival of eight mice after tumor implant in da.ys)

TUMOR I)YE Without dye With dye

No light I,ight No light Light

Sarcoma 180 Thionine 15 14 14 >67 (500,000 cells, I.P.) Rose bengal 15 14 15 >67 Proflavine 14 14 14 >67

Ehrlich ascites Thionine 14 18 15 >35 (500,000 cells, I.P.) Methylene blue 14 18 lfZ >85 Rose bengal 14 13 14 >85 Proflavine 18 18 18 >85

Number of days untiltumor palpable Mammary a denocar- cinoma (dbrB) Thionine 11 10 14 >67 (~.6 X 10 Gcells s.c.) Eosin Y 11 10 11 >67

Dyes used in 10 -5 M concentration.

TABLE 8 PItOTODYNAMIC INACTIVATION ()F 6C8HED CELLS in Vitro: EFFECT OF VARYING EXPOSURE TIME AND DYE C{)NCENTRATION

EXPOSURE TO LIGHT (*IIN.)

CONCEN TRA TION o I 511o DY~ I 3ol (M) I I Tumor viability (days until tumor palpable)

None 9 m 10

Methylene blue 10-~ 9 14 >80 >80 >80 >80 s 10 >80 5X 10 -5 14 >80

Proflavine 10-5 10 18 >80 >30 >80 >80 ~. 5 X 10 -5 18 >80 5X10-5 10 >80

Rose bengal 10-5 >80 >80 >80 >80

500,000 tumor cells implanted; dashes indicate not tested.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. B ELLIN et al.--Photoinactivation of Tumor Cells 1869 cent of the dye in solution), or, since the suspen- methane dye), and thioflavine TG (a thiazole dye) sion contained 113.:3 X 106 cells/ml, 2.34 X 10 -~6 to act as photosensitizers, whereas janus green B moles were absorbed per cell. The results are sum- and ~,6-dichloroindophenol would not be expected marized in Table 4. "Vital" dyes, such as acridine to sensitize. However, the fact that these particu- orange, did not stain light-inactivated cells to any lar dyes are able to inactivate tumor cells in the greater extent than they did those cells which had absence of light precludes the observation of a been kept in the dark. Congo red (which in no possible photodynamic effect. It is possible that at way affects the tumor-producing capacity of the lower concentrations this dark effect might be cells) was able to stain the tumor cells at least as abolished. The literature contains many references well as did the photosensitizing dyes. (5, 1~) to the in w;tro effects of redox indicators

TABLE 4 BINDING OF DYES TO 6C3HED TUMOR CELLS

DYE REMAEglNG IN CELLS DYE EXTRACTED DrE ROUND AFTER ONE WASII IN ONE WASH PHOTO- DYNAMIC OXIDA TION (% of dye ACTION DYE (Moles dye bound/cell) X 10t6 REDUCTION originally bound) (+) on POTENTIAL , DARK AC- I riot" (D) Dark Light Dark Light Dark Light Ex )osed Exposed Exposed

I Eosin Y 2.34 2.75 1.24 1.78 47.0 35.3 + Methylene blue 5.66 3.67 5.32 2.19 6.0 40.4 + 9005 Thionine 3.29 4.60 1.75 2.85 46.8 38.1 + +. 063 Proflavine 4.6~2 4.42 3.55 3.96 23.2 10.4 + .5 Acridine orange 4.93 5.41 3.77 4.18 23.5 23.7 + Neutral red 5.18 6.17 5.55 4.82 4.4 21.8 + 9320 Rose bengal 7.04 6.03 6.36 6.02 10.2 0 D .15 Crystal violeL 5.85 5.88 , 4.38 4.39 25.1 25.4 D Thioflavine TG 2.66 2.43 D Janus green B 3.78 5.90 : 2.45 5.60 64.9 5.1 D I --. 275 Methyl green 5.76 6.31 5.02 5.79 12.9 8.~ D 2,6-Dichloroindophenol D + .19 Congo red 5.73 6.02 4.62 4.49 19.4 $5.4

DISCUSSION such as methylene blue, thionine, and o,,6-dichloro- The capacity of dyes to sensitize the phoLody- indophenol on the respiration and glycolysis of namic inactivation of tumor cells parallels their tumor and other tissues. It was stated (1~) that capacity to sensitize the photodynamic oxidation the redox potentials of the dyes are important in of organic amines (18) and of transforming prin- this respect. However, our results show no con- ciple (1). Thus, eosin Y (a xanthene dye), methyl- nection between the redox potential of the dyes ene blue and thionine (thiazine dyes), proflavine and their capacity to inactivate tumor cells in the and acridine orange (acridine dyes), and neutral dark. red (an azine dye) are able to inactivate photo- The in vivo tumor-inhibiting effect of Various dynamically para-toluenediamine and transform- dyes has becn investigated by many authors. ing principle as well as tumor cells. By contrast, Thus, Brooks (13) found that, of many dyes tested, congo red (an azo dye) is unable to photosensitize only methylene blue showed some tumor-inhibit- the inactivation of any of these substrates. As ing effect and that, since the injection of Ringer pointed out in previous publications (1, 18), the solution caused almost the same inhibition, this capacity of dyes to sensitize photodynamic in- effect was not very convincing. Lewis et al. (113, activation is correlated positively with their 14) and other authors have reported that, of capacity to undergo photoreduction and hence to many acridine compounds tested, acridine orange form electronically excited molecules in a long- was the most effective in causing tumor regression. lived metastable state. On the basis of previous Similarly, Goldie et al. (9) have found that the experiments (1, 18) one would expect rose bengal. addition of 10 -3 M acriitavine (of which proflavine (a xanthene dye), crystal violet (a triphenyl- is the major constituent) to Sarcoma 180 cells

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. 1870 Cancer Research Vol. ~1, November 1961 in vitro prevented their subsequent proliferation. oxygen complex (photoperoxide) (18); (b) oxida- The dye concentrations used in all these studies tion of the tumor cells as a consequence of their were many times higher than those employed here. electron-donating action to light excited dye mole- Nevertheless, it is worth noting that no such cules; (c) oxidation of the tumor cells by hydroxyl tumor-inhibiting effect of acridine orange or pro- radicals formed by (dark) oxidation of leuco dye flavine was observed by us. The tumor-inhibiting (17). effect of rose bengal which we have noted has The capacity of the dyes to bind to, and to been previously reported in the literature (r penetrate, the tumor cells may be a necessary but The capacity of the photosensitizing dyes to not sufficient condition for their capacity to inactivate tumor cells is a nonspecific phenome- photosensitize the inactivation of the cells (cf. non. Those dyes which act as scnsitizers do so on Table 4) : congo red (inactive as a photosensitizer) a wide variety of tumor cells (cf. Tables 1, ~). The is bound as strongly to the cells as is thionine (a photodynamic action takes place rapidly, since sensitizer). The values found for the amount of all the tumor cells in suspension are inactivated in dye binding to Gardner lymphoma cells are in the 10 minutes (cf. Table S). The addition of 10-5M same range as those reported for the binding of (lye (which gives an optical density of 1.36 in the rose bengal to erythrocytcs (6 X 10 -16 moles case of methylene blue, and of 0.~9 for proflavine bound per cell) (8), and for acriflavine to Ehrlich at their respective absorption maxima) is thus suf- ascites cells (~0 X 10 -16 moles bound per cell) (4). ficient to produce a rapid inactivation of the The capacity of cells to exclude certain dyes has tumor cells. Greater concentration of dye or a been used in measuring their viability (6, 7, ~0, more prolonged period of illumination is not ~). When Ehrlich ascites cells are inactivated by necessary. depletion or by treatment with poisons or viruses, In a simple chemical system photodynamic ac- their capacity to elicit tumor growth was dimin- tion is a photo-oxidation. In such a system con- ished at a greater rate than their capacity to taining, for example, an organic amine as a sub- exclude eosine and trypan blue. Pappenhcimer strate, this can easily be demonstrated, because in (~0) reported that, when hematoporphyrin and such a system the exclusion of ox57gen abolishes chlorophyll were used as sensifizers, the photo- the photodynamic effect. It can be shown that, dynamic injury of human lymphocytes greatly since a concentration of only 5 X ]0-SM oxygen increased their permeability to :Evans blue and is necessary for a maximum rate of photo-oxida- trypan blue. In all the above studies, dye con- tion, the dye molecules participating must be centrations used were 10-~ to 10 -3 M. We have long-lived species (18). The addition of a mild re- found no significant difference in the amount of ducing agent such as glutathione or cysteine tem- dyes bound to viable or to photoinactivated tumor porarily abolishes photo-oxidation of the amine, cells in solutions containing 10 -5 ~t dyes. We have because photo-oxidation of the more easily oxidiz- no explanation for the fact that in some cases the able reducing agent takes precedence over photo- dyes bound to light-exposed cells are readily oxidation of the amine. In the present system it is eluted with saline. This is true, for example, in not feasible to exclude trace amounts of oxygen the case of methylene blue and neutral red (cf. from the cells, and the addition of glutathione or Table 4). In other cases, e.g., janus green B, the cysteine in amounts which would be sufficient to reverse is true, and in yet other instances no dif- inhibit photodynamic inactivation of the tumor ferences are noted in dye binding strength between cells is injurious to the cells. light-exposed cells and dark controls. These obser- It seems probable that, during the photo- vations may, perhaps, be related to the affinities sensitized inactivation of tumor cells, photo- of dyes to specific cell constituents. autoxidation (involving the cells as the oxidized REFERENCES substrate) is not the only process taking place; in 1. BELLIN, J. S., and OSTER, G. Photodynamic Inactivation the case of very heavy cell suspensions (about 10 s of Transforming Principle. Biochim. et Biophys. acta, cells/ml) and containing 10 -5 M thionine or 42: 533-35, 1960. methylene blue, photobleaching of the dye is ob- ~. BLUM, H. F. Photodynamic Action and Diseases Caused by Light. New York: Reinhold Publishing Corp., 1941. served. Upon cessation of illumination the leuco 3. BnOOKS, M. M. The Effects of Methylene Blue and Other dye is re-oxidized by dissolved oxygen. It is prob- Redox Indicators on Experimental Tumors: Univ. Cal. able, therefore, that some or all of the following Publ. 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Judith S. Bellin, Steven C. Mohos and Gerald Oster

Cancer Res 1961;21:1365-1371.

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