JOURNAL
OF SURGICAL
RESEARCH
48, 337-340 (1990)
/n vitro Killing of Human Malignant Mesothelioma by Photodynamic Therapy’ STEVEN M. KELLER, M.D., DOUGLAS D. TAYLOR, PH.D., AND JAMES L. WEESE, M.D. The Fox Chase Caricer Center, Philadelphia, Presented at the Annual Meeting of the Association
for Academic Surgery, Louisville,
Photodynamic therapy was investigated as a potential new modality for the treatment of human malignant mesothelioma (HMM) utilizing the H-MESOHMM cell line and the photosensitizing agent, Photofrin-II (PF-II). Uptake of PF-II by H-MESOwas documented by incubating H-MESOcells with PF-II and measuring the flourescence at 625 nm following excitation at 400 nm. Cytotoxicity of photodynamic therapy was determined by incubating H-MESOcells (2 X 10’) in microtiter plates for 24 hr with concentrations of PF-II varying from 0 to 10 pg/ml. The wells were exposed to gold vapor laser light (628 nm) in doses ranging from 0 to 24,000 J/m’. Twenty-four hours following treatment, [3H]thymidine (1 @i) was added to each well. Cells were harvested 24 hr later and counted for tritium incorporation. Five replicates were performed for each combination of light and drug. Peak absorption of PF-II by HMESO- 1 was reached within 8 hr. Maximal doses of light alone caused minimal cell killing. PF-II without light was cytotoxic only at the highest concentrations. However, the combination of PF-II at concentrations at or above 2.5 pg/ml and light produced a significant increase in cytotoxicity. These data demonstrate that photodynamic therapy can effectively kill human malignant mesothe0 1990 Academic Press, Inc. lioma cells in uitro.
INTRODUCTION The causal relationship between asbestos exposure and pleural mesothelioma was first identified two decades ago [l-4]. The interval between exposure and disease may vary from 10 to 30 years [l-3, 5, 61. Because federal guidelines restricting industrial asbestos exposure are relatively recent [ 71, malignant mesothelioma will remain a serious or significant clinical problem into the next century. Current therapy has been disappointing. Mean survival from diagnosis ranges from 4 to 21 months [8,9]. Death usually results from pulmonary insufficiency caused by local extension [9]. Distant metastases occur in a minority 1 This work was supported by a grant from the American sociation.
Pennsylvania
Lung As-
337
19111 Kentucky,
November
15-l&1989
of patients late in the disease [9, lo]. Successful treatment or palliation of this disease requires control of disease in the pleural cavity. This study was designed to evaluate the sensitivity of human malignant pleural mesothelioma to photodynamic therapy (PDT). PDT utilizes a light-activated photosensitizer to produce tumor necrosis. PDT was first utilized for the treatment of human tumors early in this century [ 111. During the last two decades interest in PDT has been rekindled and its effectiveness documented in both laboratory experimentation and clinical trials. METHODS The human malignant mesothelioma cell line H-MESO 1 was obtained from Biomeasure, Inc. (Hopkinton, MA). H-MESOoriginated from a patient with documented asbestos exposure and a classic pleural mesothelioma [ 121. The tumor was established in cell culture utilizing Dulbecco’s modified Eagle’s media and 10% fetal bovine serum under 5% COZ humidified atmosphere. Photofrin II (PF-II) was obtained from Quadrilogic Technologies (Van Couver, BC). A gold vapor laser (Metalaser Technologies, Pleasanton, CA) was utilized to provide light at 628 nm, the wavelength necessary to activate PF-II. The laser light was transmitted through a quartz fiber and the output measured at the fiber tip in watts with a Laserguide power meter (Santa Barbara, CA). The duration of exposure time in seconds required to deliver the desired amount of light was determined by desired joules/laser
output in watts = exposure duration
in seconds.
Uptake of PF-II by H-MESOwas documented by plating 5 X lo5 H-MESOcells into 35mm wells and incubating with PF-II 10 pg ml-’ for intervals of 4, 8, 24, and 48 hr. The cells were harvested, washed, and sonicated twice (10 set each) in a Branson Model 185 sonifer cell disruptor. The cellular debris was centrifuged at 2000 rpm for 5 min and the supernatant assayed for flourescence in an Aminco-Bowman spectrophotofluorometer (American Instrument Co., Inc., Silver Spring, MD). An exci002%4804/90
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
338
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 48, NO. 4, APRIL
1990
20 10 1
ot \
\
**I
12.000 0
I
I
20.0
30.0
I 40.0
by H-MESO-
24,000
3.000 1.500
1
6,000
LIGHT
JIM*
50.0
HOURS
FIG. 1. Near maximum uptake of PF-II vitro is achieved following an 8-hr incubation.
10
cells in
tation wavelength of 400 nm was utilized and flourescence measured at 625 nm. Drug uptake was calculated by comparison of measured flourescence with a flourescence curve constructed from known concentrations of the drug. Cytotoxicity of PDT was assessed utilizing a 3H incorporation assay. H-MESOcells were placed on microtiter plates (2 X lo5 cell/well) and incubated for 24 hr with concentrations of PF-II varying from 0 to 10 pg ml-‘. The wells were exposed to gold vapor laser light (628 nm) in doses ranging from 0 to 24,000 J rnM2. Twenty-four hours following treatment 3H (1 &i) was added to each well. Cells were harvested 24 hr later and 3H incorporation was counted. Five replicates were performed for each combination of light and drug. Results are expressed as a percentage of [3H]thymidine incorporation of each drug and light combination compared to a no light and no drug control. A two-tailed Student’s t test for paired observations was utilized to calculate the significance of differences between the mean values of control and treated wells.
FIG. 2. Thymidine uptake vs drug and illumination. Three-dimensional representation of PDT effect on H-MESOcytotoxicity. Note dark toxicity of PF-II (see text).
drug and light dosages. Furthermore, the addition of more drug or light increases the cytotoxicity until there are virtually no viable cells remaining. A sustained statistical difference between the points illustrated in Fig. 2 and the no light-no drug controls was reached when the concentration of PF-II exceeded 1.25 fig/ml (Table 1). DISCUSSION
Once diagnosed, pleural mesothelioma is almost always fatal. Law et al. [13] reported on 116 patients with Stage I (limited to one hemithorax) malignant pleural mesothelioma who were assigned according to physician preference to receive either no treatment or aggressive treatment. Among the 52 aggressively treated patients, 28 underwent subtotal pleurectomy, 12 received chemotherapy, and 12 were treated with radiation therapy. The median survival was 20, 19, and 18 months respectively. The median survival of the 64 patients who received no treatment was 18 months. Although not randomized, this report calls
RESULTS TABLE
PF-II is rapidly absorbed by H-MESOcells (Fig. 1). Fifty percent of maximum uptake is attained by 2 hr. A plateau is reached after 8 hr with little increase occurring at 48 hr. Following a 24-hr incubation, the concentration of Photofrin II is 1.375 pg/5 X lo4 H-MESOcells. The cytotoxicity of light and drug administered individually, as well as when given in combination, is illustrated in Fig. 2. Light alone causes minimal killing of HMESOcells. Incubation of H-MESOcells with PFII alone results in cytotoxicity proportional to drug concentration (dark toxicity). However, the combination of PF-II and light results in increased cytotoxicity at all
Thymidine
Uptake
1
of H-MESO[Photofrin
following
PDT
II, mg/ml]
Light
(J/m’) 0
1,500 3,000 6,000 12,000 24,000
0
0.625
1.25
2.5
5
10
100 75 71 81 93 a2
106 65 58* 86 88 89
94 74 73 65 78 63*
85 65* 45 67’ 53* 32*
43* 32* 34* 29* 13* 5*
13’ I* 5* 3* 2* 1*
* P < 0.001, the two-tailed
Student t test.
KELLER,
TAYLOR,
AND
WEESE:
KILLING
into question the efficacy of even the most aggressive therapy. Truly, a new approach is necessary for the treatment of malignant pleural mesothelioma. Hematoporphyrin derivative and the more purified Photofrin II are the most commonly utilized photosensitizers. Both are impure compounds consisting of hematoporphyrin, dihematoporphyrin ester, and acetylatedhematoporphyrin products [ 141. The photosensitizer normally exists in the singlet (paired electron spin) ground state. Exposure to the appropriate wavelength of light (laser or filtered white light) results in production of a short-lived excited singlet state. Due to quantum mechanical considerations the molecule undergoes a spin inversion and assumes the more stable triplet state (unpaired electron spin) [ 151. A sensitizer in the excited triplet state is capable of producing radical compounds via two mechanisms [16]. The Type I reaction involves the excited sensitizer reacting directly with a substrate, exchanging either a hydrogen atom or an electron, and producing a radical. This charged radical then interacts with oxygen, producing an oxygen radical which in turn can accept protons to form hydrogen peroxide or hydroxy radicals. The former is known to cause chromosomal abnormalities [17]. Hydroxy radicals are highly reactive. Among their many documented biological effects is peroxidation of membrane lipids which results in increased permeability and cell lysis [ 171. The Type II reaction occurs when the excited sensitizer transfers its energy directly to an oxygen molecule producing singlet oxygen. The categories of singlet oxygen reactions with biologically crucial molecules have been summarized by Foote [ 161. Oxidation affects nucleic acids and converts sulfides to sulfoxides, olefins to hydroperoxides, and heterocycles to endoperoxides. This results in membrane lysis as well as chromosomal aberrations. Cell death is thought to be mediated by singlet oxygen produced as a result of a Type II reaction [18]. Damage to cellular membranes [19], mitochondria [20, 211, and cellular respiration [22] has been documented to occur following PDT. However, clonogenic assays demonstrate that tumor cells remain viable following exposure to tumoricidal doses of light and photosensitizer [23]. Tumor necrosis correlates best with PDT-induced destruction of tumor vasculature [23-251. Damage to the microfibrils of tumor capillary subendothelium has been demonstrated [26]. Oxygen concentration appears to play a major role in PDT-mediated cell death. Hypoxic cells are not killed as efficiently by PDT as those exposed to higher oxygen concentrations [27-301. Dark toxicity, cell killing as a result of exposure to a photosensitizing agent in the absence of directed light, has been noted by a number of investigators [31,32]. The mechanism(s) has not been elucidated but may be related to activation of the drug by ambient light or a direct toxic effect of the drug [ 331. PDT has proved useful in treating human cutaneous and visceral malignancies. Complete responses in such varied tumors as metastatic breast cancer, basal cell car-
OF HUMAN
339
MESOTHELIOMA
cinema, Kaposi’s sarcoma, and Bowen’s disease have been documented. In esophageal and lung cancers, PDT has found a role in both relieving obstruction and providing an alternative form of treatment in medically inoperable and surgically incurable but obstructed patients [ 141. The therapy of intraluminal carcinoma in situ has also been reported [34]. Other human tumors treated with PDT include multicentric superficial bladder tumors, cancers of the head and neck, brain tumors, and intraocular tumors. Prior to proposing a clinical trial of PDT on human malignant mesothelioma, the following criteria must be demonstrated: (1) in vitro effectiveness of PDT; (2) successful in uivo therapy utilizing an animal model; (3) the ability to deliver adequate doses of light to all surfaces of the pleural cavity; (4) lack of PDT-induced toxicity to thoracic organs. This report represents the first documentation of in vitro cytotoxicity of PDT on human malignant mesothelioma. The H-MESOcell line grows well in the flank of nude mice and, therefore, provides an opportunity to assess the in uiuo effects of PDT. Early evidence indicates that surface [35-361 illumination of the tumor can result in tumor necrosis. Uniform tumoricidal light doses have been achieved in the smaller and more regularly shaped urinary bladder by diffusing the light in liquid media [37]. Whether this can be achieved in the larger irregular pleural space remains to be demonstrated. The intraabdominal organs seem unaffected by therapeutic PDT [38]. However, no data exist on the PDT tolerance of lung parenchyma, heart, esophagus, and diaphragm. Studies to answer these remaining questions are in progress and will hopefully allow a Phase I human study to follow.
REFERENCES
1.
Newhouse, M. L., and Thompson, H. Mesothelioma of pleura and peritoneum following exposure to asbestos in the London area. Brit. J. Inch. Med. 22: 261, 1965.
2.
Selikoff, I. J., Churg, J., Hammond, neoplasia. JAMA 188: 142, 1964.
3.
Selikoff, I. J., Churg, J., and Hammond, exposure to asbestos and mesothelioma. 560,1965.
4.
Wagner, J. C., Sleggs, C. A., and Marchand, P. Diffuse pleural mesothelioma and asbestos exposure in the North Western Cape Province. &it. J. Znd. Med. 17: 260, 1960.
5.
Selikoff, I. J. Lung cancer and mesothelioma during prospective surveillance of 1249 asbestos insulation workers, 1963-1974. Ann. N. Y. Acad. Sci. 271: 448, 1976.
6.
Whitwell, F., and Rawcliffe, R. M. Diffuse malignant pleural mesothelioma and asbestos exposure. Thorax 26: 6, 1971.
7.
Antman, K. H., and Corson, 3. M. Benign and malignant mesothelioma. Clin. Chest Med. 6: 127, 1985.
8.
Martini, N., McCormack, P. M., Bains, M. S., Kaiser, L. R., Burt, M. E., and Hilaris, B. S. Pleural mesothelioma. Ann. Thorac. Surg. 43: 113, 1987.
E. C. Asbestos exposure and E. C. Relation between N. Engl. J. Med. 272:
pleural
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9.
Antman, K. H., Blum, R. H., Greenberger, J. S., Flowerdew, G., Skarin, A. T., and Canellos, G. P. Multimodality therapy for malignant mesothelioma based on a study of natural history. Amer. J. Med. 68: 356,198O.
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Elmes, P. C., and Simpson, M. J. C. The clinical sothelioma. Q. J. Med. 45: 427, 1976.
11.
Tappeiner, H., and Jesionek. Therapeutische Versuche mit Fluoreszierenden Stoffen. Muench. Med. Wochenschr 1: 2042, 1903.
12.
Reale, F. R., Griffin, T. W., Compton, J. M., Graham, S., Townes, P. L., and Bogden, A. Characterization of a human malignant mesothelioma cell line (H-MESO-1): A biphasic solic and ascitic tumor model. Cancer Res. 47: 3199, 1987.
13.
25.
aspects of me-
Law, M. R., Gregor, A., Hodson, M. E., Bloom, H. J. G., and TurnerWarwick, M. Malignant mesothelioma of the pleura: A study of 52 treated and 64 untreated patients. Thorax 39: 255, 1984. of malignant
24.
26. 27.
28.
14.
Dougherty, T. J. Photosensitization Surg. Oncol. 2: 24, 1986.
tumors. Semin.
15.
Cannistraro, S., Jori, G., and Van De Vorst, A. Quantum yield of electron transfer and of singlet oxygen production by porphyrins: An ESR study. Photobiochem. Photobiophys. 3: 353, 1982.
16.
Foote, C. S. Mechanisms of photooxygenation. In D. R. Doiron and C. J. Gomer (Eds.), Phorphyrin Localization and Treatment of Tumors. New York: A. R. Liss, 1984. Pp. 3-18.
30.
17.
Grisham, M. D., and McCord, J. Chemistry and cytotoxicity of reactive oxygen metabolites. In A. E. Taylor, S. Matalon, and P. A. Ward (Eds.), Physiology of Oxygen Radicals. Baltimore: Williams & Wilkins, 1986. Pp. 1-8.
31.
29.
32.
18.
Weishaupt, K. R., Gomer, C. J., and Dougherty, T. J. Identification of singlet oxygen as the cytotoxic agent in photo-inactivation of a murine tumor. Cancer Res. 36: 2326,1976.
19.
Kessel, D. Sites of photosensitization by derivatives porphyrin. Photo&em. Photobiol. 44: 489, 1986.
20.
Singh, G., Jeeves, W. P., Wilson, B. C., and Jang, D. Mitochondrial photosensitization by Photofrin II. Photochem. Photobiol. 46: 645, 1987.
34.
Hilf, R., Musant, R. S., Narayanan, U., and Gibson, S. L. Relationship of mitochondrial fraction of cellular adenosine triphosphate levels to hematoporphyrin derivative-induced photosensitization in R3230 AC mammary tumors. Cancer Res. 46: 211,1986.
35. 36.
Hilf, R., Gibson, S. L., Penney, D. P., et al. Early biochemical responses to photodynamic therapy monitored by NMR spectroscopy. Photo&em. Photobiol. 46: 809, 1987.
37.
21.
22.
23.
of hemato-
Henderson, B. W., Waldow, S. M., Mang, T. S., et al. Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy. Cancer Res. 46: 572, 1985.
33.
38.
VOL. 48, NO. 4, APRIL
1990
Selman, S. H., Kreimer-Birnbaum, M., Klauny, J. E., et al. Blood flow in transplantable bladder tumors treated with hematoporphyrin derivative and light. Cancer Res. 44: 1924, 1984. Star, W. M., Marijnissen, H. P., van den Berg Blok, A. E., et al. Destruction of rat mammary tumor and normal tissue microcirculation by hematophorphyrin derivative photoradiation observed in vivo in sandwich observation chambers. Cancer Res. 46: 2532, 1986. Nelson, J. S., Liaw, L. H., and Berns, M. W. Tumor destruction in photodynamic therapy. Photochem. Photobiol. 46: 829, 1987. Mitchell, J. B., McPherson, S., DeGraff, W., Gamson, J., Zabell, A., and Russo, A. Oxygen dependence of hematoporphyrin derivative-induced photoinactivation of Chinese hamster cells. Cancer Res. 46: 2008,1985. Moan, J., and Sommer, S. Oxygen dependence of the photosensitizing effect of hematoporphyrin derivative in NHIK 3025 cells. Cancer Res. 45: 1608,1985. Gibson, S. L., and Hilf, R. Interdependence of fluence, drug dose and oxygen on hematoporphyrin derivative induced photosensitization of tumor mitochondria. Photochem. Photobiol. 42: 367, 1985. Henderson, B. W., and Fingar, V. H. Relationship of tumor hypoxia and response to photodynamic treatment in an experimental mouse tumor. Cancer Res. 47: 3110, 1987. Gomer, C. J., Rucker, N., and Murphree, A. L. Differential cell photosensitivity following porphyrin photodynamic therapy. Cancer Res. 46: 4539, 1988. Girotti, A. L., and Hussa, R. 0. Phototoxic effects of hematoporphyrin derivative and its chromatographic fractions on hormone producing human malignant trophoblast cells in vitro. In D. Kessel (Ed.), Methods in Porphyrin Photosensitization. New York: Plenum, 1985. Pp. 1299145. Boekelheide, K., Eveleth, J., Tatum, A. H., and Winkelman, J. W. Microtubule assembly inhibition by porphyrins and related compounds. Photo&em. Photobiol. 46: 657, 1987. Cortese, D. A., and Kinsey, J. H. Hematoporphyrin derivative phototherapy in the treatment of bronchogenic carcinoma. Chest 86: 8, 1984. Keller, S. M., Taylor, D. D., and Weese, J. L. Unpublished data. Feins, R. H., Hilf, R., Ross, H., and Gibson, S. Photodynamic therapy for the treatment of human malignant mesothelioma in a nude mouse model. J. Surg. Res. 48. Star, W. M., Marijnissen, H. P. A., Jansen, H., Keijzer, M., and van Gemert, M. J. Light dosimetry for photodynamic therapy by whole bladder wall irradiation. Photo&em. Photobiol. 46: 619,1987. Dougherty, T. Photodynamic therapy. In H. R. Withers and L. J. Peters (Eds.), Medical Radiology Innovations in Radiation Oncology. Berlin/Heidelberg: Springer-Verlag, 1988. P. 177.
OF SURGICAL
RESEARCH
48, 337-340 (1990)
/n vitro Killing of Human Malignant Mesothelioma by Photodynamic Therapy’ STEVEN M. KELLER, M.D., DOUGLAS D. TAYLOR, PH.D., AND JAMES L. WEESE, M.D. The Fox Chase Caricer Center, Philadelphia, Presented at the Annual Meeting of the Association
for Academic Surgery, Louisville,
Photodynamic therapy was investigated as a potential new modality for the treatment of human malignant mesothelioma (HMM) utilizing the H-MESOHMM cell line and the photosensitizing agent, Photofrin-II (PF-II). Uptake of PF-II by H-MESOwas documented by incubating H-MESOcells with PF-II and measuring the flourescence at 625 nm following excitation at 400 nm. Cytotoxicity of photodynamic therapy was determined by incubating H-MESOcells (2 X 10’) in microtiter plates for 24 hr with concentrations of PF-II varying from 0 to 10 pg/ml. The wells were exposed to gold vapor laser light (628 nm) in doses ranging from 0 to 24,000 J/m’. Twenty-four hours following treatment, [3H]thymidine (1 @i) was added to each well. Cells were harvested 24 hr later and counted for tritium incorporation. Five replicates were performed for each combination of light and drug. Peak absorption of PF-II by HMESO- 1 was reached within 8 hr. Maximal doses of light alone caused minimal cell killing. PF-II without light was cytotoxic only at the highest concentrations. However, the combination of PF-II at concentrations at or above 2.5 pg/ml and light produced a significant increase in cytotoxicity. These data demonstrate that photodynamic therapy can effectively kill human malignant mesothe0 1990 Academic Press, Inc. lioma cells in uitro.
INTRODUCTION The causal relationship between asbestos exposure and pleural mesothelioma was first identified two decades ago [l-4]. The interval between exposure and disease may vary from 10 to 30 years [l-3, 5, 61. Because federal guidelines restricting industrial asbestos exposure are relatively recent [ 71, malignant mesothelioma will remain a serious or significant clinical problem into the next century. Current therapy has been disappointing. Mean survival from diagnosis ranges from 4 to 21 months [8,9]. Death usually results from pulmonary insufficiency caused by local extension [9]. Distant metastases occur in a minority 1 This work was supported by a grant from the American sociation.
Pennsylvania
Lung As-
337
19111 Kentucky,
November
15-l&1989
of patients late in the disease [9, lo]. Successful treatment or palliation of this disease requires control of disease in the pleural cavity. This study was designed to evaluate the sensitivity of human malignant pleural mesothelioma to photodynamic therapy (PDT). PDT utilizes a light-activated photosensitizer to produce tumor necrosis. PDT was first utilized for the treatment of human tumors early in this century [ 111. During the last two decades interest in PDT has been rekindled and its effectiveness documented in both laboratory experimentation and clinical trials. METHODS The human malignant mesothelioma cell line H-MESO 1 was obtained from Biomeasure, Inc. (Hopkinton, MA). H-MESOoriginated from a patient with documented asbestos exposure and a classic pleural mesothelioma [ 121. The tumor was established in cell culture utilizing Dulbecco’s modified Eagle’s media and 10% fetal bovine serum under 5% COZ humidified atmosphere. Photofrin II (PF-II) was obtained from Quadrilogic Technologies (Van Couver, BC). A gold vapor laser (Metalaser Technologies, Pleasanton, CA) was utilized to provide light at 628 nm, the wavelength necessary to activate PF-II. The laser light was transmitted through a quartz fiber and the output measured at the fiber tip in watts with a Laserguide power meter (Santa Barbara, CA). The duration of exposure time in seconds required to deliver the desired amount of light was determined by desired joules/laser
output in watts = exposure duration
in seconds.
Uptake of PF-II by H-MESOwas documented by plating 5 X lo5 H-MESOcells into 35mm wells and incubating with PF-II 10 pg ml-’ for intervals of 4, 8, 24, and 48 hr. The cells were harvested, washed, and sonicated twice (10 set each) in a Branson Model 185 sonifer cell disruptor. The cellular debris was centrifuged at 2000 rpm for 5 min and the supernatant assayed for flourescence in an Aminco-Bowman spectrophotofluorometer (American Instrument Co., Inc., Silver Spring, MD). An exci002%4804/90
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
338
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 48, NO. 4, APRIL
1990
20 10 1
ot \
\
**I
12.000 0
I
I
20.0
30.0
I 40.0
by H-MESO-
24,000
3.000 1.500
1
6,000
LIGHT
JIM*
50.0
HOURS
FIG. 1. Near maximum uptake of PF-II vitro is achieved following an 8-hr incubation.
10
cells in
tation wavelength of 400 nm was utilized and flourescence measured at 625 nm. Drug uptake was calculated by comparison of measured flourescence with a flourescence curve constructed from known concentrations of the drug. Cytotoxicity of PDT was assessed utilizing a 3H incorporation assay. H-MESOcells were placed on microtiter plates (2 X lo5 cell/well) and incubated for 24 hr with concentrations of PF-II varying from 0 to 10 pg ml-‘. The wells were exposed to gold vapor laser light (628 nm) in doses ranging from 0 to 24,000 J rnM2. Twenty-four hours following treatment 3H (1 &i) was added to each well. Cells were harvested 24 hr later and 3H incorporation was counted. Five replicates were performed for each combination of light and drug. Results are expressed as a percentage of [3H]thymidine incorporation of each drug and light combination compared to a no light and no drug control. A two-tailed Student’s t test for paired observations was utilized to calculate the significance of differences between the mean values of control and treated wells.
FIG. 2. Thymidine uptake vs drug and illumination. Three-dimensional representation of PDT effect on H-MESOcytotoxicity. Note dark toxicity of PF-II (see text).
drug and light dosages. Furthermore, the addition of more drug or light increases the cytotoxicity until there are virtually no viable cells remaining. A sustained statistical difference between the points illustrated in Fig. 2 and the no light-no drug controls was reached when the concentration of PF-II exceeded 1.25 fig/ml (Table 1). DISCUSSION
Once diagnosed, pleural mesothelioma is almost always fatal. Law et al. [13] reported on 116 patients with Stage I (limited to one hemithorax) malignant pleural mesothelioma who were assigned according to physician preference to receive either no treatment or aggressive treatment. Among the 52 aggressively treated patients, 28 underwent subtotal pleurectomy, 12 received chemotherapy, and 12 were treated with radiation therapy. The median survival was 20, 19, and 18 months respectively. The median survival of the 64 patients who received no treatment was 18 months. Although not randomized, this report calls
RESULTS TABLE
PF-II is rapidly absorbed by H-MESOcells (Fig. 1). Fifty percent of maximum uptake is attained by 2 hr. A plateau is reached after 8 hr with little increase occurring at 48 hr. Following a 24-hr incubation, the concentration of Photofrin II is 1.375 pg/5 X lo4 H-MESOcells. The cytotoxicity of light and drug administered individually, as well as when given in combination, is illustrated in Fig. 2. Light alone causes minimal killing of HMESOcells. Incubation of H-MESOcells with PFII alone results in cytotoxicity proportional to drug concentration (dark toxicity). However, the combination of PF-II and light results in increased cytotoxicity at all
Thymidine
Uptake
1
of H-MESO[Photofrin
following
PDT
II, mg/ml]
Light
(J/m’) 0
1,500 3,000 6,000 12,000 24,000
0
0.625
1.25
2.5
5
10
100 75 71 81 93 a2
106 65 58* 86 88 89
94 74 73 65 78 63*
85 65* 45 67’ 53* 32*
43* 32* 34* 29* 13* 5*
13’ I* 5* 3* 2* 1*
* P < 0.001, the two-tailed
Student t test.
KELLER,
TAYLOR,
AND
WEESE:
KILLING
into question the efficacy of even the most aggressive therapy. Truly, a new approach is necessary for the treatment of malignant pleural mesothelioma. Hematoporphyrin derivative and the more purified Photofrin II are the most commonly utilized photosensitizers. Both are impure compounds consisting of hematoporphyrin, dihematoporphyrin ester, and acetylatedhematoporphyrin products [ 141. The photosensitizer normally exists in the singlet (paired electron spin) ground state. Exposure to the appropriate wavelength of light (laser or filtered white light) results in production of a short-lived excited singlet state. Due to quantum mechanical considerations the molecule undergoes a spin inversion and assumes the more stable triplet state (unpaired electron spin) [ 151. A sensitizer in the excited triplet state is capable of producing radical compounds via two mechanisms [16]. The Type I reaction involves the excited sensitizer reacting directly with a substrate, exchanging either a hydrogen atom or an electron, and producing a radical. This charged radical then interacts with oxygen, producing an oxygen radical which in turn can accept protons to form hydrogen peroxide or hydroxy radicals. The former is known to cause chromosomal abnormalities [17]. Hydroxy radicals are highly reactive. Among their many documented biological effects is peroxidation of membrane lipids which results in increased permeability and cell lysis [ 171. The Type II reaction occurs when the excited sensitizer transfers its energy directly to an oxygen molecule producing singlet oxygen. The categories of singlet oxygen reactions with biologically crucial molecules have been summarized by Foote [ 161. Oxidation affects nucleic acids and converts sulfides to sulfoxides, olefins to hydroperoxides, and heterocycles to endoperoxides. This results in membrane lysis as well as chromosomal aberrations. Cell death is thought to be mediated by singlet oxygen produced as a result of a Type II reaction [18]. Damage to cellular membranes [19], mitochondria [20, 211, and cellular respiration [22] has been documented to occur following PDT. However, clonogenic assays demonstrate that tumor cells remain viable following exposure to tumoricidal doses of light and photosensitizer [23]. Tumor necrosis correlates best with PDT-induced destruction of tumor vasculature [23-251. Damage to the microfibrils of tumor capillary subendothelium has been demonstrated [26]. Oxygen concentration appears to play a major role in PDT-mediated cell death. Hypoxic cells are not killed as efficiently by PDT as those exposed to higher oxygen concentrations [27-301. Dark toxicity, cell killing as a result of exposure to a photosensitizing agent in the absence of directed light, has been noted by a number of investigators [31,32]. The mechanism(s) has not been elucidated but may be related to activation of the drug by ambient light or a direct toxic effect of the drug [ 331. PDT has proved useful in treating human cutaneous and visceral malignancies. Complete responses in such varied tumors as metastatic breast cancer, basal cell car-
OF HUMAN
339
MESOTHELIOMA
cinema, Kaposi’s sarcoma, and Bowen’s disease have been documented. In esophageal and lung cancers, PDT has found a role in both relieving obstruction and providing an alternative form of treatment in medically inoperable and surgically incurable but obstructed patients [ 141. The therapy of intraluminal carcinoma in situ has also been reported [34]. Other human tumors treated with PDT include multicentric superficial bladder tumors, cancers of the head and neck, brain tumors, and intraocular tumors. Prior to proposing a clinical trial of PDT on human malignant mesothelioma, the following criteria must be demonstrated: (1) in vitro effectiveness of PDT; (2) successful in uivo therapy utilizing an animal model; (3) the ability to deliver adequate doses of light to all surfaces of the pleural cavity; (4) lack of PDT-induced toxicity to thoracic organs. This report represents the first documentation of in vitro cytotoxicity of PDT on human malignant mesothelioma. The H-MESOcell line grows well in the flank of nude mice and, therefore, provides an opportunity to assess the in uiuo effects of PDT. Early evidence indicates that surface [35-361 illumination of the tumor can result in tumor necrosis. Uniform tumoricidal light doses have been achieved in the smaller and more regularly shaped urinary bladder by diffusing the light in liquid media [37]. Whether this can be achieved in the larger irregular pleural space remains to be demonstrated. The intraabdominal organs seem unaffected by therapeutic PDT [38]. However, no data exist on the PDT tolerance of lung parenchyma, heart, esophagus, and diaphragm. Studies to answer these remaining questions are in progress and will hopefully allow a Phase I human study to follow.
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