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The effect of glucose administration on the uptake of Photofrin II in a human tumor xenograft
Cancer Letters, 58 (1991)
29
29-35
Elsevier Scientific Publishers Ireland Ltd.
The effect of glucose administration on the uptake of Photofrin II in a human tumor xenograft Q. Peng,
J. Moan and L-S. Cheng
Department
of Biophysics,
(Received 8 January (Revision received (Accepted
5 March
Institute for Cancer Research,
Montebello,
Oslo (Norway)
1991)
5 March
1991)
1991)
Summary
Introduction
Athymic BALB/c nude mice, bearing a human melanoma LOX, were given the photosensitizing drug Photofrin 11 (10 mg/kg body wt.) intraperitoneally. The mice were also given one of the following chemicals intraperitoneally: glucose, galactose and glucose plus nordihydroguaiaretic acid (NDGA) which is an inhibitor of glycolysis. Multiple injections of glucose (3 g/kg body wt. given at - 1, 0, + 1 and + 3 h relative to the injection of 10 mg/kg of Photofrin II at time 0) resulted in a significant increase in the uptake of Photofrin II
Photofrin II (P-II) is a porphyrin photosensitizer which is being used in clinical trials of photodynamic therapy (PDT) of cancer [l-5]. It is selectively taken up and/or retained in tumors compared to many normal tissues where tumors frequently grow [6-71. The reason for the high tumor uptake of P-II is not known. However, it has been proposed that the low pH value of tumors may play a role [&lo]. In vitro haematoporphyrin and many of its derivatives may get protonated and thus more lipophilic when the pH in its surroundings is reduced from 7.4 to 6.5. This results in an increased cellular uptake of the dye [8, ll- 131. Furthermore, it is also well known that the pH in the interstitium is significantly lower in tumor than in nonmalignant tissues. This is due to the fact that tumor tissue, being usually poorly vascularised, has to provide a significant fraction of its energy through anaerobic glycolysis, leading to production of lactic acid [8,14- 151. This may offer an explanation for the high tumor uptake of P-II. It is possible to test this hypothesis by injecting glucose in tumorbearing animals, which is known to be able to reduce the extracellular pH in tumor tissue selectively [14,16- 181. Indeed, such an experiment has been carried out with rhabdomyosarcoma-
in the tumor
4 h after a Photofrin
II injection,
while the uptake of Photofrin II in the other tissues remained unchanged. Administration of galactose had no significant effect on the uptake of Photofrin II in the tissues studied (tumor, muscle, skin and liver). NDGA seemed to abolish the effect of glucose injection.
photodynamic Keywords: Photofrin II; glucose; glycolysis; inhibitor; nordihydroguaiaretic acid
Correspondence Institute.. for
to:
Cancer
Q.
Per-g,
Research,
Norway.
0304-3835/91/$03.50
0
1991
Published and Printed in Ireland
therapy; glycolysis
Department
of
Montebello,
0310
Biophysics, Oslo
3,
Elsevier Scientific Publishers Ireland Ltd.
30
bearing rats [19]. It was found that multiple 3 g/kg injections of glucose resulted in a 3.4 times increase in the porphyrin fluorescence of the tumor tissue 48 h after an injection of 10 mg/kg of HPD-A (the tumorlocalising fraction of hematoporphyrin derivative). The liver uptake of HPD-A was not influenced by the glucose injection. We found these observations so interesting and clinically relevant that we decided to perform similar experiments with a human tumor model in nude mice. In the present work we report the results from the experiments of injecting P-II combined with either glucose, galactose or glucose plus a glycolysis inhibitor nordihydroguaiaretic acid (NDGA) in nude mice carrying the human melanoma tumor LOX. Materials
and Methods
Chemicals P-II was obtained as a gift from Lederle Inc. (U.S.A.) as an aqueous solution at a concentration of 2.5 mg/ml and stored at -20°C. HPLC runs to test the composition of P-II was carried out as described elsewhere [20]. All other chemicals used were of the highest purity commercially available. Animals Female BALB/c athymic nude mice were purchased from the Laboratory Breeding and Research Center, Gl. Bomholt Gaard, Ry., Denmark. At the start of the experiments the mice were 7-8 weeks old weighing 18-25 g. Three mice were housed per cage with autoclaved filter covers in a room with subdued light at constant temperature (24”26°C) and humidity (30-50%). Food and bedding were sterilized and the mice were given tap water ad libitum in sterilized bottles. Tumor model The human tumor line LOX was established in 1987 as an S.C. xenograft in nude mice from an amelanotic axillary lymph node metastasis
of a patient with malignant melanoma [21]. The LOX cells have shown a remarkable stability and similarity to the cells of the patient’s tumor with respect to morphology, karyotype and chemosensitivity. The LOX tumor was propagated by serial transplantation into BALB/c nude mice. Nonnecrotic tumor material for inoculation was obtained by sterile dissection of large flank tumors from syngeneic nude mice. Macroscopically viable tumor tissue was gently minced with a pair of scissors and forced repeatedly through an 20-gauge sterile needle to form a tumor tissue suspension, 0.02 ml of which was injected S.C. on the right flank of the mice. The rate of successful transplantation in this study was 100%. Minimal or no spontaneous necrosis was found in tumors with a transverse diameter of 6-8 mm. When the tumor had reached an appropriate size (as indicated above) 10 mg/kg body weight of P-II was administrated i.p. to each tumor-bearing mouse. At the following time points, - 1, 0, + 1, + 3 h (relative to injection of P-II at zero time), the animals received separate i.p. injections of glucose (40%, w/v) or of galactose (40%) w/v) (3.0 g/kg body wt. for both at each time point). Some tumorbearing mice were given only 10 mg/kg of PII. One group of animals were killed 4 h after P-II injection. In a separate group injections were as for the 4 h-group but followed by injections at + 5, + 7 and + 10 h before killing after 48 h. Furthermore, in two other animal groups only single injections of glucose (3.0 g/kg body wt.) or multiple doses of glucose plus a single dose of a glycolysis inhibitor, NDGA (0.4 g/kg body wt.) [22,23]. NDGA, which was dissolved in a mixture of cremophor EL and Dulbecco’s phosphate-buffered saline (PBS, 1:6 by vol.) and sonicated for 2 min before use, was given i.p. at the same time as injection of P-II. The following tissues were sampled either 4 or 48 h after P-II injection: tumor LOX, muscle tissue surrounding the tumor, skin overlying the tumor and liver tissue. Immediately after
31
collection the tissue samples were rinsed twice in PBS, blotted dry on clean paper, weighed and stored at - 20°C until used for determination of P-II concentration.
of the emission spectra was unchanged by the addition of internal standard; only the fluorescence intensity increased. The concentrations are given in pg P-II per g wet tissue.
Determination
Results
of P -11 concentrations
in tissues
The tissue samples were thawed and brought into suspension by means of a Ystral mechanical homogeniser (Dottingen, West Germany). The tissue suspension was finally homogenized once more by means of the Potter Elvehjem homogenizer. This procedure was judged to be better than just using the Potter Elvehjem homogenizer alone. Several solvents were tried for extracting the drug, and the solvent that brought a maximum of the drug into the supernatant was chosen as earlier described [6]. A solution of 1% sodium dodecylsulfate in 1 N perchloric acid/methanol (1: 1, v/v) was chosen in the present study t241. After homogenization the tissue suspensions were frozen, thawed, sonicated for 30 s, diluted 1:lOO in the same solvent, sonicated once more, centrifuged at 3000 rev./min for 10 min and finally the supernatants were collected. As judged by absorption and fluorescence measurements, it was possible to extract more than 75% of P-II from the tissue suspension with this method. Drug levels in the supernatants were quantitatively determined by recording fluorescence emission spectra of the samples using a PerkinElmer LS-5 Luminescence Spectrofluorimeter. The excitation wavelength was 404 nm, the slit width was to give a resolution of 5.0 nm, and the emission wavelength was scanned from 550 to 700 nm. A cut-off filter was used to remove scattered light of wavelengths shorter than 545 nm from the light reaching the detection system of the spectrometer. The background fluorescence from control samples was subtracted. P-II levels in the samples were determined by adding a known amount of the drug, comparable to that already present in the extraction medium, and recording the emission spectra once more. In all cases the shape
The sampling times as well as the dose of PII were selected after a series of introductory experiments had been carried out. Figure 1 shows the kinetics of the accumulation of P-II in the tumor LOX and in the liver. In agreement with earlier work [25] the concentration of the dye in the liver as well as in tumor approached the maximal values at 24-48 h (Fig. 1). In view of this and the work of Thomas and Girotti [19] the sampling times 4 h and 48 h were chosen in the present study. The concentrations of P-II in the tumor and liver were determined 48 h after injection of different drug doses (Fig. 2a and b). This experiment was performed in order to check that no saturation effects occurred at the drug dose of 10 mg/kg which was chosen for the further experiments. As demonstrated by Fig. 2, no saturation occurs since the tumor and liver up-
2‘
Time lhoursi
L8
12
96
after 1.p. injectton (IOmglkgl
Fig. 1. The kinetics of the accumulation and retention of P-11 in human LOX melanoma tumor tissue and in liver tissue of nude mice.
32
I5
I
.-S s J=-f wlow= g
I
,
I
I
-
Table 1. Effect of glucose injection on the selective uptake of P-II in tumor LOX xenografts.
I
Tumor LOX J+8h post-injection
0
.;
P-11 only /I
=‘; n-3
I
i
ZP5/
gs L r;
*/I
W
0.
0
I
1
I
I
1
I
5
IO
15
20
25
30
35
1.p. injected dose (mglkg) 80 -
Liver
P-II + galactose (multiple doses) P-II + glucose (single dose) P-II + glucose (multiple doses) P-II + glucose (multiple) + NDGA (single)
4h
48 h
2.47 zt 0.61 (6) 2.95 zt 0.16 (4) 3.42 zt 1.00 (5) 4.17”’ f 0.83 (10) 2.37 f 0.62 (5)
3.45
l
(6) 3.48
zt 0.32
(6) 3.75
zt 0.23
(5) 3.86
zt 1.06
(10) 3.22 (5)
0.58
zt 0.84
48 h post-injpcfmn 60
-
20 -
0 0
I
I
I
I
I
1
5
10
I5
20
25
30
35
1.p. injected dose (mglkg)
Fig. 2. The concentration of P-II in LOX tumors (a) and in livers (b) of nude mice 48 h after injection of different doses of P-II.
Tumor-bearing nude mice were given P-II i,p. 10 mg/kg and multiple doses of galactose (3 g/kg body wt. each time), single or multiple doses of glucose (3 g/kg body wt. each time), multiple doses of glucose plus a single dose of NDGA (0.4 g/kg body wt.) or without any injection as controls as described in Materials and Methods. Photofrin II accumulation in tumors were extracted at 4 and 48 h after administration. Indicated values are expressed as c(g P-II per g wet tumor tissue (mean f S.D.). Numbers of tumor-bearing animals in each group are shown in parentheses. Significant difference between each group and its time-corresponding P-II only control group was statistically analysed by Student’s t-Test. Significant difference only between the group (P-II + glucose, multiple doses at 4 h) and its time-corresponding P-II only control group was found (’ * lP c 0.01).
take is a linear function of the injected doses at least up to 30 mg/kg. The influence of injection(s) of glucose and galactose on the uptake of P-II in tumor, muscle, skin and liver is shown in Tables I-IV. None of the treatments had any influence on the porphyrin uptake in muscle, skin and liver. A single injection of glucose had a slight but not significantly potentiating effect on the tumor uptake of P-II at 4 h. Multiple injections of glucose had a significantly potentiating effect on the tumor uptake of P-II at 4 h but not at 48 h. NDGA seemed to have an inhibiting effect on the uptake of P-II by the tumor at 4 h after multiple doses of glucose compared to that in the group not receiving NDGA. This may be due to the inhibition of glycolysis by NDGA in the tumor.
Table II. Effect of glucose injection on the selective uptake of P-II in livers of nude mice bearing human tumor LOX. 4h P-II only P-II + galactose (multiple doses) P-II + glucose (single dose) P-II + glucose (multiple doses) P-II + glucose (multiple) +
14.97 (6) 16.56 (4) 16.07 (5) 16.40 (10) 12.12 (5)
48 h f
3.66
21.44
f 4.28
zt 4.17
(6) 23.03
zt 5.51
f
2.11
(6) 24.41
+z 3.29
C+Z 4.10
(5) 21.14
zt 6.37
f
0.51
(10) 17.50 (5)
ztz 3.22
NDGA (single) The experimental conditions legend to Table I.
are those
given
in the
33
Table 111. Effect of glucose injection on the selective uptake of P-II in muscle surrounding the tumor in nude mice. 48 h
4h P-II only P-II + galactose (multiple doses) P-II + glucose (single dose) P-H + glucose (multiple doses) P-II + glucose (multiple) +
0.58
+ 0.11
1.83
zt 0.39
zt 0.17
(6) 1.93 f
(4) 0.48
+ 0.12
(6) 1.99 zt 0.38
(5) 0.56
+ 0.18
(5) 2.0 f
f
(10) 1.75 f
(6) 0.62
(10) 0.49
0.13
0.41
0.53 0.44
(5)
(5)
NDGA (single) The experimental conditions legend to Table 1.
are those
given
in the
Discussion Following an injection of glucose, the feedback systems for control of blood glucose returns the glucose concentration rapidly back to the control level. Under normal conditions, the blood glucose concentration in a range is usually strictly controlled through increased insulin secretion and increased glucagon secreTable IV. Effect of glucose injection on the selective uptake of P-H in skin overlying human tumor LOX in nude mice.
P-II only P-II + galactose (multiple doses) P-II + glucose (single dose) P-II + glucose (multiple doses) P-II + glucose (multiple) +
4h
48 h
1.65 + 0.51
4.08
ztz 0.97
(6) 1.88 (4) 1.91 (5) 1.96 (10) 1.34 (5)
(6) 4.73
f
+ 0.60 f
0.68
ziz 0.57 zt 0.42
(6) 4.92 (5) 4.16 (10) 3.23 (5)
1.35
zt 1.50 + 1.68 ztz 0.94
NDGA (single) The experimental conditions legend to Table I.
are those
given
in the
tion. It has been shown that following an injection of glucose in rats bearing tumors, the blood glucose levels increased rapidly for about 4 h, and then returned to normal levels within about 6 h. Also the lactate concentrations in the tumors peaked about 4 h before returning to normal by 6 h [14,16,26]. Thomas and Girotti [19] observed that after an injection of glucose in tumorbearing rats, the pH in the tumor decreased for about 45 min and then returned to the control level within about 1 h. Since they allowed 32 h from the last glucose injection to the measurement of the HPD-A concentration in the tumor, one may tentatively propose that it is an increased uptake rather than an increased retention that constitutes the basis for their observation of the glucose-induced increase in the tumor content of the drug. Also our results show that glucose injection enhances the tumor uptake of P-II selectively (Table I). Quantitatively, however, our results are different from those of Thomas and Girotti [19]. They found that multiple glucose injections (carried out identically with our procedure) potentiated the tumor uptake of HPD-A at 48 h by a factor 3.4, while we found no potentiation at this time and a potentiating factor of only 1.7 at 4 h. Our results seem to indicate that the retention of P-II is lower in the tumor where the initial uptake was enhanced by glucose injection. Thus, at 4 h the tumor uptake of P-II was increased by 70% by glucose injection, while at 48 h no increase was observed. It may, therefore, seem that in our tumor model, glucose has an effect on the concentration of P-II in the tumor only as long as it has an effect on the pH in the tumor. The discrepancy between our results and those of Thomas and Girotti may be related to either the difference in species (mice vs. rats), tumor model (human melanoma vs. animal rhabdomyosarcoma), or tumor size (6-8 mm vs. 15-20 mm in diameter), but not to difference in the composition of the drug since the tumor-localizing fraction of HPD has been considered to be equivalent to P-II [27]. The fact that the size of the tumor used by Thomas and Girotti was 2-2.5 times larger than that
34
used by us may be of importance. Thus, the vascular drainage may decrease significantly with increasing size of the tumors. This may influence the microcirculation and the efficiency of interstitial transport in the tumor and thus affect both the availability of glucose to the tumor cells and the clearance of lactic acid. Galactose injection had no effect on the uptake of P-II in the tumor and the other tissues analysed (Tables I-IV). This is in good agreement with the work of Thomas et al. [19] and Calderwood et al. [ 161. The latter investigators found that galactose was not metabolized by tumors and did not result in production of lactic acid in the tumor. The abolishing effect of NDGA on the effect of glucose at 4 h further provides the evidence that hyperglycemia may promote selectively the uptake of porphyrins by tumors. Acknowledgements
8
9
10
Moan, J. and Christensen. T. (1981) Cellular uptake and photodynamic effect of hematoporphyrin. Photobiochem. Photobiophys.. 2.291-299. Bohmer. R.M. and Morstyn. G. (1985) Uptake of hematoporphyrin derivative by normal and malignant cells: Effect of serum, pH. temperature, and cell size. Cancer Res.. 45.5328-5334. Eden, M., Haines. B. and Kahler, H. (1955) The pH of rat
13
15
17 Moan, J. (1986) Porphyrin-sensitized photodynamic tivation of cells: a review. Lasers Med. Sci. 1.5-12.
inac-
2
Moan, J. (1986) Porphyrin photosensitization and phototherapy. Photochem. Photobiol., 43.681-690. Dougherty, T.J. (1989) Introduction, In: Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use,
18
pp. 1-3. Editors: G. Bock and S. Harnett. Wiley. Chichester (Ciba Foundation Symposium 146). Ash, D. and Brown, S.B. (1989) Photodynamic therapyachievements and prospects. Br. J. Cancer. 60,151
19
20
6
Gomer, C.J., Rucker. N.. Ferrario. A. and Wong. S. (1989) Review : Properties and application of photodynamic therapy. Radiat. Res.. 120. l-18. Peng, Q., Evensen. J-F.. Rimington. C. and Moan. J.
7
(1987) A comparison of different photosensitizing dyes with respect to uptake C3H-tumors and tissues of mice. Cancer Lett., 36. l-10. Belliner. D.A., Ho. Y-K, Pandey. R.K., Missert. J.R. and Dougherty, T.J. (1989) Distribution and elimination of Photofrin II in mice. Photochem. Photobiol 50.221228.
Pottier, R.H. (1990) Localization phenomena: pH effects. In: Photodynamic Therapy of Neoplastic Disease. Vol. Il. pp. 63-77. Editor: D. Kessel. CRC Press. Barrett, A.J.. Kennedy, J.C., Jones, R.A. and Pollier.
12
1
5
A.
Moan, J., Smedshammer. L. and Christensen. T. (1980) Photodynamic effects on human cells exposed to light m the presence of hematoporphyrin. pH effects. Cancer Lett., 9, 327-332.
References
4
K.. Western,
11
16
3
J.F..Berg.
R.H. (1990) The effect of tissue and cellular pH on the selective biodistribution of porphyrin-type photochemotherapeutic agents: A volumetric titration study. J. Photochem. Photobiol., B: Biol.. 6.309323.
14
The present work was supported by the Association for International Cancer Research. We would like to thank Dr. 8. Fodstad for providing the human melanoma LOX.
Moan, J., Peng, Q.. Evensen,
and Rimington, C. (1987) Photosensitizing efficiencies, tumor- and cellular uptake of different photosensitizing drugs relevant for photodynamic therapy of cancer. Photochem. Photobiol., 46, 713-721.
tumors measured in vivo. J. Natl. Cancer Inst.. 16,541-556. Gullino. P.M., Grantham, F.H.. Smith, S.H. and Haggerty. A.C. (1965) Modification of the acid-base status of the internal milieu of tumors. J. Natl. Cancer Inst.. 34.857869. Calderwood, S.K. and Dickson. J.A. (1982) Inhibition of tumor blood flow at high blood sugar levels : Effects on tumor pH and hyperthermia Natl. Cancer Inst. Monogr., 61.221-223. Jain. R.K., Shah, S.A. and Finney. P.L. (1984) Continuous noninvasive monitoring of pH and temperature in rat Walker 256 carcinoma during normoglycemia and hyperglycemia. J. Natl. Cancer Inst.. 73.429-436 Thistlethwaite, A.J.. Alexander. G-A.. Moylan, D J. and Leeper, D.B. (1987) Modification of human tumor pH by elevation of blood glucose. Int J. Radiat. Oncol. Biol. Phys.. 13.603-610. Thomas, J.P and Girotti. A.W. (1989) Glucose administration augments in viva uptake and phototoxicity of the tumor-localizing fraction of hematoporphyrin derivative. Photochem. Photobiol., 49.241-247. Sommer, S.,Rimington, C. and Moan, J. (1987) Porphyrin derivative having physical and chemical characteristics similar to those of the active components of hematoporphyrin derivative and with very strong photosensitizing effects. J. Photochem. Photobiol. B: Bi01.. 1,241-246.
21
Fodstad, 0.. Aamdal. S.. McMenamin. M. and Pihl, A. (1988) A new experimental metastasis model in athymic nude mice, the human malignant melanoma LOX. Int. J. Cancer, 41.442-449.
22
23
24
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Anholt. H. photodynamic
Burk. D. and Woods M. (1963) Hydrogen peroxide, catalase. glutathione peroxidase , quinones, nordihydroguaiaretic acid, and phosphopyridine nucleotides in relation to x-ray action on cancer cells. Radiat. Res.(suppl.), 3,212-246. Slater, T.F. (1972) Free-radical scavengers. In: Free radica mechanisms in tissue injury, pp. 55. Editor : T.F. Slater. Pion Limited, London. Gomer. C.J.. Jester, J.V., Razum, N.J., Szirth, B.C. and Murphree. A.L. (1985) Photodynamic therapy of intraocular tumors examination of hematoporphyrin derivative distribution and long-term damage in rabbit ocular tissue. Cancer Res., 45.3718-3725. Peng, Q.. Moan, J.. Kongshaug,
M., Evensen,
J.F.,
and Rimington, C. therapy of cancer:
(1991) Sensitizer for A comparison of the
26
tissue distribution of P-II and aluminum phthalocyanine tetrasulfonate in nude mice bearing a human malignant tumor. Int. J. Cancer, 48, in press. Dickson, J.A. and Calderwood. S.K. (1979) Effects of
27
hyperglycemia and hyperthermia on the pH. glycolysis. and respiration of the Yoshlda sarcoma in viva. J. Natl. Cancer Inst.. 63. 1371-1381. Thomas. J.P.. Hall, R.D. and Girotti. A.W (1987) Singlet oxygen intermediacy in the photodynamic action of membrane-bound hematoporphyrin derivative. Cancer Lett.. 35. 295-302.
29
29-35
Elsevier Scientific Publishers Ireland Ltd.
The effect of glucose administration on the uptake of Photofrin II in a human tumor xenograft Q. Peng,
J. Moan and L-S. Cheng
Department
of Biophysics,
(Received 8 January (Revision received (Accepted
5 March
Institute for Cancer Research,
Montebello,
Oslo (Norway)
1991)
5 March
1991)
1991)
Summary
Introduction
Athymic BALB/c nude mice, bearing a human melanoma LOX, were given the photosensitizing drug Photofrin 11 (10 mg/kg body wt.) intraperitoneally. The mice were also given one of the following chemicals intraperitoneally: glucose, galactose and glucose plus nordihydroguaiaretic acid (NDGA) which is an inhibitor of glycolysis. Multiple injections of glucose (3 g/kg body wt. given at - 1, 0, + 1 and + 3 h relative to the injection of 10 mg/kg of Photofrin II at time 0) resulted in a significant increase in the uptake of Photofrin II
Photofrin II (P-II) is a porphyrin photosensitizer which is being used in clinical trials of photodynamic therapy (PDT) of cancer [l-5]. It is selectively taken up and/or retained in tumors compared to many normal tissues where tumors frequently grow [6-71. The reason for the high tumor uptake of P-II is not known. However, it has been proposed that the low pH value of tumors may play a role [&lo]. In vitro haematoporphyrin and many of its derivatives may get protonated and thus more lipophilic when the pH in its surroundings is reduced from 7.4 to 6.5. This results in an increased cellular uptake of the dye [8, ll- 131. Furthermore, it is also well known that the pH in the interstitium is significantly lower in tumor than in nonmalignant tissues. This is due to the fact that tumor tissue, being usually poorly vascularised, has to provide a significant fraction of its energy through anaerobic glycolysis, leading to production of lactic acid [8,14- 151. This may offer an explanation for the high tumor uptake of P-II. It is possible to test this hypothesis by injecting glucose in tumorbearing animals, which is known to be able to reduce the extracellular pH in tumor tissue selectively [14,16- 181. Indeed, such an experiment has been carried out with rhabdomyosarcoma-
in the tumor
4 h after a Photofrin
II injection,
while the uptake of Photofrin II in the other tissues remained unchanged. Administration of galactose had no significant effect on the uptake of Photofrin II in the tissues studied (tumor, muscle, skin and liver). NDGA seemed to abolish the effect of glucose injection.
photodynamic Keywords: Photofrin II; glucose; glycolysis; inhibitor; nordihydroguaiaretic acid
Correspondence Institute.. for
to:
Cancer
Q.
Per-g,
Research,
Norway.
0304-3835/91/$03.50
0
1991
Published and Printed in Ireland
therapy; glycolysis
Department
of
Montebello,
0310
Biophysics, Oslo
3,
Elsevier Scientific Publishers Ireland Ltd.
30
bearing rats [19]. It was found that multiple 3 g/kg injections of glucose resulted in a 3.4 times increase in the porphyrin fluorescence of the tumor tissue 48 h after an injection of 10 mg/kg of HPD-A (the tumorlocalising fraction of hematoporphyrin derivative). The liver uptake of HPD-A was not influenced by the glucose injection. We found these observations so interesting and clinically relevant that we decided to perform similar experiments with a human tumor model in nude mice. In the present work we report the results from the experiments of injecting P-II combined with either glucose, galactose or glucose plus a glycolysis inhibitor nordihydroguaiaretic acid (NDGA) in nude mice carrying the human melanoma tumor LOX. Materials
and Methods
Chemicals P-II was obtained as a gift from Lederle Inc. (U.S.A.) as an aqueous solution at a concentration of 2.5 mg/ml and stored at -20°C. HPLC runs to test the composition of P-II was carried out as described elsewhere [20]. All other chemicals used were of the highest purity commercially available. Animals Female BALB/c athymic nude mice were purchased from the Laboratory Breeding and Research Center, Gl. Bomholt Gaard, Ry., Denmark. At the start of the experiments the mice were 7-8 weeks old weighing 18-25 g. Three mice were housed per cage with autoclaved filter covers in a room with subdued light at constant temperature (24”26°C) and humidity (30-50%). Food and bedding were sterilized and the mice were given tap water ad libitum in sterilized bottles. Tumor model The human tumor line LOX was established in 1987 as an S.C. xenograft in nude mice from an amelanotic axillary lymph node metastasis
of a patient with malignant melanoma [21]. The LOX cells have shown a remarkable stability and similarity to the cells of the patient’s tumor with respect to morphology, karyotype and chemosensitivity. The LOX tumor was propagated by serial transplantation into BALB/c nude mice. Nonnecrotic tumor material for inoculation was obtained by sterile dissection of large flank tumors from syngeneic nude mice. Macroscopically viable tumor tissue was gently minced with a pair of scissors and forced repeatedly through an 20-gauge sterile needle to form a tumor tissue suspension, 0.02 ml of which was injected S.C. on the right flank of the mice. The rate of successful transplantation in this study was 100%. Minimal or no spontaneous necrosis was found in tumors with a transverse diameter of 6-8 mm. When the tumor had reached an appropriate size (as indicated above) 10 mg/kg body weight of P-II was administrated i.p. to each tumor-bearing mouse. At the following time points, - 1, 0, + 1, + 3 h (relative to injection of P-II at zero time), the animals received separate i.p. injections of glucose (40%, w/v) or of galactose (40%) w/v) (3.0 g/kg body wt. for both at each time point). Some tumorbearing mice were given only 10 mg/kg of PII. One group of animals were killed 4 h after P-II injection. In a separate group injections were as for the 4 h-group but followed by injections at + 5, + 7 and + 10 h before killing after 48 h. Furthermore, in two other animal groups only single injections of glucose (3.0 g/kg body wt.) or multiple doses of glucose plus a single dose of a glycolysis inhibitor, NDGA (0.4 g/kg body wt.) [22,23]. NDGA, which was dissolved in a mixture of cremophor EL and Dulbecco’s phosphate-buffered saline (PBS, 1:6 by vol.) and sonicated for 2 min before use, was given i.p. at the same time as injection of P-II. The following tissues were sampled either 4 or 48 h after P-II injection: tumor LOX, muscle tissue surrounding the tumor, skin overlying the tumor and liver tissue. Immediately after
31
collection the tissue samples were rinsed twice in PBS, blotted dry on clean paper, weighed and stored at - 20°C until used for determination of P-II concentration.
of the emission spectra was unchanged by the addition of internal standard; only the fluorescence intensity increased. The concentrations are given in pg P-II per g wet tissue.
Determination
Results
of P -11 concentrations
in tissues
The tissue samples were thawed and brought into suspension by means of a Ystral mechanical homogeniser (Dottingen, West Germany). The tissue suspension was finally homogenized once more by means of the Potter Elvehjem homogenizer. This procedure was judged to be better than just using the Potter Elvehjem homogenizer alone. Several solvents were tried for extracting the drug, and the solvent that brought a maximum of the drug into the supernatant was chosen as earlier described [6]. A solution of 1% sodium dodecylsulfate in 1 N perchloric acid/methanol (1: 1, v/v) was chosen in the present study t241. After homogenization the tissue suspensions were frozen, thawed, sonicated for 30 s, diluted 1:lOO in the same solvent, sonicated once more, centrifuged at 3000 rev./min for 10 min and finally the supernatants were collected. As judged by absorption and fluorescence measurements, it was possible to extract more than 75% of P-II from the tissue suspension with this method. Drug levels in the supernatants were quantitatively determined by recording fluorescence emission spectra of the samples using a PerkinElmer LS-5 Luminescence Spectrofluorimeter. The excitation wavelength was 404 nm, the slit width was to give a resolution of 5.0 nm, and the emission wavelength was scanned from 550 to 700 nm. A cut-off filter was used to remove scattered light of wavelengths shorter than 545 nm from the light reaching the detection system of the spectrometer. The background fluorescence from control samples was subtracted. P-II levels in the samples were determined by adding a known amount of the drug, comparable to that already present in the extraction medium, and recording the emission spectra once more. In all cases the shape
The sampling times as well as the dose of PII were selected after a series of introductory experiments had been carried out. Figure 1 shows the kinetics of the accumulation of P-II in the tumor LOX and in the liver. In agreement with earlier work [25] the concentration of the dye in the liver as well as in tumor approached the maximal values at 24-48 h (Fig. 1). In view of this and the work of Thomas and Girotti [19] the sampling times 4 h and 48 h were chosen in the present study. The concentrations of P-II in the tumor and liver were determined 48 h after injection of different drug doses (Fig. 2a and b). This experiment was performed in order to check that no saturation effects occurred at the drug dose of 10 mg/kg which was chosen for the further experiments. As demonstrated by Fig. 2, no saturation occurs since the tumor and liver up-
2‘
Time lhoursi
L8
12
96
after 1.p. injectton (IOmglkgl
Fig. 1. The kinetics of the accumulation and retention of P-11 in human LOX melanoma tumor tissue and in liver tissue of nude mice.
32
I5
I
.-S s J=-f wlow= g
I
,
I
I
-
Table 1. Effect of glucose injection on the selective uptake of P-II in tumor LOX xenografts.
I
Tumor LOX J+8h post-injection
0
.;
P-11 only /I
=‘; n-3
I
i
ZP5/
gs L r;
*/I
W
0.
0
I
1
I
I
1
I
5
IO
15
20
25
30
35
1.p. injected dose (mglkg) 80 -
Liver
P-II + galactose (multiple doses) P-II + glucose (single dose) P-II + glucose (multiple doses) P-II + glucose (multiple) + NDGA (single)
4h
48 h
2.47 zt 0.61 (6) 2.95 zt 0.16 (4) 3.42 zt 1.00 (5) 4.17”’ f 0.83 (10) 2.37 f 0.62 (5)
3.45
l
(6) 3.48
zt 0.32
(6) 3.75
zt 0.23
(5) 3.86
zt 1.06
(10) 3.22 (5)
0.58
zt 0.84
48 h post-injpcfmn 60
-
20 -
0 0
I
I
I
I
I
1
5
10
I5
20
25
30
35
1.p. injected dose (mglkg)
Fig. 2. The concentration of P-II in LOX tumors (a) and in livers (b) of nude mice 48 h after injection of different doses of P-II.
Tumor-bearing nude mice were given P-II i,p. 10 mg/kg and multiple doses of galactose (3 g/kg body wt. each time), single or multiple doses of glucose (3 g/kg body wt. each time), multiple doses of glucose plus a single dose of NDGA (0.4 g/kg body wt.) or without any injection as controls as described in Materials and Methods. Photofrin II accumulation in tumors were extracted at 4 and 48 h after administration. Indicated values are expressed as c(g P-II per g wet tumor tissue (mean f S.D.). Numbers of tumor-bearing animals in each group are shown in parentheses. Significant difference between each group and its time-corresponding P-II only control group was statistically analysed by Student’s t-Test. Significant difference only between the group (P-II + glucose, multiple doses at 4 h) and its time-corresponding P-II only control group was found (’ * lP c 0.01).
take is a linear function of the injected doses at least up to 30 mg/kg. The influence of injection(s) of glucose and galactose on the uptake of P-II in tumor, muscle, skin and liver is shown in Tables I-IV. None of the treatments had any influence on the porphyrin uptake in muscle, skin and liver. A single injection of glucose had a slight but not significantly potentiating effect on the tumor uptake of P-II at 4 h. Multiple injections of glucose had a significantly potentiating effect on the tumor uptake of P-II at 4 h but not at 48 h. NDGA seemed to have an inhibiting effect on the uptake of P-II by the tumor at 4 h after multiple doses of glucose compared to that in the group not receiving NDGA. This may be due to the inhibition of glycolysis by NDGA in the tumor.
Table II. Effect of glucose injection on the selective uptake of P-II in livers of nude mice bearing human tumor LOX. 4h P-II only P-II + galactose (multiple doses) P-II + glucose (single dose) P-II + glucose (multiple doses) P-II + glucose (multiple) +
14.97 (6) 16.56 (4) 16.07 (5) 16.40 (10) 12.12 (5)
48 h f
3.66
21.44
f 4.28
zt 4.17
(6) 23.03
zt 5.51
f
2.11
(6) 24.41
+z 3.29
C+Z 4.10
(5) 21.14
zt 6.37
f
0.51
(10) 17.50 (5)
ztz 3.22
NDGA (single) The experimental conditions legend to Table I.
are those
given
in the
33
Table 111. Effect of glucose injection on the selective uptake of P-II in muscle surrounding the tumor in nude mice. 48 h
4h P-II only P-II + galactose (multiple doses) P-II + glucose (single dose) P-H + glucose (multiple doses) P-II + glucose (multiple) +
0.58
+ 0.11
1.83
zt 0.39
zt 0.17
(6) 1.93 f
(4) 0.48
+ 0.12
(6) 1.99 zt 0.38
(5) 0.56
+ 0.18
(5) 2.0 f
f
(10) 1.75 f
(6) 0.62
(10) 0.49
0.13
0.41
0.53 0.44
(5)
(5)
NDGA (single) The experimental conditions legend to Table 1.
are those
given
in the
Discussion Following an injection of glucose, the feedback systems for control of blood glucose returns the glucose concentration rapidly back to the control level. Under normal conditions, the blood glucose concentration in a range is usually strictly controlled through increased insulin secretion and increased glucagon secreTable IV. Effect of glucose injection on the selective uptake of P-H in skin overlying human tumor LOX in nude mice.
P-II only P-II + galactose (multiple doses) P-II + glucose (single dose) P-II + glucose (multiple doses) P-II + glucose (multiple) +
4h
48 h
1.65 + 0.51
4.08
ztz 0.97
(6) 1.88 (4) 1.91 (5) 1.96 (10) 1.34 (5)
(6) 4.73
f
+ 0.60 f
0.68
ziz 0.57 zt 0.42
(6) 4.92 (5) 4.16 (10) 3.23 (5)
1.35
zt 1.50 + 1.68 ztz 0.94
NDGA (single) The experimental conditions legend to Table I.
are those
given
in the
tion. It has been shown that following an injection of glucose in rats bearing tumors, the blood glucose levels increased rapidly for about 4 h, and then returned to normal levels within about 6 h. Also the lactate concentrations in the tumors peaked about 4 h before returning to normal by 6 h [14,16,26]. Thomas and Girotti [19] observed that after an injection of glucose in tumorbearing rats, the pH in the tumor decreased for about 45 min and then returned to the control level within about 1 h. Since they allowed 32 h from the last glucose injection to the measurement of the HPD-A concentration in the tumor, one may tentatively propose that it is an increased uptake rather than an increased retention that constitutes the basis for their observation of the glucose-induced increase in the tumor content of the drug. Also our results show that glucose injection enhances the tumor uptake of P-II selectively (Table I). Quantitatively, however, our results are different from those of Thomas and Girotti [19]. They found that multiple glucose injections (carried out identically with our procedure) potentiated the tumor uptake of HPD-A at 48 h by a factor 3.4, while we found no potentiation at this time and a potentiating factor of only 1.7 at 4 h. Our results seem to indicate that the retention of P-II is lower in the tumor where the initial uptake was enhanced by glucose injection. Thus, at 4 h the tumor uptake of P-II was increased by 70% by glucose injection, while at 48 h no increase was observed. It may, therefore, seem that in our tumor model, glucose has an effect on the concentration of P-II in the tumor only as long as it has an effect on the pH in the tumor. The discrepancy between our results and those of Thomas and Girotti may be related to either the difference in species (mice vs. rats), tumor model (human melanoma vs. animal rhabdomyosarcoma), or tumor size (6-8 mm vs. 15-20 mm in diameter), but not to difference in the composition of the drug since the tumor-localizing fraction of HPD has been considered to be equivalent to P-II [27]. The fact that the size of the tumor used by Thomas and Girotti was 2-2.5 times larger than that
34
used by us may be of importance. Thus, the vascular drainage may decrease significantly with increasing size of the tumors. This may influence the microcirculation and the efficiency of interstitial transport in the tumor and thus affect both the availability of glucose to the tumor cells and the clearance of lactic acid. Galactose injection had no effect on the uptake of P-II in the tumor and the other tissues analysed (Tables I-IV). This is in good agreement with the work of Thomas et al. [19] and Calderwood et al. [ 161. The latter investigators found that galactose was not metabolized by tumors and did not result in production of lactic acid in the tumor. The abolishing effect of NDGA on the effect of glucose at 4 h further provides the evidence that hyperglycemia may promote selectively the uptake of porphyrins by tumors. Acknowledgements
8
9
10
Moan, J. and Christensen. T. (1981) Cellular uptake and photodynamic effect of hematoporphyrin. Photobiochem. Photobiophys.. 2.291-299. Bohmer. R.M. and Morstyn. G. (1985) Uptake of hematoporphyrin derivative by normal and malignant cells: Effect of serum, pH. temperature, and cell size. Cancer Res.. 45.5328-5334. Eden, M., Haines. B. and Kahler, H. (1955) The pH of rat
13
15
17 Moan, J. (1986) Porphyrin-sensitized photodynamic tivation of cells: a review. Lasers Med. Sci. 1.5-12.
inac-
2
Moan, J. (1986) Porphyrin photosensitization and phototherapy. Photochem. Photobiol., 43.681-690. Dougherty, T.J. (1989) Introduction, In: Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use,
18
pp. 1-3. Editors: G. Bock and S. Harnett. Wiley. Chichester (Ciba Foundation Symposium 146). Ash, D. and Brown, S.B. (1989) Photodynamic therapyachievements and prospects. Br. J. Cancer. 60,151
19
20
6
Gomer, C.J., Rucker. N.. Ferrario. A. and Wong. S. (1989) Review : Properties and application of photodynamic therapy. Radiat. Res.. 120. l-18. Peng, Q., Evensen. J-F.. Rimington. C. and Moan. J.
7
(1987) A comparison of different photosensitizing dyes with respect to uptake C3H-tumors and tissues of mice. Cancer Lett., 36. l-10. Belliner. D.A., Ho. Y-K, Pandey. R.K., Missert. J.R. and Dougherty, T.J. (1989) Distribution and elimination of Photofrin II in mice. Photochem. Photobiol 50.221228.
Pottier, R.H. (1990) Localization phenomena: pH effects. In: Photodynamic Therapy of Neoplastic Disease. Vol. Il. pp. 63-77. Editor: D. Kessel. CRC Press. Barrett, A.J.. Kennedy, J.C., Jones, R.A. and Pollier.
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1
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A.
Moan, J., Smedshammer. L. and Christensen. T. (1980) Photodynamic effects on human cells exposed to light m the presence of hematoporphyrin. pH effects. Cancer Lett., 9, 327-332.
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14
The present work was supported by the Association for International Cancer Research. We would like to thank Dr. 8. Fodstad for providing the human melanoma LOX.
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