0022-5347/90/1442-03$0$02.00/0 THEJOURNAL OF UROLOGY Copyright 0 1990 by AMERICANUROLOGICAL ASSOCIATION, INC.

Vol. 144, August Printed in Lr.S.A.

A COMBINED THERAPY OF HYPERTHERMIA AND TUMOR NECROSIS FACTOR FOR NUDE MICE BEARING KK-47 BLADDER CANCER TOSHIYASU AMANO,* KAZUTO KUMINI, KAZUYOSHI NAKASHIMA, TADAO UCHIBAYASHI HARUO HISAZUMI

AND

From the Department of Urology, School of Medicine, Kanazawa Uniuersity, Kanazaura, Japan

ABSTRACT

A combined therapy of hyperthermia (43.5 C) and tumor necrosis factor (lo3 and lo4 units) for the treatment of experimental bladder carcinoma KK-47 in athymic mice was studied. Briefly, mice were injected subcutaneously with lo7 disaggregated cells. When the tumors were 250 mm3 in size, tumor necrosis factor was administered, either intravenously or by intratumor injection. Intravenous injection was lo3 to lo4 units tumor necrosis factor in the tail vein and intratumor was lo3 to lo4 units injected directly into the center of the tumor. Immediately following injection, the tumor bearing leg was placed in a 43.5 C water bath for 20 minutes. Tumor size was monitored once a week for seven weeks and the animals were divided into control, hyperthermia alone, tumor necrosis factor alone and combined therapy. Results of the study showed no significant difference in lo3 units of tumor necrosis factor intravenously versus control but a significant regression in hyperthermia alone. Anti-tumor effects significantly increased in hyperthermia plus lo3 units tumor necrosis factor versus hyperthermia alone. Similar results were seen with lo4 units tumor necrosis factor intravenously though in the combination group of hyperthermia and tumor necrosis factor, eight mice of eight died one to three days following treatment. In those receiving intratumor injections, there was no difference between tumor necrosis factor or control. Tumor necrosis factor with hyperthermia had the approximate same characteristics as hyperthermia alone and therefore there was no synergistic finding. These results reflect on the suggestion that the combination therapy of hyperthermia and systemic administration of the proper dosage of tumor necrosis factor may produce synergistic anti-cancer effects in bladder cancer patients. (J.Urol., 144: 370-374, 1990) As a multidisciplinary treatment for cancer, a combined use of different anticancer modalities, for instance, hyperthermia and anticancer chemotherapy, has been advocated in the literengineering development of radiofrequency hya t ~ r e . ' - The ~ perthermia has enabled us to treat not only superficial but also deep-seated cancers.'-4 B u t satisfactory anticancer effects by hyperthermia alone have not been reported. Tumor necrosis factor (TNF) is one of the biological response modifiers, which was reported to induce hemorrhagic necrosis in Meth-A sarcoma^.^ T h e cytotoxicity of T N F was enhanced a t high temperatures in ~ i t r o . ~Synergistic ,' cytotoxic effects combined with T N F and hyperthermia were also reported using Meth-A fibrosarcomas in vivo.' The antitumor effects of T N F for transitional cell carcinomasg-'l have also been discovered. We have investigated the combined antitumor effects of hyperthermia and T N F in vivo using nude mice bearing KK-47 bladder carcinomas.12 MATERIALS AND METHODS

Tumor necrosis factor. Human recombinant T N F (PT-050, Dainippon Pharmaceutical Co., LTD. Osaka, Japan) is a protein with a molecular weight of approximately 17,700 units consisting of 155 amino acids. Tumor model. KK-47 bladder cancer cells which were derived from a human transitional cell carcinoma, established and Accepted for publication March 12, 1990. *Requests for reprints: Department of Urology, School of Medicine, Kanazawa University, 13-1Takara-machi, Kanazawa 920, Japan. Supported by Grant-in-Aid for Cancer Research Program (59-5) of the Japanese Ministry of Health and Warfare, and Grants-in-Aid for Cancer Research 58010032, 59010030, 60010035, 63010046 and Scientific Research 63570742 from the Japanese Ministry of Education, Science and Culture.

maintained in our department since 1977." 1 x lo7 disaggregated KK-47 cancer cells suspended in saline were injected into the legs of female nude mice subcutaneously. The size of the tumor was measured with a caliper in three dimensions, length, width and height, and the volume was calculated according to the following equation: V = 413 T (d1/2) (d2/2) (d3/2) = T /6 X didzd3. T N F administration and hyperthermia. When the tumors reached approximately 250 mm.3 in size, usually within two weeks after inoculation, T N F was administered by either intravenous (I. V.) or intratumor injection (I. T.) under pentobarbital sodium (Nembutal, Abbott Laboratories, Ill., USA, 10 mg./kg. intraperitoneal) anesthesia. For I. V. administration, 10,000 or 1,000 units of T N F in 0.2 ml. normal saline were injected through a tail vein of the nude mice. For I. T . injection, 10,000 or 1,000 units of T N F in 0.05 ml. normal saline were injected into the center of the tumor with a 26 gauge needle. Then immediately after injection, the mouse was fixed in the plastic holder and the tumor-bearing leg was immersed into a water bath (Super Labo, Hirasawa Co., Tokyo, Japan) without a proximal tourniquet. The temperature of the rectum, the tumor of the mice and the water bath was monitored by a digital thermometer (M5501, Sibauradenshi Co., Saitama, Japan). Our preliminary data showed that the temperature in the center of the tumor was stabilized a t 0.2 C under that in the water bath three minutes after heating. The rectal temperature of the mice increased about 3 C during the hyperthermia treatment. To heat the tumor with 43.5 C for 20 minutes, the tumor-bearing leg was immersed into a 43.7 C water bath for 23 minutes. Each of the experimental groups consisted of five to eight nude mice. Anticancer effects. Tumor size was measured once a week for

371

H Y P E R T H E R M I A A N D T U M O R N E C R O S I S FACTOR F O R B L A D D E R C A N C E R

TABLE1. T u m o r size rate i n each erouw one to seven w e e k after treatment Control HPT(43.5"C,20 min):TNF(-) HPT(-) :TNF(IV. 1,000) HPTi-) :TNF(IV.10,000) HPT(-) :TNF(IT. 1.000)

Values are expressed as mean f standard deviation. *: All eight mice died one t o three days after the treatment. T N F = tumor necrosis factor. H P T = hyperthermia.

*

*

Control

-0HPT

+

TNF(IV.~,OOO)

-A-

HPT&TNF(IV.1,000)

Control

-0-HPT +:mice died

++

TNF(IV.IO,OOO)

-v- HPT&TNF(IV. 10,000)

loot

Weeks after the treatments

Weeks after the treatments

FIG. 2. No antitumor effect was seen in T N F (10,000 units/mouse) FIG. 1. Significant antitumor effect was observed in hyperthermia alone group (0) compared with control group ( 0 )Moreover, . antitumor I. V. administration (.I. But all eight mice died one to three days after effect was significantly increased in combination with hmerthermia "mbined therapy with hyperthermia and TNF (10,000 units/mouse, and T N F 11.600 unitsjmouse. I. V.)(A).But no s i g n i f i c a n ~ ~ m o r i c i d a l 1. V.1 (U). effect was'observed in T N F ~1,000u'nits/mouse,I ~ v . alone ) (A).

seven weeks, and compared with the size just before treatment. The tumor size was calculated, then tumor growth curves were obtained in control, hyperthermia alone, T N F alone and combined groups. Anticancer effects were analyzed using these curves. Histological studies were also performed on hematoxylin-eosin preparation. RESULTS

The mean volume of the tumors and standard deviations in all the groups were shown in table 1. There was no substantial difference in tumor growth between the control and T N F 1,000 units I. V, administration groups. On the other hand, a significant tumor regression was seen in the hyperthermia alone group (P < 0.01, Student's t test) a t all times after treatment compared with the control group. Moreover, antitumor effects were significantly increased in the combination group of hyperthermia and T N F (I. V., 1,000 units/mouse) as compared

with that of the hyperthermia alone group (p < 0.05), though tumor regrowth was seen four weeks after treatment (fig. 1). In the T N F 10,000 units I. V. administration group, there was no difference in the tumor group curve compared with that of the control group. All eight mice in the combination group of hyperthermia and T N F (I. V., 10,000 units/mouse) died one to three days after treatment (fig. 2). Dose response study of T N F (I. V.) with the combination of the hyperthermia was performed. Lethal rate in the group of 5,000,4,000, 3,000 and 2,000 units/mouse of T N F and hyperthermia (43.5 C, 20 min.) was loo%, 67%, 33% and 0% respectively. Edema and congestion of intestine in dead mice were remarkable. Therefore, T N F dosage is important factor in the acute toxicity in the combination of T N F (I. V.) and hyperthermia. On the other hand, in the group of I. T . administration, either 1,000 or 10,000 units/mouse, there was no different tumor growth between the control and TNF-alone groups. Furthermore, the growth curves of the combination group of hyper-

AMANO AND ASSOCIATES -+-

Control

-0HPT L TNF(IT. 1,000) -A-

HPT&TNF(IT.l,OOO)

Weeks after the treatments FIG. 3. No antitumor effect was noted with intratumoral T N F (1,000 units/mouse) (A). Synergistic effect of hyperthermia and T N F (1,000 units/mouse, I. T.) (A) was not obtained either.

+

Control

-0HPT +

-0-

TNF(IT. 10,000) TNF&HPT(IT.10,000)

FIG. 5. Histological finding of transplanted KK-47 bladder cancer before treatment. Vivid transitional cell carcinoma cells were seen. H & E. reduced from ~ 1 0 0 .

thermia and T N F I. T. administration (both 1,000 and 10,000 units/mouse) were almost the same as that of the hyperthermia alone group (figs. 3, 4). Therefore, the synergistic effect of hyperthermia and T N F (I. T.) was not observed. Histological studies of the combination group of hyperthermia and T N F (I. V., 1,000 units/mouse) revealed remarkable hemorrhage and necrotic changes of the tumor tissue which was almost the same as the changes in the hyperthermia alone group. On the other hand, vivid cells were dominant in the control group (figs. 5, 6). DISCUSSION

Weeks after the treatments FIG. 4. No antitumor effect was noted with intratumoral TNF (10,000 units/mouse) (4).Synergistic effect of hyperthermia and T N F (10,000 units/mouse, I. T.) (0)was not obtained either.

I t has been possible to heat cancer tissues in the deep sites of the body, including renal and prostate cancers, due to the development of the radiofrequency hyperthermia technique.' The combination therapy of hyperthermia and chemotherapy or radiation has been reported to be much more effective, compared with hyperthermia alone. The combination therapy of T N F and other anticancer modalities, such as interferon and chemotherapy, has been known ' ~ - ' ~cytotoxic effects of to enhance tumor d e s t r u ~ t i o n ~ ~ The T N F have also been known to increase with increasing temperature~.~-' The effects of the combination of hyperthermia and T N F were demonstrated against Meth-A fibrosarcoma cells in mice.' The Meth-A cell line has a high sensitivity for TNF.' An antitumor effect on MBT-2 transitional cell carcinomas of the bladder both in vitro and in vivo has also been reported.'-''

HYPERTHERMIA A N D T U M O R NECROSIS FACTOR FOR BLADDER CANCER

37'3

combination therapy of hyperthermia and a systemic administration of a certain small dose of T N F could produce synergistic antitumor effects even against tumors with very low sensitivity for T N F alone. All mice in the combined group of hyperthermia and high dose T N F (10,000 and 5,000 units/mouse) died one to three days after treatment. The lethal rate increased according to the increase of T N F dose. When the T N F dose increased, the vascular damage would happen even in normal tissues, with edema and congestion of intestine in dead mice. This may suggest that even when the proper TNF dosage for I. V. single administration is given in combination with hyperthermia, the development of lethal side effects has to be considered. The mechanism of the cytotoxicity of T N F has not been determined yet. Furthermore, the mechanism of combination of T N F and hyperthermia has not been determined. Niitsu and associates reported that the synergistic effect of hyperthermia and T N F was shown to be related to an accelerated turnover rate of TNF-receptor complex in mouse tumorigenic fibroblast cells.16 This present study suggests that vascular damage may play an important role in tumor regression with the combined therapy of T N F and hyperthermia. As the combination therapy with high dose T N F and hyperthermia could be dangerous, the administration dose of TNF should be decided very carefully. But the results suggest that the combined therapy with a certain dose of T N F and hyperthermia might be clinically very useful. REFERENCES

FIG. 6. Histological finding of transplanted KK-47 bladder cancer four weeks after combined therapy of hyperthermia and T N F (1,000 units/mouse, I. V.). Remarkable hemorrhage and necrotic change was observed. H & E, reduced from X100.

The sensitivity of KK-47 cells treated with T N F has not yet been determined either in vitro or in vivo. Our data showed neither antitumor effects nor synergistic effects with hyperthermia combined with I. T. administration of TNF. The present paper also showed no effects for I. V. administration of T N F alone. These may not indicate that the KK-47 would have a high sensitivity for TNF. Hyperthermia induced obstruction and hemorrhage of tumor vessels, followed by hypoxia and low pH condition in which cancer cells were killed.'"l7 Anti-cancer effect was observed in hyperthermia alone group and histological findings showed massive hemorrhage and necrosis. These data supports that the vessel damage is a n important factor on hyperthermia treatment. T N F itself might not be able to kill KK-47 cells, but synergistic tumoricidal effects were seen in by the combination therapy of hyperthermia and small doses (1,000 units/mouse) of T N F I. V, administration. Histological reports after the combination therapy revealed hemorrhage and necrosis which was same as that after hyperthermia alone. Hemorrhage and congestion of tumor vessels were observed after T N F alone treatment." This observation suggested that T N F might also damage to endothelium cells of vessels. Therefore, there is a possibility that the synergistic anti-cancer effect with T N F and hyperthermia is based on tumor vessel damage, rather than direct tumor cytotoxicity of T N F and hyperthermia. Thus, the

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14. Regenass, U., Muller, M., CurschelIas, E. and Matter, A,: Antitumor effects of tumor necrosis factor in combination with chemotherapeutic agents. Int. J. Cancer 39: 266, 1987. 15. Aggarwal, B. B., Essalu, T . E. and Hass, P. E.: Characterization of receptors for human tumor necrosis factor and their regulation by ?-interferon. Nature (Lond.), 318: 665, 1985. 16. Eddy, H. A,: Alterations in tumor microvasculature during hyperthermia. Radiology, 137: 515, 1980.

17. Song, C., Kang, M. S., Rhee, J. G. and Levitt, S. H.: The effect of hyperthermia on vascular function, pH, and cell survival. Radiology, 137: 795, 1980. 18. Niitu, Y., Watanabe, N., Umeno, H., Sone, H., Neda, H., Yamauchi, N., Maeda, M, and Urushizaki, I.: Synergistic effects of recombinant human tumor necrosis factor and hyperthermia o n in vitro cytotoxicity and artificial metastasis. Cancer Res., 48: 654, 1988.

A combined therapy of hyperthermia and tumor necrosis factor for nude mice bearing KK-47 bladder cancer.

A combined therapy of hyperthermia (43.5 C) and tumor necrosis factor (10(3) and 10(4) units) for the treatment of experimental bladder carcinoma KK-4...
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