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Antitumor Effects of Angiogenesis Inhibitor O-(Chloroacetyl-Carbamoyl) Fumagillol (TNP-470) against Murine Renal Cell Carcinoma
~22-5347/96/1555-1775$03.00/0 JOURNAL OF U R O ~ Y
Copyright 0 1996 by
AMERICAN
Vol. 165, 1775-1778, May 1996 Printed in U . S A
UROLOGICAL ASS~CIATION, INC.
ANTITUMOR EFFECTS OF ANGIOGENESIS INHIBITOR 0(CHLOROACETYL-CARBAMOYL) FUMAGILLOL (TNP-470)AGAINST MURINE RENAL CELL CARCINOMA TOMOAKT F'UJIOKA,* MICHIHIKO HASEGAWA, KAZUNORI OGIU, YASUSHI MATSUSHITA, MASATSUGU SAT0 AND T W H I KUBO From the Department of Urology,Iwate Medical University School of Medicine, Iwate, Japan
ABSTRACT
Purpose: The effect of 0-(chloroacetyl-carbamoyl)fumagillol (TNP-470) on tumor growth and metastasis is investigated. Materials and Methods: BALB/c mice were inoculated with the Renca murine tumor and graded doses of TNP-470 were given subcutaneously every other day beginning on day 1 and ending on day 9. Tumor angiogenesis was measured quantitatively by a colorimetric assay. Results: TNP-470inhibited tumor growth and prolonged the life span of Renca-bearing mice in a dose-dependent manner. Body weight-loss was not observed in the mice given less than 30 mg.Asg./day. When the treatment was delayed on day 6, TNP-470 did not inhibit tumor growth, pointing to the importance of the timing of drug administration in relation to disease stage. Tumor angiogenesis was inhibited 33 to 62% of the control level by TNP-470.F'urthermore, TNP-470 reduced pulmonary and hepatic metastatic foci of intravenously i n d a t e d &nca and of the tumor inoculated in the spleen. Conclusion: These data suggest that TNP-470 may be effective as a treatment of r e d cell carcinoma, especially when micrometastases are involved. KEYWORDS: angiogeneisinhibitor, TNP-470, murine renal tumor
Patients with renal cell carcinoma (RCC) have metastatic disease at the time of diagncsis in 25 to 57% of cases and develop metastases within 10 years in approximately 60% of cases after curative nephrectomy.12 Metastatic RCC is one of the most difficult urological neoplasms to cure because it is chemotherapy-resistantand is not radiosensitive. Recently, an e h c y rate as high as 30% has been reported for immune modulation therapy in treating advanced RCC.Successful use of biological respom modifiers, induding interferons and interleukin 2, as well as the adoptive transfer of lymphokineactivated killer cells and tumor-infiltratinglymphocytes has been reported to date.= However, these therapeutic modalities appear to be successful only in a relatively short term,and many authors have emphasized the need for more defhitive treatment. In 1971,Folkman introduced the concept of angiogenesis inhibition for cancer treatment, and a few substances have been reported to inhibit angiogenesis and suppress established solid tumors in animala.6.7 It has also been demonstrated that the growth of micrometastases in target organs and tissues depended on angiogenesis and that there was a significant correlation between angiogenic properties and metastatic potential of human breast cancer and prostate c81'Cinoma.8-10 Therefore, there seem to be a reasonably good possibility that inhibition of angiogenesis is clinically useful as an adjunctive modality in the treatment of solid tumors. 0-(chlomacetyl-carbamoyl)fumagillol (TNP-470), a s p thetic analogue of fumagillin isolated h m Aspergillus fumigatus, has recently been shown to possess potent antiangiogenic and antitumoral activities against some types of malignant neoplasm.11 In this study, we examined the inhibitory activities of TNP-470 against in vivo growth, angiogen-
esis and metastasis of murine renal cell carcinoma, on which no information has been available to date. MATERIALS AND METHODS
A n i d . Female, euthymic BALBlc mice were h o d in a special pathogen-fhe facility and were used routinely at 8 to 10 weeks of age when they weighed 22 to 24 g. Tumor. "he Renca cell line, a renal cortical adenocarcinoma syngeneic in B W c mice,kindly provided by Dr.R. H. Wiltrout W.C.I., Frederick, Maryland), was maintained in vivo by serial intanrenal passage. This tumor line WFIS shown to be mycoplasma-free (Flow Laboratories, Mckan, Virginia). Drug. TNP-470 was synthesized and provided by Takeda Chemical Industries (Osaka, Japan) for the purpose of clinical trial. Stock solutions of TNP-470were prepared in absolute ethanol and suspended in 5% gum arabic and normal saline. Thempeutic regimen. In Rema-indated mice, graded doses of TNP-47W0.2 ml. were administered eubcutaneously every other day begimhg on day 1 and continuing until day 9,or beginning on day 6 and continued until day 14. Control mice were administered subcutaneous iqjeetiona of 0.2ml. vehicle. Tumor growth assay. The tumor was aseptically excised h m a tumor-bearing m o w , and single cell suspensione were prepared by mechanical dissociation and filtration of the tumor cells through a &-mesh stainless-steel sieve. Routinely, injection of 6 X lo6 Renca cells into the subcapsular space of the leR kidney was performed through a flanh incision, and 1 X lo6 tumor cells were i n d a t e d subcutaneouely on the back. Tumor weight and body weight were measured on day 21 following the tumor inoculation. The subcutaxle$ccepted for publication November 7,1996. Requests for re rink D e p q e n t of Urology, Iwate M e d i d owly inoculaM tumors were measured 2 times a week fhm day 1 to the time when gross ulceration of the tumors develuniversity, 19-1 U&aru, Monoka 020, Japan. 1776
1776
TNP-470FOR RENCA
oped in the control mice. The tumor volume was calculated by the following formula: tumor volume (mm?) = 0.5 X a
X
b2
(1)
where a is the longest diameter and b is the shortest diameter. In the experiment examining the effect of TNF-470 on survival, tumor-inoculated mice were treated with TNP-470 every other day beginning on day 1 and continuing until day 9. All experiments were performed at least twice with similar results. Experimental metastasis assay. The single cell suspensions were prepared as described. Fifty thousand cells in 200 J. of serum free RPMI medium (Flow Laboratories) were injected into the lateral tail vein. In addition, injection of 5 x lo5 Renca cells under the splenic capsules was given through a flank incision. The lungs and liver were excised and fued on day 14 and day 21, respectively. Metastatic foci on the lung or spleen surface were counted under a dissecting microscope. All experiments were performed a t least twice with similar results. Tumor angiogenesis assay. Inhibition of tumor angiogenesis was assayed by the quantitative colorimetric method of Lee et a1.12 Renca cells were inoculated intradermally on day 0 at 4 sites on the back of each recipient mouse. Each site was injected with 2 x lo6 cells in 50 pl. by a 26-gauge needle. TNP-470 was injected subcutaneously into the abdominal region in graded doses every day beginning a t 4 hours on day 0 and ending on day 2 after tumor inoculation. The control treatment consisted of injection of vehicle only. The extent of angiogenesis was assayed on day 3 by measuring the total vascular volume in disks of skin from each of the 4 tumor injection sites. This was assessed by injecting 0.25%Evan’s blue (Sigma, St. Louis, Missouri) in normal saline via the tail vein, followed by a colorimetric measurement of the dye in the skin disks. The animals were sacrificed by cervical dislocation 2 minutes after the dye injection, and a midline skin incision was made to allow stretching free the involved skin areas. Then the sites of tumor cell inoculation were punched out, and the pairs of hemilateral skin disks were pooled and cut into small pieces. Normal skin was obtained from noninoculated sites and used for background measurement in the assay. The minced tissue was suspended in 1.5 ml. sodium sulfatelacetone solution and incubated at room temperature for 24 hours with occasional shaking. Then the absorbance of the supernatant was determined at 620 nm. and the percent angiogenesis was calculated by the following formula: % angiogenesis = (A-B)/(C-B) X 100,
Wilcoxon test using the same software. Differences were considered statistically significant when the P value was 5% or less.
(2)
Where A, B, and C represent the b2,, values of treated tumor, background skin and control tumor. Statistical analysis. All values are expressed as the mean 5 standard deviation (SD). Data were analyzed by generalized Wilcoxon test using Fisher statistical software. The analysis of the survival studies was performed by plotting survival curves and calculating by the Kaplan-Meier or the
RESULTS
In vivo antitumor effect of TNP-470 treatment against Renca tumor. When subcutaneous injection of TNP-470 began on day 1 following the cell inoculation under the renal capsule, daily doses of 90,30, or 10 mg.kg. resulted in dosedependent inhibitions (p <0.01) of the growth of Renca tumor as assessed on day 21. The body weight of the inoculated mice was not affected by TNP-470 below the daily dose of 30 mg.kg. However, significant weight loss occurred in the mice given more than 90 mg.kg./day of TNP-470 (table 1). The growth of the subcutaneously inoculated tumor was also inhibited (p <0.01) in a dose-related manner by TNP-470 (fig. 1). #en the administration of 30 mg.kg. TNP-470 began on day 6 after subcutaneous or renal capsular inoculation, it was entirely ineffective. In another set of experiments, TNP-470 given subcutaneously in daily doses of 10,30, or 90 mg.kg. beginning on day 1after tumor inoculation significantly prolonged the survival period compared with the untreated tumor-bearing controls (fig. 2). All control mice died by day 28. In contrast, 50%,60%, or 90%survival was seen on day 32 in the mice treated with 10, 30, or 90 mg./kg. daily. Effect of TNP-470 on experimental metastasis of Renca tumor. #en TNP-470 was injected subcutaneously at a daily dose of 10,30, or 90 mg./kg. beginning day 1 after tumor inoculation, the number of metastatic foci in the lung was significantly reduced in a dose-dependent manner as assessed on day 14. TNP-470 also reduced the number of metastatic foci in the liver when examined on day 21 after the tumor inoculation (table 2). These treatments apparently reduced the size of individual metastatic foci in both lung and liver as well (data not shown). Angiogenesis inhibition of Renca tumor by TNP-470. The effect of TNP-470 treatment on the angiogenesis of Renca tumor is shown in table 3. The background blood volume in 33.2 mm.2 skin was 0.22 5 0.08 pl. The blood volume in the tumor injection sites was 0.95 2 0.16 pl. in the controls and 0.61 ? 0.10 pl., 0.46 2 0.05 pl., or 0.32 ? 0.06 pl. in the TNP-470-treated tumor-bearing groups given 10, 30, or 90 mg./kg. daily (control versus each treated group: p <0.01). There were significant differences (p <0.05) among the 3 groups given different doses of the drug. There was a significant inhibition of the tumor angiogenesis down to 33% to 62% of the controls by TNP-470 (p <0.01) (table 2). DISCUSSION
TNP-470 is a very potent inhibitor of endothelial cell proliferation in vitro,l3.14 and the agent has also been shown to inhibit angiogenesis in a variety of in vivo assays, utilizing the chick chorioallantoic membrane, rat and mouse implanted sponges, and rabbit or rat cornea.15 An inhibitory activity against some murine tumor growth was also reported with this agent,13.16 but the antiangiogenic activity was not thoroughly studied. In this study, we examined the
TABLE1. Tumor growth inhibition
of Renca tumor by TNP-470 (1)
Tumor Dose (mg.ikg.)
No. of mice
mg.
T i c (lo)
Body wt. TIC (5%)
P value ~~~
Control 3.9 z 0.3” 10 100 ion 10 TNP-470 10 2.3 % 0.2 59 p
1777
TNP-470 FOR RENCA
k
M Control
h
*--s &--A
v
-
0
7
TNP-470(10mg/kg) TNP-470(30me/kg)
10
14
17
21
24
Days after cell inoculation
TABLE2. Effect of TNP-470 on metastasis Dm3
No of Dose Img./kg.) mice .- .
of Rettra tumor
-__
No of metastatic fococl
mn
I
P value
T/C 1%)
~~
Experiment of lung metastases 10 1 1 7 Z 14" 100 Control TNP-470 10 10 48? 4 41 p c 0 . 0 1 TNP-470 30 10 29 t 3 25 pc0.01 TNP-470 90 10 11 - 2 9 p
FIG. 1. Tumor growth inhibition of Renca tumor by TNP-470.2 BALBlc mice were i n d a t e d subcutaneously with Renca tumor on day 0 and received TNP-470 subcutaneously on days 1 , 3 , 5 , 7 and 9. Tumors were measured 3 times a week. Control versus "-470 (10 mg./kg.) p <0.01; control versus TNP-470 (30 mg.kg.) p cO.01; control versus TNP-470 (90 mg.kg.) p CO.01.
TABLE3. Angiogenesis inhibition of Renca tumor by TNP-470 Drug
No. of Dose fmgflrg.) mice
'asdm volumeb (&I,)
% angiogenesis
P value
100 10 0.95 -C 0.16b Control TNP-470 10 10 0.61 2 0.10 62 p <0.01 0.46 2 0.05 48 p <0.01 TNP-470 30 10 90 10 0.32 i 0.06 33 p <0.01 TNP-470 The B W c mice were inoculated with Rencn tumor subcutaneously at 4 sites of back on day 0 and received TNP-470 subcutaneously on daya 0 , l and 2. The tumor vascular volume on day 3 represents the blood volume of 2 skin disks extraded by the mean background blood volume. a plf33.2 nun.*; mean +- SD
FIG. 2. Extension of survival time of Renca-bearing mice by TNP470. BALB/c mice were inoculated with Renca tumor under capsule of leR kidney on day 0 and received TNP-470 subcutaneously on days 1 , 3 , 5 , 7 and 9. Control versus TNP-470 (10 m ./kg ) p cO.01; control versus TNP-470 (30 mg.kg.) p <0.01; contr8 versus TNP-470 (90 mg.kg.1 p cO.01.
antiangiogenic as well as antitumoral activity of TNP-470 using the murine RCC Renca tumor. TNP-470 showed antitumoral activity against Renca cells and effects were dose dependent. The body weight of the tumor-bearing mice was only minimally affected at a dose of 90 mg./kg./day of T"470. The optimal effective dose appeared to be 30 mg.kg./day in BALBlc mice. TNP-470 prolonged the life span of Rencabearing mice and the toxicity attributable to this agent seems to be low. However, in our experiments, TNP-470 did not inhibit tumor growth when treatment was delayed to day 6 following Renca inoculation, an indication of the importance of the timing of drug administration in relation to disease stage. From our experiments, it appears that greater effects could be expected if intervention with this drug were to take place earlier. Although it is difficult to make a meaningful correlation between the 6-day delay in the drug intervention in our animal model and the clinical stages in actual patients, our results may indicate that the drug is more u s e l l as a preventive modality than as a suppressive therapy.
Tumor metastasis is a multifactorial and multi-stage process.8 In particular, tumor angiogenesis is considered an important factor that greatly influences the growth of micrometastases in the target organs.17.18 Theoretically angiogenesis inhibitors should effectively inhibit tumor metastases based on the concept that tumors require newly formed blood vessels for their growth at both primary and secondary sites and for dissemination. Potent antimetastatic action of angiogenesis inhibitors was reported in metastatic models involving B16BL6 melanoma and M5076 reticulum cell sarcoma.lg.20 Our present data demonstrating the inhibitory effect by TNP-470 on experimental Renca metastasis are consistent with these studies. Both the number and the size of the metastatic foci in the lungs and the liver were reduced by TNP-470 treatment in our studies. Tumor angiogenesis is generally estimated by the degree of neovascularization around the tumor (or tumor extracts), and the inhibition assays can be classified into 2 groups depending on the route of administration of the test agents. The first group includes chorioallantoic membrane and rabbit (or rat) cornea assays, which are similar to bacterial ~ ' ~ ~ are implanted into the chemosensitivity t e ~ t s . 6 .Tumors hosts, test agents are simultaneously inoculated near the tumor, and the area of avascular zone is semiquantitated visually. The second group consists of rodent skin assays involving the dorsal air-sac methods and the colorimetric assay employed in our studies.**-14In these assays the tumor cells (or the Millipore chamber loaded with tumor cell suspension) are implanted subcutaneously or intradermally into the host and the test agents are given systemically. In our current studies, we adopted a colorimetric assay because the dye-perfusion technique has the advantage of providing a n objective endpoint with relatively small assay variations, as reflected in the small SD values. When the degree of angiogenesis was quantitated by measuring vascular volume in the tumor, the potent antiangiogenic action of TNP-470 was demonstrated in BALBlc mice inoculated with Renca tumor in a dose-dependent manner.
TNP-470 FOR RENCA
1778 CONCLUSIONS
Thus, the present observation obtained in mice indicated that the angiogenesis inhibitor TNP-470 has strong inhibitory activity against in Viva growth and metastasis ofmurine renal cell carcinoma. TNP-470 seems to be a promising candidate for development as an antitumor and antimetastatic agent in RCC. REFERENCES
1. Silverberg, E. and Lubera, J.: Cancer statistics. CA Cancer J. Clin., 2. 1987. 2. Potes, J. E.: Adjunctive treatment of renal cell carcinoma. Int. Adv. Surg. Oncol., 6: 309, 1983. 3. Belldegrun, A. and Rosenberg, S. A.: Adoptive immunotherapy of urologic tumors. Cancer Treat. Res., 4 6 213, 1989. 4. Robertson, C. N., Linehan, W. M., Pass, H. I., Gomella, L. G., Haas, G. P., Berman, A., Merino, M. and Rosenberg, S. A.: Preparative cytoreductive surgery in patients with metastatic renal cell carcinoma treated with adoptive immunotherapy with interleuhn-2 or interleukin-2 plus lyrnphokine activated killer cells. J. Urol., 144: 614, 1990. 5. Lotze, M. T., Custer, M. C., Bolton, E. S., Wiebke, E. A., Kawakami, Y. and Rosenberg, S. A.: Mechanisms of immunologic antitumor therapy: lessons from the laboratory and clinical applications. Hum. Immunol., 2 8 198, 1990. 6. Folkman, J.: Tumor angiogenesis: therapeutic implications. N. Engl. J. Med., 285: 1, 1971. 7. Rastinejad, F., Polverini, P. J. and Bouck, N. P.: Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell, 56: 345, 1989. 8. Folkman, J.: How is blood vessel growth regulated in normal and neoplastic tissue? Cancer Res., 4 6 467, 1986. 9. Weidner, N., Semple, J. P., Welch, W. R. and Folkman, J.: Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N. Engl. J. Med., 324: 1, 1991. 10. Weidner, N., Carroll, P. R., Flax, J., Blumenfeld, W. and Folkman, J.:Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am. J. Pathol., 143: 401, 1993.
11. Ingber, D., Fujita, T., Kishimoto, S., Sudo, K., Kawamura, T., Brem, H. and Folkman, J.: Synthetic analogues of furnagillin that inhibit angiogenesis and suppress tumor growth. Nature, 348:555, 1990. 12, h e , K., Iwamura, M. and Cockett, A. T. K.: Cortisone inhibition oftumor angiogenesis measured by a quantitative colorimetric assay in mice. Cancer Chemother. Pharmacol., 2 6 461, 1990. 13. Kusaka, M., Sudo, K, Matsutani, E., Kozai, y . , Marui, S., Fujita, T., Ingber, D. and Folkman, J.: Cytostatic inhibition of endothelial cell growth by the angiogenesis inhibitor TNP-470 (AGM-l470)-Br. J. Cancer, 6 9 212, 1994. 14. Antoine, N., Creimers, R., Roanne, C. D., Kusaka, M., Heinen, E., Simar, L. J. and Castronovo, V.: AGM-1470, a potent angiogenesis inhibitor, prevents the entry of normal but not transformed endothelial cells into the GI phase of the cell cycle. Cancer Res., 5 4 2073, 1994. 15. Kusaka, M., Sudo, K., Fujita, T., Marui, S., Itoh, F., Ingher, D. and Folkman, J.: Potent anti-angiogenic action of AGM-1470: comparison to the fumagillin parent. Biochem. Biophys. Res. Commun., 174: 1070, 1991. 16. Kreisle, R. and Ershler, W. B.: Investigation of tumor angiogenesis in a n Id mouse model: role of host-tumor interactions. J. Natl. Cancer Inst., 80: 849, 1988. 17. Sakamoto, N., Tanaka, N., Tohgo, A. and Ogawa, H.: Inhibitory effects of heparin plus cortisone acetate on endothelial cell growth both in cultures and in tumor masses. J. Natl. Cancer Inst., 7 8 518, 1987. 18. Liotta, L. A., Steeg, P. S. and Stetler-Stevenson, W. G.: Cancer metastasis and angiogenesis: a n imbalance of positive and negative regulation. Cell, 64:327, 1991. 19. Blood, C. H. and Zetter, B. R.: Tumor interactions with the vasculature: angiogenesis and tumor metastasis. Biochim. Biophys. Acta, 1032 89, 1990. 20. Yamaoka, M., Yamamoto, T., Masaki, T., Ikeyama, S., Sudo, K. and Fujita, T.: Inhibition of tumor growth and metastasis of rodent tumor by the angiogenesis inhibitor 04chloroacetylcarbamoyl)fumagillol (TNP-470; AGM-1470). Cancer Res., 53: 4267, 1993.
Copyright 0 1996 by
AMERICAN
Vol. 165, 1775-1778, May 1996 Printed in U . S A
UROLOGICAL ASS~CIATION, INC.
ANTITUMOR EFFECTS OF ANGIOGENESIS INHIBITOR 0(CHLOROACETYL-CARBAMOYL) FUMAGILLOL (TNP-470)AGAINST MURINE RENAL CELL CARCINOMA TOMOAKT F'UJIOKA,* MICHIHIKO HASEGAWA, KAZUNORI OGIU, YASUSHI MATSUSHITA, MASATSUGU SAT0 AND T W H I KUBO From the Department of Urology,Iwate Medical University School of Medicine, Iwate, Japan
ABSTRACT
Purpose: The effect of 0-(chloroacetyl-carbamoyl)fumagillol (TNP-470) on tumor growth and metastasis is investigated. Materials and Methods: BALB/c mice were inoculated with the Renca murine tumor and graded doses of TNP-470 were given subcutaneously every other day beginning on day 1 and ending on day 9. Tumor angiogenesis was measured quantitatively by a colorimetric assay. Results: TNP-470inhibited tumor growth and prolonged the life span of Renca-bearing mice in a dose-dependent manner. Body weight-loss was not observed in the mice given less than 30 mg.Asg./day. When the treatment was delayed on day 6, TNP-470 did not inhibit tumor growth, pointing to the importance of the timing of drug administration in relation to disease stage. Tumor angiogenesis was inhibited 33 to 62% of the control level by TNP-470.F'urthermore, TNP-470 reduced pulmonary and hepatic metastatic foci of intravenously i n d a t e d &nca and of the tumor inoculated in the spleen. Conclusion: These data suggest that TNP-470 may be effective as a treatment of r e d cell carcinoma, especially when micrometastases are involved. KEYWORDS: angiogeneisinhibitor, TNP-470, murine renal tumor
Patients with renal cell carcinoma (RCC) have metastatic disease at the time of diagncsis in 25 to 57% of cases and develop metastases within 10 years in approximately 60% of cases after curative nephrectomy.12 Metastatic RCC is one of the most difficult urological neoplasms to cure because it is chemotherapy-resistantand is not radiosensitive. Recently, an e h c y rate as high as 30% has been reported for immune modulation therapy in treating advanced RCC.Successful use of biological respom modifiers, induding interferons and interleukin 2, as well as the adoptive transfer of lymphokineactivated killer cells and tumor-infiltratinglymphocytes has been reported to date.= However, these therapeutic modalities appear to be successful only in a relatively short term,and many authors have emphasized the need for more defhitive treatment. In 1971,Folkman introduced the concept of angiogenesis inhibition for cancer treatment, and a few substances have been reported to inhibit angiogenesis and suppress established solid tumors in animala.6.7 It has also been demonstrated that the growth of micrometastases in target organs and tissues depended on angiogenesis and that there was a significant correlation between angiogenic properties and metastatic potential of human breast cancer and prostate c81'Cinoma.8-10 Therefore, there seem to be a reasonably good possibility that inhibition of angiogenesis is clinically useful as an adjunctive modality in the treatment of solid tumors. 0-(chlomacetyl-carbamoyl)fumagillol (TNP-470), a s p thetic analogue of fumagillin isolated h m Aspergillus fumigatus, has recently been shown to possess potent antiangiogenic and antitumoral activities against some types of malignant neoplasm.11 In this study, we examined the inhibitory activities of TNP-470 against in vivo growth, angiogen-
esis and metastasis of murine renal cell carcinoma, on which no information has been available to date. MATERIALS AND METHODS
A n i d . Female, euthymic BALBlc mice were h o d in a special pathogen-fhe facility and were used routinely at 8 to 10 weeks of age when they weighed 22 to 24 g. Tumor. "he Renca cell line, a renal cortical adenocarcinoma syngeneic in B W c mice,kindly provided by Dr.R. H. Wiltrout W.C.I., Frederick, Maryland), was maintained in vivo by serial intanrenal passage. This tumor line WFIS shown to be mycoplasma-free (Flow Laboratories, Mckan, Virginia). Drug. TNP-470 was synthesized and provided by Takeda Chemical Industries (Osaka, Japan) for the purpose of clinical trial. Stock solutions of TNP-470were prepared in absolute ethanol and suspended in 5% gum arabic and normal saline. Thempeutic regimen. In Rema-indated mice, graded doses of TNP-47W0.2 ml. were administered eubcutaneously every other day begimhg on day 1 and continuing until day 9,or beginning on day 6 and continued until day 14. Control mice were administered subcutaneous iqjeetiona of 0.2ml. vehicle. Tumor growth assay. The tumor was aseptically excised h m a tumor-bearing m o w , and single cell suspensione were prepared by mechanical dissociation and filtration of the tumor cells through a &-mesh stainless-steel sieve. Routinely, injection of 6 X lo6 Renca cells into the subcapsular space of the leR kidney was performed through a flanh incision, and 1 X lo6 tumor cells were i n d a t e d subcutaneouely on the back. Tumor weight and body weight were measured on day 21 following the tumor inoculation. The subcutaxle$ccepted for publication November 7,1996. Requests for re rink D e p q e n t of Urology, Iwate M e d i d owly inoculaM tumors were measured 2 times a week fhm day 1 to the time when gross ulceration of the tumors develuniversity, 19-1 U&aru, Monoka 020, Japan. 1776
1776
TNP-470FOR RENCA
oped in the control mice. The tumor volume was calculated by the following formula: tumor volume (mm?) = 0.5 X a
X
b2
(1)
where a is the longest diameter and b is the shortest diameter. In the experiment examining the effect of TNF-470 on survival, tumor-inoculated mice were treated with TNP-470 every other day beginning on day 1 and continuing until day 9. All experiments were performed at least twice with similar results. Experimental metastasis assay. The single cell suspensions were prepared as described. Fifty thousand cells in 200 J. of serum free RPMI medium (Flow Laboratories) were injected into the lateral tail vein. In addition, injection of 5 x lo5 Renca cells under the splenic capsules was given through a flank incision. The lungs and liver were excised and fued on day 14 and day 21, respectively. Metastatic foci on the lung or spleen surface were counted under a dissecting microscope. All experiments were performed a t least twice with similar results. Tumor angiogenesis assay. Inhibition of tumor angiogenesis was assayed by the quantitative colorimetric method of Lee et a1.12 Renca cells were inoculated intradermally on day 0 at 4 sites on the back of each recipient mouse. Each site was injected with 2 x lo6 cells in 50 pl. by a 26-gauge needle. TNP-470 was injected subcutaneously into the abdominal region in graded doses every day beginning a t 4 hours on day 0 and ending on day 2 after tumor inoculation. The control treatment consisted of injection of vehicle only. The extent of angiogenesis was assayed on day 3 by measuring the total vascular volume in disks of skin from each of the 4 tumor injection sites. This was assessed by injecting 0.25%Evan’s blue (Sigma, St. Louis, Missouri) in normal saline via the tail vein, followed by a colorimetric measurement of the dye in the skin disks. The animals were sacrificed by cervical dislocation 2 minutes after the dye injection, and a midline skin incision was made to allow stretching free the involved skin areas. Then the sites of tumor cell inoculation were punched out, and the pairs of hemilateral skin disks were pooled and cut into small pieces. Normal skin was obtained from noninoculated sites and used for background measurement in the assay. The minced tissue was suspended in 1.5 ml. sodium sulfatelacetone solution and incubated at room temperature for 24 hours with occasional shaking. Then the absorbance of the supernatant was determined at 620 nm. and the percent angiogenesis was calculated by the following formula: % angiogenesis = (A-B)/(C-B) X 100,
Wilcoxon test using the same software. Differences were considered statistically significant when the P value was 5% or less.
(2)
Where A, B, and C represent the b2,, values of treated tumor, background skin and control tumor. Statistical analysis. All values are expressed as the mean 5 standard deviation (SD). Data were analyzed by generalized Wilcoxon test using Fisher statistical software. The analysis of the survival studies was performed by plotting survival curves and calculating by the Kaplan-Meier or the
RESULTS
In vivo antitumor effect of TNP-470 treatment against Renca tumor. When subcutaneous injection of TNP-470 began on day 1 following the cell inoculation under the renal capsule, daily doses of 90,30, or 10 mg.kg. resulted in dosedependent inhibitions (p <0.01) of the growth of Renca tumor as assessed on day 21. The body weight of the inoculated mice was not affected by TNP-470 below the daily dose of 30 mg.kg. However, significant weight loss occurred in the mice given more than 90 mg.kg./day of TNP-470 (table 1). The growth of the subcutaneously inoculated tumor was also inhibited (p <0.01) in a dose-related manner by TNP-470 (fig. 1). #en the administration of 30 mg.kg. TNP-470 began on day 6 after subcutaneous or renal capsular inoculation, it was entirely ineffective. In another set of experiments, TNP-470 given subcutaneously in daily doses of 10,30, or 90 mg.kg. beginning on day 1after tumor inoculation significantly prolonged the survival period compared with the untreated tumor-bearing controls (fig. 2). All control mice died by day 28. In contrast, 50%,60%, or 90%survival was seen on day 32 in the mice treated with 10, 30, or 90 mg./kg. daily. Effect of TNP-470 on experimental metastasis of Renca tumor. #en TNP-470 was injected subcutaneously at a daily dose of 10,30, or 90 mg./kg. beginning day 1 after tumor inoculation, the number of metastatic foci in the lung was significantly reduced in a dose-dependent manner as assessed on day 14. TNP-470 also reduced the number of metastatic foci in the liver when examined on day 21 after the tumor inoculation (table 2). These treatments apparently reduced the size of individual metastatic foci in both lung and liver as well (data not shown). Angiogenesis inhibition of Renca tumor by TNP-470. The effect of TNP-470 treatment on the angiogenesis of Renca tumor is shown in table 3. The background blood volume in 33.2 mm.2 skin was 0.22 5 0.08 pl. The blood volume in the tumor injection sites was 0.95 2 0.16 pl. in the controls and 0.61 ? 0.10 pl., 0.46 2 0.05 pl., or 0.32 ? 0.06 pl. in the TNP-470-treated tumor-bearing groups given 10, 30, or 90 mg./kg. daily (control versus each treated group: p <0.01). There were significant differences (p <0.05) among the 3 groups given different doses of the drug. There was a significant inhibition of the tumor angiogenesis down to 33% to 62% of the controls by TNP-470 (p <0.01) (table 2). DISCUSSION
TNP-470 is a very potent inhibitor of endothelial cell proliferation in vitro,l3.14 and the agent has also been shown to inhibit angiogenesis in a variety of in vivo assays, utilizing the chick chorioallantoic membrane, rat and mouse implanted sponges, and rabbit or rat cornea.15 An inhibitory activity against some murine tumor growth was also reported with this agent,13.16 but the antiangiogenic activity was not thoroughly studied. In this study, we examined the
TABLE1. Tumor growth inhibition
of Renca tumor by TNP-470 (1)
Tumor Dose (mg.ikg.)
No. of mice
mg.
T i c (lo)
Body wt. TIC (5%)
P value ~~~
Control 3.9 z 0.3” 10 100 ion 10 TNP-470 10 2.3 % 0.2 59 p
1777
TNP-470 FOR RENCA
k
M Control
h
*--s &--A
v
-
0
7
TNP-470(10mg/kg) TNP-470(30me/kg)
10
14
17
21
24
Days after cell inoculation
TABLE2. Effect of TNP-470 on metastasis Dm3
No of Dose Img./kg.) mice .- .
of Rettra tumor
-__
No of metastatic fococl
mn
I
P value
T/C 1%)
~~
Experiment of lung metastases 10 1 1 7 Z 14" 100 Control TNP-470 10 10 48? 4 41 p c 0 . 0 1 TNP-470 30 10 29 t 3 25 pc0.01 TNP-470 90 10 11 - 2 9 p
FIG. 1. Tumor growth inhibition of Renca tumor by TNP-470.2 BALBlc mice were i n d a t e d subcutaneously with Renca tumor on day 0 and received TNP-470 subcutaneously on days 1 , 3 , 5 , 7 and 9. Tumors were measured 3 times a week. Control versus "-470 (10 mg./kg.) p <0.01; control versus TNP-470 (30 mg.kg.) p cO.01; control versus TNP-470 (90 mg.kg.) p CO.01.
TABLE3. Angiogenesis inhibition of Renca tumor by TNP-470 Drug
No. of Dose fmgflrg.) mice
'asdm volumeb (&I,)
% angiogenesis
P value
100 10 0.95 -C 0.16b Control TNP-470 10 10 0.61 2 0.10 62 p <0.01 0.46 2 0.05 48 p <0.01 TNP-470 30 10 90 10 0.32 i 0.06 33 p <0.01 TNP-470 The B W c mice were inoculated with Rencn tumor subcutaneously at 4 sites of back on day 0 and received TNP-470 subcutaneously on daya 0 , l and 2. The tumor vascular volume on day 3 represents the blood volume of 2 skin disks extraded by the mean background blood volume. a plf33.2 nun.*; mean +- SD
FIG. 2. Extension of survival time of Renca-bearing mice by TNP470. BALB/c mice were inoculated with Renca tumor under capsule of leR kidney on day 0 and received TNP-470 subcutaneously on days 1 , 3 , 5 , 7 and 9. Control versus TNP-470 (10 m ./kg ) p cO.01; control versus TNP-470 (30 mg.kg.) p <0.01; contr8 versus TNP-470 (90 mg.kg.1 p cO.01.
antiangiogenic as well as antitumoral activity of TNP-470 using the murine RCC Renca tumor. TNP-470 showed antitumoral activity against Renca cells and effects were dose dependent. The body weight of the tumor-bearing mice was only minimally affected at a dose of 90 mg./kg./day of T"470. The optimal effective dose appeared to be 30 mg.kg./day in BALBlc mice. TNP-470 prolonged the life span of Rencabearing mice and the toxicity attributable to this agent seems to be low. However, in our experiments, TNP-470 did not inhibit tumor growth when treatment was delayed to day 6 following Renca inoculation, an indication of the importance of the timing of drug administration in relation to disease stage. From our experiments, it appears that greater effects could be expected if intervention with this drug were to take place earlier. Although it is difficult to make a meaningful correlation between the 6-day delay in the drug intervention in our animal model and the clinical stages in actual patients, our results may indicate that the drug is more u s e l l as a preventive modality than as a suppressive therapy.
Tumor metastasis is a multifactorial and multi-stage process.8 In particular, tumor angiogenesis is considered an important factor that greatly influences the growth of micrometastases in the target organs.17.18 Theoretically angiogenesis inhibitors should effectively inhibit tumor metastases based on the concept that tumors require newly formed blood vessels for their growth at both primary and secondary sites and for dissemination. Potent antimetastatic action of angiogenesis inhibitors was reported in metastatic models involving B16BL6 melanoma and M5076 reticulum cell sarcoma.lg.20 Our present data demonstrating the inhibitory effect by TNP-470 on experimental Renca metastasis are consistent with these studies. Both the number and the size of the metastatic foci in the lungs and the liver were reduced by TNP-470 treatment in our studies. Tumor angiogenesis is generally estimated by the degree of neovascularization around the tumor (or tumor extracts), and the inhibition assays can be classified into 2 groups depending on the route of administration of the test agents. The first group includes chorioallantoic membrane and rabbit (or rat) cornea assays, which are similar to bacterial ~ ' ~ ~ are implanted into the chemosensitivity t e ~ t s . 6 .Tumors hosts, test agents are simultaneously inoculated near the tumor, and the area of avascular zone is semiquantitated visually. The second group consists of rodent skin assays involving the dorsal air-sac methods and the colorimetric assay employed in our studies.**-14In these assays the tumor cells (or the Millipore chamber loaded with tumor cell suspension) are implanted subcutaneously or intradermally into the host and the test agents are given systemically. In our current studies, we adopted a colorimetric assay because the dye-perfusion technique has the advantage of providing a n objective endpoint with relatively small assay variations, as reflected in the small SD values. When the degree of angiogenesis was quantitated by measuring vascular volume in the tumor, the potent antiangiogenic action of TNP-470 was demonstrated in BALBlc mice inoculated with Renca tumor in a dose-dependent manner.
TNP-470 FOR RENCA
1778 CONCLUSIONS
Thus, the present observation obtained in mice indicated that the angiogenesis inhibitor TNP-470 has strong inhibitory activity against in Viva growth and metastasis ofmurine renal cell carcinoma. TNP-470 seems to be a promising candidate for development as an antitumor and antimetastatic agent in RCC. REFERENCES
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