Prostaglandin: Increased Production by Renal Cell Carcinoma

Vol. 118, November Printed in U.S.A.

THE JOURNAL OF UROLOGY

Copyright © 1977 by The Williams & Wilkins Co.

PROSTAGLANDIN: INCREASED PRODUCTION BY RENAL CELL CARCINOMA KENNETH B. CUMMINGS*

AND

R. PAUL ROBERTSON

From the Department of Surgery/Urology, The Mason Clinic and Virginia Mason Research Center, Department of Medicine, University of Washington, Veterans Administration Hospital, Seattle, Washington

ABSTRACT

The syndrome of hypercalcemia in patients with renal cell carcinoma without metastasis to bone, in association with elevated levels of immunoreactive prostaglandin E and normal parathyroid hormone levels, prompted the investigation of an etiologic relationship of increased prostaglandin in this syndrome. Ethyl acetate extracts of tissue culture effluents, primary and metastatic renal cell carcinoma, and plasma were chromatographed on silicic acid colu"'llns and assayed by double antibody immunoprecipitative methods for immunoreactive prostaglandins A and E. Increased levels of immunoreactive prostaglandins A and E were found 1) to be generated in parallel with cell growth during a period of time by renal cell carcinoma in monolayer growth, 2) in extracts of primary and metastatic renal cell carcinoma tissue and 3) in the venous effluent of a kidney bearing a renal cell carcinoma. These findings support the hypothesis that renal cell carcinoma can produce prostaglandins. Furthermore, reported syndromes of patients with renal cell carcinoma associated with elevated prostaglandin levels may result from the autonomous production ofprostaglandins in vivo by the tumor. Neoplastic cells are known to produce biologically active hormone-like materials. It is reasonable to expect that a cell that has undergone malignant transformation may still retain its capability to produce substances that it formed before oncogenesis. However, in the malignant state the transformed cell might be expected to generate these substances autonomously, that is without counter-regulatory controls. Tumor extracts have been shown to possess biological activity compatible with adrenocorticotropin, melanocyte-stimulating hormones, antidiuretic hormones, parathyroid hormones, insulin, glucagon, gastrin, serotonin and erythropoietin. 1 Renal cell carcinoma, in particular, has been associated with the secretion of erythropoietin,2 renin3 and parathyroid hormone. 4 Recent reports of syndromes associated with renal cell carcinoma include 1) hypercalcemia with suppressed parathyroid hormone levels, no bony metastasis and elevated circulating immunoreactive prostaglandin E, 5 and 2) longstanding hypertension reverting to the normotensive state in association with elevated plasma levels of immunoreactive prostaglandin A. 6 These observations prompted us to investigate the possibility that renal cell carcinoma produced prostaglandins. BACKGROUND

In 1930 Kurzrok and Lieb, 2 American gynecologists, observed that seminal fluid contracts human uterine strips. 7 The term prostaglandin was coined by von Euler, who found this activity to be present in the acidic lipids extracted from the seminal vesicles of man and sheep. 8 This misnomer has continued, despite the fact that prostaglandins have been found in most human tissues. In recent years they also have been reported to be present in the blood of patients with Accepted for publication November 5, 1976. Read at annual meeting of Western Section, American Urological Association, Coronado, California, February 22-26, 1976. Supported by Virginia Mason Medical Center Restricted Cancer Fund Grant 185 and a Clinical Investigatorship, United States Veterans Administration. *Requests for reprints: The Mason Clinic, 1100 Ninth Ave., Seattle, Washington 98101. 720

medullary carcinoma of the thyroid," pheochromocytoma, 10 bronchogenic carcinoma 10 and renal cell carcinoma. 5 ' 6 Structurally and functionally there appear to be 3 subgroups of physiologically important prostaglandins - prostaglandins A, E and F. The basic structure is the same for all prostaglandins, consisting of a 5-carbon ring with 2 aliphatic side chains. Individual variation is determined by various degrees of unsaturation and substitution in the cyclopentane ring or the aliphatic side chains. The prostaglandins also are grouped into mono-unsaturated, bis-unsaturated and tris-unsaturated classes, that is E 1 , E 2 and E,3 , according to the number of carbon-to-carbon bonds in the parent prostaglandin. The structural formulas of biologically significant prostaglandins are illustrated in figure 1. Unlike classical hormones the prostaglandins are neither synthesized by specialized types of cells nor stored in the tissues that form them. The prostaglandins extracted from a tissue probably represent material recently synthesized but not yet metabolized. Prostaglandins may act as local mediators or modulators at the site of release rather than as circulating hormones. Evidence for this comes from the findings that prostaglandins E 2 and F 2 , while stable in blood, are almost completely inactivated on passage across the lungs. 11 By contrast, prostaglandins of the A series are not degraded by the lungs and, therefore, might function as circulating hormones in addition to their local tissue-modulating effects. 12 Current interest in the syndromes in patients with renal cell carcinoma and elevated prostaglandin levels prompted us to examine prostaglandin levels in 1) tissue culture effluents of renal cell carcinoma, 2) surgical specimens of renal cell carcinoma compared to those of normal kidneys, 3) renal cell carcinoma metastases compared to normal tissue in which the metastases occurred and 4) the venous effluent of renal cell carcinoma-bearing kidneys at the time of nephrectomy. If it could be shown that renal cell carcinoma in tissue culture generated prostaglandin, and primary and metastatic renal cell carcinoma had radioimmunoassayable levels of prostaglandin, the relation of prostaglandin as cause and effect in the syndromes reported could be strengthened.

PROSTAGLANIHNS IN RENAL CELL CARCINOMA

H F'!G. J. Structmre of

A, E andF

MATERIALS AND ME7HODS

materials were obtained for 1) effluents of renal cell carcinoma 2) renal cell carcinoma tissue

and uninvolved carcinoma, normal liver spEicn:nerts and 4) blood from a tumor vein, main renal vein vein. Tissue cultures. Renal cell carcinom.a obtained at an operation was established in tissue culture as described J:J Cellular was confirmed by NYrn,·.c,,rrn of the tumor to cells 2). Renal carcinoma at a lQ
Culture media per cent heat-inactivated fetal calf serum) served as control. Tissues. Tumor and control tissues were obtained an or autopsy and iu,u.,ccuo"'"·'J' frozen and stored at minus 70C. Blood. Blood uuCH>ii-'"'•U

The residue is then dissolved acetate and~nn~I~c:~,"I"'kt~ncH and which sepa-· cct 1~1eunu,,,, A, E and F from one another. Each nr•,m,,q,, and dissolved in buffer. for tritiated prostato calculate recovery the column. The remainder for prostaglandin content the use of antisera directed at either the or F and the tritiated vav,u.,-.w.,,,~u.,. v,,JVU,F,
reactivity with prostaglandins E 1 and F2 the prostaglandin E antiserum has a 15 per cent l per cent cross-reactivity with A and F?. and the p,rosta,g1.1u1a1n antiserum has less

722

CUMMINGS AND ROBERTSON

1 per cent crossreactivity with prostaglandins A 1 and E 1 . All antisera are raised from immunized guinea pigs. After a minimum of 2 hours of incubation with anti-prostaglandin antisera normal guinea pig serum and rabbit anti-guinea pig antisera are added to separate bound from unbound prostaglandin. After overnight incubation at 40C the supernatant is decanted, the precipitate is dissolved in Bray's solution and the bound 3 H prostaglandin is quantified in a liquid scintillation counter. A standard curve for prostaglandins A,, E, and F 2 alpha is included in each assay. The values for the patient sample are estimated by use of the standard curve that inversely and logarithmically relates prostaglandin concentration to precipitated counts. Patient samples for plasma are expressed in pg. per ml. and tumor samples are expressed in ng. per gm. tissue. The assays for the prostaglandins A, E and F have a sensitivity of 50 pg., accuracy (recovery of prostaglandin added to plasma) more than 90 per cent and intra-assay and interassay coefficients of variation of less than 12 per cent.

60

*

ng/g

0

0

500

~,--,I PGA I

I

I

i'.)..

I

PGE

I

iPG

I I

250 pg/ml

I I

I

I

j'3 /

/

&"

I

I

0 0.5

~

Normal Renal Cortex

t r-, Renal Cell CA

Fm. 4. Comparison of extractable immunoreactive prostaglandin E material from normal and tumor tissue. Each set of symbols represents replicate determinations from 1 patient. TABLE 1. Immunoreactive prostaglandin content of renal cell carcinoma liver metastases, normal liver and normal kidney cortex

Immunoreactive Prostaglandin A (ng./gm. tissue)*

Liver metastases Normal liver Kidney cortex

2.54 ± 0.36 0.22 ± 0.02 0.34 ± 0.02

Immunoreactive Prostaglandin E (ng./gm. tissue)* 13.59 ± 0.18 1.97 ± 0.14 3.24 ± 0.83

* Mean plus or minus standard deviation.

DISCUSSION

The premise that prostaglandin is related etiologically in the syndromes associated with renal cell carcinoma has been strengthened by our observations. Renal cell carcinoma in tissue culture has been shown to generate immunoreactive prostaglandins E and A with progressively increasing levels as monolayers approach confluency. Furthermore, renal cell carcinoma primary and metastatic tumors have been shown to contain increased levels of immunoreactive prostaglandins

(cum.)

TISSUE EXTRACTS

iPGE

RESULTS

There was a progressive accumulation of immunoreactive prostaglandins A and E in effluents from renal cell carcinoma growing in monolayer (fig. 3) and greater amounts of immunoreactive prostaglandin E were found in renal cell carcinoma than in normal renal cortex (fig. 4). The levels ofimmunoreactive prostaglandins A and E were elevated in renal cell carcinoma liver metastases as compared to normal liver and renal cortex (table 1). Plasma separated from intraoperative blood samples drawn from tumor, main renal and antecubital veins exhibited progressively diminishing values of immunoreactive prostaglandins A and E (table 2).

~

2

4

Days Fm. 3. Increments in immunoreactive prostaglandins A and E during 4 days immediately after plating for parallel effluents of renal cell carcinoma. Each point is mean plus or minus standard error of prostaglandin concentration of media in 4 flasks.

2. Immunoreactive prostaglandins A and E concentrations change from tumor vein to main renal vein to antecubital vein

TABLE

Antecubital vein Main renal vein Tumor vein

Immunoreactive Prostaglandin A (pg./ml.)

Immunoreacti ve Prostaglandin E (pg./ml.)

93 214

61 195

E and A as compared to normal renal cortex and the tissue in which the metastases occurred. The observations suggest that one possible source of prostaglandin in renal cell carcinoma is the tumor tissue itself. Compared to metastatic renal cell carcinoma, adjacent uninvolved tissue (liver) contained one-tenth the amount of prostaglandin A (0.22 plus or minus 0.02 versus 2.54 plus or minus 0.36 ng. per gm. tissue) and immunoreactive prostaglandin E (1.97 plus or minus 0.14 versus 13.59 plus or minus 0.18 ng. per gm. tissue). Therefore, it is possible that increased prostaglandin levels were the result of tumor production and not the reaction of the surrounding normal tissue to the presence of tumor. The exact mode by which prostaglandins exert their effects is not known. However, prostaglandin E is known to release previously labeled calcium from bone 15 and osteoclastic effects have been produced by prostaglandins. 16 In the 2 studies cited hypercalcemia was associated with renal cell carcinoma in the absence of bony metastases. 5 ' 17 Both of these patients had pulmonary metastases and elevated levels of prostaglandin E were noted in 1 patient. 5 Considering that prostaglandin E is nearly completely degraded on passage through the lungs, one might not expect elevated peripheral plasma levels except in those cases with pulmonary metastases in which prostaglandin E is released directly into the pulmonary venous bed and subsequently delivered to the systemic circula-

PROSTAGLANDINS IN RENAL CELL CARCINOMA

tion at elevated levels. It is noteworthy that in 1 case referred to indomethacin treatment effectively lowered serum calcium to normal calcemic levels" and in the other there were elevated circulating immunoreactive prostaglandin E levels. 0 Jndomethacin treatment may be effective even in the treatment of hypercalcemia in patients with metastatic bone disease. It has been shown that certain tumors produce prostaglandins (predominantly prostaglandin E) 18 and our studies have shown that metastatic renal cell carcinoma has this ability. 19 Because of the rapid degradation of prostaglandin E it might not be possible to demonstrate that prostaglandin E by bone metastases might contribute to localized Extramustine phosphate is a drug that has been 1r,c.1nCYru"" to be effective in the treatment of mets.static prostatic carcinoma refractory to conventional forms oftherapy. 20 Alleviation of bone pain and regression of metastases have been the ascribed benefits of treatment. This anti-tumor drug has been shown to inhibit in vitro prostaglandin production by bovine seminal vesicles and may owe its in vivo effectiveness in part to a blockade of prostaglandin production by the tumor.~ 1 The syndrome cited of a patient with longstanding diastolic reverting to the normotensive state coincident of renal cell carcinoma, and associated peripheral plasma levels ofimmunoreactive prostaglandin was considered to be consequent to prostaglandin production by the tumor. Subsequent to surgical removal of the tumor the patient again had fixed hypertension." Lee and associates, studying the cardiovascular effects of prostaglandins, observed that prostaglandin A is a potent antihypertensive agent.2 2 • 23 The antihypertensive effects are considered to be mediated local effects within the kidney (alteration of intrarenal hemodynamics with increased cortical blood flow and diminished sodium resorption) and by effects (diminished peripheral vascular resistance). observations that prostaglandin A is generated by renal cell carcinoma in tissue culture and that elevated levels of immunoreactive prostaglandin A are found in primary and metastatic renal cell carcinoma support the consideration that prostaglandin A production by the tumor reversed the to the normotensive state in the case cited. REFERENCES

Amatruda, T. T., Jr.: Nonendocrine secreting tumors. In: Duncan's Disease of Metabolism, 6th ed. Edited by P. K. Bondy and I. E. Rosenberg. Philadelphia: W. B. Saunders Co., vol. 2, pp. 1227-1244, 1969. 2. Bennington, J. L. and Kradjian, R. M.: Renal Carcinoma. Philadelphia: W. B. Saunders Co., 1967. 3. Eddy, R. L. and Sanchez, S. A.: Renin-secreting renal neoplasm and hypertension with hypokalemia. Ann. Intern. Med., 75: 725, 1971. 4. Plimpton, C. H. and Gellhorn, A.: Hypercalcemia in malignant disease without evidence of bone destruction. Amer. J. Med., 21: 750, 1956.

5. Robertson, R. P., Baylink, D. J., Marini, J. J. and Adkison, W.: Elevated prostaglandins and suppressed parathyroid hormone associated with hypercalcemia and renal cell carcinoma, J. Clin. Endocr., 41: 164, 1975. 6. Zusman, R. M., Snider, J. J., Cline, A., Caldwell, B. Speroff, L.: Antihypertensive function of a renal cell carcl· noma. Evidence for a prostaglandin-A-secreting tumor. New Engl. J. Med., 290: 843, 1974. 7. Kurzrok, R. and Lieb, C. C.: Biochemical studies of hurnan semen. IL The action of semen on the human uterus. Pree. Soc. Exp. Biol. Med., 28: 268, 1930. 8. von Euler, U.S.: On the specific vaso-dilating and plain muscle stimulating substances from accessory genital glands in and certain animals (prostaglandin and vesiglandin). J. Physiol., 88: 213, 1937. 9. Williams, E. D., Karim, S. M. and Sandler, M.: Prostaglandin secretion by medullary carcinoma of the thyroid. A possible cause of the associated diarrhoea. Lancet, 1: 22, 1968. 10. Sandler, M., Karim, S. M. and Williams, E. D.: in amine-peptide-secreting tumours. Lancet, 2: 1053, 11. Ferreira, 8. H. and Vane, J. R.: Prostaglandins: their ct1,;ai:1pearance from and release into the circulation. Nature, 1967. 12. McGiff, J. C., Terragno, N. A., Strand, J. C., Lee, J. B. and Lonigro, A. J.: Selective passage of prostaglandins across the lung. Nature, 223: 742, 1969. 13. Cummings, K. B., Peter, J. B. and Kaufman, J. J .. Cell-. mediated immunity to tumor antigens in patients with renal cell carcinoma. J. Urol., 110: 31, 1973. 14. Zusman, R. M., Caldwell, B. V., Speroff, L. and Behrman, H. R.: Radioimmunoassay of the A prostaglandins. Prostaglan·· dins, 2: 41, 1972. 15. Klein, D. C. ad Raisz, L. G .. Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology, 86: 14,J6, 1970. 16. Tashjian, A. H., Jr., Voelkel, E. F., Goldhaber, P. and Levine, L.: Prostaglandins, calcium metabolism and cancer . Fed. Proc., 33: 81, 1974. 17. Brereton, H, D., Halushka, P. V., Alexander, R. W., Mason, D. M., Keiser, H. R. and DeVita, V. T., J·r.: Indomethacinresponsive hypercalcemia in a patient with renal-cell adenocarcinoma. New Engl. J. Med., 291: 83, 1974. 18. Jaffe, B. NL, Parker, C. W. and Philpott, G. W.: Immunochemical measurement ofprostaglandin or prostaglandin-like activ ity from normal and neoplastic cultured tissue. Surg. Forum. 22: 90, 1971. 19. Cummings, K. B., Wheelis, R. F. and Robertson, R. P .. Prostaglandin: increased production by renal cell carcinoma. Surg. Forum, 26: 572, 1975. 20. Jonsson, G. and Hogberg, B.: Treatment of advanced carcinoma with Estracyt. A preliminary report. Urol. Nephrol., 5: 103, 1971. 21. Perklev. T.: Blockade of prostaglamlin synthesis by an antitumor drug, Estracyt. Acta Endocr., 68: 219, 1971. 22. Lee, J., Kannegiesser, H., O'Toole, J. and Westura, E.: tension and renomedullary prostaglandins: a human the antihypertensive effects of PGA 1 . Ann. N. Y. Acad. 180: 218, 1971. 23. Lee, J. B., McGiff, J. C., Kannegiesser, H., Aykent, Y. Y., Mudd, J. G. and Frawley, T. F.: Prostaglandin A,: tensive and renal effects. Ann. Intern. Med., 74: 703,