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Testolactone, sulindac, warfarin, and vitamin k1 for unresectable desmoid tumors
Testolactone, Sulindac, Warfarin, and Vitamin K1 for Unresectable Desmoid Tumors William R. Waddell,
MD,
TUCSON,
Arizona,
Ten patients with large inoperable desmoid tumors in various body locations were treated with testolactone. Four tumors (40%) responded with major . . regresstons, i.e., more than 50% reduction in volume. Eight patients received nonsteroid anti-inflammatory drugs (indomethacin, sulindac, or sulindac with warfarin and vitamin Kl [Mephyton]) for periods of 2 to 91 months. There was one major regression, one partial regression, and three instances of tumor growth arrest over periods up to 8 years. Seven patients were treated with nonsteroid antiinflammatory drugs concurrent with or after testolactone or tamoxifen. There were five major regressions and one partial regression with extensive central necrosis of an enormous intra-abdominal tumor. The last patient has been treated for only 12 months, with no change in tumor volume. It appears that estrogens function as growth factors for desmoid tumors, and that minimization of these effects inhibits tumor growth in some, but not all, cases. In those instances where antiestrogens were not effective as single agents, the tumors usually responded to subsequent nonsteroid anti-inflammatory drug therapy. Withdrawal of estrogen may be followed by inhibition of transcription of genes that support tumor cell proliferation, and sulindac and indomethatin may augment these effects by inhibiting prostaglandin and cyclic AMP synthesis and the activity of protein kinase C. Warfarin may function as a protonophore to acidify the cytoplasm and prevent the alkalinization that is necessary to initiate DNA synthesis and cell cycle progression, again an impairment of the transcription process.
Wolff M. Kirsch,
MD, Loma Linda, California
he management of inoperable desmoid tumors is a T difficult problem for which there is no single or welltested solution. There is general agreement that small tumors should be excised with wide margins [I]. Large intra-abdominal tumors are often unresectable, and this is also true of tumors in locations such as the mediastinum, neck, thigh, pelvis, and shoulder girdle. Radiation has been reported to control some tumors [2], but this requires large doses that carry the risks of damage to adjacent viscera. Endocrine manipulations have resulted in some dramatic responses. Tamoxifen [3,4], clomiphene [5], progesterone [a, and testolactone [7,8] have all been used successfully, and, as with other modes, there have been frequent failures. This project began in an attempt to control the growth of an inoperable intra-abdominal desmoid tumor in a young woman with Gardner’s syndrome. The tumor had developed while the patient was taking birth control pills, suggesting some relationship to the reproductive endocrine system [7]. At that time, testolactone was used occasionally for disseminated breast cancer [9,10] as an analogue of testosterone. Its use for desmoid tumors was founded on early experimental work suggesting that estrogens were involved in the development of fibrous tumors in guinea pigs [II ,121. After resolution of the desmoid tumor in the original patient, other patients were treated, but there were frequent unexplained failures. Until recently, there was little appreciation of the original assumptions that prompted this clinical approach to an often insoluble problem. This report presents therapeutic recommendations to minimize the effects of estrogens and to inhibit prostaglandin and CAMP synthesis, both of which influence growth and proliferation of fibroblasts [13-181. PATIENTS AND METHODS
The observations were made over a period of 17 years. Eight patients were members of families with Gardner’s syndrome, and 11 had tumors that arose sporadically. In the former group, the tumors were located preponderantly in the bowel mesentery and abdominal wall of patients who had undergone surgery (Tables I and II). All but one of the patients were between 21 and 40 years of age. Six additional patients were excluded from this analysis because recurrent tumors had formed about Marlex mesh (in five patients) or methacrylate (in one patient) replacements of abdominal and thoracic walls. These tumors behave differently. Large foreign bodies may have initiated and perpetuated mitogenic action in susceptible indiFrom the Department of Surgery, University of Arizona, Tucson, Arividuals. zona, and the Division of Neurosuraerv. Loma Linda University Medical School, Loma Linda, California. The responses of the tumors were measured by whatRequests for reprints should be addressed to William R. Waddell, ever means were appropriate. The tumors were very MD, Department of Surgery, University of Arizona Health Sciences_ large, and in most, the presenting parts could be palpated Center, Tucson, Arizona 85724. and measured. In other instances, computed tomography Manuscript submitted July 11, 1990, and accepted in revised form (CT) and, lately, magnetic resonance imaging (MRI) October 10, 1990. 416
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--_ TABLE I Testolactone’
Age
sex
Gardner’s Syndrome
Location of Tumor
Duration of Treatment (mo)
Outcome
Reference
laT 2 3 4 5
23 24 21 22 27
F F F F M
Yes No No No Yes
12 2 2 48 7
Complete regression Progression Progression Major regression Progression
7
6 7
44 36
M M
Yes No
18 3
Major regression Progression
a
38
M
No
9 10
44 33
M M
Yes No
Small & large bowel mesentery Posterior thigh & popliteal space Abdominal wall Pelvic wall 8 retroperitoneum Small & large bowel mesentery, retroperitoneum Small bowel mesentery Abdominal wall, small bowel mesentery Mediastinum Sternum Small bowel mesentery Mandible, neck, upper chest
Patient
5
-
Progression
60 6
4. 31 8
4. 31
Complete regression Progression
8
* These results pertain only to the period during which testolactone was administered(not the eventual outcome). Dose of testolactone was 200 mg/day. The response rate was 40 % 7Estrogen-containingcontraceptives omitted immediately prior to start.
TABLE II Indomethacln, Sulindac, or Sullndac with Warfarln’ Dose
Duration
(mg/d)
(mo)
Sulindac
300
29
Major regression
-
Wafarin
7.5
Axilla, chest wall
Sulindac
400
12
(central necrosis with fistulas) Progression
-
Warfarin Sulindac Sulindac
10 400 400
12 29
Partial regression Growth arrest
-
Warfarin lndomethacin Sulindac Sulindac Sulindac lndomethacin Sulindac
7.5 300 375 300 300 75 300
53
Growth arrest
91 2 9
Growth arrest Progression Major regression
Patient
Age
Sex
Gardner’s Syndrome
Location of Tumor(s)
11
22
F
Yes
Small bowel mesentery, retroperitoneum
12
34
F
No
13 14
40 39
M M
Yes No
Bowel mesentery Groin & pelvis
15
34
M
No
Axilla
16 17 16
36 25 76
M F F
Yes Yes No
Bowel mesentery Small bowel mesentery Chest wall
Drugs
Outcome
Reference
4
4 4, 31
* The patients in this grouping had not received prior antiestrogens. Countinggrowth arrest, the response rate was 62%.
have been used for evaluation of size and local effects. Repeated observations over very long periods minimize errors of significance to this study. The protracted course of desmoid tumor growth permitted establishment of pretreatment tumor status and growth patterns as the best feasible controls. Tumor responses were characterized as progression, arrested growth, partial regression (less than 50%), major regression (more than 50%), and complete regression. When significant regression or arrest of growth occurred, the medication was usually continued indefinitely, in the first patient now for 17 years. These patients and their families were fully informed about their disease and the experimental nature of the treatment. All of them had previous operations, and many had other forms of chemotherapy and/or radiotherapy, all of which were unsuccessful. THE AMERICAN
One group of 10 patients received testolactone (Table I). A second group of eight patients treated with indomethacin, sulindac, or sulindac with warfarin and vitamin KI (Mephyton) is presented in Table II. The third group of seven patients shown in Table III was treated with indomethacin, sulindac, or sulindac with warfarin and Mephyton, after or concurrently with testolactone or tamoxifen. The currently recommended regimen is, in effect, a summation of a clinical experience extending over the past 17 years, consisting of: 1. Testolactone (TeslacQ) 750 mg daily (250 mg three times a day); 2. Sulindac (Clinorile) 300 mg daily (150 mg twice a day), then 200 mg twice a day if tolerated, starting after 1 month on lower dose; 3. Warfarin (CoumadirP) 5, 7.5 or 10 mg daily as tolerated. Start with lower dose and work up to 10 mg. JOURNAL
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TABLE
III
Antiestrogen wlth Indomethacln, Sullndac, or Sulindac with Warfarln Location of Tumor(s)
‘atlent
Age
Sex
lb
23
F
Abdominal wall
17
25
F
2
24
F
Small bowel mesentery Thigh 8 popliteal space
3 12
21 34
F F
5
27
M
8
38
M
Endocrine Adjustment
Other Drugs
Duration of Treatment (mo)
lndomethacin Warfarin Sullndac
29
Major regression
4, 7
64
Major regression
4
Piroxicam Sulindac Warfarin lndomethacin Sullndac Warfarin Sulindac Warfarin
52
Major regression
92 4
Major regression No change in 12 months Progression (eventual central necrosis and enteric fistula) Subsequent death due to uncontrolled sepsis Major regression (complete)
Antiestrogen’
Testolactone Tamoxifen Tamoxifen
Concurrent
Testolactone
Before
Abdominal wall Axilla, chest wall, arm Small 8 large bowel mesentery, retroperitoneum
Testolactone Testolactone
Before Concurrent
Testolactone
Before
Mediastinum, sternum
Testolactone
Before
Concurrent
53
1
lndomethacin
Outcome
Reference
-
7, 31
-
7, 31
* Under the “Antiestrogen” heading, the lowering of estrogen levels occurred “before” the administration of other drugs or “concurrently.” Patient 12 received 750 mg testolactone daily. In those patients who received antiestrogens before other drugs were added, the subsequent responses occurred while the patients were taking sulindac, indomethacin, or sulindac and warfarin.
Weekly measurement of prothrombin times until a steady tolerable dose established, then only occasionally; 4. Vitamin K1 (Mephytona), 5 or 10 mg daily to keep the prothrombin time slightly above normal. Drugs: Testolactone, 13-hydroxy-3-oxo- 13,17-secoandrosta- 1,Cdien- 17-oic acid 8-lactone, is not androgenie, estrogenic, progestational, or antiprogestational in the usual sense. Its main known biochemical effect is noncompetitive and irreversible inhibition of aromatase, a rate-controlling enzyme present in most tissues that catalyzes the final hydroxylations and A-ring desaturation in the modification of testosterone to estradiol and androstenedione to estrone [ 19,211. It acts synergistically with dibutyryl cyclic AMP in causing “reverse transformation” of cultured Chinese hamster ovary cells [22]. Its main clinical use has been in the treatment of advanced breast cancer, with doses from 100 to 2,000 mg daily. Most of the patients in the present study received 200 mg daily. The optimal dose is obviously not known. Tumoxifen, (Z)2-4-(l,Zdiphentl-l-butenyl)phenoxy-N,N-dimethylethanamine 2-hydroxyl- 1,2,3-propanetricarboxylite (l:), is a nonsteroid antiestrogen which competes with estrogen for binding sites in target tissues. It is widely used to treat breast adenocarcinoma in postmenopausal women. Antitumor effects are thought to be the result of binding to estrogen receptors and the consequences thereof. The patients in this study received 20 to 40 mg per day. Indomethacin, 1-p-chlorobenzoyl-5-methoxy-2methylindole-3-acetic acid, is a substituted 3-indolylacetic acid whose principal effect is inhibition of cyclooxygenase, the enzyme that catalyzes the oxidation of arachidonic acid to prostaglandin precursors [23-251.
Sulinduc, (Z)-5-fluoro-2-methyl-l-p-(methylsulfinyl)phenylmethylene-lH-indene-3-acetic acid, inhibits the cyclooxygenase that catalyzes the oxidation of arachidonic acid to prostaglandin precursors. It is a long-acting analogue of indomethacin, with similar biochemical actions [2q. It undergoes enterohepatic circulation, and is excreted through the intestinal tract and in urine. The compound is ingested as the inactive oxidized form and is inhibitory only after enzymatic reduction to the sulfide [27-291. Wurfarin, 3-(acetonylbenzyl)-Chydroxycoumarin, inhibits vitamin K epoxide reductase, interrupts the “vitamin K cycle,” and leads to inhibition of vitamin K-dependent protein carboxylation and formation of prothrombin. Warfarin in concentrations as low as low8 M inhibits DT diaphorase and uncouples oxidative phosphorylation in mitochondria [30]. Warfarin has other effects on tumor cells, including inhibition of DNA synthesis and, more particularly, the salvage pathway for DNA synthesis. The patients took 5, 7.5 or 10 mg daily. Mephyton is vitamin K1 or phylloquinone (Zmethyl3-phytyl- 1,4_naphthoquinone). It is an essential cofactor for the hepatic microsomal enzyme complex involved in the carboxylation of glutamic acid residues in preprothrombin to yield active prothrombin. This drug also inhib its the salvage pathway to DNA synthesis and acts additively with warfarin in this regard. Mephyton antagonizes the anticoagulation effects of warfarin without affecting the cytotoxic action. Some patients took 10 mg of Mephyton daily, but in others a dose of 10 mg two or three times weekly was established as adequate to maintain slightly elevated prothrombin times. In these trials, Mephyton was given to permit higher warfarin dosage.
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RESULTS In Table I, the 10 patients treated with testolactone for 2 to 60 months are listed, along with other patient information. Two patients responded within the first few months, and this was sustained. At the other extreme, the tumors of two patients stopped growing shortly after beginning testolactone, but did not show any signs of regression until 36 months, whereupon they regressed completely over the ensuing year. The tumors in the remaining six patients never stopped growing, and the drug was stopped after trial periods of 2 to 7 months. In Table II, the eight patients that received indomethacin, sulindac, or sulindac, warfarin, and Mephyton as the initial treatment are listed, none of whom had previously been treated with testolactone. There was one major and one partial regression; in three patients, tumor growth was arrested for periods of 29,53, and 91 months. Counting growth arrest as a form of success, the response rate was 62%; otherwise it was 25%. Table III summarizes the treatment of seven patients who received nonsteroid anti-inflammatory and antiestrogen drugs. Major regressions occurred in six. The only complication of testolactone was menorrhagia due to hyperplastic endometrium in one patient after receiving testolactone for 1 year. This was followed by long-lasting amenorrhea and menopausal symptoms after the drug was stopped. Sometimes pain in the area of tumor correlated with softening of the tumor and purplish discoloration of overlying skin. Three patients developed fistulas between small bowel and the necrotic interior of large intra-abdominal desmoids. Upper gastrointestinal dysfunction attributable to gastritis occurred occasionally and required reduction or brief omission of indomethacin or sulindac. There were no gastric or duodenal ulcers. Pregnancy should be avoided pending outcome of therapy. Genetic counseling should be strongly advised. If pregnancy occurs, the diagnosis should be made early and medications stopped immediately. Birth defects can occur in babies of mothers taking Coumadin, and nonsteroid anti-inflammatory drugs absorbed across the placenta could lead to premature closure of the ductus arteriosus. New babies should be fed formula, and the mothers should resume medication after delivery. Postpartum exacerbation of tumors has occurred. Estrogencontaining contraceptives should be avoided. COMMENTS Desmoid tumors are relatively slow-growing sarcomas that do not metastasize but may invade locally. Fifty-five percent of the tumors were in patients with Gardner’s syndrome, an inherited disease with propensity to form benign osteomas, desmoid tumors, intestinal tract polyps, and ultimately cancer, mainly involving the colon. Desmoid tumors arising on a sporadic basis appear identical to those of genetic origin, except that they are not associated with other manifestations of Gardner’s syndrome, and the majority arise in the fibrous tissues of the musculoskeletal system, particularly around the shoulder girdle, pelvis, and groin. THE AMERICAN
This recounting of the results of a 17-year experience is presented to unify the fragmentary information that developed from early trials with testolactone [7,8] and nonsteroid anti-inflammatory drugs [4,31,32] and to record that near-complete control of the growth of desmoid tumors can be achieved in patients with advanced tumors by combining the two modes of therapy. From the literature [3-8,11-141 and this experience, it is concluded that estrogen is trophic for these tumors, and that withdrawal of this stimulus results in resolution in about half the cases. In the experience with testolactone here and elsewhere (on our recommendation), only low doses were used. There appears to be an effect of dosage so that the response rate reported in Table I (40%) is probably lower than it would have been with doses in the range of 750 to 2,000 mg per day [9,10]. The low doses might also account for the long delay between the initiation of therapy and the response of some tumors. The molecular mechanism by which estrogen stimulates growth of fibroblasts to form tumors is not known. An obvious possibility, judging from the inherited tendency to develop desmoid tumors in patients with Gardner’s syndrome and polyposis coli is that the growth of these tumors results from maintenance of or reversion to a genetic set in which estrogen is important 1331.In estrogen-sensitive tissues, the hormone-receptor complex reacts with chromatin at promotor or enhancer sites upstream to the genes that are activated [34]. It may be that in mutated tumor cells, the estrogen-receptor unit functions as a constitutive molecule with continuous activation of a family of genes that influence growth and proliferation. Among such genes is that for ornithine decarboxylase (ODC), which in the present context can be regarded as a marker for the overall process. It is established that ODC induction is stimulated by estrogen [35], and it seems logical to speculate that estrogen withdrawal would inhibit synthesis of this as well as other enzymes and lead to restriction of DNA synthesis and growth. It is also known that inhibition of prostaglandin synthesis by indomethacin is accompanied by impaired ODC activity [ 131. Incorporation of warfarin and Mephyton into the regimen for patients with desmoid tumors was prompted by the report of decreased ribosomai RNA in subcutaneous mouse hepatoma transplants that had been exposed to warfarin in ho. There was no effect on normal livers from the same animals [36]. However, this effect of warfarin does not appear to offer a satisfactory explanation, because in vivo concentrations completely out of the attainable range were required. An exception to this is the sensitivity of DT diaphorases that are inhibited by warfarin concentrations as low as 10-s M [3U]. Exactly how inhibition of these enzymes would affect tumor cells is problematic. Another possible explanation of the effect of warfarin might also apply to sulindac. Both drugs uncouple oxidative phosphorylation [37] and might function cooperatively as lipid-soluble, weakly acidic compounds, the undissociated forms or dimeric complexes of the anionic and undissociated forms of which penetrate cell membranes freely and then function as protonophores to JOURNAL
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dissipate the proton gradient across inner mitochondrial membranes [38]. This effect could lead to acidification of the cytoplasm and prevent or minimize the alkalinization that characterizes tumor cells [39] and is requisite for initiation of DNA synthesis and cell proliferation [40]. These effects of warfarin may accentuate those of estrogen withdrawal and sulindac on the transcription of involved genes. On the basis of the results reported herein, which introduce a new and different type of chemotherapy for desmoid tumors, it is suggested that some other conditions arising from fibroplasia associated with acute and chronic inflammatory conditions such as cirrhosis, the late effects of adult respiratory distress syndrome, idiopathic retroperitoneal fibrosis, esophagitis, palmar fasciitis, Dupuytren’s contracture, and keloid and peritoneal adhesion formation after operations might be altered by the same measures. Neurofibromatosis and meningioma are tumors that might be susceptible to this form of therapy. Tamoxifen is recommended in preference totestolactone (Teslcc) because of cost, availability, and convenience for the patient. Testolactone is manufactured only in 50-mg tablets. The decision to allow reference to individual patients in this paper is in contrast to the policy of the last few years and of the for-seeable future. However, it seems to me that if we are to properly convey the uncommon and anecdotal, but biologically important, nature of these observations, it had to be done on a patient-by-patient basis. The paper should be read with this reservation in mind. The Editor REFERENCES 1. Hayry P, Scheinin TM. The desmoid (Reitamo) syndrome: etiology, manifestations, pathogenesis and treatment. Curr Probl Surg 1980; 25: 225-320. 2. Leibel SA, Wara WM, Hill DR, er al. Desmoid tumors: local control and patterns of relapse following radiation therapy. J Radiat Gncol Phys 1983; 9: 1167-71. 3. Kinzbrunner B, Ritter S, Domingo J, Rosenthal CJ. Remission of rapidly growing desmoid tumors after tamoxifen therapy. Cancer 1983; 52: 2201-4. 4. Waddell WR, Gerner RE, Reich MP. Nonsteroid anti-inflammatory drugs and tamoxifen for desmoid tumors and carcinoma of the stomach. J Surg Oncol 1983; 22: 197-211. 5. Perez HA, Patraca G, Parada EA. Fibromatosis musculoaponeurotica (tumor desmoide extra-abdominal). Reporte de un case tratado con el Clomifeno. Rev Invest Clin 1976; 28: 45-51. 6. Lanari A. Effect of progesterone on desmoid tumors (aggressive tibromatcsis). N Engl J Med 1983; 309: 1523. 7. Waddell WR. Treatment of intra-abdominal and abdominal wall desmoid tumors with drugs that effect the metabolism of cyclic 3,5-adenosine monophosphate. Ann Surg 1975; 181: 229-302. 8. Franz RC, Becker H, Botha PAG. The treatment of the fibromatoses with agents that inhibit the action of adenosine monophosphate d&erase. Proc 12th Biennial Congress of the Association of Surgeons of South Africa, Cape Town, South Africa, April 1980 and personal communication. 9. Segaloff A, Weeth JB, Meyer KK, Rongone EL, Cuningham MEG. Hormonal therapy in cancer of the breast. XIX. Effect of oral administration of At-testololactone on clinical course and hormonal excretion. Cancer 1962; 15: 633. 420
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10. Segaloff A. Al-testololactone: clinical trials. Cancer Res (suppl) 1982; 42: 3387s. 11. Lipschutz A. Bases endocrinas experimentale de la tumorigenesis epitelial y conjunctiva en la esfera genital femenina. Medicina (B Aires) 1942, 2: 404-38. 12. Lipschutz A. Steroid hormones and tumors. Baltimore: Williams & Wilkins, 1950. 13. Verma AK, Ashendel CL, Boutwell RK. An inhibition by prostaglandin, and tumor promotion caused by 12-O-tetradecanoxylphorbol-13-acetate. Cancer Res 1980; 40: 308-15. 14. Fischer SM, Mills GD, Slaga TJ. Inhibition of mouse skin tumor promotion by several inhibitors of arachadonic acid metabolism. Carcinogenesis 1982; 3: 1243-5. 15. Furstenberger G, Marks F. Indomethacin inhibition of cell proliferation induced by the phorbolester TPA is reversed by prostaglandin Ez in mouse epidermis. Biochem Biophys Res Comm 1978; 84: 1103-l 1. 16. Rozengurt E, Legg A, Strang G, Courtney-Luck N. Cyclic AMP: a mitogenic signal for Swiss 3T3 cells. Proc Nat1 Acad Sci USA 1981; 78: 4392-6. 17. Rozengurt E. Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of CAMP levels, DNA synthesis and cell division. EXD Cell Res 1982: 139: 71-8. 18. Rozengurt E. Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and nrowth factors in mouse 3T3 cells. J Cell Physiol 1982; 112: 24330. 19. Thompson EA, Siiteri PK. The involvement of human placental microsomal cytochrome P-450 in aromatization. J Biol Chem 1974: 249: 5373. 20. Siiteri PK, Thompson EA. Studies of human placental aromatase. J Steroid Biochem 1975: 6: 317. 21. Barone RM, Shamonki IM, Siiteri PK, Judd HL. Inhibition of peripheral aromatization of androstenedione to estrone in postmenopausal women with breast cancer using A’-testolactone. JClin Endocrinol Metab 1979: 49: 672-6. 22. Puck TT, Wenger L: Substitution of testolactone for testosterone in the “reverse transformation” of Chinese hamster cells. IRCS International Communications System, June 1973. 23. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 1971; 231: 232-5. 24. Flower JF, Vane JR. Commentary. Inhibition of prostaglandin synthesis. Biochem Pharmacol 1974; 23: 1439-50. 25. Samuelson B. From studies of biochemical mechanism to novel biological mediators: prostaglandin endoperoxides, thromboxanes, and leukotrienes. Biosci Rep 1983; 3: 791-813. 26. Shen T, Winter CA. Chemical and biological studies on indomethacin, sulindac and their analogs. Adv Drug Res 1977; 12: 89245. 27. Hucker HB, Stauffer SC, White SD, et al. Physiologic disposition and metabolic fate of a new anti-inflammatory agent, cis-5fluoro-2-methyl-pp(methylsulfinyl)-benzylidenyl-indene-3-acetic acid in the rat, dog, rhesus monkey and man. Drug Metab Dispos 1973; 1: 721-36. 28. Duggan DE, Hooke KF, Risley EA, et al. Identification of the biologically active form of sulindac. J Pharmacol Exp Therap 1977; 201: 8-13. 29. Duggan DE, Hare LE, Ditzler CA, er al. The disposition of sulindac. Clin Pharmacol Therap 1977; 21: 326-35. 30. Ernster L, Danielson L, Ljunggren M. DT Diaphorase. I. Purification from the soluble fraction of rat-liver cytoplasm and properties. Biochem Biophys Acta 1962; 58: 171-88. 3 1. Waddell WR, Gerner RE. Indomethacin and ascorbate inhibit desmoid tumors. J Surg Oncol 1980; 15: 85-90. 32. Jones IT, Fazio VW, Weakley FL, et al. Desmoid tumors in familial polyposis coli. Ann Surg 1986; 204: 94-7. 33. Darnell J, Lodish H, Baltimore D. Molecular cell biology. New York: Scientific American Books, 1986: 698-9. 34. Evans RM. The steroid and thyroid receptor superfamily. Science 1988; 240: 889-95. 35. Cohen S, G’Malley BW, Stastny M. Estrogenic induction of
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ornithine decarboxylase in vivo and in vitro. Science 1970; 170: 336-8. 36. VanBuskirk JJ, Kirsch WM. Loss of hepatoma ribosomal RNA during warfarin therapy. Biochem Biophys Res Comm 1973; 52: 562-8. 37. Whitehouse MW. Uncoupling of oxidative phosphorylation by some arylacetic acids. Nature 1964; 201: 629-30.
38. McLaughlin SGA, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev 1980; 60: 825-63. 39. Oberhaensli RD, Hilton-Jones D, Bore PJ, et al. Biochemical investigation of human tumours with phosphorus-3 1 magnetic resonance spectroscopy. Lancet 1986; 2: 8- 11. 40. Roos A, Boron WF. Intracellular pH. Physiol Rev 198 1; 61: 296-434.
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MD,
TUCSON,
Arizona,
Ten patients with large inoperable desmoid tumors in various body locations were treated with testolactone. Four tumors (40%) responded with major . . regresstons, i.e., more than 50% reduction in volume. Eight patients received nonsteroid anti-inflammatory drugs (indomethacin, sulindac, or sulindac with warfarin and vitamin Kl [Mephyton]) for periods of 2 to 91 months. There was one major regression, one partial regression, and three instances of tumor growth arrest over periods up to 8 years. Seven patients were treated with nonsteroid antiinflammatory drugs concurrent with or after testolactone or tamoxifen. There were five major regressions and one partial regression with extensive central necrosis of an enormous intra-abdominal tumor. The last patient has been treated for only 12 months, with no change in tumor volume. It appears that estrogens function as growth factors for desmoid tumors, and that minimization of these effects inhibits tumor growth in some, but not all, cases. In those instances where antiestrogens were not effective as single agents, the tumors usually responded to subsequent nonsteroid anti-inflammatory drug therapy. Withdrawal of estrogen may be followed by inhibition of transcription of genes that support tumor cell proliferation, and sulindac and indomethatin may augment these effects by inhibiting prostaglandin and cyclic AMP synthesis and the activity of protein kinase C. Warfarin may function as a protonophore to acidify the cytoplasm and prevent the alkalinization that is necessary to initiate DNA synthesis and cell cycle progression, again an impairment of the transcription process.
Wolff M. Kirsch,
MD, Loma Linda, California
he management of inoperable desmoid tumors is a T difficult problem for which there is no single or welltested solution. There is general agreement that small tumors should be excised with wide margins [I]. Large intra-abdominal tumors are often unresectable, and this is also true of tumors in locations such as the mediastinum, neck, thigh, pelvis, and shoulder girdle. Radiation has been reported to control some tumors [2], but this requires large doses that carry the risks of damage to adjacent viscera. Endocrine manipulations have resulted in some dramatic responses. Tamoxifen [3,4], clomiphene [5], progesterone [a, and testolactone [7,8] have all been used successfully, and, as with other modes, there have been frequent failures. This project began in an attempt to control the growth of an inoperable intra-abdominal desmoid tumor in a young woman with Gardner’s syndrome. The tumor had developed while the patient was taking birth control pills, suggesting some relationship to the reproductive endocrine system [7]. At that time, testolactone was used occasionally for disseminated breast cancer [9,10] as an analogue of testosterone. Its use for desmoid tumors was founded on early experimental work suggesting that estrogens were involved in the development of fibrous tumors in guinea pigs [II ,121. After resolution of the desmoid tumor in the original patient, other patients were treated, but there were frequent unexplained failures. Until recently, there was little appreciation of the original assumptions that prompted this clinical approach to an often insoluble problem. This report presents therapeutic recommendations to minimize the effects of estrogens and to inhibit prostaglandin and CAMP synthesis, both of which influence growth and proliferation of fibroblasts [13-181. PATIENTS AND METHODS
The observations were made over a period of 17 years. Eight patients were members of families with Gardner’s syndrome, and 11 had tumors that arose sporadically. In the former group, the tumors were located preponderantly in the bowel mesentery and abdominal wall of patients who had undergone surgery (Tables I and II). All but one of the patients were between 21 and 40 years of age. Six additional patients were excluded from this analysis because recurrent tumors had formed about Marlex mesh (in five patients) or methacrylate (in one patient) replacements of abdominal and thoracic walls. These tumors behave differently. Large foreign bodies may have initiated and perpetuated mitogenic action in susceptible indiFrom the Department of Surgery, University of Arizona, Tucson, Arividuals. zona, and the Division of Neurosuraerv. Loma Linda University Medical School, Loma Linda, California. The responses of the tumors were measured by whatRequests for reprints should be addressed to William R. Waddell, ever means were appropriate. The tumors were very MD, Department of Surgery, University of Arizona Health Sciences_ large, and in most, the presenting parts could be palpated Center, Tucson, Arizona 85724. and measured. In other instances, computed tomography Manuscript submitted July 11, 1990, and accepted in revised form (CT) and, lately, magnetic resonance imaging (MRI) October 10, 1990. 416
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--_ TABLE I Testolactone’
Age
sex
Gardner’s Syndrome
Location of Tumor
Duration of Treatment (mo)
Outcome
Reference
laT 2 3 4 5
23 24 21 22 27
F F F F M
Yes No No No Yes
12 2 2 48 7
Complete regression Progression Progression Major regression Progression
7
6 7
44 36
M M
Yes No
18 3
Major regression Progression
a
38
M
No
9 10
44 33
M M
Yes No
Small & large bowel mesentery Posterior thigh & popliteal space Abdominal wall Pelvic wall 8 retroperitoneum Small & large bowel mesentery, retroperitoneum Small bowel mesentery Abdominal wall, small bowel mesentery Mediastinum Sternum Small bowel mesentery Mandible, neck, upper chest
Patient
5
-
Progression
60 6
4. 31 8
4. 31
Complete regression Progression
8
* These results pertain only to the period during which testolactone was administered(not the eventual outcome). Dose of testolactone was 200 mg/day. The response rate was 40 % 7Estrogen-containingcontraceptives omitted immediately prior to start.
TABLE II Indomethacln, Sulindac, or Sullndac with Warfarln’ Dose
Duration
(mg/d)
(mo)
Sulindac
300
29
Major regression
-
Wafarin
7.5
Axilla, chest wall
Sulindac
400
12
(central necrosis with fistulas) Progression
-
Warfarin Sulindac Sulindac
10 400 400
12 29
Partial regression Growth arrest
-
Warfarin lndomethacin Sulindac Sulindac Sulindac lndomethacin Sulindac
7.5 300 375 300 300 75 300
53
Growth arrest
91 2 9
Growth arrest Progression Major regression
Patient
Age
Sex
Gardner’s Syndrome
Location of Tumor(s)
11
22
F
Yes
Small bowel mesentery, retroperitoneum
12
34
F
No
13 14
40 39
M M
Yes No
Bowel mesentery Groin & pelvis
15
34
M
No
Axilla
16 17 16
36 25 76
M F F
Yes Yes No
Bowel mesentery Small bowel mesentery Chest wall
Drugs
Outcome
Reference
4
4 4, 31
* The patients in this grouping had not received prior antiestrogens. Countinggrowth arrest, the response rate was 62%.
have been used for evaluation of size and local effects. Repeated observations over very long periods minimize errors of significance to this study. The protracted course of desmoid tumor growth permitted establishment of pretreatment tumor status and growth patterns as the best feasible controls. Tumor responses were characterized as progression, arrested growth, partial regression (less than 50%), major regression (more than 50%), and complete regression. When significant regression or arrest of growth occurred, the medication was usually continued indefinitely, in the first patient now for 17 years. These patients and their families were fully informed about their disease and the experimental nature of the treatment. All of them had previous operations, and many had other forms of chemotherapy and/or radiotherapy, all of which were unsuccessful. THE AMERICAN
One group of 10 patients received testolactone (Table I). A second group of eight patients treated with indomethacin, sulindac, or sulindac with warfarin and vitamin KI (Mephyton) is presented in Table II. The third group of seven patients shown in Table III was treated with indomethacin, sulindac, or sulindac with warfarin and Mephyton, after or concurrently with testolactone or tamoxifen. The currently recommended regimen is, in effect, a summation of a clinical experience extending over the past 17 years, consisting of: 1. Testolactone (TeslacQ) 750 mg daily (250 mg three times a day); 2. Sulindac (Clinorile) 300 mg daily (150 mg twice a day), then 200 mg twice a day if tolerated, starting after 1 month on lower dose; 3. Warfarin (CoumadirP) 5, 7.5 or 10 mg daily as tolerated. Start with lower dose and work up to 10 mg. JOURNAL
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III
Antiestrogen wlth Indomethacln, Sullndac, or Sulindac with Warfarln Location of Tumor(s)
‘atlent
Age
Sex
lb
23
F
Abdominal wall
17
25
F
2
24
F
Small bowel mesentery Thigh 8 popliteal space
3 12
21 34
F F
5
27
M
8
38
M
Endocrine Adjustment
Other Drugs
Duration of Treatment (mo)
lndomethacin Warfarin Sullndac
29
Major regression
4, 7
64
Major regression
4
Piroxicam Sulindac Warfarin lndomethacin Sullndac Warfarin Sulindac Warfarin
52
Major regression
92 4
Major regression No change in 12 months Progression (eventual central necrosis and enteric fistula) Subsequent death due to uncontrolled sepsis Major regression (complete)
Antiestrogen’
Testolactone Tamoxifen Tamoxifen
Concurrent
Testolactone
Before
Abdominal wall Axilla, chest wall, arm Small 8 large bowel mesentery, retroperitoneum
Testolactone Testolactone
Before Concurrent
Testolactone
Before
Mediastinum, sternum
Testolactone
Before
Concurrent
53
1
lndomethacin
Outcome
Reference
-
7, 31
-
7, 31
* Under the “Antiestrogen” heading, the lowering of estrogen levels occurred “before” the administration of other drugs or “concurrently.” Patient 12 received 750 mg testolactone daily. In those patients who received antiestrogens before other drugs were added, the subsequent responses occurred while the patients were taking sulindac, indomethacin, or sulindac and warfarin.
Weekly measurement of prothrombin times until a steady tolerable dose established, then only occasionally; 4. Vitamin K1 (Mephytona), 5 or 10 mg daily to keep the prothrombin time slightly above normal. Drugs: Testolactone, 13-hydroxy-3-oxo- 13,17-secoandrosta- 1,Cdien- 17-oic acid 8-lactone, is not androgenie, estrogenic, progestational, or antiprogestational in the usual sense. Its main known biochemical effect is noncompetitive and irreversible inhibition of aromatase, a rate-controlling enzyme present in most tissues that catalyzes the final hydroxylations and A-ring desaturation in the modification of testosterone to estradiol and androstenedione to estrone [ 19,211. It acts synergistically with dibutyryl cyclic AMP in causing “reverse transformation” of cultured Chinese hamster ovary cells [22]. Its main clinical use has been in the treatment of advanced breast cancer, with doses from 100 to 2,000 mg daily. Most of the patients in the present study received 200 mg daily. The optimal dose is obviously not known. Tumoxifen, (Z)2-4-(l,Zdiphentl-l-butenyl)phenoxy-N,N-dimethylethanamine 2-hydroxyl- 1,2,3-propanetricarboxylite (l:), is a nonsteroid antiestrogen which competes with estrogen for binding sites in target tissues. It is widely used to treat breast adenocarcinoma in postmenopausal women. Antitumor effects are thought to be the result of binding to estrogen receptors and the consequences thereof. The patients in this study received 20 to 40 mg per day. Indomethacin, 1-p-chlorobenzoyl-5-methoxy-2methylindole-3-acetic acid, is a substituted 3-indolylacetic acid whose principal effect is inhibition of cyclooxygenase, the enzyme that catalyzes the oxidation of arachidonic acid to prostaglandin precursors [23-251.
Sulinduc, (Z)-5-fluoro-2-methyl-l-p-(methylsulfinyl)phenylmethylene-lH-indene-3-acetic acid, inhibits the cyclooxygenase that catalyzes the oxidation of arachidonic acid to prostaglandin precursors. It is a long-acting analogue of indomethacin, with similar biochemical actions [2q. It undergoes enterohepatic circulation, and is excreted through the intestinal tract and in urine. The compound is ingested as the inactive oxidized form and is inhibitory only after enzymatic reduction to the sulfide [27-291. Wurfarin, 3-(acetonylbenzyl)-Chydroxycoumarin, inhibits vitamin K epoxide reductase, interrupts the “vitamin K cycle,” and leads to inhibition of vitamin K-dependent protein carboxylation and formation of prothrombin. Warfarin in concentrations as low as low8 M inhibits DT diaphorase and uncouples oxidative phosphorylation in mitochondria [30]. Warfarin has other effects on tumor cells, including inhibition of DNA synthesis and, more particularly, the salvage pathway for DNA synthesis. The patients took 5, 7.5 or 10 mg daily. Mephyton is vitamin K1 or phylloquinone (Zmethyl3-phytyl- 1,4_naphthoquinone). It is an essential cofactor for the hepatic microsomal enzyme complex involved in the carboxylation of glutamic acid residues in preprothrombin to yield active prothrombin. This drug also inhib its the salvage pathway to DNA synthesis and acts additively with warfarin in this regard. Mephyton antagonizes the anticoagulation effects of warfarin without affecting the cytotoxic action. Some patients took 10 mg of Mephyton daily, but in others a dose of 10 mg two or three times weekly was established as adequate to maintain slightly elevated prothrombin times. In these trials, Mephyton was given to permit higher warfarin dosage.
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RESULTS In Table I, the 10 patients treated with testolactone for 2 to 60 months are listed, along with other patient information. Two patients responded within the first few months, and this was sustained. At the other extreme, the tumors of two patients stopped growing shortly after beginning testolactone, but did not show any signs of regression until 36 months, whereupon they regressed completely over the ensuing year. The tumors in the remaining six patients never stopped growing, and the drug was stopped after trial periods of 2 to 7 months. In Table II, the eight patients that received indomethacin, sulindac, or sulindac, warfarin, and Mephyton as the initial treatment are listed, none of whom had previously been treated with testolactone. There was one major and one partial regression; in three patients, tumor growth was arrested for periods of 29,53, and 91 months. Counting growth arrest as a form of success, the response rate was 62%; otherwise it was 25%. Table III summarizes the treatment of seven patients who received nonsteroid anti-inflammatory and antiestrogen drugs. Major regressions occurred in six. The only complication of testolactone was menorrhagia due to hyperplastic endometrium in one patient after receiving testolactone for 1 year. This was followed by long-lasting amenorrhea and menopausal symptoms after the drug was stopped. Sometimes pain in the area of tumor correlated with softening of the tumor and purplish discoloration of overlying skin. Three patients developed fistulas between small bowel and the necrotic interior of large intra-abdominal desmoids. Upper gastrointestinal dysfunction attributable to gastritis occurred occasionally and required reduction or brief omission of indomethacin or sulindac. There were no gastric or duodenal ulcers. Pregnancy should be avoided pending outcome of therapy. Genetic counseling should be strongly advised. If pregnancy occurs, the diagnosis should be made early and medications stopped immediately. Birth defects can occur in babies of mothers taking Coumadin, and nonsteroid anti-inflammatory drugs absorbed across the placenta could lead to premature closure of the ductus arteriosus. New babies should be fed formula, and the mothers should resume medication after delivery. Postpartum exacerbation of tumors has occurred. Estrogencontaining contraceptives should be avoided. COMMENTS Desmoid tumors are relatively slow-growing sarcomas that do not metastasize but may invade locally. Fifty-five percent of the tumors were in patients with Gardner’s syndrome, an inherited disease with propensity to form benign osteomas, desmoid tumors, intestinal tract polyps, and ultimately cancer, mainly involving the colon. Desmoid tumors arising on a sporadic basis appear identical to those of genetic origin, except that they are not associated with other manifestations of Gardner’s syndrome, and the majority arise in the fibrous tissues of the musculoskeletal system, particularly around the shoulder girdle, pelvis, and groin. THE AMERICAN
This recounting of the results of a 17-year experience is presented to unify the fragmentary information that developed from early trials with testolactone [7,8] and nonsteroid anti-inflammatory drugs [4,31,32] and to record that near-complete control of the growth of desmoid tumors can be achieved in patients with advanced tumors by combining the two modes of therapy. From the literature [3-8,11-141 and this experience, it is concluded that estrogen is trophic for these tumors, and that withdrawal of this stimulus results in resolution in about half the cases. In the experience with testolactone here and elsewhere (on our recommendation), only low doses were used. There appears to be an effect of dosage so that the response rate reported in Table I (40%) is probably lower than it would have been with doses in the range of 750 to 2,000 mg per day [9,10]. The low doses might also account for the long delay between the initiation of therapy and the response of some tumors. The molecular mechanism by which estrogen stimulates growth of fibroblasts to form tumors is not known. An obvious possibility, judging from the inherited tendency to develop desmoid tumors in patients with Gardner’s syndrome and polyposis coli is that the growth of these tumors results from maintenance of or reversion to a genetic set in which estrogen is important 1331.In estrogen-sensitive tissues, the hormone-receptor complex reacts with chromatin at promotor or enhancer sites upstream to the genes that are activated [34]. It may be that in mutated tumor cells, the estrogen-receptor unit functions as a constitutive molecule with continuous activation of a family of genes that influence growth and proliferation. Among such genes is that for ornithine decarboxylase (ODC), which in the present context can be regarded as a marker for the overall process. It is established that ODC induction is stimulated by estrogen [35], and it seems logical to speculate that estrogen withdrawal would inhibit synthesis of this as well as other enzymes and lead to restriction of DNA synthesis and growth. It is also known that inhibition of prostaglandin synthesis by indomethacin is accompanied by impaired ODC activity [ 131. Incorporation of warfarin and Mephyton into the regimen for patients with desmoid tumors was prompted by the report of decreased ribosomai RNA in subcutaneous mouse hepatoma transplants that had been exposed to warfarin in ho. There was no effect on normal livers from the same animals [36]. However, this effect of warfarin does not appear to offer a satisfactory explanation, because in vivo concentrations completely out of the attainable range were required. An exception to this is the sensitivity of DT diaphorases that are inhibited by warfarin concentrations as low as 10-s M [3U]. Exactly how inhibition of these enzymes would affect tumor cells is problematic. Another possible explanation of the effect of warfarin might also apply to sulindac. Both drugs uncouple oxidative phosphorylation [37] and might function cooperatively as lipid-soluble, weakly acidic compounds, the undissociated forms or dimeric complexes of the anionic and undissociated forms of which penetrate cell membranes freely and then function as protonophores to JOURNAL
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dissipate the proton gradient across inner mitochondrial membranes [38]. This effect could lead to acidification of the cytoplasm and prevent or minimize the alkalinization that characterizes tumor cells [39] and is requisite for initiation of DNA synthesis and cell proliferation [40]. These effects of warfarin may accentuate those of estrogen withdrawal and sulindac on the transcription of involved genes. On the basis of the results reported herein, which introduce a new and different type of chemotherapy for desmoid tumors, it is suggested that some other conditions arising from fibroplasia associated with acute and chronic inflammatory conditions such as cirrhosis, the late effects of adult respiratory distress syndrome, idiopathic retroperitoneal fibrosis, esophagitis, palmar fasciitis, Dupuytren’s contracture, and keloid and peritoneal adhesion formation after operations might be altered by the same measures. Neurofibromatosis and meningioma are tumors that might be susceptible to this form of therapy. Tamoxifen is recommended in preference totestolactone (Teslcc) because of cost, availability, and convenience for the patient. Testolactone is manufactured only in 50-mg tablets. The decision to allow reference to individual patients in this paper is in contrast to the policy of the last few years and of the for-seeable future. However, it seems to me that if we are to properly convey the uncommon and anecdotal, but biologically important, nature of these observations, it had to be done on a patient-by-patient basis. The paper should be read with this reservation in mind. The Editor REFERENCES 1. Hayry P, Scheinin TM. The desmoid (Reitamo) syndrome: etiology, manifestations, pathogenesis and treatment. Curr Probl Surg 1980; 25: 225-320. 2. Leibel SA, Wara WM, Hill DR, er al. Desmoid tumors: local control and patterns of relapse following radiation therapy. J Radiat Gncol Phys 1983; 9: 1167-71. 3. Kinzbrunner B, Ritter S, Domingo J, Rosenthal CJ. Remission of rapidly growing desmoid tumors after tamoxifen therapy. Cancer 1983; 52: 2201-4. 4. Waddell WR, Gerner RE, Reich MP. Nonsteroid anti-inflammatory drugs and tamoxifen for desmoid tumors and carcinoma of the stomach. J Surg Oncol 1983; 22: 197-211. 5. Perez HA, Patraca G, Parada EA. Fibromatosis musculoaponeurotica (tumor desmoide extra-abdominal). Reporte de un case tratado con el Clomifeno. Rev Invest Clin 1976; 28: 45-51. 6. Lanari A. Effect of progesterone on desmoid tumors (aggressive tibromatcsis). N Engl J Med 1983; 309: 1523. 7. Waddell WR. Treatment of intra-abdominal and abdominal wall desmoid tumors with drugs that effect the metabolism of cyclic 3,5-adenosine monophosphate. Ann Surg 1975; 181: 229-302. 8. Franz RC, Becker H, Botha PAG. The treatment of the fibromatoses with agents that inhibit the action of adenosine monophosphate d&erase. Proc 12th Biennial Congress of the Association of Surgeons of South Africa, Cape Town, South Africa, April 1980 and personal communication. 9. Segaloff A, Weeth JB, Meyer KK, Rongone EL, Cuningham MEG. Hormonal therapy in cancer of the breast. XIX. Effect of oral administration of At-testololactone on clinical course and hormonal excretion. Cancer 1962; 15: 633. 420
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10. Segaloff A. Al-testololactone: clinical trials. Cancer Res (suppl) 1982; 42: 3387s. 11. Lipschutz A. Bases endocrinas experimentale de la tumorigenesis epitelial y conjunctiva en la esfera genital femenina. Medicina (B Aires) 1942, 2: 404-38. 12. Lipschutz A. Steroid hormones and tumors. Baltimore: Williams & Wilkins, 1950. 13. Verma AK, Ashendel CL, Boutwell RK. An inhibition by prostaglandin, and tumor promotion caused by 12-O-tetradecanoxylphorbol-13-acetate. Cancer Res 1980; 40: 308-15. 14. Fischer SM, Mills GD, Slaga TJ. Inhibition of mouse skin tumor promotion by several inhibitors of arachadonic acid metabolism. Carcinogenesis 1982; 3: 1243-5. 15. Furstenberger G, Marks F. Indomethacin inhibition of cell proliferation induced by the phorbolester TPA is reversed by prostaglandin Ez in mouse epidermis. Biochem Biophys Res Comm 1978; 84: 1103-l 1. 16. Rozengurt E, Legg A, Strang G, Courtney-Luck N. Cyclic AMP: a mitogenic signal for Swiss 3T3 cells. Proc Nat1 Acad Sci USA 1981; 78: 4392-6. 17. Rozengurt E. Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of CAMP levels, DNA synthesis and cell division. EXD Cell Res 1982: 139: 71-8. 18. Rozengurt E. Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and nrowth factors in mouse 3T3 cells. J Cell Physiol 1982; 112: 24330. 19. Thompson EA, Siiteri PK. The involvement of human placental microsomal cytochrome P-450 in aromatization. J Biol Chem 1974: 249: 5373. 20. Siiteri PK, Thompson EA. Studies of human placental aromatase. J Steroid Biochem 1975: 6: 317. 21. Barone RM, Shamonki IM, Siiteri PK, Judd HL. Inhibition of peripheral aromatization of androstenedione to estrone in postmenopausal women with breast cancer using A’-testolactone. JClin Endocrinol Metab 1979: 49: 672-6. 22. Puck TT, Wenger L: Substitution of testolactone for testosterone in the “reverse transformation” of Chinese hamster cells. IRCS International Communications System, June 1973. 23. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 1971; 231: 232-5. 24. Flower JF, Vane JR. Commentary. Inhibition of prostaglandin synthesis. Biochem Pharmacol 1974; 23: 1439-50. 25. Samuelson B. From studies of biochemical mechanism to novel biological mediators: prostaglandin endoperoxides, thromboxanes, and leukotrienes. Biosci Rep 1983; 3: 791-813. 26. Shen T, Winter CA. Chemical and biological studies on indomethacin, sulindac and their analogs. Adv Drug Res 1977; 12: 89245. 27. Hucker HB, Stauffer SC, White SD, et al. Physiologic disposition and metabolic fate of a new anti-inflammatory agent, cis-5fluoro-2-methyl-pp(methylsulfinyl)-benzylidenyl-indene-3-acetic acid in the rat, dog, rhesus monkey and man. Drug Metab Dispos 1973; 1: 721-36. 28. Duggan DE, Hooke KF, Risley EA, et al. Identification of the biologically active form of sulindac. J Pharmacol Exp Therap 1977; 201: 8-13. 29. Duggan DE, Hare LE, Ditzler CA, er al. The disposition of sulindac. Clin Pharmacol Therap 1977; 21: 326-35. 30. Ernster L, Danielson L, Ljunggren M. DT Diaphorase. I. Purification from the soluble fraction of rat-liver cytoplasm and properties. Biochem Biophys Acta 1962; 58: 171-88. 3 1. Waddell WR, Gerner RE. Indomethacin and ascorbate inhibit desmoid tumors. J Surg Oncol 1980; 15: 85-90. 32. Jones IT, Fazio VW, Weakley FL, et al. Desmoid tumors in familial polyposis coli. Ann Surg 1986; 204: 94-7. 33. Darnell J, Lodish H, Baltimore D. Molecular cell biology. New York: Scientific American Books, 1986: 698-9. 34. Evans RM. The steroid and thyroid receptor superfamily. Science 1988; 240: 889-95. 35. Cohen S, G’Malley BW, Stastny M. Estrogenic induction of
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ornithine decarboxylase in vivo and in vitro. Science 1970; 170: 336-8. 36. VanBuskirk JJ, Kirsch WM. Loss of hepatoma ribosomal RNA during warfarin therapy. Biochem Biophys Res Comm 1973; 52: 562-8. 37. Whitehouse MW. Uncoupling of oxidative phosphorylation by some arylacetic acids. Nature 1964; 201: 629-30.
38. McLaughlin SGA, Dilger JP. Transport of protons across membranes by weak acids. Physiol Rev 1980; 60: 825-63. 39. Oberhaensli RD, Hilton-Jones D, Bore PJ, et al. Biochemical investigation of human tumours with phosphorus-3 1 magnetic resonance spectroscopy. Lancet 1986; 2: 8- 11. 40. Roos A, Boron WF. Intracellular pH. Physiol Rev 198 1; 61: 296-434.
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