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© 2000 American Society for Clinical Oncology Fenretinide Therapy in Prostate Cancer: Effects on Tissue and Serum Retinoid ConcentrationFrom the Department of Biochemistry, Matsunaga-Conte Prostate Cancer Research Center, and Scott Department of Urology, Baylor College of Medicine, Houston, TX. Address reprint requests to Dov Kadmon, MD, Scott Department of Urology, Baylor College of Medicine, 6560 Fannin St, Suite 2100, Houston, TX 77030.
PURPOSE: To examine the feasibility of using fenretinide (4-HPR) for the prevention and treatment of prostate cancer. MATERIALS AND METHODS: We measured the impact of 4-HPR therapy on retinoid concentrations in vivo, in a mouse model of prostate cancer and clinically, in patients with prostate cancer who were given oral 4-HPR (200 mg/d) or placebo for 4 weeks before undergoing a radical prostatectomy. RESULTS: Prostate tumors in mice treated with 4-HPR contained high levels of 4-HPR and of all-trans-retinoic acid (RA) and reduced levels of retinol (ROH). Patients given 4-HPR were found to have significantly higher concentrations of 4-HPR in the cancerous prostate as compared with the serum levels (463 nmol/L v 326 nmol/L; P = .049), but they were only 1/10 the levels found in mice and were far below the concentrations reported in human breast tissue. Serum and tissue ROH levels were reduced to less than half the concentrations found in untreated controls. RA concentrations in human serum and in cancerous prostates were not significantly affected by 4-HPR treatment, in contrast with the findings in mice. CONCLUSION: The standard oral dose of 4-HPR proposed for breast cancer (200 mg/d) achieved only modest drug levels in the prostate and is unlikely to be effective for prostate cancer prevention or treatment. Higher doses need to be explored.
PROSTATE CANCER, A formidable health problem in the United States, is the most commonly diagnosed cancer in men. The American Cancer Society estimates that 180,400 new cases of prostate cancer will be diagnosed in 2000.1 Because the incidence of prostate cancer increases sharply with each decade of life past the age of 50 years and because life expectancy in the United States is climbing, the high incidence of detection is expected to continue. Treatment options for prostate cancer currently include radical prostatectomy and radiation therapy for cure of localized disease and androgen ablation therapy for palliation of locally advanced or metastatic disease. Gene therapy is in its infancy, and standard chemotherapy does not result in any cures or prolonged survival. An alternative approach would be to address the disease not from a therapeutic standpoint but rather from a preventive perspective, and in recent years, the concept of chemoprevention has come of age. Retinoids (natural and synthetic analogs of vitamin A) are the most investigated class of chemopreventive drugs. They have been demonstrated to inhibit carcinogenesis in epithelial cells in animal models and to reverse oral, skin, and cervical premalignancies. Concomitantly, they have been found to upregulate the expression of retinoic acid receptors in these tissues.2-8 The prostate was shown to contain endogenous all-trans-retinol and its active metabolite all-trans-retinoic acid (RA), but their concentrations were markedly diminished in prostate carcinoma tissues. Consequently, abnormalities of retinoid metabolism may be implicated in the development of prostate cancer,9 and hence the rationale for using retinoids in prostate cancer prevention and therapy. Fenretinide (4-HPR), a synthetic retinoid, has been shown to exert promising preclinical effects on animal models of prostate cancer and has demonstrated its safety in a large breast chemoprevention trial.10,11 Its exact mechanism of action, however, remains largely unknown. A prerequisite for efficient action of 4-HPR in the prostate is achieving high concentrations of this synthetic retinoid in the target organ and in cancerous cells. In a preclinical study, we evaluated the levels of retinoids in an oncogene-induced mouse model of prostate cancer after feeding the mice either a 4-HPR–containing diet or a control diet. We then measured retinoid concentrations in the serum and prostate of patients treated with either 200 mg/d of 4-HPR or with placebo orally for 28 days before undergoing radical prostatectomy.
Study in Mice Mouse prostate reconstitution (MPR) tissues were generated as reported previously.12,13 Briefly, urogenital sinus tissue was isolated from 17-day-old C57BL/6 mouse fetuses, and the anterior and posterior regions were trimmed, leaving only that portion which gives rise to the prostate gland. After a 2-hour digestion in 1.0% trypsin, standard microdissection techniques were used to separate the urogenital sinus epithelium (UGE) from the urogenital sinus mesenchyme (UGM) compartment. After the separation, both UGE and UGM were treated with 0.1% collagenase and washed, and an aliquot of the UGM suspension was counted. UGE cell numbers were extrapolated using the UGM:UGE cell ratio that has been shown previously to be 7:3. Transduction of the ras and myc oncogenes was accomplished via the replication deficient recombinant retrovirus Zipras/myc 9.12 Zipras/myc 9 was derived from a Moloney murine leukemia virus–derived recombinant retroviral vector and contains the v-Ha-ras gene as well as a fragment from MC29 carrying the v-gagmyc oncogene, both under the transcriptional regulation of the viral long terminal repeat promoter. Supernatants containing helper virus-free Zipras/myc 9 (5 x 105 focus forming units/mL) mixed with polybrene were added to UGE alone in these experiments at a multiplicity of infection of approximately 1.0. Two hours after infection, the UGE and UGM cells were pelleted. Then, 1.5 x 106 cells were mixed at the appropriate cell ratios to reconstitute the fetal urogenital sinus tissue and resuspended in collagen for an overnight incubation. The next morning, the collagen suspension (MPRs) was grafted under the right renal capsule of adult isogenic male hosts. Four weeks later, in this model, 100% of the animals were found to have developed prostate cancers in the MPR tissues. Eight MPRs were generated for this experiment, each implanted in a different male host. Three of the animals were fed a 4-HPR–containing diet for 7 weeks, and the other animals were fed a control diet. Tumors were collected, homogenized and extracted, and extracts were subjected to high-performance liquid chromatography (HPLC). Specifically, extracts were suspended in 2 mL of stabilizing buffer (phosphate-buffered saline containing 9 mmol/L EDTA, 25 mmol/L sodium ascorbate, and 21 mmol/L sodium sulfate, pH 6.5) and disrupted by ultrasound. Conditioned medium was diluted 1:1 with stabilizing buffer. As internal standard, 60 pg of [11,12-3H]all-trans-retinoic acid (3HRA, New England Nuclear, Boston, MA; specific activity, 50.7 Ci/mmol.) was added to each sample and a mixture of ethyl acetate and methyl acetate (volume-to-volume, 8:1) was used to extract retinoids and 4-HPR metabolites by vigorous shaking for 45 minutes at 4°C. The organic and aqueous phases were separated by centrifugation (7 minutes at 3,300 rpm at 4°C) The organic phase containing the compounds of interest was dried down in a stream of nitrogen and redissolved in 30 µL of ethanol. The extracts were fractionated on a reverse-phase HPLC column (C18 Microsorb; Beckman, Westbury, NY; 4.6 mm x 250 mm, Rainin, using methanol:acetonitril: 0.1 mol/L ammonium acetate pH 6.8 64:18:18) at a flow rate of 0.8 mL/min as mobile phase. A Shimadzu ultraviolet detector (Shimadzu Scientific Instruments, Columbia, MD) was used to monitor optical density at 350 nm and 280 nm, and 0.8-mL fractions were collected and counted in a scintillation counter. To determine the amounts of various retinoids present in a sample, the integrated peak area at optical density 350 nm for a particular retinoid was divided by area units per nanogram for this retinoid. Recovery for each extract was calculated using the amount of 3HRA measured after the HPLC run.
Study in Humans
Statistical Analysis
Study in Mice Prostate tumor tissues from 4-HPR–fed mice contained 458 nmol/L of ROH, a peak coeluting with authentic 4-HPR corresponding to 5,033 nmol/L of 4-HPR, and a peak coeluting with RA containing 557 nmol/L of RA. These values must be compared with 955 nmol/L of ROH, 0 nmol/L of 4-HPR, and 165 nmol/L of RA found in prostate tumor tissues from the control group (Table 1).
Study in Humans Twenty-six patients were recruited for this study (12 in the placebo group and 14 in the treatment group). The preoperative clinical and postoperative pathologic characteristics of the two groups are described in Table 2. There were no differences in any characteristics between placebo and treatment group patients, except for biopsy Gleason scores preoperatively (P = .009) and tumor sizes postoperatively (P = .036).
Treatment with 4-HPR results in the production of its derivative, 4-MPR.11 By day 28 of the treatment, the serum and benign prostatic tissue concentrations of both compounds were about the same (P = .283 and P = .897; Table 3). Apparently, there is no concentration of the drug in benign prostatic tissue when compared with serum levels. By contrast, the cancerous tissues were significantly enriched with 4-HPR as compared with serum levels (P = .049). In these tissues, high concentrations of 4-MPR were also found, but because of the wide variations, it did not reach a statistical significance (P = .398). The tissue concentration of 4-HPR in humans was approximately 10 times lower than in mice. In the patients treated with 4-HPR, the serum and tissue levels of ROH were depressed by a factor of two as compared with serum and tissue levels in the control group (Table 4). No differences were found between the two groups in pretreatment serum ROH levels (P = .713) or in serum and tissue RA concentrations.
Retinoids have demonstrated effectiveness in both prevention and treatment of certain human cancers. The most impressive results clearly are in acute promyelocytic leukemia patients who achieve a complete remission after treatment with high doses of retinoic acid.16-18 Preclinical and clinical studies have documented the efficacy of retinoids in chemoprevention of oral leukoplakia and cervical cancer and in the prevention of second primary tumors in head and neck neoplasia.2-8 Retinoids exert their actions on tissues through nuclear retinoic acid receptors (RARs) and retinoid X receptors. Studies have shown that loss or diminished expression of some of these receptors occurs in premalignant and malignant tissues and that expression of these receptors can be upregulated by retinoid therapy. These observations suggest that a dysregulation of retinoid metabolism may be causally implicated in carcinogenesis. Endogenous ROH and its biologically active metabolite RA were found to be present in normal prostate, benign prostatic hyperplasia, and prostatic carcinoma. Prostate cancer, however, contained five to eight times less RA than normal prostate or benign prostatic hyperplasia.9 Thus retinoids may play a role also in the pathophysiology of prostate cancer. The unique biology of prostate cancer is highlighted by (1) a high frequency of histologic cancers in autopsy series but a much lower incidence of clinical disease,19-21 and (2) an exceedingly slow rate of progression in early disease, with a doubling time estimated to be in excess of 2 years.22 These features suggest that prostate cancer may be highly amenable to a chemopreventive approach. 4-HPR, a synthetic retinoid, has been widely studied as a possible chemopreventive agent for prostate cancer. It was shown to inhibit cell proliferation and induce apoptosis in some human prostate cancer cell lines as well as to prevent the development of methylnitrosourea-induced prostate malignancies and inhibit metastases of PA-III cells in Lobund-Wistar rats.23-26 Dietary 4-HPR also decreased the tumor incidence by 49% and tumor mass by 52% of ras+myc-induced carcinomas in the mouse prostate reconstitution model system.10 The mechanisms by which 4-HPR induces these effects are not well understood. No direct evidences exist to prove that 4-HPR binds to any of the known retinoid receptors, although Sun et al27 in a recent study have demonstrated that RAR expression could be reduced in LNCaP cells treated with 4-HPR. The same authors found that 4-HPR may lead to apoptosis in LNCaP cells (which are androgen-sensitive cells) by increasing the reactive oxygen species activity and expression of p53, p21, and c-jun genes and decreasing the expression of the c-myc gene. On the other hand, RARs may be involved in 4-HPR–induced apoptosis in human androgen independent cells concomitantly with an increase in p53, p21, and c-myc gene activities.27 As in any drug therapy, to achieve a biologic effect, retinoids must achieve therapeutic concentrations in the target organ. In a study performed on breast cancer patients after 7 days of treatment with 4-HPR at a dose of 200 mg/d, the concentrations of 4-HPR achieved in the target organ were 1.4 to 8.2 times higher than those found in plasma.11 The concentration of its metabolite, 4-MPR, was even higher, reaching levels 15- to 36-fold greater then those found in plasma. In the current study, we examined the uptake of 4-HPR in a murine model of prostate cancer and then in patients undergoing a radical prostatectomy. In mice, 4-HPR caused a decline in the endogenously present ROH from 955 nmol/L to 458 nmol/L. By contrast, RA levels increased by a factor of nearly eight. 4-HPR accumulated in the tumor of treated animals to greater than 5,000-nmol/L concentrations. The marked effects of 4-HPR on tumor size and incidence in mice documented in a previous study10 and the significant enrichment of the drug in prostate tumors of treated animals prompted us to initiate this comparative study in patients with localized prostate cancer. By day 28 of treatment, the serum and benign prostatic tissue concentrations of both 4-HPR and 4-MPR were approximately equal. In the cancerous tissue, 4-HPR reached significantly higher concentrations as compared with serum levels, but it was far below the difference in concentrations achieved between serum and tissues in mice and in breast cancer patients as reported by Formelli et al.11 4-HPR also did not have an impact on the RA concentrations in either serum or prostate. Possible explanations for these differences are that the duration of treatment was insufficient or dosage of 4-HPR was too low. The time to achieve equilibrium of drug concentrations in plasma and tissue may vary depending on the type of tissue examined and the ratio of surface area to volume of the tissue fluid compartment. This may explain why it may take a shorter time and a lower dosage to reach an equilibrium of drug concentrations in plasma and breast tissue and, later, the enrichment of the tissue with the drug, than to reach the same equilibrium in plasma and the prostate. Moreover, it is well known that the CNS, eye, and prostate are fed with nonfenestrated capillary beds that act like a barrier to the diffusion of certain drugs.28 This, in addition to the different pH partition in the prostate gland, may contribute to the insufficient 4-HPR enrichment in the human prostate. In our mouse prostate reconstitution model system, the tumor cells were not orthotopic in the prostate; this may have facilitated the accumulation of 4-HPR in these tumors. Although the concentrations of 4-HPR reached in human cancerous tissues were only 10% of those achieved in mice, they were significantly higher than levels achieved in human benign prostatic tissue. This may be due to either an enhanced absorption or a reduced metabolism of 4-HPR in cancerous prostate tissue. 4-HPR also caused the reduction of tissue levels of ROH in mice and of plasma and tissue ROH levels in humans, a phenomenon previously demonstrated and attributed to concomitant reduction in retinol-binding protein.11 Low expression of retinol-binding protein has been reported in some breast cancer patients.29 This reduction is reversible after drug cessation. In conclusion, there is some enrichment of 4-HPR in prostate cancer tissue in patients treated with 200 mg/d of 4-HPR for 28 days before radical prostatectomy. RA concentrations in human serum and in cancerous prostates were not affected by the treatment with 4-HPR. These preliminary findings suggest that higher doses of 4-HPR need to be explored for the achievement of therapeutic concentrations of this drug in the prostate. In studies examining toxicity, the dose of 200 mg/d of 4-HPR given in cycles of 1 month with a 3-day drug holiday at the end of each cycle was found to be well tolerated during long-term administration.30 Our study suggests that in prostate cancer, higher doses of 4-HPR may be required (600 to 1,200 mg/d or more). If 4-HPR is given in such high doses, then shorter pulses with longer drug-free intervals may need to be considered.
Supported by the National Cancer Institute (SPORE P50–58204) and General Clinical Research Center grant no. MOI, RR-00188.
1. Greenlee RT, Murray T, Bolden S, et al: Cancer statistics 2000. CA Cancer J Clin 50: 7-33, 2000[Abstract] 2. Chopra DP, Wilkoff LJ: Inhibition and reversal by beta-retinoic acid of hyperplasia induced in cultured mouse prostate tissue by 3-methylcholantrene or N-methyl-N-nitro-N-nitrosoguanidine. J Natl Cancer Inst 56: 583-587, 1976
3.
Lasnitzki I, Goodman DS: Inhibition of the effects of methylcholantrene on mouse prostate in organ culture by vitamin A and its analogs. Cancer Res 34: 1564-1571, 1974 4. Hong WK, Endicott J, Itri LM, et al: 13-Cis retinoic acid in the treatment of oral leukoplakia. N Engl J Med 315: 1501-1505, 1986[Abstract]
5.
Lippman SM, Batsakis JG, Toth BB, et al: Comparison of low-dose isotretinoin with beta carotene to prevent oral carcinogenesis. N Engl J Med 328: 15-20, 1993
6.
Meyskens FL, Surwit E, Moon TE, et al: Enhancement of regression of cervical intraepithelial neoplasia II (moderate dysplasia) with topically applied all-trans-retinoic acid: A randomized trial. J Natl Cancer Inst 86: 539-543, 1994 7. Hong WK, Lippman SM, Itri LM, et al: Prevention of second primary tumors with isotretinoin in squamous-cell carcinoma of the head and neck. N Engl J Med 323: 795-801, 1990[Abstract]
8.
Bemmer S, Pajak TF, Lippman SM, et al: Prevention of second primary tumors with isotretinoin in patients with squamous-cell carcinoma of the head and neck: Long term follow-up. J Natl Cancer Inst 86: 140-141, 1994 9. Pasquali D, Thaller C, Eichele G: Abnormal level of retinoic acid in prostate cancer tissues. J Clin Endocrinol Metab 8: 2186-2191, 1996
10.
Slawin K, Kadmon D, Park SH, et al: Dietary fenretinide, a synthetic retinoid, decreases the tumor incidence and the tumor mass of ras+myc-induced carcinomas in the mouse prostate reconstitution model system. Cancer Res 53: 4461-4465, 1993
11.
Formelli F, Clerici M, Campa T, et al: Five-year administration of fenretinide: Pharmacokinetics and effects on plasma retinol concentrations. J Clin Oncol 11: 2036-2042, 1993 12. Thompson TC, Southgate J, Kitchener G, et al: Multistage carcinogenesis induced by ras+myc oncogenes in a reconstituted organ cell. Cell 56: 917-930, 1989[Medline] 13. Merz VW, Miller GJ, Krebs T, et al: Elevated transforming growth factor-beta1 and beta3 mRNA levels are associated with ras+myc-induced carcinomas in reconstituted mouse prostate: Evidence for a paracrine role during progression. Mol Endocrinol 5: 503-513, 1991[Abstract] 14. Costa A, Malone W, Perloff M, et al: Tolerability of the synthetic retinoid fenretinide (HPR). Eur J Cancer Clin Oncol 25: 805-808, 1989[Medline] 15. Wheeler TM, Lebovitz RM: Fresh tissue harvest for research from prostatectomy specimens. Prostate 25: 274-279, 1994[Medline]
16.
Borrow J, Goddard AD, Sheer D, et al: Molecular analysis of acute promyelocytic leukemia breakpoint cluster region on chromosome 17. Science 249: 1577-1580, 1990 17. Kakizuka A, Miller WH Jr, Umesono K, et al: Chromosomal translocation t(15,17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell 66: 663-674, 1991[Medline] 18. de The H, Lavau C, Marchio A, et al: The PML-RAR alpha fusion mRNA generated by the t(15,17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 66: 675-684, 1991[Medline] 19. Franks LM: Latent carcinoma of the prostate. J Pathol Bacteriol 68: 603-616, 1954[Medline] 20. Mc Neal JE: Origin and development of carcinoma of the prostate. Cancer 23: 24-34, 1969[Medline] 21. Seidman H, Mushinski MH, Geib SK: Probabilities of eventually developing or dying of cancer: United States 1985. Cancer 35: 35-56, 1985 22. Berges RR, Vukanovic J, Epstein JI, et al: Implication of cell kinetic changes during the progression of human prostatic cancer. Clin Cancer Res 1: 473-480, 1995[Abstract] 23. Igawa M, Tanabe T, Chodak GW, et al: N-(4-hydroxyphenyl) retinamide induces cell cycle specific growth inhibition in PC3 cells. Prostate 24: 299-305, 1994[Medline] 24. Hsieh T-C, Wu JM: Effects of fenretinide (4-HPR) on prostate LNCaP cell growth, apoptosis, and prostate-specific gene expression. Prostate 33: 97-104, 1997[Medline] 25. Roberson KM, Penland SN, Padilla GM, et al: Fenretinide: Induction of apoptosis and endogenous transforming growth factor beta in PC-3 prostate cancer cells. Cell Growth Diff 8: 101-111, 1997[Abstract]
26.
Pollard M, Luckert PH, Sporn MB: Prevention of primary prostate cancer in Lobund-Wistar rats by N-(4-hydroxyphenyl) retinamide. Cancer Res 51: 3610-3611, 1991
27.
Sun S-Y, Yue P, Lotan R: Induction of apoptosis by N-(4-hydroxyphenyl) retinamide and its association with reactive oxygen species, nuclear retinoic acid receptors and apoptosis-related genes in human prostate carcinoma cells. Mol Pharmacol 55: 403-410, 1999 28. Barza M: Anatomical barriers for antimicrobial agents. Eur J Clin Microbiol Infect Dis 12: S31-35, 1993 (suppl 1)
29.
Kuppumbatti YS, Bleiweiss IJ, Mandeli JP, et al: Cellular retinol-binding protein expression and breast cancer. J Natl Cancer Inst 92: 475-480, 2000 30. Costa A, De Palo G, Decensi A, et al: Retinoids in cancer chemoprevention: Clinical trials with the synthetic analogue fenretinide. Ann N Y Acad Sci 768: 148-162, 1995[Medline] Submitted February 23, 2000; accepted June 19, 2000. This article has been cited by other articles:
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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TOP
ABSTRACT
INTRODUCTION
