B J Long

University of Maryland Medical Center, Baltimore, Maryland, United States

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Publications (28)156.82 Total impact

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    ABSTRACT: Ovariectomized mice bearing tumor xenografts grown from aromatase-transfected estrogen receptor (ER)-positive human breast cancer cells (MCF-7Ca) were injected s.c. with 10 microg/d letrozole for up to 56 weeks. Western blot analysis of the tumors revealed that ERs (ERalpha) were increased at 4 weeks but decreased at weeks 28 and 56. Expression of erbB-2 and p-Shc increased throughout treatment, whereas growth factor receptor binding protein 2 (Grb2) increased only in tumors proliferating on letrozole (weeks 28 and 56). In cells isolated from tumors after 56 weeks and maintained as a cell line (LTLT-Ca) in 1 micromol/L letrozole, ERalpha was also decreased whereas erbB-2, adapter proteins (p-Shc and Grb2), and the signaling proteins in the mitogen-activated protein kinase (MAPK) cascade were increased compared with MCF-7Ca cells. Growth was inhibited in LTLT-Ca cells but not in MCF-7Ca cells treated with MAPK kinase 1/2 inhibitors U0126, and PD98059 (IC(50) approximately 25 micromol/L). PD98059 (5 micromol/L) also reduced MAPK activity and increased ERalpha to the levels in MCF-7Ca cells. Epidermal growth factor receptor kinase inhibitor, gefitinib (ZD1839) inhibited growth of LTLT-Ca cells (IC(50) approximately 10 micromol/L) and restored their sensitivity to tamoxifen and anastrozole. In xenografts, combined treatment with ER down-regulator fulvestrant and letrozole, prevented increases in erbB-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results indicate that blocking both ER- and growth factor-mediated transcription resulted in the most effective inhibition of growth of ER-positive breast cancer cells.
    Cancer Research 07/2005; 65(12):5380-9. · 8.65 Impact Factor
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    ABSTRACT: A number of models have been used to study the development and treatment of breast cancer. Since most breast cancer patients are postmenopausal and treated with hormone therapy, we developed a model to simulate this type of patient. Tumors of human estrogen receptor (ER)-positive breast cancer cells transfected with aromatase (MCF-7Ca) are grown in immune suppressed mice. In this model, we have explored a number of strategies to optimize the antitumor efficacy of treatment such as combining the non-steroidal aromatase inhibitor letrozole with the antiestrogens, tamoxifen. This combination resulted in tumor suppression similar to the antiestrogen alone, but was less effective than letrozole alone. Clinical findings with the non-steroidal inhibitor anastrozole in combination with tamoxifen (ATAC trial) were consistent with our results. Although letrozole was the most effective single agent tested in the model, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors adapt to growth during letrozole treatment, we determined the expression of signaling proteins in tumors during the course of letrozole treatment compared to the tumors of control mice. We found that tumors initially up regulated the ER, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the MAPK cascade (p-Raf, p-Mek1/2, and p-MAPK), but not Akt, were increased in tumors no longer responsive to letrozole. The results suggest that tumor cells adapt to estrogen deprivation during letrozole treatment by activation of alternate signaling pathways to increase transcription. When letrozole was combined with the pure antiestrogen fulvestrant, to down regulate ER, the combination was more effective than either letrozole or fulvestrant alone. Tumors regressed by 45% and were maintained without growth for the duration of the experiment (29 weeks). Down regulation of ER by fulvestrant may prevent cross talk between these signaling pathways. The results suggest that achieving more complete estrogen blockade may delay development of hormone-independent signaling pathways regulating proliferation.
    The Journal of Steroid Biochemistry and Molecular Biology 06/2005; 95(1-5):41-8. · 3.98 Impact Factor
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    ABSTRACT: To study the long-term effects of estrogen deprivation on breast cancer, MCF-7Ca human estrogen receptor-positive breast cancer cells stably transfected with human aromatase gene were cultured in the steroid-depleted medium for 6 to 8 months until they had acquired the ability to grow. Proliferation of these cells (UMB-1Ca) was accompanied by increased expression of human epidermal growth factor receptor 2, increased activation of AKT through phosphorylation at Ser473 and Thr308, and increased invasion compared with parental MCF-7Ca cells. Estrogen receptor expression was also increased 5-fold. Although growth was inhibited by the antiestrogen fulvestrant, the IC50 was 100-fold higher than for parental MCF-7Ca cells. Aromatase inhibitor letrozole also inhibited growth at 10,000-fold higher concentration than required for MCF-7Ca cells, whereas anastrozole, exemestane, formestane, and tamoxifen were ineffective at 100 nmol/L. Growth of UMB-1Ca cells was inhibited by phosphatidylinositol 3-kinase inhibitor wortmannin (IC50 approximately 25 nmol/L) and epidermal growth factor receptor kinase inhibitor gefitinib (ZD 1839; IC50 approximately 10 micromol/L) whereas parental MCF-7Ca cells were insensitive to these agents. Concomitant treatment of UMB-1Ca cells with the signal transduction inhibitors and anastrozole and tamoxifen restored their growth inhibitory effects. These studies show that estrogen deprivation results in up-regulation of growth factor signaling pathways, which leads to a more aggressive and hormone refractory phenotype. Cross-talk between ER and growth factor signaling was evident as inhibition of these pathways could restore estrogen responsiveness to these cells.
    Cancer Research 06/2005; 65(9):3903-10. · 8.65 Impact Factor
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    ABSTRACT: To optimize treatment strategies for postmenopausal breast cancer patients, we investigated the efficacy of the steroidal aromatase inhibitor exemestane alone or in combination with the antiestrogen tamoxifen in a xenograft model of postmenopausal breast cancer. We also determined the effects of these agents in sequential second-line therapy and the effect of the nonsteroidal aromatase inhibitor letrozole on tumors that progressed on the above treatments. Experimental: Aromatase-transfected human estrogen receptor-positive breast cancer cells (MCF-7Ca) were grown as tumors in ovariectomized athymic mice. Animals received subcutaneous injection with vehicle, tamoxifen, exemestane, tamoxifen plus exemestane, and letrozole. Tumor volumes were measured weekly. All treatments were effective initially in suppressing tumor growth as first-line therapy compared with vehicle treatment. Exemestane suppressed tumor growth to a greater extent than tamoxifen. However, the combination of tamoxifen plus exemestane was more effective than either drug alone. After tumor volumes doubled on initial treatment, the mice were crossed over to receive exemestane or tamoxifen. Tumor growth slowed briefly in mice treated with tamoxifen and crossed over to exemestane, but tumor growth continued unabated in those changed from exemestane to tamoxifen. However, letrozole was effective in both groups as third-line therapy for a limited period. Letrozole as initial single agent was the best overall treatment in terms of the degree of tumor suppression and the length of effectiveness of treatment. Exemestane was more effective in controlling tumor growth than tamoxifen. In addition, the combination of exemestane plus tamoxifen was clearly more effective than sequential use of these agents in the tumor model. However, the nonsteroidal aromatase inhibitor letrozole as first-line therapy was overall the most effective treatment in controlling tumor growth.
    Clinical Cancer Research 12/2004; 10(21):7375-81. · 7.84 Impact Factor
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    ABSTRACT: A facile preparation of azolyl steroids, VN/85-1 and VN/87-1 (potent inhibitors of CYP17) has been developed. This process without tedious chromatographic separations improved the overall yields from 55 and 45% to 70 and 65% for VN/85-1 and VN/87-1, respectively. Pharmacokinetic studies of VN/85-1 were conducted in male SCID mice. Following subcutaneous (s.c.) administration of 100mg/kg of VN/85-1, peak plasma level of 16.73 microg/ml occurred after 45 min, and the compound was cleared rapidly with a t(1/2) of 52.34 min. The bioavailability of VN/85-1 after s.c. administration was 83.0%. VN/85-1 was also rapidly metabolized to the corresponding 3-oxo-4-ene analog, 17-(1H-imidazol-1-yl)androsta-4,16-diene-3-one (VN/108-1). In our attempt to optimize the anti-tumor efficacy of these two CYP17 inhibitors, we studied their anti-tumor efficacies in male SCID mice bearing LNCaP tumor xenografts, utilizing various drug doses and drug scheduling. Three times a day dose regimen (3 x dose regimen) of VN/85-1 was more effective than a once daily dose. In contrast, 3 x dose regimen doses of VN/87-1 were less effective than the once daily dose. However, at their effective dosage regimes, VN/85-1 and VN/87-1 were each as effective as castration and more effective than finasteride or casodex, an anti-androgen used for prostate cancer (PC) therapy. For all of the treatments, there was a strong correlation between the tumor volumes and other associated parameters, such as, tumor weights, and serum testosterone (T) and PSA levels. These results indicate that VN/85-1 or VN/87-1 may be useful in the treatment of hormone-dependent prostate cancer.
    The Journal of Steroid Biochemistry and Molecular Biology 11/2004; 92(3):155-65. · 3.98 Impact Factor
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    ABSTRACT: The antiestrogen tamoxifen has potent activity against estrogen receptor-positive breast cancer, but two nonsteroidal aromatase inhibitors, letrozole and anastrozole, show considerable advantages over tamoxifen with respect to patient survival and tolerability. To determine the optimal way to use letrozole and tamoxifen, we studied their effects on a breast tumor xenograft model, MCF-7Ca, that is responsive to both antiestrogens and aromatase inhibitors. Female ovariectomized BALB/c athymic nude mice carrying xenograft tumors were treated daily subcutaneously with one of the following first-line therapies for varying durations: no drug (control), tamoxifen (100 microg/day) alone, letrozole (10 microg/day) alone, both drugs at the same time, or alternating 4-week courses of each drug (beginning with a course of tamoxifen or beginning with a course of letrozole). Tumor volumes and weights were estimated using linear mixed-effects models. The time to tumor doubling was calculated, and tumor weights in the treatment groups were compared, with adjustments for multiple comparisons being made with either Tukey's or Dunnett's procedure. Second-line therapies (with tamoxifen, letrozole, or fulvestrant) were initiated when tumors doubled in size under first-line therapies. All statistical tests were two-sided. The times for doubling of tumor volume were as follows: control, 3-4 weeks; tamoxifen alone, 16 weeks; tamoxifen alternating with letrozole, 17-18 weeks; tamoxifen plus letrozole, 18 weeks; letrozole alternating with tamoxifen, 22 weeks; letrozole alone, 34 weeks. First-line treatment with letrozole was superior to treatment with tamoxifen alone or with the two drugs combined (at week 16, both P<.001). Alternating tamoxifen and letrozole and alternating letrozole and tamoxifen were also not as effective as letrozole alone (at week 16, P =.002 and P<.001, respectively). Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole compared with those remaining on tamoxifen at the end of treatment (week 28, P<.001), whereas tumors progressing on letrozole were unaffected by second-line treatment with the antiestrogens tamoxifen or fulvestrant. First-line letrozole therapy extends time for tumor progression in this model relative to the other treatment regimens tested. However, further studies are needed to determine the most effective second-line therapy for tumors that progress on letrozole.
    CancerSpectrum Knowledge Environment 03/2004; 96(6):456-65. · 14.07 Impact Factor
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    ABSTRACT: Aromatase inhibitors have recently been reported to be more effective than the antiestrogen tamoxifen (Tam) in treating breast cancer. Here, we studied the mechanisms and signaling pathways of cell growth, cell cycle progression, and apoptosis induced by three aromatase inhibitors: letrozole (Let), anastrozole, and 4-hydroxyandrostenedione in comparison with estrogen withdrawal (E2W) and antiestrogens Tam and faslodex. Estrogen-dependent human breast cancer cells stably transfected with aromatase (MCF-7Ca) were used. All treatments induced growth suppression and cell cycle arrest at the G(0)-G(1) phase that was associated with up-regulation of p53 and p21 protein and mRNA levels and down-regulation of cyclin D1 and c-myc mRNA. The apoptotic index was increased 4-7 fold, Bcl-2 protein expression decreased, Bax increased, and caspase-9, caspase-6, and caspase-7 were activated but not caspase-3 and caspase-8. Let and E2W caused regression of tumors of MCF-7Ca cells grown in nude mice and increased the number of cells undergoing apoptosis. In contrast, Tam and faslodex did not induce tumor regression and a lower number of apoptotic cells was detected. Cleavage of poly(ADP-ribose) polymerase was detected. Treatment with Let, Tam, or E2W resulted in a dose- and time-dependent increase in active caspase-7 and up-regulation of p53 and p21 protein. Although the mechanisms involved appeared to be similar for antiestrogens and aromatase inhibitors, the most significant effects occurred with Let, which were significantly greater than with E2W and consistent with marked effects of Let on tumor and cell growth.
    Cancer Research 12/2003; 63(22):8037-50. · 8.65 Impact Factor
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    ABSTRACT: Aromatase inhibitors have now been approved as first-line treatment options for hormone-dependent advanced breast cancer. When compared to tamoxifen, these aromatase inhibitors provide significant survival and tolerability advantages. However, the optimal use of an aromatase inhibitor and tamoxifen remains to be established. To date, the intratumoral aromatase xenograft model has proved accurate in predicting the outcome of clinical trials. Utilizing this model, we performed long-term studies with tamoxifen and letrozole to determine time to disease progression with each of the treatment regimens. Aromatase-transfected MCF-7Ca human breast cancer cells were grown as tumor xenografts in female ovariectomized athymic nude mice in which androstenedione was converted to estrogen and stimulated tumor growth. When tumor volumes were approximately 300 mm3, the animals were grouped for continued supplementation with androstenedione only (control) or for treatment with letrozole 10 microg per day (long-term), tamoxifen 100 microg per day (long-term), letrozole alternating to tamoxifen (4-week rotation), tamoxifen alternating to letrozole (4-week rotation), or a combination of the two drugs. Tumors of control mice had doubled in volume in 3-4 weeks. In mice treated with tamoxifen and the combination, tumor doubling time was significantly shorter (16 and 18 weeks, respectively) than with letrozole (34 weeks). Furthermore, alternating letrozole and tamoxifen treatment every 4 weeks was less effective than letrozole alone. Tumors doubled in 17-18 weeks when the starting treatment was tamoxifen and in 22 weeks when the starting treatment was letrozole. Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole (10 microg per day). However, when mice with letrozole-resistant tumors were switched to antiestrogen treatment, tumors did not respond to tamoxifen (100 microg per day) or faslodex (1 mg per day). This suggests that advanced breast cancers treated with letrozole may be insensitive to subsequent second-line hormonal agents. Thus, although letrozole was determined to be an effective second-line treatment option for tumors progressing on tamoxifen, antiestrogen therapy does not appear to be effective for tumors progressing on letrozole. However, response to second-line treatment was observed in a model where tumors that had progressed on letrozole were transplanted to new mice. These tumors had been allowed to grow in the presence of supplemented androstenedione but absence of letrozole. This suggests that resistance to letrozole may be reversible, allowing tumors to respond to subsequent antiestrogens and letrozole.
    The Journal of Steroid Biochemistry and Molecular Biology 10/2003; 86(3-5):283-8. · 3.98 Impact Factor
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    ABSTRACT: We have developed a tumor model for studying the effects of aromatase inhibitors and antiestrogens in vivo. The model simulates postmenopausal breast cancer patients with estrogen-dependent tumors. This model utilizes human estrogen-dependent breast cancer cells transfected with the aromatase gene (MCF-7(CA)), which are inoculated in Matrigel s.c. into ovariectomized nude mice. The estrogen receptor-positive cells produce sufficient estrogen by aromatization to stimulate their proliferation and the formation of tumors in the mice. Using several different strategies, we have compared the aromatase inhibitors (letrozole and anastrozole) with antiestrogens (tamoxifen and fulvestrant). Aromatase inhibitors were more effective than tamoxifen and were more effective alone than when combined with antiestrogens. In addition, letrozole had a longer duration of effect than tamoxifen. The possibility of delaying the development of resistance to antiestrogens and aromatase inhibitors was investigated by alternating letrozole with tamoxifen every 4 weeks. However, although alternating treatment proved more effective than tamoxifen alone (tumors doubled in 16 weeks), tumors doubled in volume at about 18 weeks when treatment began with tamoxifen. When treatment started with letrozole and alternated with tamoxifen, tumors doubled in volume in about 22 weeks. However, tumors of mice treated with letrozole alone did not double in size until 35 weeks. These results demonstrate that this aromatase inhibitor is more effective and has a longer duration of response as a single agent than tamoxifen or in combination with tamoxifen.
    Clinical Cancer Research 02/2003; 9(1 Pt 2):455S-9S. · 7.84 Impact Factor
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    ABSTRACT: A tumor xenograft model has been established that more closely relates to hormone-dependent breast cancer than tumors induced with the carcinogens in the rat. The MCF-7Ca tumors are developed from MCF-7 cells transfected with aromatase. These cells synthesize estrogens and are responsive to the effects of estrogen via estrogen receptors. Tumor growth is inhibited by both aromatase inhibitors and antiestrogens. The purpose of these studies was to determine the effectiveness of several strategies of treatment with aromatase inhibitors and antiestrogens. Experiments have shown that tumor growth can be inhibited more effectively with aromatase inhibitors such as letrozole than with tamoxifen. Furthermore, the model demonstrated that an aromatase inhibitor alone is more effective than in combination with tamoxifen administered either together or sequentially. This result predicted the outcome of such clinical trials as ATAC (Arimidex and Tamoxifen Alone or in Combination) and suggests that the model is a good predictor of responses observed in human breast cancer patients. Thus, the model could be useful to guide the design of future clinical trials in patients with hormone-dependent breast cancer.
    American Journal of Cancer 12/2002; 2:1-6.
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    ABSTRACT: The aromatase inhibitors letrozole and anastrozole have been approvedrecently as first-line treatment options for hormone-dependent advanced breast cancer. Although it is established that a proportion of patients who relapse on first-line tamoxifen therapy show additional responses to aromatase inhibitors, it has not been determined whether tumors that acquire resistance to aromatase inhibitors in the first line remain sensitive to second-line therapy with antiestrogens. The aim of this study was to determine whether aromatase-transfected and hormone-dependent MCF-7Ca human breast cancer cells remain sensitive to antiestrogens after: (a) long-term growth in steroid-depleted medium in vitro; and (b) long-term treatment with the aromatase inhibitor letrozole in vivo. In the first approach, a variant of the MCF-7Ca human breast cancer cell line was selected that had acquired the ability to grow in estrogen-depleted medium after 6-8 months of culture. Steroid-deprived UMB-1Ca cells were analyzed for aromatase activity levels, hormone receptor levels, and sensitivity to estrogens and antiestrogens in vitro and in vivo. In the second approach, established MCF-7Ca breast tumor xenografts were treated with letrozole 10 microg/day for 12 weeks followed by 100 microg/day for 25 weeks until tumors acquired the ability to proliferate in the presence of the drug. Long-term letrozole-treated tumors were then transplanted into new mice, and the effects of antiestrogens and aromatase inhibitors on tumor growth were determined. Steroid-deprived UMB-1Ca breast cancer cells continued to express aromatase activity at levels comparable with the parental cell line. However, compared with MCF-7Ca cells, UMB-1Ca cells expressed elevated levels of functionally active estrogen receptor. The growth of UMB-1Ca cells in vitro was inhibited by the antiestrogens tamoxifen and faslodex and tumor growth in vivo was inhibited by tamoxifen. In the second approach, the time for MCF-7Ca tumor xenografts to approximately double in volume after being treated sequentially with the increasing doses of letrozole was thirty-seven weeks. Long-term letrozole-treated tumors continued to express functionally active aromatase. When transplanted into new mice, growth of the long-term letrozole-treated tumors was slowed by tamoxifen and inhibited more effectively by faslodex. Tumor growth was refractory to the aromatase inhibitors anastrozole and formestane but, surprisingly, showed sensitivity to letrozole. Steroid-deprived UMB-1Ca human breast cancer cells selected in vitro and long-term letrozole-treated MCF-7Ca breast tumor xenografts remain sensitive to second-line therapy with antiestrogens and, in particular, to faslodex. This finding is associated with increased expression of functionally active estrogen receptor after steroid-deprivation of MCF-7Ca human breast cancer cells in vitro.
    Clinical Cancer Research 08/2002; 8(7):2378-88. · 7.84 Impact Factor
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    A Brodie, B Long
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    ABSTRACT: Aromatase is the key enzyme in the synthesis of estrogens and mediates the conversion of androstenedione and testosterone to estrone and estradiol. Because of the importance of estrogen in stimulating breast cancers, the inhibition of estrogen synthesis is a logical approach to treatment. Aromatase is an excellent target for inhibition, because it is the last step in steroid biosynthesis, and, therefore, there are no important downstream enzymes to be affected. In addition, although aromatase is a P-450 enzyme and shares common features with other enzymes in this class, such as liver metabolizing enzymes and steroidogenic enzymes, it has unique features in the aromatizing reaction, features that are amenable to the development of selective inhibition. The approach we took to develop the first aromatase inhibitors was to design substrate analogues based on the structure of androstenedione. Some of these inhibitors, such as 4-hydroxyandrostenedione [4-OHA (later known as formestane)], also cause enzyme inactivation. Instead of being released at the end of the reaction, the substrate analogue remains bound. Therefore, the inhibitor is not required to be present at all times to maintain inhibition, and it has high enzyme specificity. Subsequently, other investigators have taken a different approach to developing compounds based on inhibitors of P-450 enzymes. High selectivity has been achieved with some of these reversible inhibitors. We have developed a unique animal model with human tumors to compare the antitumor efficacy of antiestrogens and aromatase inhibitors and to optimize their use in sequence and combination as a guide for future clinical trials.
    Clinical Cancer Research 01/2002; 7(12 Suppl):4343s-4349s; discussion 4411s-4412s. · 7.84 Impact Factor
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    ABSTRACT: We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E2 is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE2 produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.
    The Journal of Steroid Biochemistry and Molecular Biology 12/2001; 79(1-5):41-7. · 3.98 Impact Factor
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    ABSTRACT: An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10-30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress.
    The Journal of Steroid Biochemistry and Molecular Biology 03/2001; 76(1-5):199-202. · 3.98 Impact Factor
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    ABSTRACT: Pregnenolone (P(5)), a common precursor of many steroids, is present in the blood of normal adult men at concentrations of 1-3 nM. In vitro, P(5) was found to stimulate LNCaP-cell proliferation 7-8-fold at a physiological concentration (2 nM), and 3-4-fold at a subphysiological concentration (0.2 nM). Growth stimulation at the 2-nM concentration was comparable with that of the androgen, dihydrotestosterone at its physiological concentration (0.5 nM; 9-10-fold increase in cell number). To determine whether P(5) or its metabolites were mediating this growth response, LNCaP cells were incubated with [3H]P(5) and high-performance liquid chromatography (HPLC) was performed. After a 48-h exposure, two unidentified metabolites were detected. Although, the P(5) metabolites slightly increased LNCaP-cell growth in vitro, their effect was significantly less than P(5) alone, suggesting that the growth stimulation was mediated by P(5) itself. We further showed that P(5) sustained its proliferative activity in vivo and stimulated the growth of LNCaP-tumor xenografts in intact male SCID mice as well as in castrated animals. In order to determine whether P(5) was binding to a specific site in LNCaP cells, receptor binding studies were performed. Scatchard analysis predicted for a single class of binding sites with K(d)=1.4 nM. Studies were performed to determine the effects of P(5) on transcription mediated by wild-type and LNCaP androgen receptors. P(5) was shown to activate transcription through the LNCaP androgen receptor (AR), but not the wild-type AR. This implies that P(5) most likely stimulates LNCaP-cell proliferation through binding to the cellular mutated AR present in LNCaP cells. We have also demonstrated that drugs designed to be antagonists of the androgen, progesterone and estrogen receptors, and one of our novel compounds designed to be an inhibitor of androgen synthesis, were potent inhibitors of the AR-mediated transcriptional activity induced by P(5), and were able to inhibit LNCaP-cell proliferation. These findings suggest that some prostate cancer patients who appear to become hormone-independent may have tumors which are stimulated by P(5) via a mutated AR and that these patients could benefit from treatment with antiestrogens, antiprogestins, or with some of our novel androgen synthesis inhibitors.
    The Journal of Steroid Biochemistry and Molecular Biology 01/2001; 75(1):1-10. · 3.98 Impact Factor
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    ABSTRACT: We have found that in addition to being potent inhibitors of 17alpha-hydroxylase/C17,20-lyase and/or 5alpha-reductase, some of our novel androgen synthesis inhibitors also interact with the mutated androgen receptor (AR) expressed in LNCaP prostate cancer cells and the wild-type AR expressed in hormone-dependent prostatic carcinomas. The effects of these compounds on the proliferation of hormone-dependent human prostatic cancer cells were determined in vitro and in vivo. L-2 and L-10 are delta4-3-one-pregnane derivatives. L-35 and L-37 are delta5-3beta-ol-androstane derivatives, and L-36 and L-39 are delta4-3-one-androstane-derived compounds. L-2, L-10, and L-36 (L-36 at low concentrations) stimulated the growth of LNCaP cells, indicating that they were interacting agonistically with the mutated AR expressed in LNCaP cells. L-35, L-37, and L-39 acted as LNCaP AR antagonists. To determine whether the growth modulatory effects of our novel compounds were specific for the mutated LNCaP AR, competitive binding studies were performed with LNCaP cells and PC-3 cells stably transfected with the wild-type AR (designated PC-3AR). Regardless of AR receptor type, all of our novel compounds were effective at preventing binding of the synthetic androgen methyl-trienolone[17alpha-methyl-(3H)-R1881 to both the LNCaP AR and the wildtype AR. L-36, L-37, and L-39 (5.0 microM) prevented binding by >90%, whereas L-35 inhibited binding by 30%. To determine whether the compounds were acting as agonists or antagonists, LNCaP cells and PC-3AR cells were transfected with the pMAMneoLUC reporter gene. When luciferase activity was induced by dihydrotestosterone, all of the compounds were found to be potent inhibitors of transcriptional activity, and the pattern of inhibition was similar for both receptor types. However, L-2, L-10, and L-36 were determined to be AR agonists, and L-35, L-37, and L-39 were wild-type AR antagonists. When tested in vivo, L-39 was the only AR antagonist that proved to be effective at inhibiting the growth of LNCaP prostate tumor growth. L-39 slowed tumor growth rate in LNCaP tumors grown in male SCID mice to the same level as orchidectomy, significantly reduced tumor weights (P < 0.05), significantly lowered serum levels of prostate-specific antigen (P < 0.02), and significanty lowered serum levels of testosterone (P < 0.05). L-39 also proved to be effective when tested against the PC-82 prostate cancer xenograft that expresses wild-type AR. These results show that some of our compounds initially developed to be inhibitors of androgen synthesis also interact with the human AR and modulate the proliferation of hormone-dependent prostatic cancer cells. Therefore, compounds such as L-39, which have multifunctional activities, hold promise for the treatment of androgen-dependent prostate tumors.
    Cancer Research 12/2000; 60(23):6630-40. · 8.65 Impact Factor
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    ABSTRACT: 17-(5'-Isoxazolyl)androsta-4,16-dien-3-one (L-39), a novel androstene derivative, was synthesized and evaluated in vitro and in vivo. L-39 showed potent and non-competitive inhibition of human testicular microsomal 17alpha-hydroxylase/C(17,20)-lyase with an IC50 value of 59 nM and Ki of 22 nM. L-39 also showed potent and competitive inhibition of 5alpha-reductase in human prostatic microsomes with IC50 and Ki values of 33 and 28 nM respectively. L-39 (5 microM) has also been shown to manifest anti-androgenic activity in cultures of human prostate cancer cell lines (LNCaP) by preventing the labelled synthetic androgen R1881 (5 nM) from binding to the androgen receptors. Androgen-dependent human prostate cancer xenografts (PC-82) were grown in nude mice and the effects of L-39 (50 mg kg(-1) day(-1)) on tumour growth and prostate-specific antigen (PSA) levels were determined after 28 days. L-39 significantly (P < 0.01) diminished tumour growth and wet weights to a similar extent as castration or flutamide treatment. L-39 also significantly (P < 0.01) reduced serum PSA levels by more than 80% in the mice bearing human prostate cancer xenografts. Pharmacokinetic studies were also conducted in male Balb/c mice. After subcutaneous administration of a single bolus dose, L-39 was rapidly absorbed into the systemic circulation. Peak plasma levels occurred at 0.75 h and then declined with a t(1/2) of 1.51 h. The bioavailability of L-39 after subcutaneous administration was 28.5%. These results demonstrate that L-39 is a potent inhibitor of androgen synthesis and is effective in reducing the growth of human prostate cancer xenografts in nude mice. Although improvements in the bioavailability are necessary, L-39 is a potential lead compound with this profile as an inhibitor of prostate cancer growth.
    British Journal of Cancer 08/2000; 83(1):74-82. · 5.08 Impact Factor
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    ABSTRACT: Our laboratory has been developing new inhibitors of a key regulatory enzyme of testicular and adrenal androgen synthesis 17alpha-hydroxylase/C(17,20)-lyase (P450c17), with the aim of improving prostate cancer treatment. We designed and evaluated two groups of azolyl steroids: delta5-non-competitive inhibitors (delta5NCIs), VN/63-1, VN/85-1, VN/87-1 and their corresponding delta4 derivatives (delta4NCIs), VN/107-1, VN/108-1 and VN/109-1. The human P450c17 gene was transfected into LNCaP human prostate cancer cells, and the resultant LNCaP-CYP17 cells were utilized to evaluate the inhibitory potency of the new azolyl steroids. VN/85-1 and VN/108-1 had the lowest IC50 values of 1.25 +/- 0.44 nM and 2.96 +/- 0.78 nM respectively, which are much lower than that of the known P450 inhibitor ketoconazole (80.7 +/- 1.8 nM). To determine whether the compounds had direct actions on proliferation of wild-type LNCaP cells, cell growth studies were performed. All of the delta5NCIs and VN/108-1 blocked the growth-stimulating effects of androgens. In steroid-free media, the delta5NCIs decreased the proliferation of LNCaP cells by 35-40%, while all of the delta4NCIs stimulated LNCaP cells growth 1.5- to 2-fold. In androgen receptor (AR) binding studies, carried out to determine the mechanism of this effect, all of the delta4NCIs (5 microM) displaced 77-82% of synthetic androgen R1881 (5 nM) from the LNCaP AR. The anti-androgen flutamide and the delta5NCIs displaced 53% and 32-51% of R1881 bound to AR respectively. These results suggested that the delta5NCIs may also be acting as anti-androgens. We further evaluated our inhibitors in male severe combined immunodeficient mice bearing LNCaP tumour xenografts. In this model VN/85-1 was as effective as finasteride at inhibiting tumor growth (26% and 28% inhibition, respectively) and the inhibitory effect of VN/87-1 was similar to that of castration (33% and 36% inhibition respectively). These results suggest that VN/85-1 and VN/87-1 may be potential candidates for treatment of prostate cancer.
    British Journal of Cancer 11/1999; 81(4):622-30. · 5.08 Impact Factor
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    ABSTRACT: We have previously established a model for postmenopausal, hormone-dependent breast cancer in nude mice which is responsive to both antiestrogens and aromatase inhibitors. In this model, MCF-7 human breast carcinoma cells transfected with the aromatase gene (MCF-7CA) synthesize sufficient estrogen to form tumors in ovariectomized nude mice. In the present study we used this intratumoral aromatase model to investigate the effects on tumor growth of the new nonsteroidal aromatase inhibitors letrozole (CGS 20,267) and anastrozole (ZD 1033) and the antiestrogens tamoxifen (ICI 47,474) and faslodex (ICI 182,780). Furthermore, we determined whether the inhibition of estrogen synthesis together with inhibition of estrogen action would be more effective in controlling breast tumor growth. The results of our studies indicate that the aromatase inhibitors anastrozole and letrozole, as well as the new pure antiestrogen faslodex, have potent antitumor effects in the mouse model. In the treatment of mice with mammary tumors, letrozole was more effective in suppressing tumor growth than anastrozole. This was consistent with the Ki values of these inhibitors against placental aromatase and the IC50 values in cell culture (MCF-7CA), which indicated the greater potency of letrozole as an aromatase inhibitor. Letrozole also had greater antitumor effects than tamoxifen and faslodex. The antitumor effect of letrozole was substantial, making it difficult to detect any additional effect on the tumors when letrozole was combined with the antiestrogens. However, the combined treatment of anastrozole + tamoxifen and anastrozole + faslodex also did not increase efficacy compared to the aromatase inhibitor alone. In addition, combining the two antiestrogens did not suppress tumor growth more effectively than faslodex alone. Our results show that treatment with the combinations of aromatase inhibitors with either tamoxifen or faslodex are not more effective in blocking estrogen stimulation of tumor growth than the aromatase inhibitors alone.
    Breast Cancer Research and Treatment 10/1999; 57(2):183-92. · 4.47 Impact Factor
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    A Brodie, Q Lu, Y Liu, B Long
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    ABSTRACT: The potential of aromatase (estrogen synthetase) within the breast to provide a significant source of estrogen mediating tumor proliferation is suggested by studies reporting 4- to 6-fold higher estrogen levels in tumors than in plasma of postmenopausal patients with breast cancer. Recent studies in our laboratory have identified aromatase and its mRNA in tumor epithelial cells using immunocytochemistry and in situ hybridization. In addition, significant aromatase activity, which was stimulated 7-fold by dexamethasone, was measured in metastatic cells isolated from a breast cancer patient. Increase in proliferation, as measured by proliferating cell nuclear antigen immunostaining in tumor sections and by thymidine incorporation into DNA in response to testosterone, was observed in histocultures of breast cancer samples. This latter effect could be inhibited by 4-hydroxyandrostenedione. These results imply that intratumoral aromatase has functional significance and may be an important target for successful inhibitor treatment of breast cancer patients. To investigate treatment strategies with aromatase inhibitors and antiestrogens, we developed an intratumoral aromatase model to simulate the hormone responsive postmenopausal breast cancer patient. Tumors of estrogen receptor positive human breast carcinoma cells (MCF-7) transfected with the human aromatase gene are grown in ovariectomized nude mice. These cells synthesize sufficient estrogen to stimulate tumor formation. We have utilized this model to investigate the effects on tumor growth of the antiestrogens, tamoxifen and ICI 182780, and the aromatase inhibitors, letrozole and anastrozole (arimidex), alone and in combination. Both the aromatase inhibitors and the antiestrogens were effective in suppressing tumor growth. However, letrozole was significantly more effective than the antiestrogens. When the aromatase inhibitors were combined with the antiestrogen, tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Furthermore, the results do not suggest any benefit from combining tamoxifen with the pure antiestrogen, ICI 182780. Thus sequential use of these agents is likely to be more advantageous to the patient in terms of longer duration of effective treatment.
    Endocrine Related Cancer 07/1999; 6(2):205-10. · 5.26 Impact Factor

Publication Stats

998 Citations
156.82 Total Impact Points

Institutions

  • 2004
    • University of Maryland Medical Center
      Baltimore, Maryland, United States
  • 1998–2004
    • University of Maryland, Baltimore
      • • Department of Pharmacology
      • • Department of Medicine
      Baltimore, MD, United States
  • 1999
    • Loyola University Maryland
      Baltimore, Maryland, United States