Rajeshwar Rao Tekmal

Overexpression of Aromatase Leads to Development of Testicular Leydig Cell Tumors : An in Vivo Model for Hormone-Mediated Testicular Cancer

Despite recent advances in diagnosis and treatment of testicular cancer, its causes remain unknown. The most common conditions known to be associated with testicular cancer are cryptorchidism, infertility, and overexposure to pesticides or radiation. Recent studies also indicate hormones may play a crucial role in testicular tumorigenesis. Our studies show that about half of the male transgenic mice overexpressing aromatase in testis were infertile and/or had larger than normal testicles. Gross pathology and histological analysis showed the mice to have Leydig cell tumors, unilaterally or bilaterally. Serum estradiol levels for transgenic mice were at least twice as high as those for nontransgenic mice. Expression of aromatase and estrogen receptor were also very high in testicular tissue of transgenic mice compared to nontransgenic mice. Consistent with increased estrogenic activity in the testicular tissue, we also saw an increase in the levels of genes involved in cell cycle that are regulated by the estrogen. To obtain a better understanding of the biological significance of testicular tumorigenesis, a reliable animal model is necessary to clarify the mechanisms and correlations associated with human cancers. Here we describe such a model, which shows that overexpression of aromatase results in increased estrogen production and a changed hormone milieu, leading to the induction of testicular cancer (Leydig cell tumors). This predictable and useful model is a potential tool for the study of testicular tumorigenesis, hormonal carcinogenesis, synergistic action of other carcinogens on hormone-induced tumors, and tumor dependency on endocrine factors.

Suppression of aromatase (estrogen synthetase) by red wine phytochemicals

Estrogen promotes the proliferation of breast cancer cells. Aromatase is the enzyme that converts androgen to estrogen. In tumors, the expression of aromatase is upregulated compared to surrounding non-cancerous tissue. In this study, we found that wine contains phytochemicals that are capable of suppressing aromatase. Red wine was shown to be much more effective than white wine in the suppression of aromatase activity. Whole wine, lyophilized wine, and heat-treated extracts were examined for aromatase inhibition in a human placenta microsomal assay. C18 Sep-Pak cartridge (Waters Co.) separation of red wine extracts under an increasing acetonitrile (ACN) gradient found that the most active components were in the 20% ACN fraction, in that they inhibited the wild-type human placenta aromatase, wild-type porcine placenta and blastocyst aromatase in a dose-dependent fashion. The 20% ACN active fraction was heat stable and inhibited aromatase in a non-competitive manner. The aromatase-inhibitory action of red wine extracts was also examined with a transgenic mouse model in which aromatase is over-expressed in the mammary tissues. It was found that the intake of the 20% ACN fraction by gavage completely abrogated aromatase-induced hyperplasia and other changes in the mammary tissue. This is the first report demonstrating that wine, especially red wine, contains phytochemicals that can inhibit aromatase.

Overexpression of aromatase in transgenic male mice results in the induction of gynecomastia and other biochemical changes in mammary glands.

Our previous studies have shown that overexpression of aromatase in mammary glands results in the induction of hyperplastic and dysplastic changes in female transgenic mice. In this study we show that overexpression of aromatase in male transgenic mice results in increased mammary growth and histopathological changes similar to gynecomastia. Increased estrogenic activity also results in an increase in estrogen and progesterone receptor expression in the mammary glands of transgenic males as compared to the nontransgenic males, as well as an increase in the expression of various genes involved in cell cycle and cell proliferation. We have also observed an increase in certain growth factors, such as bFGF and TGFbeta, as a result of aromatase overexpression in the male transgenic mammary glands. In order to obtain a better understanding of the biological significance of gynecomastia, a reliable model is necessary to explain the mechanisms and correlations associated with human cancers. This model, can potentially serve as a predictable and useful tool for studying gynecomastia, hormonal carcinogenesis and action of other carcinogens on hormone induced cancers.

Overexpression of Macrophage Colony-Stimulating Factor (CSF-I) and Its Receptor, c-fms, in Normal Ovarian Granulosa Cells Leads to Cell Proliferation and Tumorigenesis

Objective: To investigate the interdependent role of macrophage colony-stimulating factor (CSF-1) and its receptor (c-fms) on their induction and their role in granulosa cell tumorigenesis. Methods: Normal ovarian granulosa cells were used to develop stable transfectants that overexpress CSF-1 or CSF-1/c-fms. CSF-1 was expressed under the control of tissue/cell specific αinhibin promoter, and c-fms was expressed constitutively using a viral promoter. Stable transfectants were used to examine the effect of overexpression of these molecules on the proliferation, induction of autocrine loop, and tumorigenesis. Results: Expression vectors were developed for CSF-1 and its receptor, c-fms, and used to generate stable transfects overexpressing these genes in granulosa cells. Data show that overexpression of CSF-1 leads to the induction of its receptor. Stable transfectants that overexpress CSF-1 show about a 2.5-fold increase in cell proliferation compared with normal granulosa cells, and these cells are also converted to anchorage-independent and tumorigenic phenotype. Using an antisense RNA approach, we also demonstrated that the increased cell proliferation is CSF-1 specific. Concomitant overexpression of CSF-1 and c-fms further results in increased cell proliferation (sixfold), rapid anchorage-independent growth, and aggressive tumor formation. Conclusion: CSF-1 is capable of inducing its own receptor, and, similarly, the CSF-1 receptor, c-fms, can also induce its growth factor ligand. These studies also demonstrate the interdependent role of these genes in transformation of nromal ovarian granulosa cells to a tumorigenic phenotype and suggest the possibility of a similar role for these genes in progression of ovarian cancer.

Use of letrozole as a chemopreventive agent in aromatase overexpressing transgenic mice.

Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in mammary glands, and gynecomastia and testicular cancer in male aromatase transgenic mice. Our studies also have shown that aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of letrozole, an aromatase inhibitor without any effect on normal physiology. In the present study, we have examined the effect of prior low dose letrozole treatment on pregnancy and lactation. We have also investigated the effect of low dose letrozole treatment on subsequent mammary growth and biochemical changes in these animals. There was no change in the litter size, birth weight and no visible birth defects in letrozole-treated animals. Although, there was an insignificant increase in mammary growth in aged animals after 6 weeks of letrozole treatment, the levels of expression of estrogen receptor, progesterone receptor and genes involved in cell cycle and cell proliferation remained low compared to control untreated animals. These observations indicate that aromatase inhibitors such as letrozole can be used as chemopreventive agents without effecting normal physiology in aromatase transgenic mice.

Acceleration of mammary neoplasia in aromatase transgenic mice by 7,12-dimethylbenz[a]anthracene.

Our studies using the aromatase transgenic mice model have shown that early exposure of mammary epithelium to in situ estrogen as a result of overexpression of aromatase predispose mammary tissue to preneoplastic changes. Here, we hypothesize that the preneoplastic changes induced by mammary estrogen in aromatase transgenic females may be susceptible to environmental carcinogens like 7,12-dimethylbenz[a]anthracene (DMBA), and may result in the acceleration and/or increase in the incidence of breast cancer. Results presented in this study show that tumors appeared in 25% of the mice that were treated with DMBA and all treated transgenic animals had microscopic evidence of neoplastic progression. Control non-transgenic females did not have significant changes even after treatment with DMBA. Consistent with increased neoplastic changes in DMBA-treated aromatase mice, we have seen an increase in the expression of genes involved in cell proliferation and cell cycle. We have also seen changes in the expression of oxidative stress markers and changes in estrogen-mediated growth factors. These studies indicate that more than one event is required for tumor formation, and that early estrogen exposure leading to preneoplastic changes in the mammary epithelial cells increases susceptibility to environmental carcinogens that may result in acceleration and/or an increase in the incidence of breast cancer.

EM012, a microtubule-interfering agent, inhibits the progression of multidrug-resistant human ovarian cancer both in cultured cells and in athymic nude mice.

Drug resistance, in particular multidrug resistance, is a serious problem that impedes the effectiveness of chemotherapy. Multidrug resistance results mainly from an enhanced efflux of drugs by drug pumps located on the cell membrane such as P-glycoprotein. In the study reported here we showed that EM012, a microtubule-interfering agent, is a weak substrate for P-glycoprotein and inhibited the proliferation of A2780/ADR human ovarian cancer cells, which possess multidrug resistance due to P-glycoprotein overexpression. A2780/ADR cells treated with EM012 exhibited pronounced mitotic arrest, developed large multilobed nuclei, and eventually died through the initiation of apoptosis. Intraperitoneal treatment of A2780/ADR xenograft tumors in athymic nude mice with EM012 significantly inhibited tumor progression through triggering apoptosis and conferred an apparent survival advantage. Furthermore, EM012 treatment did not cause detectable toxicity to normal tissues. These findings suggest that EM012 may serve as a novel chemotherapeutic agent for the treatment of multidrug-resistant human ovarian cancer.

Aromatase overexpression transgenic mice model: cell type specific expression and use of letrozole to abrogate mammary hyperplasia without affecting normal physiology

Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in female mammary glands and gynecomastia and testicular cancer in male aromatase transgenic mice. Both aromatase mRNA and protein are overexpressed in transgenic mammary glands and its expression is not limited to epithelial cells. However, it is more in epithelial than in stromal cells. Our results also indicate aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of the aromatase inhibitor, letrozole. Low concentration of letrozole had no effect on normal physiology as indicated by no significant change in the circulating levels of estradiol and follicle stimulating hormone as well as no change in estrogen responsive genes such as the progesterone receptor and lactoferrin in the uterine tissue. These observations indicate that the expression of aromatase in both epithelial and stromal cells can influence the complex interactions of biochemical pathways leading to mammary carcinogenesis and that the aromatase inhibitor, letrozole can be used as chemopreventive agents without affecting normal physiology.

ROLE OF MMTV INTEGRATION LOCUS CELLULAR GENES IN BREAST CANCER

Mouse mammary tumorigenesis as a result of mouse mammary tumor virus (MMTV) integrations has helped to identify a wide variety of interesting genes that play a role in mammary development and tumorigenesis. Several such genes int1/wnt1, wnt3, wnt 10B, int2/fgf3, fgf4, int3/notch and int6 have been shown to be genetically altered in naturally formed mammary tumors as a consequence of MMTV integration. Some of these genes have been well characterised and examined in in vivo breast cancer transgenic models for their potential for tumorigenesis. Overexpression of one or more of these genes have resulted in a striking proliferation of mammary gland epithelium of both female and male transgenic mice. Our own studies have demonstrated overexpression of int5/aromatase in mammary glands of virgin and postlactational females leads to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer, that may, in turn, increase the risks for developing breast cancer. Therefore, further understanding of these genes should provide new insights to their involvement and mechanism of action in breast cancer.

Anti-aromatase chemicals in red wine

Abstract: Estrogen synthesized in situ plays a more important role in breast cancer cell proliferation than does circulating estrogen. Aromatase is the enzyme that converts androgen to estrogen and is expressed at a higher level in breast cancer tissue than in surrounding noncancer tissue. A promising route of chemoprevention against breast cancer may be through the suppression of in situ estrogen formation using aromatase inhibitors. A diet high in fruits and vegetables may reduce the incidence of breast cancer, because they contain phytochemicals that can act as aromatase inhibitors. In our previous studies, we found that grapes and wine contain potent phytochemicals that can inhibit aromatase. We show that red wine was more effective than white wine in suppressing aromatase activity. Interestingly, our results from white wine studies suggest a weak inductive effect of alcohol on aromatase activity. On the other hand, the potent effect of anti‐aromatase chemicals in red wine overcomes the weak inductive effect of alcohol in wine. Several purification procedures were performed on whole red wine to separate active aromatase inhibitors from non‐active compounds. These techniques included liquid‐liquid extraction, silica gel chromatography, various solid phase extraction (SPE) columns, and high performance liquid chromatography. An active Pinot Noir red wine SPE C18 column fraction (20% acetonitrile:water) was more effective than complete Pinot Noir wine in suppressing aromatase assay. This red wine extract was further analyzed in a transgenic mouse model in which aromatase was over‐expressed in mammary tissue. Our gavaged red wine extract completely abrogated aromatase‐induced hyperplasia and other neoplastic changes in mammary tissue. These results suggest that red wine or red wine extract may be a chemopreventive diet supplement for postmenopausal women who have a high risk of breast cancer. Further research is underway to purify and characterize the active compounds in red wine that are responsible for the inhibition of aromatase.