Publications by authors named "Alicia Goyeneche"

24 Publications

  • Page 1 of 1

Acetylsalicylic Acid Exerts Potent Antitumor and Antiangiogenic Effects in Cutaneous and Uveal Melanoma Cell Lines.

Ocul Oncol Pathol 2020 Dec 4;6(6):442-455. Epub 2020 Nov 4.

Henry C. Witelson Ocular Pathology Laboratory, McGill University, Montreal, Québec, Canada.

Introduction: Acetylsalicylic acid (ASA) has been investigated for a potential anticancer role in several cancers, such as colorectal, ovarian, and endometrial cancer. Moreover, ASA has been shown to abrogate various processes that contribute to tumor growth and progression.

Objective: The aim of this study was to evaluate the effects of ASA on cutaneous melanoma (CM) and uveal melanoma (UM).

Methods: Human CM and UM cells were treated with 5 mM ASA and assessed for changes in cellular functions. Antiangiogenic effects of ASA were determined using an ELISA-based assay for 10 proangiogenic cytokines, and then validated by Western blot. Finally, proteomic analysis of ASA-treated cells was performed to elucidate the changes that may be responsible for ASA-mediated effects in melanoma cells.

Results: Treatment with ASA significantly inhibited the proliferation, invasion, and migration capabilities, and caused a significant decrease in angiogenin and PIGF secretion in both CM and UM. Mass spectrometry revealed 179 protein changes associated with ASA in the CM and UM cell lines.

Conclusions: These results suggest that ASA may be effective as an adjuvant therapy in metastatic CM and UM. Future studies are needed to determine the regulating targets that are responsible for the antitumor effects of ASA.
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http://dx.doi.org/10.1159/000510582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772880PMC
December 2020

Prenatal testosterone exposure induces insulin resistance, uterine oxidative stress and pro-inflammatory status in rats.

Mol Cell Endocrinol 2021 01 23;519:111045. Epub 2020 Oct 23.

Laboratorio de Fisio-Patología Ovárica, Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Medicina, Universidad de Buenos Aires (UBA), Argentina.

Prenatal androgen excess is considered one of the main causes of the development of polycystic ovary syndrome. In this study, we investigated the effect of prenatal hyperandrogenization (PH) on the physiology of the adult uterine tissue using a murine model of fetal programming caused by androgen excess in adult female rats. Pregnant rats were hyperandrogenized with testosterone and female offspring were studied when adult. Our results showed that PH leads to hyperglycemia and hyperinsulinemia. Consequently, PH developed insulin resistance and a systemic inflammatory state reflected by increased C-reactive protein. In the uterine tissue, levels of PPAR gamma-an important metabolic sensor in the endometrium-were found to be impaired. Moreover, PH induced a pro-inflammatory and an unbalanced oxidative state in the uterus reflected by increased COX-2, lipid peroxidation, and NF-κB. In summary, our results revealed that PH leads to a compromised metabolic state likely consequence of fetal reprogramming.
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http://dx.doi.org/10.1016/j.mce.2020.111045DOI Listing
January 2021

The Capacity of High-Grade Serous Ovarian Cancer Cells to Form Multicellular Structures Spontaneously along Disease Progression Correlates with Their Orthotopic Tumorigenicity in Immunosuppressed Mice.

Cancers (Basel) 2020 Mar 16;12(3). Epub 2020 Mar 16.

Experimental Pathology Unit, Department of Pathology, Faculty of Medicine, McGill University, Montreal, QC H3A 2B4, Canada.

Many studies have examined the biology, genetics, and chemotherapeutic response of ovarian cancer's solid component; its liquid facet, however, remains critically underinvestigated. Floating within peritoneal effusions known as ascites, ovarian cancer cells form multicellular structures, creating a cancer niche in suspension. This study explores the pathobiology of spontaneously formed, multicellular, ovarian cancer structures derived from serous ovarian cancer cells isolated along disease evolution. It also tests their capacity to cause peritoneal disease in immunosuppressed mice. Results stem from an analysis of cell lines representing the most frequently diagnosed ovarian cancer histotype (high-grade serous ovarian cancer), derived from ascites of the same patient at distinct stages of disease progression. When cultured under adherent conditions, in addition to forming cellular monolayers, the cultures developed areas in which the cells grew upwards, forming densely packed multilayers that ultimately detached from the bottom of the plates and lived as free-floating, multicellular structures. The capacity to form foci and to develop multicellular structures was proportional to disease progression at the time of ascites extraction. Self-assembled in culture, these structures varied in size, were either compact or hollow, irregular, or spheroidal, and exhibited replicative capacity and an epithelial nature. Furthermore, they fully recreated ovarian cancer disease in immunosuppressed mice: accumulation of malignant ascites and pleural effusions; formation of discrete, solid, macroscopic, peritoneal tumors; and microscopic growths in abdominal organs. They also reproduced the histopathological features characteristic of high-grade serous ovarian cancer when diagnosed in patients. The following results encourage the development of therapeutic interventions to interrupt the formation and/or survival of multicellular structures that constitute a floating niche in the peritoneal fluid, which in turn halts disease progression and prevents recurrence.
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http://dx.doi.org/10.3390/cancers12030699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140084PMC
March 2020

Prenatally androgenized female rats develop uterine hyperplasia when adult.

Mol Cell Endocrinol 2020 01 4;499:110610. Epub 2019 Oct 4.

Laboratorio de Fisio-Patología Ovárica, Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Medicina, Universidad de Buenos Aires (UBA), Argentina.

Prenatal hyperandrogenization (PH) is hypothesized as one of the main factors contributing to the development of polycystic ovary syndrome (PCOS). In this study, we aimed to investigate the impact of prenatal exposure to androgen excess on the uterus when animals reach their adulthood. We found that PH altered the morphology of the uteri that show a hyperplastic morphology with increased total uterine thickness as well as luminal epithelium thickness, with both enhanced and altered distribution of glands as compared with controls. Morphological alterations were associated with an unbalanced homeostasis as assessed by the expression of regulators of cell cycle progression and cell death dynamics. PH also causes disturbances in the cell cycle of the uterine tissue and dysregulates cell death and survival pathways leading to the development of uterine hyperplasia. These findings suggest that PH may have a deleterious effect on the uterus.
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http://dx.doi.org/10.1016/j.mce.2019.110610DOI Listing
January 2020

Beta-blockers exert potent anti-tumor effects in cutaneous and uveal melanoma.

Cancer Med 2019 12 7;8(17):7265-7277. Epub 2019 Oct 7.

Cancer Research Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.

Background: Melanoma is a life-threatening group of cancers mainly affecting the skin (cutaneous melanoma, CM) and the eyes (uveal melanoma, UM). Nearly half of patients with UM develop liver metastases regardless of the primary treatment. For this reason, adjuvant therapy to prevent disease progression is essential to improve survival of patients with melanoma. Beta-adrenoceptors (β-AR) have emerged as novel targets to inhibit tumor growth and dissemination in CM, but have not been investigated in UM.

Methods: The aim of this study was to comprehensively evaluate the effects of a non-selective β-blocker in UM and CM. Propranolol was tested on four UM and two CM cell lines to determine the effects of this beta-blocker. The expression of β-AR in UM was assessed in enucleated eyes of 36 patients.

Results: The results showed that propranolol exerts potent anti-proliferative effects, attenuates migration, reduces VEGF and induces cell cycle arrest and apoptosis in both UM and CM in a dose-dependent manner. Furthermore, levels of cell-free DNA released from the cells correlated to propranolol treatment and may be an indicator of treatment response. Finally, immunohistochemical analysis revealed the expression of β1 and β2 adrenoceptors in all UM patients, with higher expression seen in the more aggressive epithelioid versus less aggressive spindle cells.

Conclusions: Collectively our data suggest that a nonselective beta-blocker may be effective against melanoma. For the first time, we show potent anti-tumor effects in UM cells following propranolol administration and expression of β1 and β2 adrenoceptors in patient tissue.
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http://dx.doi.org/10.1002/cam4.2594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885887PMC
December 2019

Advanced assessment of migration and invasion of cancer cells in response to mifepristone therapy using double fluorescence cytochemical labeling.

BMC Cancer 2019 Apr 24;19(1):376. Epub 2019 Apr 24.

Experimental Pathology Unit, Department of Pathology, Faculty of Medicine, McGill University, 3775 University Street, Montreal, Qc, H3A 2B4, Canada.

Background: Previous work in our laboratory demonstrated that antiprogestin mifepristone impairs the growth and adhesion of highly metastatic cancer cells, and causes changes in their cellular morphology. In this study, we further assess the anti-metastatic properties of mifepristone, by studying whether cytostatic doses of the drug can inhibit the migration and invasion of various cancer cell lines using a double fluorescence cytochemical labeling approach.

Methods: Cell lines representing cancers of the ovary (SKOV-3), breast (MDA-MB-231), glia (U87MG), or prostate (LNCaP) were treated with cytostatic concentrations of mifepristone. Wound healing and Boyden chamber assays were utilized to study cellular migration. To study cellular invasion, the Boyden chamber assay was prepared by adding a layer of extracellular matrix over the polycarbonate membrane. We enhanced the assays with the addition of double fluorescence cytochemical staining for fibrillar actin (F-actin) and DNA to observe the patterns of cytoskeletal distribution and nuclear positioning while cells migrate and invade.

Results: When exposed to cytostatic concentrations of mifepristone, all cancer cells lines demonstrated a decrease in both migration and invasion capacities measured using standard approaches. Double fluorescence cytochemical labeling validated that mifepristone-treated cancer cells exhibit reduced migration and invasion, and allowed to unveil a distinct migration pattern among the different cell lines, different arrays of nuclear localization during migration, and apparent redistribution of F-actin to the nucleus.

Conclusion: This study reports that antiprogestin mifepristone inhibits migration and invasion of highly metastatic cancer cell lines, and that double fluorescence cytochemical labeling increases the value of well-known approaches to study cell movement.
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http://dx.doi.org/10.1186/s12885-019-5587-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480622PMC
April 2019

High-Grade Serous Ovarian Cancer: Basic Sciences, Clinical and Therapeutic Standpoints.

Int J Mol Sci 2019 Feb 22;20(4). Epub 2019 Feb 22.

Experimental Pathology Unit, Department of Pathology, McGill University, Montreal, QC H3A 2B4, Canada.

Among a litany of malignancies affecting the female reproductive tract, that of the ovary is the most frequently fatal. Moreover, while the steady pace of scientific discovery has fuelled recent ameliorations in the outcomes of many other cancers, the rates of mortality for ovarian cancer have been stagnant since around 1980. Yet despite the grim outlook, progress is being made towards better understanding the fundamental biology of this disease and how its biology in turn influences clinical behaviour. It has long been evident that ovarian cancer is not a unitary disease but rather a multiplicity of distinct malignancies that share a common anatomical site upon presentation. Of these, the high-grade serous subtype predominates in the clinical setting and is responsible for a disproportionate share of the fatalities from all forms of ovarian cancer. This review aims to provide a detailed overview of the clinical-pathological features of ovarian cancer with a particular focus on the high-grade serous subtype. Along with a description of the relevant clinical aspects of this disease, including novel trends in treatment strategies, this text will inform the reader of recent updates to the scientific literature regarding the origin, aetiology and molecular-genetic basis of high-grade serous ovarian cancer (HGSOC).
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http://dx.doi.org/10.3390/ijms20040952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412907PMC
February 2019

Mifepristone inhibits non-small cell lung carcinoma cellular escape from DNA damaging cisplatin.

Cancer Cell Int 2018 15;18:185. Epub 2018 Nov 15.

1Experimental Pathology Unit, Department of Pathology, Faculty of Medicine, McGill University, 3775 University Street, Montreal, QC H3A 2B4 Canada.

Background: Lung cancer is the leading cause of cancer deaths in the world. The major histopathological subtype of lung cancer is non-small cell lung cancer (NSCLC). Platinum-based therapy is the standard of care for patients with advanced stage NSCLC. However, even with treatment, most patients will die of this disease within 5 years and most of these deaths are due to recurrence. One strategy to inhibit recurrence is to use cytostatic compounds following courses of lethal chemotherapy. We have shown in various cancer cell types that mifepristone (MF), an anti-progestin/anti-glucocorticoid, is a powerful cytostatic anti-cancer agent. Thus, in this work we tested the hypothesis that MF should be efficacious in inducing cytostasis and preventing repopulation of NSCLC following cisplatin (CDDP) therapy.

Methods: We established an in vitro approach wherein human NSCLC cells with different genetic backgrounds and sensitivities to CDDP (A549 and H23) were exposed to rounds of lethal concentrations of CDDP for 1 h followed or not by MF monotherapy. Every 2 days, cell number, cell viability, and colony-forming ability of viable cells were studied.

Results: CDDP killed the majority of cells, yet there were remnant cells escaping CDDP lethality and repopulating the culture, as evidenced by the improved clonogenic survival of viable cells. In contrast, when cells exposed to CDDP where further treated with MF following CDDP removal, their number and clonogenic capacity were reduced drastically.

Conclusion: This study reports that there is repopulation of NSCLC cells following a lethal concentration of CDDP monotherapy, that NSCLC cells are sensitive to the growth inhibition properties of MF, and that MF abrogates the repopulation of NSCLC cells following CDDP therapy. Our study supports further evaluating MF as an adjuvant therapy for NSCLC.
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http://dx.doi.org/10.1186/s12935-018-0683-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238342PMC
November 2018

Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors.

Mol Oncol 2016 08 17;10(7):1099-117. Epub 2016 May 17.

Division of Basic Biomedical Sciences, Sanford School of Medicine of The University of South Dakota, Vermillion, SD 57069, USA; Department of Pathology, Faculty of Medicine, McGill University, Montreal, QC H3A 2B4, Canada. Electronic address:

The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic-lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors.
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http://dx.doi.org/10.1016/j.molonc.2016.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240778PMC
August 2016

Long-term primary culture of a clear cell ovarian carcinoma reveals an epithelial-mesenchymal cooperative interaction.

Cancer Cell Int 2015 24;15:88. Epub 2015 Sep 24.

Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, Vermillion, SD USA ; Department of Obstetrics and Gynecology, Sanford School of Medicine, The University of South Dakota, Sioux Falls, SD USA ; Department of Pathology, Faculty of Medicine, McGill University, 3775 University Street, Montreal, QC H3A 2B4 Canada.

Background: We studied a primary culture developed from a biopsy of a clear cell carcinoma of the ovary (O-CCC) by (a) assessing its capacity to retain in vitro pathological features of the tumor of origin; (b) characterizing the main cells released from the complex mass without forced purification of any particular cellular entity; and (c) investigating its long-term proliferative capacity.

Methods: A primary cell culture was developed from a pelvic mass diagnosed as an O-CCC. The morphological analysis of the cell culture was carried out by phase contrast microscopy. Markers of epithelial, mesenchymal, and tumor initiating cells were evaluated by immunocytochemistry. Cell proliferation was studied by detection of bromodeoxyuridine (BrdU) incorporated into newly synthesized DNA. As a biomarker of O-CCC, we assessed the expression of hepatocyte nuclear factor (HNF) 1β.

Results: We show that cells with epithelial morphological features express E-cadherin and expand with time in culture, a fact that the incorporation of BrdU confirms. Cells with mesenchymal-like characteristics that express the mesenchymal marker vimentin, however, allocate to the edges of the epithelial compartment. Moreover, we found that some cells with epithelial features also expressed vimentin. At the beginning of incubation, over 60 % of primary cells expressed the O-CCC marker HNF1β; such percentage declined upon passaging. We show that epithelial not mesenchymal cells undergo DNA replication, and that few cells in both epithelial and mesenchymal compartments express the stem-like tumor antigen CD133.

Conclusions: We provide proof-of-principle that cells separated in bulk from a biopsy of an O-CCC can be maintained in culture for several months, and that two consistent cellular compartments-one epithelial that retains the O-CCC marker HNF1β, and another mesenchymal-persist, and seem to have a cooperative interaction leading to the multiplication of epithelial cells within a mesenchymal cellular environment.
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http://dx.doi.org/10.1186/s12935-015-0243-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4581082PMC
September 2015

Antiprogestins in gynecological diseases.

Reproduction 2015 Jan 24;149(1):R15-33. Epub 2014 Sep 24.

Division of Basic Biomedical SciencesSanford School of Medicine, The University of South Dakota, Vermillion, South Dakota 57069, USA

Antiprogestins constitute a group of compounds, developed since the early 1980s, that bind progesterone receptors with different affinities. The first clinical uses for antiprogestins were in reproductive medicine, e.g., menstrual regulation, emergency contraception, and termination of early pregnancies. These initial applications, however, belied the capacity for these compounds to interfere with cell growth. Within the context of gynecological diseases, antiprogestins can block the growth of and kill gynecological-related cancer cells, such as those originating in the breast, ovary, endometrium, and cervix. They can also interrupt the excessive growth of cells giving rise to benign gynecological diseases such as endometriosis and leiomyomata (uterine fibroids). In this article, we present a review of the literature providing support for the antigrowth activity that antiprogestins impose on cells in various gynecological diseases. We also provide a summary of the cellular and molecular mechanisms reported for these compounds that lead to cell growth inhibition and death. The preclinical knowledge gained during the past few years provides robust evidence to encourage the use of antiprogestins in order to alleviate the burden of gynecological diseases, either as monotherapies or as adjuvants of other therapies with the perspective of allowing for long-term treatments with tolerable side effects. The key to the clinical success of antiprogestins in this field probably lies in selecting those patients who will benefit from this therapy. This can be achieved by defining the genetic makeup required - within each particular gynecological disease - for attaining an objective response to antiprogestin-driven growth inhibition therapy.Free Spanish abstractA Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/149/1/15/suppl/DC1.
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http://dx.doi.org/10.1530/REP-14-0416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247796PMC
January 2015

Resistance to cisplatin and paclitaxel does not affect the sensitivity of human ovarian cancer cells to antiprogestin-induced cytotoxicity.

J Ovarian Res 2014 27;7:45. Epub 2014 Apr 27.

Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South, Dakota, 414 East Clark Street, Vermillion, SD 57069, USA.

Background: Antiprogestin compounds have been shown to be effective in blocking the growth of ovarian cancer cells of different genetic backgrounds. Herein we studied the anti-ovarian cancer effect of a series of antiprogestins sharing the chemical backbone of the most characterized antiprogestin, mifepristone, but with unique modifications in position C-17 of the steroid ring. We assessed the effect of mifepristone-like antiprogestins on the growth of ovarian cancer cells sensitive to the standard combination therapy cisplatin-paclitaxel or made double-resistant upon six cycles of pulse-selection with the drugs used at clinically relevant concentrations and exposure times.

Methods: IGROV-1 and SKOV-3 cells were pulsed with 20 μM cisplatin for 1 h followed by 100 nM paclitaxel for 3 h once a week for six weeks. The cells that did not die and repopulate the culture after the chemotherapies were termed Platinum-Taxane-EScape cells (PTES). Parental cells were compared against their PTES derivatives in their responses to further platinum-taxane treatments. Moreover, both ovarian cancer cells and their PTES siblings were exposed to escalating doses of the various antiprogestin derivatives. We assessed cell growth, viability and sub-G1 DNA content using microcapillary cytometry. Cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1) and cleavage of downstream caspase-3 substrate PARP were used to assess whether cell fate, as a consequence of treatment, was limited to cytostasis or progressed to lethality.

Results: Cells subjected to six pulse-selection cycles of cisplatin-paclitaxel gave rise to sibling derivatives that displayed ~2-7 fold reduction in their sensitivities to further chemotherapy. However, regardless of the sensitivity the cells developed to the combination cisplatin-paclitaxel, they displayed similar sensitivity to the antiprogestins, which blocked their growth in a dose-related manner, with lower concentrations causing cytostasis, and higher concentrations causing lethality.

Conclusions: Antiprogestins carrying a backbone similar to mifepristone are cytotoxic to ovarian cancer cells in a manner that does not depend on the sensitivity the cells have to the standard ovarian cancer chemotherapeutics, cisplatin and paclitaxel. Thus, antiprogestin therapy could be used to treat ovarian cancer cells showing resistance to both platinum and taxanes.
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http://dx.doi.org/10.1186/1757-2215-7-45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007005PMC
October 2015

Cytostasis and morphological changes induced by mifepristone in human metastatic cancer cells involve cytoskeletal filamentous actin reorganization and impairment of cell adhesion dynamics.

BMC Cancer 2013 Jan 26;13:35. Epub 2013 Jan 26.

Division of Basic Biomedical Science, Sanford School of Medicine of The University of South Dakota, 414 East Clark Street, Vermillion, SD 57069, USA.

Background: Changes in cell shape and plasticity in cytoskeletal dynamics are critically involved in cell adhesion, migration, invasion and the overall process of metastasis. Previous work in our laboratory demonstrated that the synthetic steroid mifepristone inhibited the growth of highly metastatic cancer cells, while simultaneously causing striking changes in cellular morphology. Here we assessed whether such morphological alterations developed in response to cytostatic concentrations of mifepristone are reversible or permanent, involve rearrangement of cytoskeletal proteins, and/or affect the adhesive capacity of the cells.

Methods: Cancer cell lines of the ovary (SKOV-3), breast (MDA-MB-231), prostate (LNCaP), and nervous system (U87MG) were exposed to cytostatic concentrations of mifepristone and studied by phase-contrast microscopy. The transient or permanent nature of the cytostasis and morphological changes caused by mifepristone was assessed, as well as the rearrangement of cytoskeletal proteins. De-adhesion and adhesion assays were utilized to determine if mifepristone-arrested and morphologically dysregulated cells had abnormal de-adhesion/adhesion dynamics when compared to vehicle-treated controls.

Results: Mifepristone-treated cells displayed a long, thin, spindle-like shape with boundaries resembling those of loosely adhered cells. Growth arrest and morphology changes caused by mifepristone were reversible in SKOV-3, MDA-MB-231 and U87MG, but not in LNCaP cells that instead became senescent. All cancer cell types exposed to mifepristone displayed greatly increased actin ruffling in association with accelerated de-adhesion from the culture plate, and delayed adhesion capacity to various extracellular matrix components.

Conclusions: Cytostatic concentrations of mifepristone induced alterations in the cellular structure of a panel of aggressive, highly metastatic cancer cells of different tissues of origin. Such changes were associated with re-distribution of actin fibers that mainly form non-adhesive membrane ruffles, leading to dysregulated cellular adhesion capacity.
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http://dx.doi.org/10.1186/1471-2407-13-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562154PMC
January 2013

Mifepristone prevents repopulation of ovarian cancer cells escaping cisplatin-paclitaxel therapy.

BMC Cancer 2012 Jun 22;12:200. Epub 2012 Jun 22.

Division of Basic Biomedical Sciences, Sanford School of Medicine of The University of South Dakota, 414 East Clark Street, Vermillion, SD, USA.

Background: Advanced ovarian cancer is treated with cytoreductive surgery and combination platinum- and taxane-based chemotherapy. Although most patients have acute clinical response to this strategy, the disease ultimately recurs. In this work we questioned whether the synthetic steroid mifepristone, which as monotherapy inhibits the growth of ovarian cancer cells, is capable of preventing repopulation of ovarian cancer cells if given after a round of lethal cisplatin-paclitaxel combination treatment.

Methods: We established an in vitro approach wherein ovarian cancer cells with various sensitivities to cisplatin or paclitaxel were exposed to a round of lethal doses of cisplatin for 1 h plus paclitaxel for 3 h. Thereafter, cells were maintained in media with or without mifepristone, and short- and long-term cytotoxicity was assessed.

Results: Four days after treatment the lethality of cisplatin-paclitaxel was evidenced by reduced number of cells, increased hypodiploid DNA content, morphological features of apoptosis, DNA fragmentation, and cleavage of caspase-3, and of its downstream substrate PARP. Short-term presence of mifepristone either enhanced or did not modify such acute lethality. Seven days after receiving cisplatin-paclitaxel, cultures showed signs of relapse with escaping colonies that repopulated the plate in a time-dependent manner. Conversely, cultures exposed to cisplatin-paclitaxel followed by mifepristone not only did not display signs of repopulation following initial chemotherapy, but they also had their clonogenic capacity drastically reduced when compared to cells repopulating after cisplatin-paclitaxel.

Conclusions: Cytostatic concentrations of mifepristone after exposure to lethal doses of cisplatin and paclitaxel in combination blocks repopulation of remnant cells surviving and escaping the cytotoxic drugs.
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http://dx.doi.org/10.1186/1471-2407-12-200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381704PMC
June 2012

Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression.

BMC Cancer 2011 May 27;11:207. Epub 2011 May 27.

Division of Basic Biomedical Sciences, Sanford School of Medicine of The University of South Dakota, Vermillion, SD, USA.

Background: Mifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR.

Methods: Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1.

Results: MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR.

Conclusions: Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased hypodiploid DNA content. Contrary to common opinion, growth inhibition of cancer cells by antiprogestin MF is not dependent upon expression of classical, nuclear PR.
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http://dx.doi.org/10.1186/1471-2407-11-207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125282PMC
May 2011

Growth inhibition induced by antiprogestins RU-38486, ORG-31710, and CDB-2914 in ovarian cancer cells involves inhibition of cyclin dependent kinase 2.

Invest New Drugs 2012 Jun 22;30(3):967-80. Epub 2011 Mar 22.

Division of Basic Biomedical Sciences, Sanford School of Medicine of The University of South Dakota, Vermillion, SD, USA.

Antiprogestins have been largely utilized in reproductive medicine, yet their repositioning for oncologic use is rapidly emerging. In this study we investigated the molecular mediators of the anti-ovarian cancer activity of the structurally related antiprogestins RU-38486, ORG-31710 and CDB-2914. We studied the responses of wt p53 OV2008 and p53 null SK-OV-3 cells to varying doses of RU-38486, ORG-31710 and CDB-2914. The steroids inhibited the growth of both cell lines with a potency of RU-38486 > ORG-31710 > CDB-2914, and were cytostatic at lower doses but lethal at higher concentrations. Antiprogestin-induced lethality associated with morphological features of apoptosis, hypodiploid DNA content, DNA fragmentation, and cleavage of executer caspase substrate PARP. Cell death ensued despite RU-38486 caused transient up-regulation of anti-apoptotic Bcl-2, ORG-31710 induced transient up-regulation of inhibitor of apoptosis XIAP, and CDB-2914 up-regulated both XIAP and Bcl-2. The antiprogestins induced accumulation of Cdk inhibitors p21(cip1) and p27(kip1) and increased association of p21(cip1) and p27(kip1) with Cdk-2. They also promoted nuclear localization of p21(cip1) and p27(kip1), reduced the nuclear abundances of Cdk-2 and cyclin E, and blocked the activity of Cdk-2 in both nucleus and cytoplasm. The cytotoxic potency of the antiprogestins correlated with the magnitude of the inhibition of Cdk-2 activity, ranging from G1 cell cycle arrest towards cell death. Our results suggest that, as a consequence of their cytostatic and lethal effects, antiprogestin steroids of well-known contraceptive properties emerge as attractive new agents to be repositioned for ovarian cancer therapeutics.
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http://dx.doi.org/10.1007/s10637-011-9655-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348464PMC
June 2012

Resistance to cisplatin does not affect sensitivity of human ovarian cancer cell lines to mifepristone cytotoxicity.

Cancer Cell Int 2009 Feb 17;9. Epub 2009 Feb 17.

Division of Basic Biomedical Sciences, Sanford School of Medicine of The University of South Dakota, Vermillion, SD 57069, USA.

Background: The prototypical antiprogestin mifepristone exhibits potent growth inhibition activity towards ovarian cancer cells in vitro and in vivo. The aim of this research was to establish whether mifepristone is capable of inhibiting cell proliferation and inducing apoptotic cell death regardless of the degree of sensitivity ovarian cancer cells exhibit to cisplatin.

Methods: OV2008, OV2008/C13, A2780, A2780/CP70, Caov-3, and SK-OV-3 cell lines exhibiting a range of sensitivities to cisplatin were used. Growth inhibition, cell viability, and sub-diploid DNA content in response to treatment with escalating doses of either mifepristone or cisplatin were assessed by microcapillary cytometry. Apoptotic cell death was evaluated by measuring genomic DNA fragmentation and cleavage of caspase-3 and poly (ADP ribose) polymerase (PARP).

Results: The sensitivities to cisplatin manifested by the cell lines were OV2008 > A2780 > Caov-3 > SK-OV-3 > OV2008/C13 > A2780/CP70. Mifepristone inhibited the growth of all six cell lines in a dose-related manner with IC50s ranging from ~6-12 muM and without significant correlation with the relative sensitivities the cells displayed for cisplatin. Moreover, at the highest concentration studied, mifepristone triggered apoptotic death in all six cell lines as evidenced by the increase in sub-diploid fragmented DNA content and cleavage of caspase-3 and of its downstream substrate PARP.

Conclusion: Mifepristone is cytotoxic towards ovarian cancer cells independent of the sensitivity exhibited by the cells to cisplatin, displaying cytostatic effects at lower concentrations and lethal effects at higher concentrations. Mifepristone monotherapy emerges as a valuable therapeutic alternative for platinum-resistant ovarian cancers.
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http://dx.doi.org/10.1186/1475-2867-9-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661041PMC
February 2009

Mifepristone abrogates repopulation of ovarian cancer cells in between courses of cisplatin treatment.

Int J Oncol 2009 Mar;34(3):743-55

Sanford School of Medicine, The University of South Dakota, Vermillion, SD 57069, USA.

Repopulation of cancer cells escaping lethal chemotherapy is a critical factor hindering treatment success. One strategy to inhibit tumor cell repopulation is the use of cytostatic compounds between courses of lethal chemotherapy. In this study, we tested the hypothesis that mifepristone (MF), a steroid compound with demonstrated growth inhibition activity in ovarian cancer, should be efficacious in inducing cytostasis and preventing repopulation of ovarian cancer cells if given among rounds of cisplatin (CDDP) treatment. We established an in vitro approach wherein ovarian cancer cells with high (OV2008) or low (SK-OV-3) sensitivity to CDDP were exposed to 3 (OV2008) or 2 (SK-OV-3) rounds of lethal doses of CDDP for 1 h, 12 (OV2008) or 24 (SK-OV-3) days apart. Every 4 or 8 days cell number, cell viability, cell cycle traverse, and colony-forming capacity of viable cells was analyzed. Although CDDP killed the vast majority of cells, there were remnant cells escaping CDDP lethality and repopulating the culture, as evidenced by increased cell number, improved clonogenic capacity of viable cells, and normalization of DNA synthesis. Conversely, when cells were exposed to CDDP for 1 h, and 5, 10 or 20 microM MF was present in the culture medium after CDDP removal, the number, clonogenic capacity, and DNA synthesis ability of the cells were reduced in a dose-dependent manner. The blockage by MF of post-CDDP repopulation was accompanied by a remarkable increase in the percentage of cells expressing the cell death marker cleaved poly(ADP-ribose) polymerase and the mitotic marker phospho-histone H3, suggesting that MF also potentiated CDDP lethality and that the cells likely die due to mitotic failure. In summary, this is the first study reporting that presence of cytostatic concentrations of MF after courses of lethal doses of CDDP prevents repopulation of remnant ovarian cancer cells surviving CDDP treatment.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2690705PMC
http://dx.doi.org/10.3892/ijo_00000200DOI Listing
March 2009

Mifepristone inhibits ovarian cancer cell growth in vitro and in vivo.

Clin Cancer Res 2007 Jun;13(11):3370-9

Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, Vermillion, South Dakota 57069, USA.

Purpose: These studies were designed to determine whether the synthetic steroid mifepristone inhibits ovarian cancer growth in vitro and in vivo and the molecular mechanisms involved.

Experimental Design: The effect of mifepristone on ovarian cancer cell growth in vitro was studied in ovarian cancer cell lines of different genetic backgrounds (SK-OV-3, Caov-3, OV2008, and IGROV-1). In addition, the growth inhibition capacity of mifepristone on ovarian carcinoma xenografts was tested in nude mice.

Results: Mifepristone inhibited ovarian cancer cell proliferation in a dose- and time-dependent manner. The cytostatic effect of mifepristone was confirmed in a clonogenic survival assay and was not linked to loss of viability. Mifepristone blocked DNA synthesis, arrested the cell cycle at the G(1)-S transition, up-regulated cyclin-dependent kinase (cdk) inhibitors p21(cip1)and p27(kip1), down-regulated transcription factor E2F1, decreased expression of the E2F1-regulated genes cdk1 (cdc2) and cyclin A, and modestly decreased cdk2 and cyclin E levels. The abrupt arrest in cell growth induced by mifepristone correlated with reduced cdk2 activity, increased association of cdk2 with p21(cip1) and p27(kip1), increased nuclear localization of the cdk inhibitors, and reduced nuclear abundance of cdk2 and cyclin E. In vivo, mifepristone significantly delayed the growth of ovarian carcinoma xenografts in a dose-dependent manner and without apparent toxic effects for the animals.

Conclusions: These preclinical studies show that mifepristone is effective as a single agent in vitro and in vivo, inhibiting the growth of human epithelial ovarian cancer cells. Mifepristone markedly reduces cdk2 activity likely due to increased association of cdk2 with the cdk inhibitors p21(cip1) and p27(kip1) and reduced nuclear cdk2/cyclin E complex availability. Acting as a cytostatic agent, mifepristone promises to be of translational significance in ovarian cancer therapeutics.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-0164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2505183PMC
June 2007

Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis.

Reproduction 2006 Jan;131(1):103-11

Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 East Clark Street, Vermillion, 57069, USA.

The corpus luteum is a transient endocrine gland specializing in the production of progesterone. The regression of the corpus luteum involves an abrupt decline in its capacity for producing progesterone followed by its structural involution, which is associated with apoptosis of the luteal cells. An in vitro experimental approach is needed to study the molecular mechanisms underlying hormonal regulation of luteal cell death under defined experimental conditions. In this study, we investigated simian virus-40-transformed luteal cells to determine whether they can be driven to apoptosis and, if so, to define the intracellular pathway involved. Luteal cells were cultured in the presence or absence of fetal bovine serum for 24 or 48 h. Under serum starvation conditions, the luteal cells underwent growth arrest accompanied by cell death as evaluated by dye exclusion, and confirmed by two-color fluorescence cell viability/cytotoxicity assay. We next studied whether serum starvation-induced death of luteal cells occurred by apoptosis. Morphologic features of apoptosis were observed in cells stained with hematoxylin after being subjected to serum starvation for 48 h. The apoptotic nature was further confirmed by in situ 3'-end labeling and fragmentation of genomic DNA. Apoptosis of serum-deprived luteal cells was dependent upon caspase activation. Serum starvation induced cleavage of poly (ADP-ribose) polymerase (PARP), suggesting that caspase-3 had been activated under the stress of withdrawal of growth factors. This was confirmed by cleavage of full-length procaspase-3. Finally, the fact that serum starvation promoted the cleavage of full-length procaspase-9 and the decrease in the expression of endogenous Bid, a BH-3-only proapoptotic protein of the Bcl-2 family, indicates that the intrinsic (i.e., mitochondrial) pathway of apoptosis was activated. In summary, we have characterized an in vitro experimental model of luteal cell death that can be utilized to evaluate the role of hormones in apoptosis of luteal cells under defined culture conditions, and to study the mechanism of luteal regression.
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http://dx.doi.org/10.1530/rep.1.00751DOI Listing
January 2006

Exogenous estradiol enhances apoptosis in regressing post-partum rat corpora lutea possibly mediated by prolactin.

Reprod Biol Endocrinol 2005 Aug 30;3:40. Epub 2005 Aug 30.

Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, South Dakota 57069, USA.

Background: In pregnant rats, structural luteal regression takes place after parturition and is associated with cell death by apoptosis. We have recently shown that the hormonal environment is responsible for the fate of the corpora lutea (CL). Changing the levels of circulating hormones in post-partum rats, either by injecting androgen, progesterone, or by allowing dams to suckle, was coupled with a delay in the onset of apoptosis in the CL. The objectives of the present investigation were: i) to examine the effect of exogenous estradiol on apoptosis of the rat CL during post-partum luteal regression; and ii) to evaluate the post-partum luteal expression of the estrogen receptor (ER) genes.

Methods: In a first experiment, rats after parturition were separated from their pups and injected daily with vehicle or estradiol benzoate for 4 days. On day 4 post-partum, animals were sacrificed, blood samples were taken to determine serum concentrations of hormones, and the ovaries were isolated to study apoptosis in situ. In a second experiment, non-lactating rats after parturition received vehicle, estradiol benzoate or estradiol benzoate plus bromoergocryptine for 4 days, and their CL were isolated and used to study apoptosis ex vivo. In a third experiment, we obtained CL from rats on day 15 of pregnancy and from non-lactating rats on day 4 post-partum, and studied the expression of the messenger RNAs (mRNAs) encoding the ERalpha and ERbeta genes.

Results: Exogenous administration of estradiol benzoate induced an increase in the number of apoptotic cells within the CL on day 4 post-partum when compared with animals receiving vehicle alone. Animals treated with the estrogen had higher serum prolactin and progesterone concentrations, with no changes in serum androstenedione. Administration of bromoergocryptine blocked the increase in serum prolactin and progesterone concentrations, and DNA fragmentation induced by the estrogen treatment. ERalpha and ERbeta mRNAs were expressed in CL of day 4 post-partum animals at levels similar to those found in CL of day 15 pregnant animals.

Conclusion: We have established that estradiol accelerates apoptosis in the CL during post-partum luteal regression through a mechanism that possibly involves the secretion of pituitary prolactin. We have also shown that the post-partum rat CL express ERalpha and ERbeta mRNAs suggesting that they can be targeted by estrogen.
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http://dx.doi.org/10.1186/1477-7827-3-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1236958PMC
August 2005

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro.

Biol Reprod 2003 Jun 5;68(6):2322-30. Epub 2003 Feb 5.

Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, South Dakota 57069, USA.

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.
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http://dx.doi.org/10.1095/biolreprod.102.013540DOI Listing
June 2003

Progesterone promotes survival of the rat corpus luteum in the absence of cognate receptors.

Biol Reprod 2003 Jan;68(1):151-8

Institute of Medicine and Experimental Biology of Cuyo, LARLAC-CONICET, 5500 Mendoza, Argentina.

Progesterone production by the corpus luteum (CL) is essential for preparation of the endometrium for implantation and for the maintenance of gestation. Progesterone modulates its own production and opposes functional luteal regression induced by exogenous agents, such as prostaglandin F(2alpha). In the present study, we evaluated whether progesterone is also capable of interfering with the process of structural luteal regression, which is characterized by a decrease in weight and size of the gland because of programmed cell death (i.e., apoptosis). We have found that a low number of luteal cells undergo apoptosis throughout gestation. On the day of parturition, but following the initial decline in endogenous progesterone production, a small increase in the number of luteal cells undergoing cell death was observed. This increase in apoptotic cells continued postpartum, reaching dramatic levels by Day 4 postpartum, and was accompanied by a marked decrease in average luteal weight. We have established that the exogenous administration of progesterone significantly reduces the decline in luteal weight observed during structural luteal regression postpartum. This effect was associated with a decrease in the number of cells undergoing apoptosis and with enhanced circulating levels of androstenedione. Furthermore, in vivo administration of progesterone delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that neither the CL of gestation nor the newly formed CL after postpartum ovulation express the classic progesterone-receptor mRNA. In summary, the present results support a protective action of progesterone on the function and survival of the CL through inhibition of apoptosis and stimulation of androstenedione production. Furthermore, this effect is carried out in the absence of classic progesterone receptors.
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http://dx.doi.org/10.1095/biolreprod.102.007898DOI Listing
January 2003

Androstenedione interferes in luteal regression by inhibiting apoptosis and stimulating progesterone production.

Biol Reprod 2002 May;66(5):1540-7

Laboratory of Reproduction and Lactation, CONICET, 5500 Mendoza, Argentina.

Androgens, in concert with lactogenic hormones, contribute to the maintenance of function of the corpus luteum (CL) in pregnant rats. Whereas some of the androgenic actions in the CL are clearly mediated by intracrine conversion to estrogen, pure androgenic effects are also implicated in the regulation of this transient endocrine gland. In this report, we have established, to our knowledge for the first time, the expression of androgen receptor (AR) mRNA and protein throughout gestation in the rat CL. We have found that the AR remains expressed in the CL of gestation on Day 4 postpartum and becomes expressed in the newly formed CL after postpartum ovulation. An AR immunoreactive protein was identified in the CL of pregnancy as well as in prostate and epididymis, which were used as positive controls. The luteal AR protein had mainly nuclear localization, yet some diffuse cytoplasmic staining was also observed. Moreover, we have established that androstenedione, the main circulating androgen in pregnant rats, significantly reduces the decline in luteal weight observed during postpartum structural regression. This effect was correlated with a decrease in the number of cells undergoing apoptosis and with enhanced levels of circulating progesterone. In addition, in vivo administration of androstenedione delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that the interference with apoptosis in vitro elicited by androstenedione is accompanied by an increased capacity of the CL to secrete progesterone. In summary, the results of this study have established that the rat CL expresses AR throughout pregnancy and after parturition, and they have defined a potential role for androstenedione in opposing postpartum luteal regression through inhibition of apoptosis and stimulation of progesterone production.
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http://dx.doi.org/10.1095/biolreprod66.5.1540DOI Listing
May 2002