Charcoal-stripped Serum Research Articles

Endometrial-engineered cell sheets for resumption of fertility

Event Abstract Back to Event Endometrial-engineered cell sheets for resumption of fertility Chien-Cheng Tai1*, Wen-Ling Cheng2* and How Tseng1, 2* 1 Taipei Medical University, Graduate Institute of Medical Sciences, Taiwan 2 Taipei Medical University, Department of Biochemistry and Molecular Cell Biology, Taiwan Introduction: Women with endometriosis or experience of abortion surgery may suffer from endometrial stromal injury, and this injury can sometimes cause female infertility. In view of this situation, we studied the use of autologous endometrial cells as a source of cells for the reconstruction of endometrium. We proposed a method to engineer cell sheets as an alternative therapy of endometrial reconstruction. Materials and Methods: Briefly, we harvested endometrial cell sheets on a transparent polyvinylidene fluoride (PVDF) membrane grafted disulfide bond-containing amino acid and biopolymer in order, and the disulfide bond can be cleaved by adding amino acidic reductant so that the cell sheet can be detached from the surface of the membrane[1]. In this study, the endometrium from the mouse carrying a luminescence gene was used as a cell source. After tissue separation, DMEM/F12 medium with 10% charcoal-stripped serum were used to culture endometrial cell on the disulfide-bonding containing membrane as mention above. After cultivation, the reductant were added, and the multi-layered cell sheets were detached and were transferred onto electrospun poly L-lactide (PLLA) carrier. After that, vital assay, haematoxylin staining and immune-histochemical staining assay were used to evaluate engineered cell sheet. Results and Discussion: The water contact angle decrease from the original untreated PVDF membrane, 70.9 ± 1.45°, to 26.1 ± 2.39° after two-stage surface modification by γ-PGA. The water contact angle was 37.8 ± 4.87° when Cysteine was used as a reducing agent to cleave the disulfide bond and release the residue γ-PGA. In addition, the ESCA analysis (shown in Figure 1) show the element changes at every response as expected, respectively. This further confirms that these membranes surface were successfully modified and by adding reducing agent, the disulfide bonds can be easily cleaved. The cultured transgenic mice endometrial cell show good biocompatibility on the modified PVDF membranes, and also normally express FVB/N-Tg (PolII-luc) Ltc gene (see Figure 2(A) and 2(C)). Furthermore, the cell sheet was successfully detached from the modified PVDF membranes (Figure 2 (B)) by adding Cysteine after culture for one week. Finally, that most of endometrial cell in the detached cell sheet shows highly survival state were confirmed by high green florescent expression and ultra-low red florescent expression in vital assay (Figure 3). Conclusion: In summary, engineered mouse endometrial cell sheet were successfully fabricated by a cultured cell sheet system that we proposed. The PLLA carrier was applied to hold the cell sheet for operation-ready, and the cell sheets were at survival state confirmed by the vital assay. Based on these results, follow-up experiments and assessments in clinical practice are needed to evaluate this method. The authors sincerely appreciate the grant were supported by MOST 104-2221-E-038-005 from Ministry of Science and Technology, ROC.

CSL regulates AKT to mediate androgen independence in prostate cancer progression

Aberrant signaling pathways leads to cancer initiation and progression. Both Notch and PI3K/AKT signaling pathways are believed to be involved in prostate cancer. How the interaction between the two pathways contributes to prostate cancer progression to androgen independence is still elusive. Prostate cancer cells were grown in RPMI 1,640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) or 10% charcoal-stripped heat-inactivated fetal bovine serum (FCS), 1% penicillin-streptomycin in 75 cm2 polystyrene flasks, and maintained at 37 °C in a humidified atmosphere with 5% CO2 . Cell proliferation, invasion were performed with cell counting, matrigel assay in vitro. Dual luciferase assays were performed using reporter plasmids with ARE (Androgen Response Element, ARE). RNA interference was applied to gene silence. Tumorigenicity of cancer cells was evaluated by mouse xenograft in vivo. A subpopulation of casodex resistant prostate cancer cells were identified with an overexpressed androgen receptor (AR) and aggressive phenotypes, characterized with high proliferation, invasion in vitro and enhanced tumorigenesis in vivo. Gene profiling for androgen-dependent LNCaP and androgen-independent LNCaP-CR revealed that both CSL and AKT gave the similar expressional pattern upon casodex treatment. Immunoblot demonstrated that CSL and AKT were dramatically suppressed in androgen dependent LNCaP cells, but slightly inhibited in LNCaP-CR cells as well as other androgen independent prostate cancer cells. Further studies indicated that CSL regulates AKT, and subsequently AR in prostate cancer cells. AKT mediates casodex resistance and androgen independence through regulation of cyclin D1. CSL-AKT-AR axis might play an important role in prostate cancer progression. Targeting CSL depleted the casodex resistant population through inhibition of the AKT, suggesting a more effective therapeutic strategy for abrogating casodex resistance in advanced prostate cancer.

Estradiol Elicits Proapoptotic and Antiproliferative Effects in Human Trophoblast Cells.

During the first trimester of pregnancy, appropriate regulation of estradiol (E2) is essential for normal placental development. Previous studies demonstrate that premature elevation in E2 concentrations can lead to abnormal placentation, but have not fully elaborated the mechanism of this effect in the first-trimester trophoblast. Our aim was to determine whether E2 elicits trophoblast cell death or inhibits proliferation. The first-trimester human cytotrophoblast cell line HTR-8/SVneo was cultured in phenol red-free medium containing charcoal-stripped serum and treated with 17beta-E2 at concentrations between 0 and 100 nM. TUNEL and invasion assays indicated that E2 significantly increased cell death and reduced cell invasion at 10 nM, and nuclear Ki67 expression revealed that it decreased cell proliferation at 1 nM. A similar effect on cell death was observed in first-trimester placental explants. The E2 antagonist fulvestrant blocked all effects of E2. Immunohistochemistry showed that protein expression of proapoptotic caspases 3, 8, and 9 increased at E2 concentrations of 25 nM and greater, whereas expression of antiapoptotic BCL2-alpha decreased at E2 concentrations of 10 nM and greater. Additionally, treatments with estrogen receptor (ER) alpha-specific and ERbeta-specific agonists at concentrations between 0 and 1000 nM indicated that only ERalpha mediates E2's effects, although immunohistochemistry and Western immunoblotting showed that HTR-8/SVneo cells and placental explants express both ERalpha and ERbeta. Taken together, these findings reveal the interplay between elevated serum E2 and apoptosis in the first trimester of pregnancy. These factors could be associated with pregnancy complications including infertility and uteroplacental insufficiency.

Abstract 2853: Investigating DNAPK as a biomarker and a novel therapeutic target in aggressive prostate cancer

Abstract Background/purpose: Despite recent advances in the development of androgen deprivation therapies (ADT), prostate cancer (PCa) remains the second leading cause of cancer death in American men. Thus, there is a critical need to improve treatment outcomes for men with aggressive PCa. The purpose of this study was to identify and functionally characterize kinases that can serve as both prognostic biomarkers and therapeutic targets in PCa. Methods: To identify kinases associated with metastatic progression of PCa, we utilized high-density oligonucleotide arrays to interrogate the expression of all kinases in 545 prostatectomy samples, obtained from high-risk patients with long-term clinical follow-up (>10 years). We ranked all kinases by their fold change in expression between PCas that subsequently metastasized versus those that did not. We then characterized the top ranked kinase in preclinical PCa models with in vitro mechanistic experiments and in vivo therapeutic studies. Results: We nominated DNA-dependent protein kinase (DNAPK) as the top kinase associated with metastatic PCa progression. Interrogation of pathways associated with DNAPK knockdown in PC3, DU145, VCaP and C4-2B, identified canonical Wnt signaling as the top DNAPK-activated pathway. In PCa cells (PC3, LNCaP C.S. {LNCaP grown under charcoal-stripped serum condition}, C4-2B and LNCaP-AR) DNAPK inhibition significantly abrogates Wnt signaling and abolishes cell proliferation, migration and invasion. Interestingly, we found that DNAPK directly modulates the Wnt signaling independent of the androgen receptor (AR), highlighting the therapeutic potential of DNAPK inhibition in cancers resistant to ADT. Indeed, in VCaP xenograft models, pharmacologic inhibition of DNAPK with NU7441 demonstrated 3.4 fold (p<0.01) reduction of tumor growth at non-toxic doses. Using an independent prostate cancer tissue cohort, we validate DNAPK expression as a biomarker for metastatic disease progression (p = 6.4 e−06, hazard ratio = 2). Conclusion: In summary, we nominate DNAPK as a potential biomarker of disease progression and as a novel therapeutic target in aggressive prostate cancer. Our data suggests that DNAPK mediates PCa progression by upregulating Wnt signaling, and demonstrates that DNAPK inhibition results in significant tumor responses in PCa xenografts. Citation Format: Vishal Kothari, Jonathan F. Goodwin, Shuang Zhao, Elai Davicioni, Jeffrey R. Karnes, Robert B. Den, Rohit Mehra, Karen E. Knudsen, Felix Y. Feng. Investigating DNAPK as a biomarker and a novel therapeutic target in aggressive prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2015-2853

Abstract P3-05-19: A new cell panel to study oestrogen receptor loss in acquired endocrine resistant breast cancer

Abstract Background: Oestrogen receptor positive (ER+) breast cancer patients can acquire endocrine resistance and 10-20% tumours have lost ER at relapse. While growth factor pathway hyperactivity and ER promoter methylation contribute to de novo ER negativity, ER loss in acquired resistance is largely unexplored. We have recently developed 11 lines from MCF7, T47D, BT474 & MDAMB361 cells to model acquired resistance emerging with prolonged (3 year) endocrine treatment both in ER+/HER2- and ER+/HER2+ disease. Here we establish prevalence of acquired ER loss in the panel, determine any association with aggressiveness, and explore ER loss mechanisms in acquired endocrine resistance. Methods: Authenticated acquired resistant models derived from endocrine responsive lines cultured for 3 years with 10-7M tamoxifen (TamR), 10-7M fulvestrant (FasR) or oestrogen deprivation (5% charcoal-stripped foetal calf serum SFCSR) were profiled for ER & PR by PCR, immunocytochemistry and Western blotting (+/- 1-2wk antihormone withdrawal), for 2nd-line endocrine responsiveness and for migration using Boyden chamber assays vs. time-matched controls. Src kinase, EGFR, HER2, MAPK & AKT activity were examined and whether their respective inhibition using saracatinib or gefitinib (1μM), trastuzumab (100nM), U0126 or LY294002 (5μM) for 1wk restored ER. ESR1 promoter methylation was examined by bisulfite modification & MethyLight PCR. Results: Substantial ER mRNA & protein loss occurred in 7/11 long-term acquired endocrine resistant lines. This was irreversible by antihormone withdrawal and paralleled by complete PR loss and endocrine growth-insensitivity. While seen in all fulvestrant resistant lines, ER loss was less frequent with tamoxifen (in MCF7TamR & MDATamR) and only seen in oestrogen deprived resistant T47DSFCSR cells. Increased migration accompanied acquired ER loss in ER+/HER2- derived MCF7TamR, MCF7FasR & T47DSFCSR cells and was saracatinib-sensitive. Src and further growth factor pathway activity increased in several acquired resistant models, and ER loss associated with increased EGFR/HER2 in the MCF7- & MDA-derived cells and with increased MAPK activity in all lines. Weak ER recovery was seen in antioestrogen resistant models treated with saracatinib (MDATamR, MCF7FasR), gefitinib (MDATamR, BT474FasR, MCF7FasR/TamR) or trastuzumab (MCF7TamR). ESR1 DNA methylation was only prominent in MDATamR and MCF7TamR cells. No inhibitor restored ER in the T47D-derived cells, including T47DSFCSR which also lacked ESR1 methylation. Conclusions: Although ER loss is very prominent in this acquired resistant cell panel, it demonstrates there is capacity of prolonged antihormones, chiefly antioestrogens, to promote receptor loss independent of initial HER2 status. Acquired ER loss clinically would be expected to confer endocrine insensitivity and poorer prognosis given the panel findings. Where ER loss emerged with antioestrogens there was some mechanistic-overlap with de novo ER negativity, including ER promoter methylation for acquired tamoxifen resistance. Our future studies will use the panel to address if targeting these mechanisms can be optimized for ER recovery or if further mechanisms also drive ER loss in acquired resistance, notably for prolonged oestrogen deprivation. Citation Format: Julia M Gee, Mei Yee Meng, Richard A McClelland, Huw J Mottram, Susan R Kyme, Pauline Finlay, Lindy Goddard, Carol M Dutkowski, Denise Barrow, Walther Parson, Heidi Fiegl. A new cell panel to study oestrogen receptor loss in acquired endocrine resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-19.

G Protein‐Coupled Estrogen Receptor Activation Attenuates Rat Aortic Smooth Muscle Cell Proliferation

Activation of the newly discovered G protein-coupled estrogen receptor (GPER) decreases blood pressure, promotes vasorelaxation, and induces protective effects in the heart and kidney. However the role of GPER in vascular remodeling, a pathology implicated in cardiovascular diseases like atherosclerosis and restenosis, has yet to be established. The objective of this study was to investigate whether GPER activation influences smooth muscle cell proliferation, one aspect of vascular remodeling. We hypothesized that GPER activation via the selective agonist G-1 inhibits serum-induced smooth muscle cell proliferation. Cultured aortic smooth muscle cells from female Lewis rats were allowed to reach 50% confluence before starving in media containing 0.5% charcoal-stripped serum (CSS) media for 48 hours. Media was then changed to 0.5% CSS + vehicle, 5% CSS + vehicle, or 5% CSS + 100 nM G-1. After 24 hours, cells were counted using a hemocytometer. Proliferation in 5% CSS + vehicle was more than 3-fold higher in comparison to 0.5% CSS + vehicle (347 ± 59%, p<0.01, n=6). Treatment with G-1 decreased proliferation to 131 ± 39% (p<0.01, n=6). We conclude that GPER activation attenuated serum-stimulated proliferation of female rat aortic smooth muscle cells. GPER activation may mediate estrogen's beneficial effects on vascular remodeling through anti-proliferative mechanisms in smooth muscle cells.

Abstract 2114: UGT2B17 is a unique co-regulator of the androgen receptor and androgen receptor splice variants in prostate cancer

Abstract Background: Expression of the steroid glucuronidating enzyme, UGT2B17, is suppressed by ligand bound full-length androgen receptor (AR-FL). However, we have shown that the AR constitutively active AR splice variants AR-V7 and ARv567es significantly increase the expression of UGT2B17 in prostate cancer cell lines and xenografts. Further, recent studies demonstrated that UGT2B17 is increased in patients with castrate recent prostate cancer. We have also shown that UGT2B17 is increased in multiple cell lines when AR splice variants (AR-Vs) increase in response to androgen deprivation therapies such as enzalutamide and abiraterone. Objective: Since UGT2B17 functions to glucuronidate dihydrotestosterone (DHT) and decrease intracellular androgen, it would be a potential mechanism for generation of constitutively active AR-Vs by androgen deprivation therapy-induced alternative splicing. In xenografts that have been shown to harbor genomic rearrangements that encode for AR-Vs, we see marked elevations of UGT2B17, an indication that it was not necessary for generation of AR-Vs but has additional activities to enhance AR-Vs signaling. The purpose of this study was to determine the interaction of UGT2B17 with AR-Vs, in cell lines that have not been shown to harbor AR genomic rearrangements. Results: In the parental LNCaP cell line, expression of UGT2B17 decreased after growth in charcoal stripped serum (CSS) supplemented with 10-9M DHT. Expression increased following growth in CSS alone and when enzalutamide was added to CSS. In contrast, in LNCaP-abl or LNCaP 95 cells, which are castration resistant sublines that express AR-Vs, basal UGT2B17 was significantly elevated compared with LNCaP baseline levels. Further, in these castrate resistant sublines, UGT2B17 exhibited less suppression by DHT and there was no increase in UGTB17 over baseline following enzalutamide treatment. ChIP assays in parental LNCaP cells stably transfected with ARv567es demonstrated binding to the UGT2B17 promoter in the absence of DHT and decreased binding in the presence of DHT. In control LNCaP cells transfected with empty vector, no binding to the UGT2B17 promoter was seen in the presence of DHT. Similar results were seen in enzalutamide resistant VCaP xenografts: UGT2B17 was elevated in the castrated state but suppressed when DHT was added. When lysates from LNCaP-abl, LNCaP 95, or VCaP cells grown in CSS were immunoprecipitated with UGT2B17 antibody then blotted with an AR-N terminal antibody, AR and AR-Vs were noted to be present. AR-negative M12 cells and LNCaP cells grown in DHT demonstrated no pull-down of AR in the UGT2B17 IP's. Interpretation: This study demonstrated that UGT2B17 is a unique co-regulator of AR-Vs activity and it is up-regulated by AR-Vs and suppressed by androgen transactivated AR-FL receptor. UGT2B17 may thus be part of an autocrine-loop driving castrate resistant prostate cancer by AR-Vs. Citation Format: Gang Liu, Shihua Sun, Kathleen Haugk, Cynthia Sprenger, Xeusen Dong, Elahe Mostaghel, Stephen R. Plymate. UGT2B17 is a unique co-regulator of the androgen receptor and androgen receptor splice variants in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2014-2114

Abstract 3391: Alteration of mammary tumor cell behavior by FLIZ1

Abstract Fetal liver zinc finger protein (FLIZ1, ZC3H8) was previously described as a negative regulator of GATA3 expression in T cells. GATA3 also is an important determinant of mammary cell fate and breast tumor cell behavior. We hypothesized that FLIZ1 also regulates GATA3 in the mammary gland, thereby influencing normal and tumor cell processes controlled by GATA3. We identified tumor cell lines with high levels of FLIZ1 expression, and transfected them with vectors driving expression of FLIZ1-interfering RNAs. Stable transfectants (Flizout cells) were evaluated for ability to migrate in a wound healing assay, and were found to have decreased mobility. Flizout cells also grew more slowly and with more pronounced epithelial morphology in media containing charcoal-stripped serum, suggesting increased dependence on steroid hormones. Flizout cells were compared to control cells in proliferation assays, and were found to grow at equivalent rates in standard media, but Flizout cells grew at decreased rates in the presence of tamoxifen. We implanted Flizout cells and controls into the mammary glands of syngeneic female mice, which were then treated with tamoxifen or peanut oil vehicle alone. Flizout cells grew more slowly in tamoxifen-treated animals, and tissue weights were significantly less than controls. In order to determine if FLIZ1 plays a role in the normal mammary gland, we assessed its expression during stages of mouse mammary gland development by immunoblot, and found FLIZ1 expression increased during mammary involution. These data are consistent with a role for FLIZ1 in regulation of mammary cell growth and tumor cell behavior. Citation Format: Torri Anderson, Christopher Cali, Katherine Bell, Keith G. Danielson, Ashley Klein, Christina Martin, Sara Radecki, Adam Santoro, John A. Schmidt, Janice E. Knepper. Alteration of mammary tumor cell behavior by FLIZ1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3391. doi:10.1158/1538-7445.AM2014-3391

Abstract 4891: Dormancy and growth of metastatic breast cancer cells in a bone-like microenvironment

Abstract We have developed a three dimensional cell culture model (bioreactor) in which cancer cells are cultured in a bone-like microenvironment. When co-cultured with MC3T3-E1 osteoblasts and the resulting bone-like matrix, MDA-MB-231 (231) human metastatic breast cancer cells attach, form “single cell files”, develop invadapodia, infiltrate and partially degrade the thick matrix. Eventually, the cells form large colonies. By contrast, the metastasis-suppressed variant, MDA-MB-231BRMS1, (231BRMS1) demonstrates a dormant phenotype in the same system. They loosely attach, and are slow to proliferate. This pattern of growth is also observed in femurs of mice inoculated with these cells. We hypothesized that changes in the cytokine microenvironment or the matrix structure would either cause the 231 cells to become dormant or the 231BRMS1 cells to proliferate. Because of anecdotal evidence that bone trauma or breakage is associated with latent metastasis, we added either a cocktail of inflammatory cytokines (IL-6, IL-8, MCP-1, VEGF, GROα ) or bone remodeling cytokines (IL-6, TNFα, IL-1β, PGE-2 ). The inflammatory cytokines had little effect on the morphology or proliferation of either cell type. In contrast, bone remodeling cytokines stimulated the 231BRMS1 cells to grow vigorously. Of the remodeling cytokines, we determined that TNFα and IL-1β were sufficient to cause the change in growth. However, we were able to block the response of the 231BRMS1 cells with indomethecin, a cyclooxygenase inhibitor or with an inhibitor to the receptor for PGE2. These data suggest that the additional cytokines lead to prostaglandin production. We also modulated the matrix by growing the osteoblasts with charcoal-stripped serum to reduced estradiol. The osteoblasts differentiated under these conditions (alkaline phosphatase positive) but did not fully mineralize the matrix (minimal von Kossa staining ). In the presence of the modified matrix, both the 231(ER-) and the 231BRMS1(ER-) cells showed increased proliferation. In contrast, MCF-7 cells (ER+) maintained a dormant phenotype and did not grow. In conclusion, our data highlight the importance of a bone-like microenvironment in maintaining breast cancer dormancy. Disrupting the microenvironment could result in dormant cells re-entering the cell cycle. We are currently determining the role of the cytokine microenvironment and the matrix alteration in the modulation of the cancer cells dormancy or growth in the bone. This work was supported by the U.S. Army Medical Research and Materiel Command under W81XWH-12-1-0127, and by the Metavivor Research Foundation. Citation Format: Yu-Chi Chen, Andrea M. Mastro, Donna M. Sosnoski, Robert J. Norgard, Cassidy D. Grove, Erwin A. Vogler. Dormancy and growth of metastatic breast cancer cells in a bone-like microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2014-4891

The steroid inhibitor trilostane decreases gonadotropin-, but not prostaglandin e2-, stimulated haluronic acid expression by macaque cumulus-oocyte-complexes during culture

To determine whether the inhibition of steroid synthesis with trilostane (TRL) affects synthesis of the cumulus expansion-associated glycosaminoglycan, haluronic acid (HA), in macaque culumus-oocyte-complexes (COCs) in the absence or presence of gonadotropins or prostaglandin E2 (PGE2). Nonhuman primate model, 3x2 factorial design, randomized, controlled study. Healthy macaque (n=5) COCs (n=3-6/group/animal) from 0.5-1.5mm small antral follicles during spontaneous menstrual cycles were cultured with or without TRL (250ng/ml) in 1) TALP media with 5% charcoal-stripped monkey serum (CON), 2) media plus gonadotropins (FSH, LH; 75 mIU/ml each), or 3) media plus PGE2 (5ng/ml) for 30 hours in 5% CO2. Following culture, COCs were fixed, immunolabeled with HA binding protein, phalloidin (actin) and Hoechst 33342 (DNA), and imaged by confocal microscopy. Non-cultured COCs were used as negative controls for HA staining. HA staining intensity for each COC was assigned a score (1 to 3) by 3 blind observers based on the percentage (%) of extracellular space occupied by HA; “1”: less than 25%, “2”: 25-75%, “3”-over 75%. Statistical significance (p≤0.05) was determined using the SAS GLM method. Non-cultured COCs exhibited no HA staining. In CON and CON+TRL groups, HA staining remained low and unchanged. Gonadotropins (p<0.05) as well as PGE2 (p<0.05) increased HA staining in cultured COCs in comparison to that of the CON. HA localized to the extracellular matrix between neighboring cumulus cells and sometimes concentrated in the outermost area of expanded COCs, resulting in a ring-like appearance surrounding the COC. However, addition of TRL reduced (p<0.01) HA staining in the FSH+LH+TRL group compared to FSH+LH alone. Interestingly, TRL did not alter HA staining (p=0.66) in the PGE2+TRL group in comparison to COCs treated with PGE2 alone. In macaque COCs, cumulus expansion and HA expression appears to be steroid-dependent during gonadotropin stimulation, but either independent of steroid hormones or downstream of the steroid hormone signaling pathway during PGE2 induction of cumulus expansion. This study supports an important role of steroid hormones in the intrafollicular environment for cumulus expansion during oocyte maturation in primates, and has implications for improving in vitro fertilization techniques and fertility preservation in humans. Ongoing studies include hormone replacement to determine which steroid hormone is required for HA synthesis.

A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells

Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells.

Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro

Alkyl esters of p-hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. As proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20 ± 2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10(-8) M 17β-oestradiol, 1-5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben. Long-term exposure (20 ± 2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and β-catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.

Rapid induction of the Growth Hormone Gene Transcription by Glucocorticoids In Vitro: Possible Involvement of Membrane Glucocorticoid Receptors and Phosphatidylinositol 3‐Kinase Activation

The regulation of transcription of the growth hormone (GH) gene by glucocorticoids was studied in MtT/S cells, a cell line derived from an oestrogen-induced mammotrophic tumour in the rat, and in the primary culture of the anterior pituitary gland of adult mice. The levels of the GH heteronuclear RNA (GH hnRNA), which are mainly determined by the transcription rate, increased by 25-fold during 24 h in response to dexamethasone (DEX; 1 μM) in MtT/S cells that were cultured in the medium containing charcoal-stripped serum for 7 days. The stimulatory effect of DEX on the GH hnRNA levels was detectable as early as 30 min. This rapid effect of DEX did not require on-going protein synthesis, whereas it was considered that DEX requires the presence of unknown cellular proteins produced independently of DEX stimulation. By contrast, on-going protein synthesis was required for DEX action when incubated for 6 h, as has been observed in the previous studies. The specific inhibitor of glucocorticoid receptor, RU486, inhibited both rapid (30 min) and delayed (6 h) the effects of glucocorticoids on GH hnRNA levels. Membrane impermeable glucocorticoid, corticosterone-bovine serum albumin conjugate (CSBSA), was found to have effects similar to those of DEX and free corticosterone (CS), suggesting that glucocorticoids regulate GH gene transcription at least in part through the membrane bound receptors. From pharmacological studies, it was suggested that phosphatidylinositol 3-kinase (PI3K) activation is involved in the rapid effects but not in the delayed effects of glucocorticoids. This also suggests that the delayed effects of glucocorticoids depend on mechanisms other than the activation of PI3-kinase. Finally, both rapid and delayed effects of CS and CSBSA were observed not only in MtT/S cells, but also in the mouse pituitary cells in primary culture. Therefore, it is possible that the membrane initiated action of glucocorticoids is involved in the regulation of GH transcription in normal pituitary cells, as well as in pituitary tumour cells.

Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism

The atherogenic effects of hypothyroidism on lipid metabolism could result, in part, from the reduced clearance of remnant lipoproteins. In this study, we investigated the expression of hepatic low-density lipoprotein receptor-related protein 1 (LRP1), a receptor for remnant lipoproteins, in hypothyroidism and the effect of 3,3',5-triiodo-L-thyronine (T3) treatment on hepatic LRP1 expression. C57BL/6 mice were fed a normal diet (control group) or a low-iodine diet supplemented with 0.15% propylthiouracil (PTU/LI group) for 4 weeks. Mice in the PTU/LI group were injected intraperitoneally with T3 (0, 30, and 150 μg/kg of body weight) for 7 days. HepG2 cells were incubated in fetal bovine serum or charcoal-stripped fetal bovine serum with various concentrations of T3. The expression and function of LRP1 in liver samples and cells were analyzed. Hypothyroidism was successfully induced in PTU/LI mice. Hepatic LRP1 protein expression was lower in the PTU/LI group than in the control group. T3 treatment upregulated hepatic LRP1 protein expression in PTU/LI mice. LRP1 expression in HepG2 cells was reduced after incubation in the medium containing charcoal-stripped fetal bovine serum, which mimics hypothyroidism in vitro, and was recovered by T3 treatment. The protein expression of LRP1 in HepG2 cells was increased by T3 treatment in a dose-dependent manner up to 2.0 nM T3. However, LRP1 mRNA transcription was not affected by hypothyroidism conditions or T3 treatment, either in liver samples or in HepG2 cells. T3 treatment on HepG2 cells increased cellular uptake of lipid-conjugated apolipoprotein E through LRP1. Our data demonstrate that hepatic LRP1 expression and function decrease in hypothyroidism and are regulated by the thyroid hormone. These results suggest that in hypothyroidism, decreased expression of hepatic LRP1 may be associated with reduced clearance of circulating remnant lipoproteins.

The direct inhibitory effect of dutasteride or finasteride on androgen receptor activity is cell line specific

Finasteride and dutasteride were developed originally as 5α-reductase inhibitors to block the conversion of testosterone to dihydrotestosterone (DHT). These drugs may possess off-target effects on the androgen receptor (AR) due to their structural similarity to DHT. A total of four human prostate cancer cell models were examined: LNCaP (T877A mutant AR), 22Rv1 (H874Y mutant AR), LAPC4 (wild-type AR), and VCaP (wild-type AR). Cells were cultured in 10% charcoal-stripped fetal bovine serum, either with or without DHT added to the medium. AR activity was evaluated using the ARE-luciferase assay or the expression of AR regulated genes. Dutasteride was more potent than finasteride in interfering with DHT-stimulated AR signaling. Disruption of AR function was accompanied by decreased cell growth. Cells that rely on DHT for protection against death were particularly vulnerable to dutasteride. Different prostate cancer cell models exhibited different sensitivities to dutasteride and finasteride. LNCaP was most sensitive, LAPC4 and VCaP were intermediate, while 22Rv1 was least sensitive. Regardless of the AR genotype, if AR was transfected into drug-sensitive cells, AR was inhibited by drug treatment; and if AR was transfected into drug-resistant cells, AR was not inhibited. The direct inhibitory effect of dutasteride or finasteride on AR signaling is cell line specific. Mutations in the ligand binding domain of AR do not appear to play a significant role in influencing the AR antagonistic effect of these drugs. Subcellular constituent is an important factor in determining the drug effect on AR function.

Suppression of metallothionein 3 gene expression by androgen in LNCaP prostate cancer cells.

Androgen deprivation therapy is the standard treatment for prostate cancer. However, tumors often progress towards a more aggressive phenotype despite treatment. Prostate tissue has a high zinc concentration, which may correlate with prostate cancer progression. Therefore, we investigated the effect of dihydrotestosterone (DHT) on the gene expression of metallothioneins (MTs) and zinc transporters in prostate cancer with quantitative real-time polymerase chain reaction (PCR). The MT3 gene expression in LNCaP cells was suppressed by DHT in a dose-dependent manner. However, it increased in a culture medium containing androgen-deficient charcoal-stripped fetal bovine serum (FBS). Bicalutamide, an androgen receptor antagonist, increased the gene expression of MT3 and partially reversed the suppression of MT3 gene expression induced by DHT. In PC-3 cells lacking androgen receptors, DHT and bicalutamide exerted no effect on MT3 gene expression. The reporter gene assay with a luciferase reporter plasmid containing the 5'-flanking region of MT3 demonstrated a decrease in luciferase activity caused by DHT that was reversed by bicalutamide. These results suggest that MT3 gene expression is downregulated by androgen.

Novel Interaction between the Co-chaperone Cdc37 and Rho GTPase Exchange Factor Vav3 Promotes Androgen Receptor Activity and Prostate Cancer Growth*

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. Vav3, a guanine nucleotide exchange factor, is up-regulated following progression to castration resistance in preclinical models and is overexpressed in a significant number of human prostate cancers. Vav3 is a novel co-activator of the AR. We sought to identify Vav3 binding partners in an effort to understand the molecular mechanisms underlying Vav3 enhancement of AR activity and to identify new therapeutic targets. The cell division cycle 37 homolog (Cdc37), a protein kinase-specific co-chaperone for Hsp90, was identified as a Vav3 interacting protein by yeast two-hybrid screening. Vav3-Cdc37 interaction was confirmed by GST pulldown and, for native proteins, by co-immunoprecipitation experiments in prostate cancer cells. Cdc37 potentiated Vav3 co-activation of AR transcriptional activity and Vav3 enhancement of AR N-terminal-C-terminal interaction, which is essential for optimal receptor transcriptional activity. Cdc37 increased prostate cancer cell proliferation selectively in Vav3-expressing cells. Cdc37 did not affect Vav3 nucleotide exchange activity, Vav3 protein levels, or subcellular localization. Disruption of Vav3-Cdc37 interaction inhibited Vav3 enhancement of AR transcriptional activity and AR N-C interaction. Diminished Vav3-Cdc37 interaction also caused decreased prostate cancer cell proliferation selectively in Vav3-expressing cells. Taken together, we identified a novel Vav3 interacting protein that enhances Vav3 co-activation of AR and prostate cancer cell proliferation. Vav3-Cdc37 interaction may provide a new therapeutic target in prostate cancer.

A dominant negative Cx43 mutant differentially affects tumorigenic and invasive properties in human metastatic melanoma cells

Previous reports have implicated connexin 43 (Cx43) as a tumor suppressor in early stages of tumorigenesis and in some cases as an enhancer of cell migration in later stages. To address the role of Cx43 in melanoma tumor progression, we utilized two melanoma cell lines derived from the same patient in pre-metastasis (WM793B) and following isolation from a lung metastasis in nude mice (1205Lu). Our results demonstrate a strikingly increased expression of Cx43 in both the pre-metastatic and metastatic melanoma cell lines that were actively migrating compared to non-migrating cells. To further investigate the role of Cx43 in these melanoma cells, we overexpressed wild type (wt) Cx43 as well as a mutant dominant negative Cx43 mutant that causes closed channels (T154A). The metastatic 1205Lu cells expressing Cx43-T154A showed a twofold decrease in colony formation on soft agar while the nonmetastatic WM793B cells showed no significant change. In invasion assays through a collagen matrix, the same Cx43-T154A 1205Lu cells demonstrated a three- to fourfold increase in the invasion index compared to either wt Cx43 or vector control cells. The increase in invasiveness was eliminated by migration towards media with charcoal-stripped serum, suggesting that migration may be directed towards a lipophilic compound(s). Our findings demonstrate that a dominant negative Cx43 mutant deficient in channel formation exhibits a dual pattern of regulation in metastatic melanoma cells with a decrease in anchorage-independent growth and an increase in invasive potential.

Phosphoenolpyruvate Carboxykinase and Glucose-6-phosphatase Are Required for Steroidogenesis in Testicular Leydig Cells

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

Mitochondrial dynamics is affected by 17β-estradiol in the MCF-7 breast cancer cell line. Effects on fusion and fission related genes

Mitochondrial dynamics, specifically fusion and fission processes, maintain mitochondria integrity and function, yet at this time, effect of estrogens on fusion and fission in breast cancer cell lines has not been studied. The aim of this study was to characterize the effect of 17β-estradiol on fusion and fission-related genes, as well as on mitochondria proliferation and function. We used MCF-7 breast cancer cell line, which is estrogen sensitive (estrogen receptor positive). Cells were grown in Dulbecco's modified Eagle medium charcoal-stripped fetal bovine serum and treated with 1nM of 17β-estradiol and with/without 100nM of ICI 182,780, a drug that caused rapid degradation of estrogen receptor. mRNA levels of fusion (mfn1, mfn2, opa1) and fission-related genes (drp1 and fis1) were examined by RT-PCR, cardiolipin content by N-acridyl-orange fluorescence and oxidative phosphorylation protein levels, as well as, the major fusion and fission related protein levels, by Western blot. mRNA expression of fusion-related genes increased after 17β-estradiol-treatment for 4h; however fis1 fission-related gene expression decreased. All these effects were not found in cells pre-treated with ICI 182,780, save for the changes in mfn-1, conferring them the effects of 17β-estradiol to estrogen receptor. The changes in protein levels were less prominent, but in the same way, than in mRNA levels, showing an increase in Mfn1 and Mfn2, as well as in Drp1, but there was no change in Fis1 protein levels. Mitochondrial biogenesis was also affected by 17β-estradiol, showing an increase in mtDNA but with no change in N-acridyl-orange fluorescence. On the whole, our results suggest an imbalance in the fusion/fission ratio, with a high fusion by 17β-estradiol-estrogen receptor action, which can affect to mitochondrial biogenesis, concretely in mitochondria proliferation. According to this information, 17β-estradiol would modify mitochondrial dynamics, biogenesis and metabolism, and thus compromise the normal development and function of mitochondria in cancer affected tissues.