Charcoal-stripped Fetal Bovine Serum Research Articles

Interaction of Sex Hormones and Dopamine D2 Receptor Polymorphisms in Human Renal Proximal Tubule Cells

The renal inflammatory response can be modulated by the dopamine D2 receptor (D2R) subtype. The DRD2 gene is highly polymorphic in humans, and single nucleotide polymorphisms (SNPs), such as rs6276 and rs6277 ( DRD2 SNPs), decrease its expression and function. We hypothesized that the response of human renal proximal tubular cells (hRPTCs) to hormones in the culture medium differs by sex and DRD2 SNPs, leading to differences in the inflammatory response. We determined the effects of dihydrotestosterone (DHT) and estradiol (ES) on hRPTCs genotyped for the presence or absence of DRD2 SNPs. We studied four cell lines from males (M) and females (F) with wild-type (WT) DRD2 (M- DRD2 WT and F- DRD2 WT) and DRD2 SNPs (M- DRD2 SNPs and F- DRD2 SNPs). The cells were cultured in a medium containing charcoal-stripped fetal bovine serum (FBS) for 24 hours, without (control group, CG) and with dihydrotestosterone (DHT, 5 nM) or estradiol (ES, 20 nM). We quantified the renal protein expression (protein of interest/GAPDH) of proinflammatory and profibrotic factors and markers of injury and proliferation. DHT decreased the expression of transforming growth factor (TGF-β) by ≍50% in both M- DRD2 WT (0.4±0.1 vs. 1.0±0.2) and M- DRD2 SNPs (1.0±0.1 vs. 1.6±0.1) (n=5-6, p<0.05). However, there was no significant effect of DHT or ES on the expression of TGF-β in F- DRD2 WT and F- DRD2 SNPs. DHT did not affect the fibronectin 1 (FN1) expression in M- DRD2 WT and M- DRD2 SNPs. In F- DRD2 WT, DHT did not affect FN1 expression but decreased in F- DRD2 SNPs (0.9±0.1 vs. 1.7±0.1, n=6, p<0.05). Similar to DHT, ES did not affect FN1 expression in M- DRD2 WT. However, ES decreased FN1 by 50% in M- DRD2 SNPs (0.5±0.1 vs. 1.1±0.1, n=6, p<0.05). Similar to the studies in males, ES did not affect FN1 expression in F- DRD2 WT but also decreased FN1 (1.0±0.1 vs. 1.7±0.1, n=6, p<0.05) in F-DRD2 SNPs. Neither DHT nor ES affected the expression of Kidney Injury Molecule-1 (KIM-1) in M- DRD2 WT. However, in M- DRD2 SNPs, both DHT and ES decreased KIM-1 (1.4±0.2 vs. 1.8±0.2 and 1.1±0.1 vs. 1.8±0.2, respectively; n=5, p<0.05). Unlike in M- DRD2-WT, in F- DRD2 WT, ES increased KIM-1 expression (1.0±0.2 vs. 0.52±0.05, n=5, p<0.05), while DHT had no significant effect. Unlike in M- DRD2 SNPs, where both DHT and ES decreased KIM-1, in F- DRD2 SNPs, neither DHT nor ES affected KIM-1. DHT did not affect the expression of Ki67 in M- DRD2 WT. However, in M- DRD2 SNPs, DHT decreased Ki67 expression (0.60±0.08 vs. 1.6±0.2, n=6, p<0.05). Similar to M- DRD2-WT, DHT did not affect Ki67 expression in F- DRD2-WT. ES also did not affect Ki67 expression in M- DRD2 WT but decreased Ki67 expression in M-DRD2 SNPs (0.91± 0.18 vs. 1.6±0.2, n=6, p<0.05). Similar to the male data, the treatment with ES did not affect the Ki67 expression in F- DRD2 WT but decreased Ki67 expression in F- DRD2 SNPs (1.5±0.2 vs. 0.9±0.1, n=6, p<0.05). Our results demonstrate similarities (FN1 and Ki67) and differences (TGF-β and KIM-1) in profibrotic and proliferation markers expressed in hRPTCs between males and females. These differences are more pronounced in cell lines expressing DRD2 SNPs than those expressing the DRD2 WT. While both DHT and ES decreased KIM-1 expression in males with DRD2 polymorphisms, they had no effect in females with DRD2 polymorphisms. These results indicate a complex interaction of the effect of hormones and DRD2 polymorphisms in regulating the inflammatory response in hRPTCs. Their roles in regulating renal physiology and blood pressure remain to be defined. R01 DK039308 P01 HL068686 R01 HL023081 R37 HL023081 R01 DK119652/ NIH HH/ US. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulation of Estrogen Receptor Alpha (ERα) and Tumor Suppressor Gene BRCA1 in Breast Cancer Cells by Bazedoxifene Acetate (BZA).

Selective estrogen receptor modulators (SERMs) are steroid analogs with dual functionality, acting as partial estrogen receptor agonists to preserve postmenopausal bone density and as estrogen receptor antagonists in breast tissue. Bazedoxifene acetate (BZA) is an FDA-approved, third-generation SERM used in the treatment of osteoporosis in women. It demonstrates potential as a therapeutic option for breast cancer patients undergoing endocrine therapy. Our study aimed to assess BZA's effects on Estrogen Receptor Alpha (ERα) and tumor suppressor gene BRCA1 in T-47D and MCF-7 breast cancer cells, using Western blots, cellular viability, apoptosis assays, and RT-qPCR. Cells were cultured in 5% charcoal-stripped fetal bovine serum for six days to deplete endogenous steroids. Following a 24 h exposure to 2 µM BZA (optimal concentration determined from 1 nM-2 µM studies), Western blot analyses revealed reduced ERα and BRCA1 protein levels in both cell lines. ERα decreased by 48-63% and BRCA1 by 61-64%, indicating sensitivity to antiestrogens. Cytolocalization of ERα and BRCA1 remained unchanged after BZA and 17-β-estradiol (E2) treatment. ESR1 mRNA expression correlated with Western blot findings. Image cytometric analysis using the stain, propidium iodide, detected decreased cellular proliferation in T-47D and MCF-7 cells following a 6-day treatment ranging from 1 nM to 2 µM BZA. BZA treatment alone led to a tenfold reduction in cellular proliferation compared to estrogen-treated cells, suggesting antiproliferative effects. Understanding BZA's modulation of BRCA1 and ERα, along with their mechanistic interactions, is vital for comprehending its impact on breast cancer tumor suppressors and hormone receptors.

Simvastatin Enhances the Radiosensitivity of Androgen-independent Prostate Cancer Cells via Inhibition of RAD51 Expression.

Statins exert antitumor effects via various mechanisms. Additionally, the recurrence rate of prostate cancer after radiation therapy is lower in patients taking statins. This study investigated the efficacy of combination therapy with statins and irradiation in androgen-independent prostate cancer cells. PC-3 and LNCaP human prostate cancer cell lines were used in this study. We developed androgen-independent LNCaP cells (LNCaP-LA) by gradually replacing fetal bovine serum (FBS) with charcoal-stripped FBS. Microarray analysis was performed, followed by Ingenuity Pathway Analysis. Cell viability was determined using the MTS assay. Simvastatin alters gene expressions in PC-3 cells. Microarray data showed that the number of differentially expressed genes was the highest in the pathway of "Role of BRCA1 in DNA Damage Response". In the validation, the expression of RAD51, listed in "Role of BRCA1 in DNA Damage Response", decreased significantly by simvastatin in PC-3 cells. Reduction in RAD51 expression following siRNA transfection increased the cytocidal effects of X-ray therapy in PC-3 and LNCaP-LA cells. The combination of simvastatin and irradiation further inhibited cell proliferation compared with monotherapy with either therapy in PC-3 or LNCaP-LA cells. In addition, compared with X-ray monotherapy, the combination of simvastatin and irradiation further enhanced the expression of γH2AX, which is reported to be one of the accurate markers of DNA damage in PC-3 cells. Simvastatin decreased the expression of RAD51 in androgen-independent prostate cancer cells. The combination of irradiation and drugs that reduce RAD51 expression can potentially affect androgen-independent prostate cancer growth.

Bovine amniotic membrane stem cell-conditioned medium improves the culture conditions for in vitro produced ovine embryos

Despite the progress in in vitro embryo production technology, the quality of embryos produced in vitro remains inferior to that of embryos produced in vivo. There have been reports highlighting the beneficial impacts of stem cells and their conditioned medium on the in vitro maturation of oocytes and the in vitro culture of embryos. In this study, we investigated the effects of adding bovine amniotic membrane stem cell-conditioned medium (bAMSC-CM) to the IVC medium of ovine embryos. Moreover, the effects of bAMSC-CM were compared with the effects of charcoal-stripped fetal bovine serum (CSS). In the first experiment, culture media refreshment was performed on the third day of IVC (day 0 = IVF). The addition of bAMSC-CM and CSS to the IVC medium was conducted in three different ways: from day 1–3 of IVC, from day 1–7 of IVC, and from day 3–7 of IVC. In the second experiment, no culture medium renewal was conducted, and CSS and bAMSC-CM were added from the first day of IVC until the seventh day (CSS and CM groups). In both experiments, a control group (Con) was included that did not receive any supplementation. The data of embryonic cleavage of various treatments was aggregated. The cleavage rate of the embryos cultured in the CM-containing medium from the first day of IVC (CM1) was higher compared with the cleavage rate of the embryos cultured in the Con medium (P < 0.05). In the first experiment, the addition of bAMSC-CM from the onset of IVC resulted in a significant improvement in the blastocyst rate, hatching rate, and blastocyst cell numbers compared with the Con group (P < 0.05). The inclusion of CSS from the first day of IVC resulted in a significant reduction in both blastocyst rate and quality (P < 0.05). The addition of CSS on the third day of IVC resulted in a significant increase in the blastocyst rate compared with the Con group (P < 0.05). In the second experiment, the blastocyst rate and blastocyst cell count in the CM group showed a significant increase compared with the CSS and Con groups (P < 0.05). In conclusion, supplementing ovine IVC medium with bAMSC-CM from the start of IVC increased blastocyst rate and improved the quality of the resulting blastocysts. Furthermore, the need to refresh the culture medium on the third day of the culture has been eliminated.

Abstract C056: Characterization of prostate cancer secretomes for therapeutic intervention

Abstract Introduction: Prostate cancer (PC) is a common malignancy among men, often driven by dysregulated androgen receptor (AR) signaling. Earlier stages of PC can be favorably treated with androgen deprivation therapies or AR signaling inhibitors. Unfortunately, metastatic PC (mPC) is currently incurable. Neuroendocrine prostate cancer (NEPC) is a subgroup of mPC that may be derived as result of transdifferentiation of adenocarcinomas, after AR-directed treatment. Previous studies using preclinical models have suggested a potentially beneficial relationship between NEPC and AR-active prostate cancer (ARPC) cells. We hypothesize that secreted factors derived from NEPC cells influence local, and distant ARPC cell types to promote treatment resistance. Methods: Five NEPC in vitro cell models: NCI-H660, MSKCC-EF1, LTL331R, LuCaP-49 and LuCaP-173.1 were maintained in serum free media for 24 hours. The conditioned media (CM) was then collected and admixed with growth medium for ARPC cells. The ARPC growth medium was either supplemented with Fetal Bovine Serum (FBS) or Charcoal Stripped fetal bovine Serum (CSS). GFP-labelled ARPC cells: LnCaP and C42B were seeded in the respective media admixed with the CM. The viability of cells was measured using a Cytation5 microplate reader. We implemented a transcriptomic (RNAseq) and mass-spectrometry based proteomic analysis to identify differentially expressing secretory proteins that could have an impact on the treatment resistance of ARPC cell types. Results: All 5 NEPC-CM showed qualitative growth promoting effects towards ARPC C42B cells grown in CSS supplemented media. Only CM from EF1 and H660 promoted the growth of ARPC C42B in FBS supplemented media. NEPC-CM did not affect growth of LnCaP ARPC cells. Mass-spectrometry for secreted proteins was performed on 3 NEPC cell models. Analysis of the proteins against the PANTHER gene ontology database shows: (i) cell communication, and (ii) cellular response to stimulus, as two important biological processes active in NEPC. Genes involved in the two pathways were further compared against a comprehensive transcriptomic dataset spanning distinct mPC phenotypes and genotypes. Neurotrophic growth factors: Brain-derived growth factor (BDNF), Nerve-growth factor (NGF), VGF and neurotrophin-3 (NT-3) emerged as important targets that were differentially expressed in NEPC versus ARPC cells. Conclusions: NEPC cells have the potential to influence ARPC phenotypes that include enhanced proliferation, AR targeting, and potentially other drug resistance programs. As mPC exhibits substantial intra-individual and intra-tumor heterogeneity, understanding a targeting heterotypic cell interactions could lead to new druggable targets and address treatment resistance resulting from complex inter-cellular interactions. (Funding: PC220408) Citation Format: Tarana Arman, Lisa A. Jones, Ilsa M. Coleman, Philip R. Gafken, Peter S. Nelson. Characterization of prostate cancer secretomes for therapeutic intervention [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C056.

SAT428 The Effects Of Diphenyl Phthalate (DP) On Estrogen Receptor Alpha (ERα;) And Tumor Suppressor Gene (BRCA-1) In Breast Cancer Cells

Abstract Disclosure: K. Whittaker: None. E. Hallman: None. A. Zanib: None. S. Pfiffner: None. R. Yaldo: None. S. Dinda: None. Phthalates are a group of compounds that have been linked to xenoestrogenic activity which can lead to the proliferation of breast cancer cells. Specifically, Diphenyl Phthalate (DP) is a compound found in cosmetics, toys, plastic containers, and medical equipment. Previous studies have linked phthalates as endocrine-disrupting compounds (EDC) which may have an effect on diseases such as breast cancer. Due to the endocrine-disrupting actions of phthalates, it is critical to further investigate DP’s potential impact on breast cancer cells. This study investigates the estrogen-like effects of DP, both alone and in combination with hormones and antihormones on T-47D and MCF-7 breast cancer cell lines. The breast cancer cells were cultured in a medium that contained 5% charcoal-stripped fetal bovine serum (FBS) to ensure depletion of any endogenous steroids or growth factors. The cells were treated with varying concentrations of DP (25-250uM) for 24 hours. Through western blot analysis, alterations in the expression of ERα; related to the varying concentrations of DP were observed. A concentration-dependent decrease of ERα; expression levels was observed in both cell lines when compared to the control. A concentration-dependent increase of BRCA1 was seen in both cells. The optimal concentration of DP was revealed to be 200uM in T-47D breast cancer cells as it exhibited the most estrogen-like effect. The optimal concentration (200uM) was then used alone and in combination with hormones and antihormones for further investigation of DP on T47-D cells. Treatment with DP, E2 and the combination of DP and E2 for 24 hours showed a significant down-regulation of ERα in T-47D cells when compared to the control. These effects were sensitive to the presence of ICI showing significant down-regulation of ERα (approximately 80%) in T-47D cells. Image cytometric analysis with propidium iodide staining was utilized to examine the effects of DP on cellular viability in T-47D cells. Cells were treated for 6 days with DP concentrations ranging from 25-250 µM, and all concentrations displayed an increase in cell proliferation compared to the control in T47D cells. DP alone and in combination with E2 showed an increase in cell proliferation that was reversed when treated in combination with anti-hormones. RT-qPCR studies in T-47D cells revealed a transcriptional expression of ESR1 and BRCA-1 mRNA levels that correlate with the translational data from western blot analyses. Confocal microscopy, cell viability, and RT-qPCR analyses are in progress as well as evaluation on the effects of DP with hormones and antihormones on MCF-7 cells. Our studies offer an intriguing direction to explore the molecular mechanisms of DP as an EDC on the steroid receptors and tumor suppressor gene in breast cancer cells. Presentation: Saturday, June 17, 2023

A New Culture Model for Enhancing Estrogen Responsiveness in HR+ Breast Cancer Cells through Medium Replacement: Presumed Involvement of Autocrine Factors in Estrogen Resistance.

Hormone receptor-positive breast cancer (HR+ BC) cells depend on estrogen and its receptor, ER. Due to this dependence, endocrine therapy (ET) such as aromatase inhibitor (AI) treatment is now possible. However, ET resistance (ET-R) occurs frequently and is a priority in HR+ BC research. The effects of estrogen have typically been determined under a special culture condition, i.e., phenol red-free media supplemented with dextran-coated charcoal-stripped fetal bovine serum (CS-FBS). However, CS-FBS has some limitations, such as not being fully defined or ordinary. Therefore, we attempted to find new experimental conditions and related mechanisms to improve cellular estrogen responsiveness based on the standard culture medium supplemented with normal FBS and phenol red. The hypothesis of pleiotropic estrogen effects led to the discovery that T47D cells respond well to estrogen under low cell density and medium replacement. These conditions made ET less effective there. The fact that several BC cell culture supernatants reversed these findings implies that housekeeping autocrine factors regulate estrogen and ET responsiveness. Results reproduced in T47D subclone and MCF-7 cells highlight that these phenomena are general among HR+ BC cells. Our findings offer not only new insights into ET-R but also a new experimental model for future ET-R studies.

42 Adrenomedullin Stimulates Proliferation, Migration and Adhesion of Porcine Trophectoderm Cells Via CALCRL-AKT-TSC2-MTORC1 Cell Signaling Pathway

Abstract Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr1) isolated from day -12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via AKT-TSC2-MTOR cell signaling pathway in pTr1 cells. The pTr1 cells were cultured in DMEM/F12 medium with 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, 0.1 mM each for nutritionally nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 4 μg/mL insulin. Opti-MEM supplied with 2.5% (vol/vol) charcoal-stripped FBS was used for siRNA-mediated knockdown targeting non-treated control (siNTC) or specific genes including ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL). Cells were starved in FBS- and insulin-free medium for 24 hours before treatment. For proliferation assay, cell numbers were determined by staining with Janus-Green B after 48 h incubation. For migration assay, cells were treated with ADM after straight scratch in 6-well plates, and area of cell migration was calculated after 12 h treatment. For adhesion assay, cells were trypsinized in T-25 flasks and allowed for seeding in 96-well plates with density of 2×105 cells/0.2 mL/well, and the numbers of attached cells were determined after 12 h incubation. Western blot analyses were used to determine the expressions of target proteins at total and phosphorylated level. Porcine ADM at 10-7 M stimulated (P &amp;lt; 0.05) pTr1 cell proliferation, migration and adhesion by 1.4%-, 1.5%- and 1.2%-folds, respectively. These ADM-induced effects were abrogated (P &amp;lt; 0.05) by siADM and siCALCRL, as well as by rapamycin, the inhibitor of mechanistic target of rapamycin (MTOR). Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph act on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.

PMON13 The Effects of Perfluorooctanesulfonic Acid (PFOS) on the Expression of Estrogen Receptor Alpha (ERα) and Tumor Suppressor Gene BRCA1 in Breast Cancer Cells

Abstract Perfluoroctanesulfonic Acid (PFOS) is a perfluorinated chemical (PFC) and endocrine-disrupting compound (EDC) found in fire-extinguishing foams, food packing, and commonly used household products such as nonstick cookware and cleaning products. PFOS is also an environmental toxicant that has polluted air, soil, and water in past years. Few studies have suggested PFOS may cause endocrine-disrupting effects, therefore it is critical to explore the effects of PFOS on breast cancer cells. This study examined the estrogenic-like effects of PFOS, alone and in combination with hormones and antihormones, on the expression of ERα and BRCA1 in T-47D and MCF-7 breast cancer cell lines by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assay and RT-qPCR analyses. The cells were cultured with 5% charcoal-stripped fetal bovine serum (FBS) for six days to deplete endogenous steroids or growth factors. In the concentration-dependency study, cells were treated with various concentrations of PFOS (300µM-800µM) for 24 hours. Western blot analysis revealed alterations in the expression of ERα and BRCA1 related with these varying concentrations of PFOS (300 µM-800µM). In comparison to the control, a concentration-dependent decrease of ERα expression levels were seen in both cell lines. A concentration-dependent increase of BRCA1 was seen in both cells. Western blot analysis revealed the optimal concentration of PFOS to be 700 µM in cells, which exhibited the maximal estrogenic-like effect. To further examine the effects of PFOS on breast cancer cells, the optimal concentration (700 µM PFOS) was then used alone and in combination with hormones and anti-hormones in T-47D cells. Treatment with PFOS, E2and the combination of PFOS and E2 for 24 hours showed a significant down-regulation of ERα in T-47D cells when compared to the control. Treatment of PFOS with antiestrogen ICI revealed a significant down-regulation of ERα (approximately 50%) in T-47D cells. Image cytometric analysis with propidium iodide staining was utilized to examine the effects of PFOS on cellular viability in T-47D cells. PFOS alone and in combination with E2 revealed an increase in cellular proliferation compared to the control in T-47D cells and these effects were sensitive to the presence of antiestrogens. RT-qPCR studies revealed a transcriptional expression of ESR1 and BRCA-1 mRNA levels that correlate with the translational data obtained via western blot analyses. Cytolocalization experiments and effects of PFOS with hormones and antihormones on MCF-7 cells studies are in progress. Our studies provide exciting leads to clearly delineate the molecular mechanisms of PFOS as an EDC on the steroid receptors and tumor suppressor gene in breast cancer cells. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ODP275 The Effects of Tris(2-Chloroethyl) phosphate (TCEP) on the Expression of Estrogen Receptor Alpha and Tumor Suppressor Gene BRCA-1 in Breast Cancer Cell Lines

Abstract TCEP is an organophosphorus flame-retardant (OPFR) that is used widely in polyurethane foams, furniture, and household products. From these products, TCEP has significant emission into its surrounding environment. Inhalation and dermal absorption are the most common routes of human exposure to OPFRs, which have been measured in human hair, breast milk, and urine. Previous studies of OPFRs have shown their endocrine disrupting actions that have influence on diseases such as cancer. Breast cancer is the second most common cancer among women in the United States, and because of this prevalence, it is imperative to investigate possible causes and treatments for this disease. Due to the endocrine disrupting nature of OPFRs, we are investigating the effects of TCEP on hormone dependent breast cancer cell lines MCF-7 and T-47D. Our study examines the effects of TCEP, alone and in combination with hormones and anti-hormones, on ERα and BRCA1 expression in MCF-7 and T-47D breast cancer cells by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assays and RT-qPCR analyses. In order to deplete any endogenous steroids or effectors, breast cancer cells were cultured in a medium containing 5% charcoal-stripped fetal bovine serum for six days. Western blot analysis revealed alterations in the expression of ER-alpha after 24 hours of treatment with varying concentrations of TCEP (1µM-2mM). A concentration-dependent decrease of ERα protein levels was noted in the T-47D cell line when compared to the control. BRCA1 protein levels also displayed an altered expression compared to the control through the various concentrations of TCEP. Through our concentration studies, optimum concentrations of TCEP were found to be 100 µM for T-47D and 2mM for MCF-7. For our hormone studies, cell lines were treated with their respective optimum concentration of TCEP as well with combinations of hormones and anti-hormones. After 24-hour treatment of E 2, TCEP, and a combination of E2 with TCEP, a decrease in ERα expression was observed when compared to the control in both MCF-7 and T-47D cell lines. A combination of TCEP with ICI treatment revealed a significant down regulation of ERα expression level compared to the control. The same treatment conditions exhibited an increase with BRCA1 expression compared to the control and these effects were sensitive to the presence of antiestrogens in both cell lines. For cell viability studies, cells were treated for 6 days with TCEP concentrations ranging from 10nm-2µM which displayed an increase (40-50%) in cell proliferation compared to the control in both cell lines. Cytolocalization of ERα remained unaltered with the above treatment conditions. Our studies provide interesting findings about the molecular mechanisms of TECP as a potential endocrine disrupting compound on the steroid receptors and tumor suppressor genes in breast cancer cells. Presentation: No date and time listed

Pregnane X receptor (PXR) represses osteoblast differentiation through repression of the Hedgehog signaling pathway

The pregnane X receptor (PXR, NR1I2) belongs to the nuclear receptor family and functions as a xenobiotic and endobiotic sensor by binding to various molecules through its relatively flexible ligand-binding domain. In addition to these well-known canonical roles, we previously reported that PXR represses osteoblast differentiation. However, the mechanisms underlying the PXR-mediated repression of osteoblast differentiation remains unknown. In this study, we analyzed the changes in global gene expression profiles induced by PXR in calvarial osteoblasts cultured in standard fetal bovine serum (in which PXR induces repression of differentiation), and in those cultured in charcoal-stripped fetal bovine serum (in which PXR does not induce repression of differentiation). The comparison revealed that PXR attenuated the Hedgehog-mediated signaling in culture conditions that induced PXR-mediated repression of differentiation. Real-time PCR analysis showed that PXR repressed the Hedgehog signaling-induced genes such as Gli1 and Hhip, and conversely induced the Hedgehog signaling-repressed genes such as Cdon, Boc, and Gas1. Activation of Smo-mediated signaling in osteoblasts following treatment with a Smo agonist (SAG) significantly restored Gli-mediated transcriptional activity and osteoblast differentiation. Our results demonstrate the osteoblast-autonomous effects of PXR and identify a novel regulation of Hedgehog signaling by nuclear receptors.

Abstract P5-06-05: Palmitate induces a senescent-like phenotype in fibroblasts resulting in altered phenotypes in cells of the breast tumor microenvironment

Abstract Background: Obese breast cancer patients face a worse prognosis, including an increased risk of recurrence and mortality. While the causative mechanisms have yet to be fully uncovered, emerging evidence implicates palmitate, increased in the obese state, in development of cellular senescence, an inflammatory state associated with proliferation, metastasis, and tumor-associated neutrophil (TAN) polarization, among other measures of carcinogenesis. However, studies have yet to investigate the impact of palmitate on induction of senescence in breast fibroblasts, and no studies have assessed the effect of senescent fibroblasts on neutrophil polarization in the breast tumor microenvironment. This said, we hypothesize that palmitate alters breast cancer cell gene expression and neutrophil phenotype via induction of a senescent-like phenotype in fibroblasts. Methods: HCA2, IMR-90, and human mammary fibroblasts were exposed to palmitate or vehicle in media supplemented with 2% charcoal-stripped fetal bovine serum, after which the cells were measured through qPCR for expression of IL-1a, IL-6 and IL-8, some of the most prominent members of the senescence-associated secretory phenotype. Palmitate-exposed fibroblasts were also subjected to chromogenic staining for senescence-associated beta-galactosidase and immunoenzymatic BrdU analysis, two well-established measures of senescence. Next, in order to study the influence of these fibroblasts on other cells of the breast tumor microenvironment, we assessed their impact on polarization of DMSO-differentiated HL-60 neutrophils by using flow cytometry to measure neutrophil expression of CD54 and CD95, differentially expressed on N1 and N2 neutrophils. Finally, we employed PCR arrays to assess the impact of palmitate-exposed fibroblasts on the expression of 84 genes in MCF-7 and 231 breast cancer cells, measuring activation of pathways related to apoptosis, cell cycle, DNA damage, senescence, telomere maintenance, metabolism, angiogenesis, and the endothelial-to-mesenchymal transition. Results and Conclusions: Palmitate induced pro-inflammatory gene expression and SA-beta-gal activity and decreased BrdU incorporation in fibroblasts. Palmitate also exhibited non-cell-autonomous effects, as palmitate-exposed fibroblasts induced phenotypic changes in both neutrophils and breast cancer cells. These findings are among the first to implicate palmitate-induced fibroblast senescence in the stimulation of non-cell-autonomous changes in the breast tumor microenvironment and will ultimately inform our understanding of the mechanistic connection between the obesity-associated factor palmitate and breast tumorigenesis. Citation Format: Brittany Susanne Harlow, Albert Davalos, Bryan McClellan, Andrew Brenner, Christopher Jolly, Stefano Tiziani, Steve Hursting, Linda deGraffenried. Palmitate induces a senescent-like phenotype in fibroblasts resulting in altered phenotypes in cells of the breast tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-05.

N-Myc promotes angiogenesis and therapeutic resistance of prostate cancer by TEM8

Although patients with early localized prostate cancer can survive longer, castration-resistant prostate cancer (CRPC) has gradually emerged with the use of androgen deprivation therapy (ADT). N-Myc and TEM8 play a vital role in the progression of several cancer types. However, the underlying mechanism of how N-Myc and TEM8 promote the progression of prostate cancer remains unclear. In this study, the expression of N-Myc and TEM8 was detected in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) tissues by immunohistochemistry (IHC). LNCaP cell lines were maintained in RPMI 1640 medium supplemented with 10% charcoal-stripped fetal bovine serum. Subsequently, R language software was used to verify our results. Tubule formation assay of human umbilical vein endothelial cell (HUVEC) was conducted to examine the effect of N-Myc and TEM8 overexpression on angiogenesis in prostate cancer cells. IHC results showed a positive correlation between the expression of N-Myc and TEM8 in prostate cancer tissues. Further analysis showed that N-Myc and TEM8 were associated with clinicopathological features and poor prognosis in prostate cancer patients. Moreover, the overexpression of N-Myc and TEM8 promoted proliferation of prostate cancer cells and angiogenesis. Additionally, N-Myc and TEM8 overexpression was associated with therapeutic resistance. We further found that N-Myc promoted angiogenesis and therapeutic resistance in prostate cancer via TEM8. Hence, targeting N-Myc/TEM8 pathway in prostate cancer would be a novel therapeutic strategy to enhance the treatment of prostate cancer patients.

Abstract 2022: Palmitate promotes breast cancer progression in vitro through induction of a senescent-like phenotype in fibroblasts

Abstract Background: Obesity confers a worse breast cancer prognosis, including an increased risk of recurrence and mortality. While the causative mechanisms have yet to be fully uncovered, emerging evidence implicates palmitate, increased in the obese state, in development of cellular senescence, an inflammatory state associated with breast tumorigenesis in preclinical models. However, studies are warranted to corroborate the impact of palmitate on induction of a cohesive senescent-like phenotype as well as the extent to which palmitate-induced senescence impacts breast tumorigenesis either in vitro or in vivo. This said, we hypothesize that palmitate exposure induces a senescent-like phenotype in fibroblasts, contributing to measures of breast cancer progression. Methods: HCA2, IMR-90, and human mammary fibroblasts were exposed to bovine serum albumin or palmitate in media supplemented with 2% charcoal-stripped fetal bovine serum, after which the cells were measured through qPCR for expression of IL-1a, IL-6 and IL-8, some of the most prominent members of the senescence-associated secretory phenotype. Palmitate-exposed fibroblasts were also stained for senescence-associated beta-galactosidase and BrdU incorporation, well-established senescence markers. Experiments were then repeated with administration of eicosapentaenoic and docosahaexanoic acids to evaluate the potential of omega-3 fatty acids to limit the effects of palmitate on fibroblast senescence. Finally, we assessed the tumor-promoting potential of these palmitate-exposed fibroblasts by culturing MCF-7 and T47D breast cancer cells in their conditioned media and assessing changes in carcinogenic measures. Results and Conclusions: Palmitate induced pro-inflammatory gene expression and SA-beta-gal positivity and decreased proliferation in fibroblasts, while omega-3 fatty acid supplementation reversed these effects. These palmitate-exposed fibroblasts also appeared to be of pathological impact, as exposure to their CM increased proliferation in breast cancer cells. These findings are important in that they support emerging evidence implicating obesity-associated factors in the exacerbation of breast cancer progression as well as indicate the potential of omega-3 fatty acids to improve outcome. Citation Format: Brittany Susanne Harlow, Albert R. Davalos, Andrew J. Brenner, Christopher Jolly, Stefano Tiziani, Stephen D. Hursting, Linda A. deGraffenried. Palmitate promotes breast cancer progression in vitro through induction of a senescent-like phenotype in fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2022.

Abstract 1935: Antitumor effect mechanism of statin in PCa cells focusing on autophagy

Abstract Antitumor effect mechanism of statin in prostate cancer cells focusing on autophagy Background:Statins have recently been studied for their proapoptotic and antimetastatic effects. However, the exact mechanisms of their anticancer actions remain unclear. We have studied the novel castration resistant prostate cancer (CRPC) treatment with statins. Using microarrays, we discovered up-regulation of ATG4B (Autophagy related 4 homolog B) in PC-3 cells after simvastatin treatment. In this study, we evaluated the effects of simvastatin focusing on androgen-independent prostate cancer cells. Methods:PC-3, LNCaP-LA, 22RV-1-LA (taht were derived from LNCaP and 22RV1 cells and cultured in 10% charcoal-stripped fetal bovine serum for 3 months), and DU145 human prostate cancer cell lines were used. Microarrays were performed using PC-3 untreated or administered with simvastatin (0 uM versus 5 uM). Proliferation, migration, and invasion were evaluated using MTS assay, Migration assay, and Invasion assay, respectively. The increases of autophagy were detected by western blot using microtubule associated protein 1 light chain 3 (LC-3) antibody and confirmed by a fluorescence microscope with staining autophagosomes by autophagy detection kit (abcam, Cambridge, UK). Results:Using microarrays, we discovered up-regulation of ATG4B in PC-3 cells after simvastatin treatment. Simvastatin inhibited the proliferation, migration, and invasion of PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. The expression level of LC-3 was up-regulated by simvastatin in PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. In PC-3 cells treated with statin, a significant increase in autophagosomes was confirmed by fluorescence microscopy. The expression of LC-3 and autophagosomes were increased in a concentration-dependent manner. Conclusions:It was found that statin administration enhances autophagy induction in a prostate cell line. We plan to study the combined effect with other drugs that induce autophagy. We would like to develop CRPC treatment by inducing autophagy cell death. Citation Format: Yoshiyuki Miyazawa, Daisuke Oka, Hiroshi Nakayama, Yoshitaka Sekine, Seiji Arai, Kazuhiro Suzuki. Antitumor effect mechanism of statin in PCa cells focusing on autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1935.

The Effects of Tert-butyl Hydroquinone (TBHQ) on Estrogen Receptor Alpha (ERα) and Tumor Suppressor Gene p53 in Breast Cancer Cells

Tert-butyl hydroquinone (TBHQ) is an aromatic compound that is commonly used as a preservative in processed food to prevent rancidity and lengthen shelf life. TBHQ is known to act as an antioxidant by protecting cells from radical oxygen species and thus preventing DNA damage. Although previous studies have found TBHQ to cause cancer cell death at high concentrations, they have also contrastingly found TBHQ, when studied in animal models, to enhance carcinogenic effects. However, the effect of TBHQ on breast cancer has not been thoroughly explored. With the prevalence of breast cancer and the wide use of TBHQ in processed food items, it is imperative that we explore their possible relationship. This study examined the effects of TBHQ, alone and in combination with hormones and anti-hormones, on ERα and p53 expression in both MCF-7 and T-47D breast cancer cell lines. To ensure treatment conditions without the presence of endogenous steroids or growth factors, the cells were cultured with a 5% charcoal-stripped fetal bovine serum (FBS) for six days. Western blot analysis revealed alterations in the expression of ERα and p53 protein levels after 24 hours of treatment with varying concentrations of TBHQ (0.005 to 1 mM). A concentration-dependent decrease of ERα protein levels was observed in both cell lines, with a 49% reduction occurring with 100 µM TBHQ as compared to the control. P53 levels portray a continued increase of expression through concentrations of TBHQ (0.005 to 1 mM), found similarly in both cell lines. To gain further insight into possible similarities between BPS and other known effectors of ERα, the optimal concentration of TBHQ (100 μM) was used in combination with hormones and anti-hormones. Down-regulation of ERα protein levels was observed after 24-hour co-treatment of T-47D & MCF-7 cells with a combination of TBHQ and E2. Antiestrogen ICI with TBHQ showed a significant down-regulation as compared to TBHQ alone, and TBHQ with TAM portrayed no significant differences. A similar trend in the effects on p53 expression was depicted in T-47D and MCF-7 cells. Image cytometric analysis with propidium iodide staining was utilized to quantify cell values and viability changes to further portray the effects of TBHQ on T-47D and MCF-7 cellular growth. The viability assay shows a biphasic effect with increasing concentrations of TBHQ, with a maximum decrease in proliferation seen at a concentration of 100 uM TBHQ. TBHQ alone and in combination with E2 and antiestrogens showed a decreased proliferation compared to the control in T-47D cells. However, cytolocalization of ERα upon treatment with estradiol and TBHQ remained unaltered. Our studies offer a unique perspective on the effects of TBHQ on two different breast cancer cell lines, and provide valuable insight for further exploration of the mechanism of action of TBHQ on tumor suppressor gene and steroid receptors.

Differential impacts of charcoal-stripped fetal bovine serum on c-Myc among distinct subtypes of breast cancer cell lines

Charcoal-stripped fetal bovine serum (CS-FBS) is frequently used in studies on hormone-responsive cancers to provide hormone-free cell culture conditions. CS-FBS may influence the growth of cancer cells; however, the underlying mechanisms remain unclear. In this study, we aimed to clarify the effects of CS-FBS on distinct subtypes of breast cancer cells. We found that the crucial oncoprotein c-Myc was significantly inhibited in estrogen receptor alpha (ER-α)-positive breast cancer cells when cultured in CS-FBS-supplemented medium, but it was not suppressed in ER-α-negative cells. The addition of 17β-estradiol (E2) to CS-FBS-supplemented medium rescued the CS-FBS-induced inhibition of c-Myc, while treatment with 5α-dihydrotestosterone (DHT) suppressed c-Myc expression. Our data demonstrated that CS-FBS may impede the growth of ER-α-positive breast cancer cells via c-Myc inhibition, and this was possibly due to the removal of estrogen. These results highlighted that the core drivers of c-Myc expression were subtype-specific depending on the distinct cell context and special caution should be exercised when using CS-FBS in studies of hormone-responsive cancer cells.

Hormone-Like Effects of Bisphenol A on p53 and Estrogen Receptor Alpha in Breast Cancer Cells.

Bisphenol A (BPA) is a polymerizing agent commonly found in plastics that has been linked to xenoestrogenic activity. In this study, we analyzed the estrogen-like effects of BPA on the expression of estrogen receptor (ER)α and p53 with hormonal and antihormonal treatments in T-47D and MCF-7 cells. Cells were cultured in medium containing 5% charcoal-stripped fetal bovine serum for 6 days to deplete any endogenous steroids or effectors. The cells were then treated for 24 h with 600 nM BPA, which was determined to be the optimal value by a concentration study of BPA from 1 nM to 2 μM. Extracted cellular proteins were quantified and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot analysis. The cell proliferation assays were quantified upon exposure to BPA. Laser confocal microscopy was performed to determine the cytolocalization of p53 and ERα upon treatment with BPA. Western blot analysis revealed that BPA caused an increase in the cellular protein p53 in a concentration-dependent manner. While treatment with BPA did not affect the cytolocalization of p53, an increase in cell proliferation was observed. Our studies provide interesting leads to delineate the possible mechanistic relationship among BPA, ER, and tumor suppressor proteins in breast cancer cells.

Upregulation of erythropoietin and erythropoietin receptor in castration-resistant progression of prostate cancer.

Hypoxia-induced erythropoietin signaling plays an important role in tumor growth and invasion. In the present study, we investigated the contribution of erythropoietin signaling pathway to castration-resistant prostate cancer and the development of a neuroendocrine phenotype. Immunohistochemical staining showed that the erythropoietin and erythropoietin receptor scores in castration-resistant prostate cancer and androgen-dependent prostate cancer were 7.55 versus 4.5 and 7.45 versus 5.9, respectively (P < 0.001). Furthermore, a cell proliferation assay was conducted, and the differential expression of erythropoietin and erythropoietin receptor in LNCaP cells and hypoxia-induced LNCaP cells was evaluated using western blot and quantitative real-time PCR. The proliferation capacity of hypoxia-induced LNCaP cells was similar in cultures of both fetal bovine serum and charcoal-stripped fetal bovine serum, suggesting that LNCaP cells acquired hypoxia-induced androgen-independent growth. After 2 weeks of hypoxic culture, LNCaP cells showed a neuroendocrine cell change and increased expression of neuron-specific enolase, erythropoietin, and erythropoietin receptor; knockdown of erythropoietin receptor reversed the hypoxia-induced upregulation of neuron-specific enolase in the LNCaP cells. In conclusion, the concurrent upregulation of erythropoietin and erythropoietin receptor in castration-resistant prostate cancer suggests that the erythropoietin/erythropoietin receptor autocrine loop plays an important role in the progression of castration resistance and is responsible for the development of a neuroendocrine phenotype.

MON-038 The Effects of Isoliquiritigenin (ISL) on the Expression of Estrogen Receptor and Tumor Suppressor Genes BRCA-1 and P53 in Breast Cancer Cells

Naturally sourced treatments are increasingly sought for health issues including breast cancer. Isoliquiritigenin (ISL), a phenolic flavonoid in licorice root, has recently received attention in public and scientific communities for its antioxidant, anti-inflammatory and tumor suppressive effects. Standard medical practice now includes determining receptor status of breast cancers and estimates show that 55-65% of women with a BRCA1 gene mutation will develop hereditary breast cancer, therefore finding treatment options for receptor-positive patients is critically important. This study examined the effects of ISL, alone and in combination with hormones and anti-hormones, on ERα, p53, and BRCA1 expression in MCF-7 and T-47D breast cancer cells by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assay and RT-qPCR analyses. To ensure treatment conditions without the presence of endogenous steroids or growth factors, cells were cultured in a medium containing 5% charcoal-stripped fetal bovine serum (FBS) for a duration of 6 days. For western blot analyses, cells were treated with various concentrations of ISL ranging from 5-100 µM for 24 hours. Compared to the control, a concentration dependent decrease of ERα (85%) and BRCA1 (60%) was noted in both cell lines, while the expression of p53 seemed to be altered based on the specific breast cancer cell lines. In our hormone studies, we used the optimal concentration of 50μM ISL alone and in combination with hormones and anti-hormones. After 24-hour treatment of ISL, E2, and E2 with ISL on the breast cancer, cells showed a decrease in ERα and BRCA-1 expression compared to the control. Combination treatment of ISL with anti-hormone ICI revealed a significant down regulation (approximately 65-85%) of ERα, BRCA-1, and p53 in T-47D cells. The same treatment conditions in MCF-7 cells revealed a significant down regulation of ERα and BRCA-1, while p53 expression was up regulated. Image cytometric analysis with propidium iodide staining was performed to examine the effects that ISL has on cellular viability in T-47D and MCF-7 cells. Cells treated for 6 days with 50 μM ISL concentration with hormone and anti-hormones displayed a decrease in cell proliferation compared to the control in both cell lines. RT-qPCR studies revealed a transcriptional expression of ESR1 and BRCA-1 mRNA levels that correlate with the translational data obtained via western blot analyses. Cytolocalization studies are in progress and apoptosis assays are currently being performed to determine the mechanism(s) of cell death. Our studies offer an intriguing direction to explore the molecular mechanisms of ISL on the steroid receptors and tumor suppressor gene in breast cancer cells.