Treatment Of T47D Cells Research Articles

Letrozole and zoledronic acid changed signalling pathways involved in the apoptosis of breast cancer cells

ObjectivesOestrogen plays a key role in the development of breast malignancies. Therefore, aromatase inhibitors (e.g. letrozole [LTZ]) are widely used in the treatment of breast cancer. On the other hand, oestrogen is important to the integrity of bone mass. Research has shown that zoledronic acid (ZLA) may prevent osteoporosis. Therefore, the present research aims to investigate the effect of a combination of LTZ and ZLA in the treatment of breast cancer and in reducing osteoporosis in patients with breast cancer. MethodsWe used immunocytochemistry and Western immunoblotting techniques in this study. ResultsWe observed that LTZ inhibited cellular growth of Michigan Cancer Foundation-7 (MCF-7) and T-47D at IC50 (70 ± 0.001) and (140 ± 0.004) nM, respectively, whereas ZLA inhibited cellular growth at IC50 (50 ± 0.005) μM and (150 ± 0.004) μM for MCF-7 and T-47D cell lines, respectively. Interestingly, the LTZ and ZLA combination down-regulated the protein expression of signal transducer and activator of transcription 3 (STAT3) and up-regulated BRCA1 protein expression in both cell lines. Moreover, a notable enhancement in the nuclear localisation of the BRCA1 protein was obtained after treatment of T-47D cells with LTZ for 24 h compared to the control cells. In contrast, there was a reduction in the nuclear localisation of STAT3 protein, which could be an attractive target for inhibition of breast cancer proliferation and progression. ConclusionOur study has shown that a combination of LTZ and ZLA enhanced apoptosis and inhibited growth of both breast cancer cell lines. This combination can be used to maintain bone integrity in women with breast cancer.

Adenosine A1 Receptor Antagonist Up-regulates Casp3 and Stimulates Apoptosis Rate in Breast Cancer Cell Line T47D

Background: Adenosine receptor family, especially A1 type is-overexpressed in breast-derived tumor cells and the P53 gene is mutant in some of these cells while the casps gene is of wild type as well. The aim of this study was to evaluate the effect of the A1 receptor function on cell programmed death or proliferation, as well as the relationship between this receptor stimulation/inhibition and caspase 3 (casp3) expression in T47D cell line that has a mutant and non-functional P53 gene. Materials and Methods: The expression of casps3 was measured by real-time polymerase chain reaction and then flow cytometery and MTT assay were used to assess the apoptotic and proliferation cell rate after the treatment of T47D cells with specific agonist N6-cyclopentyladenosine (CPA) and antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) of this receptor 24, 48, and 72 hours after treatment. Result: Our results indicated that DPCPX significantly induces apoptosis in T47D cells and the rate of survival cell after the reduction of this treatment, especially 72 hours after treatment. Finally, the expression of casp3 was up-regulated by DPCPX treatment, especially in 72 hours while CPA treatment had opposite results (P>0.05). Conclusion: In general, DPCPX could up-regulate casp3 gene expression and subsequently increase the apoptosis rate in T47D cells with casp3 expression without the P53 gene interference. Therefore, adenosine A1 receptor antagonists may be introduced as anti-cancer agents.

Visualization of Breast Cancer Metabolism Using Multimodal Nonlinear Optical Microscopy of Cellular Lipids and Redox State.

Label-free nonlinear optical microscopy (NLOM) based on two-photon excited fluorescence (TPEF) from cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+) is widely used for high-resolution cellular redox imaging. In this work, we combined three label-free NLOM imaging methods to quantitatively characterize breast cancer cells and their relative invasive potential: (i) TPEF optical redox ratio (ORR = FAD+/NADH + FAD+), (ii) coherent Raman scattering of cellular lipids, and (iii) second harmonic generation of extracellular matrix (ECM) collagen. 3D spheroid models of primary mammary epithelial (PME) cells and breast cancer cell lines (T47D and MDA-MB-231) were characterized based on their unique ORR and lipid volume fraction signatures. Treatment with 17β-estradiol (E2) increased glycolysis in both PME and T47D ER+ breast cancer acini. However, PME cells displayed increased lipid content with no effect on ECM, while T47D cells had decreased lipid storage (P < 0.001) and significant reorganization of collagen. By measuring deuterated lipids synthesized from exogenously administered deuterium-labeled glucose, treatment of T47D cells with E2 increased both lipid synthesis and consumption rates. These results confirm that glucose is a significant source for the cellular synthesis of lipid in glycolytic breast cancer cells, and that the combination of cellular redox and lipid fraction imaging endpoints is a powerful approach with new and complementary information content.Significance: These findings provide unique insight into metabolic processes, revealing correlations between cancer metastasis and cellular redox state, lipid metabolism, and extracellular matrix. Cancer Res; 78(10); 2503-12. ©2018 AACR.

Synergistic Growth Inhibitory Effects of Chrysin and Metformin Combination on Breast Cancer Cells through hTERT and Cyclin D1 Suppression

Objective:To explore the possibility of a novel chemopreventive strategy for improving breast cancer treatment, the anticancer effects of a combination two natural compounds, Chrysin and Metformin, against T47D breast cancer cells were investigated.Materials and Methods:After treatment of T47D cells with Metformin, Chrysin and the two drugs in combination, toxicity to cancer cells was evaluated by MTT assay. Real time PCR was then used to determine the expression levels of hTERT and cyclin D1 genes.Results:The MTT test findings showed that the combination of metformin and chrysin had high synergistic effects in killing cancer cells. In addition PCR demonstrated a significant decrease in cyclin D1 and hTERT gene expression in the T47D breast cancer cell line.Conclusion:The conmbination of metformin and chrysin suppressing hTERT and cyclin D1 gene expression might offer an appropriate approach for breast cancer therapy.

Cholesterol biosynthesis inhibitor RO 48-8071 reduces progesterone receptor expression and inhibits progestin-dependent stem cell-like cell growth in hormone-dependent human breast cancer cells.

Clinical trials and studies have shown that postmenopausal women undergoing combination hormone replacement therapy containing estrogen and progestin have an increased risk of breast cancer compared with women taking estrogen or placebo alone. Using animal models, we have previously shown that synthetic progestins, including medroxyprogesterone acetate (MPA), which is widely used clinically, accelerate breast cancer tumor growth and promote metastasis. Furthermore, we have found that MPA elevates CD44 protein expression and aldehyde dehydrogenase (ALDH) activity, two markers of cancer stem cells (CSCs), and increases mammosphere formation, another hallmark of stem cells, in hormone-dependent T47-D human breast cancer cells. Herein, we show that RO 48-8071 (RO), an inhibitor of cholesterol synthesis, reduced MPA-induced CD44 protein expression in two hormone-dependent human breast cancer cell lines, T47-D and BT-474. Because we have previously shown that MPA induction of CD44 is progesterone receptor (PR) dependent, we examined RO’s effects on PR protein and mRNA expressions in T47-D cells. PR mRNA levels remained unchanged after RO treatment; however, RO significantly reduced the protein expression of both PR receptor isoforms, PR-A and PR-B. Using the proteasome inhibitor MG-132, we demonstrated that RO decreases PR protein expression in T47-D cells via the proteasomal degradation pathway. Importantly, treatment of T47-D cells with RO abolished MPA-induced mammosphere formation. Based on our observations, we contend that RO may represent a novel means of preventing MPA-induced CSC expansion. RO could be used clinically to both treat and prevent hormone-dependent breast cancers, which represent the majority of human breast cancers. RO may also have clinical utility in reducing resistance to antihormone therapy.

Abstract 3003: p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines

Abstract Background: The p73 gene is a member of the p53 tumor suppressor family. A dinucleotide polymorphism (DNP) in p73 is a G4C14-to-A4T14 linked pair of transitions located in exon 2, between the P1 and P2 promoters, from which TAp73 and DeltaNp73 (DNp73) mRNA isoforms are transcribed, respectively. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and deltaNp73 (DNp73) isoforms, respectively. These p73 mRNA isoforms, TAp73 and αNp73, encode protein isoforms which differ in their N-termini. TAp73 isoforms include the full-length N-terminal sequence and are transcriptionally active. αNp73 isoforms lack the N-terminal trans-activation domain and are dominate negative. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive prostate cancer and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. Etoposide is known to induce p73 gene expression. Others recently reported treatment of T-47D cells with 5-aza-2’-deoxycytidine (5AZA) resulted in increased TAp73/DNp73 mRNA ratios. Hypothesis: We hypothesized that treatment with 5AZA increases the TAp73/DNp73 mRNA ratios in human cell lines and that the presence of the p73 DNP allele potentiates 5AZA-induced increases TAp73/DNp73 cellular ratios. Methods. We treated human cancer cell lines with 50 microM etoposide for 24 hrs and/or 20 microM 5AZA for 48 hrs. RNA was isolated, converted to cDNA and used in TaqMan RT-PCR assays to detect total p73, TAp73 or αNp73 mRNAs. Results: Cell lines wild-type for p73 DNP (DU145, T47-D, MDA-MB-468) had TAp73/DNp73 mRNA ratios that were lower than those heterozygous for p73 DNP (PC-3 and MDA-MB-231) following co-treatment with 5AZA and etoposide or with etoposide alone (p&amp;lt;0.01 for DU145 &amp; T47-D). PC-3 and MDA-MB-231 cell lines had TAp73/DNp73 mRNA ratios following 5AZA treatment (0.8546 and 0.7138, respectively) that were equal to or greater than the three cell lines wild type for p73 DNP: DU145, T47-D and MDA-MB-468 (0.8626, 0.6480, 0.3866, respectively). Conclusion: Of the five cell lines used in this study, cells that harbor a p73 DNP allele had the highest TAp73/DNp73 mRNA ratios following co-treatment with 5AZA and etoposide or with etoposide alone as compared to wild-type cell p73 DNP, which is consistent with our previously reported data. Lastly, our data in this work assessing the effect of 5AZA treatment of T47-D cells on TAp73/DNp73 mRNA ratios is not consistent with a previously reported study. Citation Format: Zachary Connelly, L. Michael Carastro. p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3003.

Bisphenol AF stimulates transcription and secretion of C-X-C chemokine ligand 12 to promote proliferation of cultured T47D breast cancer cells.

Bisphenol AF (4,4'-hexafluoroisopropylidene-2-diphenol, BPAF), an endocrine disruptor, has been shown to stimulate the proliferation of human breast cancer cells. However, the underlying mechanism has not been fully elucidated. We found that BPAF promoted the in vitro proliferation of estrogen receptor α (ERα)-positive breast cancer cells (T47D and MCF7), but not ERα-negative cells (MDA-MB-231 and MDA-MB-435s). BPAF significantly stimulated the proliferation of cultured T47D cell in a dose-dependent manner, and the half-maximal effective concentration (EC50) was approximately 123 nM. We employed lentivirus-mediated short hairpin RNA (shRNA) to knockdown ERα and ER antagonist ICI 182780 to inhibit ER activation, which resulted in the repression of BPAF-induced proliferation of T47D and MCF7 cells. We observed that C-X-C chemokine ligand 12 (CXCL12) was up-regulated in T47D cells under treatment with BPAF. Quantitative real-time PCR results showed that BPAF caused a time and dose dependent increase in mRNA level of CXCL12. Furthermore, treatment of T47D cells with BPAF increased CXCL12 secretion according to ELISA assay. BPAF-induced CXCL12 transcription and secretion was significantly attenuated by small interfering RNA (siRNA) targeting ERα and ICI 182780, indicating BPAF-induced CXCL12 expression is mediated through ERα. Notably, knockdown CXCL12 in T47D cells significantly attenuated BPAF-induced cell proliferation. We also observed that inhibition of CXCL12 binding to its receptors CXCR4 and CXCR7 by chalcone 4 blocked BPAF-induced cell growth. Our results indicated that CXCL12 facilitated BPAF-induced proliferation of T47D cells. Taken together, our data provided support that BPAF stimulated transcription and secretion of CXCL12 depending on ERα, and ERα/CXCL12 signaling positively regulated BPAF-induced proliferation of cultured T47D breast cancer cells.

In vitro and in vivo application of radiolabeled gastrin-releasing peptide receptor ligands in breast cancer.

Breast cancer (BC) consists of multiple subtypes defined by various molecular characteristics, for instance, estrogen receptor (ER) expression. Methods for visualizing BC include mammography, MR imaging, ultrasound, and nuclear medicine-based methods such as (99m)Tc-sestamibi and (18)F-FDG PET, unfortunately all lacking specificity. Peptide receptor scintigraphy and peptide receptor radionuclide therapy are successfully applied for imaging and therapy of somatostatin receptor-expressing neuroendocrine tumors using somatostatin receptor radioligands. On the basis of a similar rationale, radioligands targeting the gastrin-releasing peptide receptor (GRP-R) might offer a specific method for imaging and therapy of BC. The aim of this study was to explore the application of GRP-R radioligands for imaging and therapy of BC by introducing valid preclinical in vitro and in vivo models. GRP-R expression of 50 clinical BC specimens and the correlation with ER expression was studied by in vitro autoradiography with the GRP-R agonist (111)In-AMBA. GRP-R expression was also analyzed in 9 BC cell lines applying (111)In-AMBA internalization assays and quantitative reverse transcriptase polymerase chain reaction. In vitro cytotoxicity of (177)Lu-AMBA was determined on the GRP-R-expressing BC cell line T47D. SPECT/CT imaging and biodistribution were studied in mice with subcutaneous and orthotopic ER-positive T47D and MCF7 xenografts after injection of the GRP-R antagonist (111)In-JMV4168. Most of the human BC specimens (96%) and BC cell lines (6/9) were found to express GRP-R. GRP-R tumor expression was positively (P = 0.026, χ(2)(4) = 12,911) correlated with ER expression in the human BC specimens. Treatment of T47D cells with 10(-7) M/50 MBq of (177)Lu-AMBA resulted in 80% reduction of cells in vitro. Furthermore, subcutaneous and orthotopic tumors from both BC cell lines were successfully visualized in vivo by SPECT/CT using (111)In-JMV4168; T47D tumors exhibited a higher uptake than MCF7 xenografts. Targeting GRP-R-expressing BC tumors using GRP-R radioligands is promising for nuclear imaging and therapy, especially in ER-positive BC patients.

Cell inactivation by combined low dose-rate irradiation and intermittent hypoxia

Purpose: To investigate in detail the earlier observed combined effect of low dose-rate β-irradiation delivered at a dose-rate of 15 mGy/h and continued intermittent hypoxia that leads to extensive cell death after approximately 3–6 weeks.Material and methods: Continuous low dose-rate β-irradiation at a dose rate of 15, 1.5 or 0.6 mGy/h was given by incorporation of [3H]-labelled valine into cellular protein. The cells were cultivated in an atmosphere with 4% O2 using an INVIVO2 hypoxia glove box. Clonogenic capacity, cell-cycle distribution and cellular respiration were monitored throughout the experiments.Results: After 3–6 weeks most cells died in response to the combined treatment, giving a surviving fraction of only 1–2%. However, on continued cultivation a few cells survived and restarted proliferation as the cellular oxygen supply increased with the reduced cell number. Irradiating the T-47D cells grown in an atmosphere with 4% O2 at dose-rates 10 and 25 times lower than 15 mGy/h did not have a pronounced effect on the clonogenic capacity with surviving fractions of 60–80%.Conclusions: Treatment of T-47D cells with low dose-rate β-irradiation leads to a specific effect on intermittent hypoxic cells, inactivating more than 98% of the cells in the population. Given improved oxygen conditions, the few surviving cells can restart their proliferation.

Lithium increases radiosensitivity by abrogating DNA repair in breast cancer spheroid culture.

Lithium chloride (LiCl), a drug for bipolar disorder, has antiproliferative and apoptotic effects on certain breast cancer cell lines. This study was conducted to determine the effect of LiCl on radiosensitivity in a human breast cancer cell line in monolayer culture and the more realistic tumor model, multicellular tumor spheroid. Monolayer and spheroid cells were treated with LiCl (20 mM) for 24 hours. The clonogenic assay was used to indicate changes in survival after x-ray radiation. The percentage of apoptotic cells was determined by acridine orange/ethidium bromide double staining. The amounts of DNA damage and repair after exposure to ionizing radiation were assessed by comet assay. Mre11 mRNA level was determined by RT-PCR. GSK-3β and β-catenin protein levels were measured by Western blotting. Treatment with LiCl decreased surviving fraction at 2, 3 and 6 Gy doses of x-ray (P < 0.01). The sensitizer enhancement ratio was higher in spheroids than monolayer culture. LiCl also decreased DNA repair (P < 0.05) and Mre11 mRNA level (P < 0.01) in T47D cells. These decreases were more prominent in spheroids than monolayer culture. Treatment of T47D cells with LiCl sensitized this breast cancer cell line to ionizing radiation in monolayer and especially in the tumor-like spheroid culture. This radiosensitization was attributed, in part, to decline in DNA repair. Decrease in Mre11 mRNA level upon LiCl treatment was suggested to be an important cause for the decreased DNA repair in T47D monolayer and spheroid cells.

Antimanic drug sensitizes breast cancer cell line to ionizing radiation

Breast cancer is one of the most prevalent types of cancer among women. Lithium chloride (LiCl) is an FDA-approved drug for bipolar disorder. Breast cancer is reported to occur with higher rate in women with bipolar disorder. The effect of LiCl on the response of breast cancer cells to ionizing radiation has not been studied. We studied the effect of LiCl on the radiosensitivity of radioresistant T47D breast cancer cell line. Treatment of T47D cells with 20 mM LiCl for 24 hours decreased the radioresistance of these cells indicated by clonogenic survival assay. Comet assay demonstrated decreased DNA repair in LiCl-treated cells. LiCl treatment also decreased the meiotic recombination 11 (Mre11) mRNA level. Mre11 is an essential protein for DNA repair whose transcription is regulated by β-catenin protein. Western blot analysis indicated that the β-catenin protein level was decreased in LiCl-treated cells. LiCl increased glycogen synthase kinase-3β (GSK-3β) protein that is involved in β-catenin degradation. The results demonstrated that LiCl could radiosensitize T47D cells by decreasing DNA repair, partially through Mre11 repression. GSK-3β/β-catenin/Mre11 pathway might be the connection between LiCl treatment and the decreased DNA repair in T47D cells.

Abstract 3569: Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.

Abstract Progesterone receptors (PR) are emerging as important breast cancer drivers. Elevated mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2) signaling is common in breast cancer and these kinases drive direct PR-B, but not PR-A, Ser294 phosphorylation. Post-translational modifications, including phosphorylation and SUMOylation, dramatically impact PR transcriptional activity. Phospho-Ser294 PRs are less SUMOylated on Lys388 (i.e. a repressive modification). Thus, active mitogenic kinase signaling provides a mechanism for PR derepression (i.e. transcriptional activation) during breast cancer progression. A broad range of PR protein expression is observed clinically; PR-negative tumors are generally more aggressive. However, clinical (IHC) assays do not measure phospho-PRs, which turnover rapidly and may appear undetectable, confounding the true level of activated PR within tumors. A PR gene signature would provide a valuable marker of PR contribution to breast cancer progression. Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild type or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Target genes sensitive to changes in PR SUMOylation included those required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors were preferentially recruited to enhancer regions of derepressed genes. SUMO-deficient (i.e. phospho-Ser294) PR gene signatures were significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Furthermore, treatment with antiprogestin or MEK inhibitors abrogated expression of SUMO-sensitive PR target-genes and inhibited progestin-driven proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells. Similarly, treatment of T47D cells expressing deSUMOylated PR with ERBB2 inhibitors blocked progestin-dependent growth. Potential mechanisms of differential promoter selection were investigated. Notably, genes preferentially up-regulated by SUMO-deficient PR contain RUNX binding motifs located near their transcriptional start sites. Herein, we demonstrated that RUNX2 directly interacts with PR. Further, RUNX2 knockdown in breast cancer cells reduced SUMO-deficient PR dependent gene expression. Our studies suggest that deSUMOylated PR cooperates with RUNX2 to mediate promoter selective gene expression. We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters PR target gene selection in breast cancer cells, in part via regulation of PR interactions with gene-specific co-factors, such as RUNX2. Patients whose ER+/PR+ tumors are driven by hyperactive phospho-PRs may benefit from endocrine (antiestrogen) therapies that also contain an antiprogestin. Citation Format: Todd P. Knutson, Andrea R. Daniel, Danhua Fan, Kevin A. Silverstein, Kyle R. Covington, Suzanne A. Fuqua, Carol A. Lange. Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3569. doi:10.1158/1538-7445.AM2013-3569

Cacospongionolide and Scalaradial, Two Marine Sesterterpenoids as Potent Apoptosis-Inducing Factors in Human Carcinoma Cell Lines

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.

Abstract LB-306: U3-1287 (AMG 888), a fully human anti-HER3 mAb, inhibits HER3 activation and induces HER3 internalization and degradation

Abstract Background: Understanding the mechanism of action (MOA) of novel therapeutic agents is instrumental for their recommended use in the clinic. U3-1287 (AMG 888) is a novel, fully human mAb that targets HER3, a member of the EGFR family involved in tumor growth and resistance to anti-HER inhibitors. To understand its MOA, we evaluated HER3 internalization, degradation, and downstream signaling in tumor cell lines in response to U3-1287 (AMG 888) treatment. Methods: Confocal microscopy was used to locate antibody/receptor complexes. MDA-MB-453 and MDA-MB-175 breast cancer cells were serum starved overnight and treated on ice with 10 ug/mL of quantum dot 585 (Invitrogen) conjugated U3-1287 (AMG 888) and 2 ug/mL of quantum dot 655 (Invitrogen) conjugated HER2 Ab5 (Labvision). Images were taken at time 0 and after 1 hour incubation at 37°C. In addition, cell surface reduction of HER3 was measured by flow cytometry. T47D breast cancer cells were incubated at 37°C for 0, 1, 4 and 24 hours with either U3-1287 (AMG 888), c225, trastuzumab or c2C4 (10 µg/ml). The cells were re-stained with the same primary antibodies (10 µg/ml) at 4°C, and analyzed. To evaluate the consequence of longer term U3-1287 (AMG 888) treatment, BxPC3 pancreatic cells were treated from 0-96 hours in vitro and A549 NSCLC xenografts were treated for 2 weeks in vivo. Total HER3, pHER3 and pAKT were measured by Western blot and HER3 mRNA was measured by RT-PCR. Results: U3-1287 (AMG 888) treatment of MDA-MB-453 and MDA-MB-175 cells resulted in the internalization of HER3 but not HER2. Treatment of T47D cells with trastuzumab, c2C4 or c225, U3-1287 (AMG 888) resulted in a 8, 4, 0 and 73% reduction of cell surface HER2, HER2, HER1 and HER3, respectively at the 1 hr time point. A ∼75% reduction of HER3 was maintained during the 24 hr treatment with U3-1287 (AMG 888). Although a 60% reduction in total HER3 was not observed until 4 hours post treatment of BxPC3 cells with U3-1287 (AMG 888), pHER3 and pAKT were reduced &amp;gt;90% within 15 minutes. Neither U3-1287 (AMG 888) nor heregulin (the ligand for HER3) treatment resulted in a significant change in HER3 mRNA levels over the 96 hr treatment period. Analyses of A549 tumor xenograft tissue demonstrated a dose dependent loss of total HER3 and pHER3 in response to U3-1287 (AMG 888) administration versus IgG control treatment. Conclusions: We demonstrate that U3-1287 (AMG 888)-induced inhibition of HER3 signaling and HER3 degradation are separate events, whereby loss of HER3 receptor in response to U3-1287 (AMG 888) treatment is preceded by U3-1287 (AMG 888)-mediated inhibition of HER3 activation. HER3 transcription is not affected by U3-1287 (AMG 888) or heregulin in BxPC3 cells. These findings suggest that U3-1287 (AMG 888) mediates both inhibition of HER3 downstream signaling and HER3 degradation and that these mechanisms may contribute to the activity of U3-1287 (AMG 888) in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-306.

Caffeic acid, chlorogenic acid, EGCG가 유방암 세포 T-47D의 p16 유전자 DNA methylation에 미치는 영향

In the present investigation, we studied the modulating effects of caffeic acid, chlorogenic acid, and (-)-epigallocatechin-3-gallate(EGCG) on the methylation status of promoter regions of cell cycle regulator, p16, in human breast cancer T-47D cells. We demonstrated that treatment of T-47D cells with caffeic acid, chlorogenic acid, or EGCG partially inhibited the methylation status of the promoter regions of p16 genes determined by methylation-specific PCR. In contrast, unmethylated p16 genes were increased with the treatment of T-47D cells with 20 μM of caffeic acid or chlorogenic acid for 6 days. Treatment of T-47D cells with 5, 20 or 50 μM of EGCG increased the unmethylation status of p16 gene up to 100%, and the methylation-specific bands of this gene were decreased up to 50 % in a concentration-dependent manner. The finding of present study demonstrated that coffee polyphenols and EGCG have strong inhibitory effects of the cellular DNA methylation process through increased formation of S-adenosyl-homocysteine(SAH) during the catechol-O-methyltransferase (COMT)- mediated O-methylation of these dietary chemicals or an direct inhibition of the DNA methyltransferases. In conclusion, various dietary polyphenols could reverse the methylation status of p16 gene in human breast T-47D cells.

ErbB-4 and TNF-α converting enzyme localization to membrane microdomains

Sequential proteolytic processing of ErbB-4 occurs in response to ligand addition. Here, we assess the localization of cleavable and non-cleavable ErbB-4 isoforms to membrane microdomains using three methodologies: (1) Triton X-100-insolubility, (2) Brij98-insolubility, and (3) detergent-free density gradient centrifugation. Whereas ErbB-4 translocated to a Triton X-100-insoluble fraction upon treatment of T47D cells with heregulin, it constitutively associated with a Brij98-insoluble fraction and a lipid raft fraction isolated using detergent-free methodology. Comparison of cleavable and non-cleavable isoforms of ErbB-4 revealed that both ErbB-4 isoforms are constitutively localized to either a Triton X-100-soluble or Brij98-insoluble fraction. In contrast, addition of heregulin resulted in translocation of the cleavable isoform to a detergent-free lipid raft. Tumor necrosis factor-α converting enzyme (TACE), the ectodomain secretase for ErbB-4, was present predominantly in its mature active form in most microdomains analyzed. These data suggest the assembly of ErbB-4 ectodomain cleavage apparatus in a membrane microdomain.

Hydrogen peroxide inhibits activity of the IGF-1 receptor kinase

IGF-1 receptor (IGF1R) is a transmembrane tyrosine kinase, which is indispensable for cellular growth and differentiation. Using a recombinant GST-tagged cytosolic fragment of IGF1R (GST-IGFK), we now show that oxidation by low doses (50 μM) of hydrogen peroxide markedly inhibits maximum phosphate incorporation in autophosphorylation and substrate phosphorylation assays. A similar inhibition was observed on the activity of intact IGF1R after treatment of T-47D cells. These results are in sharp contrast to the positive influence of hydrogen peroxide on the highly homologous insulin receptor kinase, which was assayed for comparison. This reciprocal influence of physiologically relevant doses of hydrogen peroxide may have important implications on signal transduction of the closely related receptors for insulin and IGF-1.

Sulindac Sulfide and Exisulind Inhibit Expression of the Estrogen and Progesterone Receptors in Human Breast Cancer Cells

In previous studies, we found that sulindac sulfide and exisulind (sulindac sulfone, Aptosyn) cause growth inhibition, arrest cells in the G1 phase of the cell cycle, and induce apoptosis in human breast cancer cell lines. These effects were associated with decreased expression of cyclin D1. The present study focuses on the effects of sulindac sulfide and exisulind on hormone signaling components in breast cancer cells. We found that estrogen receptor (ER)-positive and progesterone receptor (PR)-positive T47D breast cancer cells were somewhat more sensitive to growth inhibition by sulindac sulfide or exisulind than ER-negative PR-negative MB-MDA-468 breast cancer cells. Further studies indicated that sulindac sulfide and exisulind caused marked down-regulation of expression of the ER and PR-A and PR-B in T47D cells. However, neither compound caused a major change in expression of the retinoic acid receptor alpha (RARalpha), RARbeta, or RARalpha in T47D cells. Sulindac sulfide and exisulind also caused a decrease in expression of the ER in estrogen-responsive MCF-7 breast cancer cells. Both compounds also markedly inhibited estrogen-stimulated activation of an estrogen-responsive promoter in transient transfection reporter assays. Treatment of T47D cells with specific protein kinase G (PKG) activators did not cause a decrease in ER or PR expression. Therefore, although sulindac sulfide and exisulind can cause activation of PKG, the inhibitory effects of these two compounds on ER and PR expression does not seem to be mediated by PKG. Our findings suggest that the growth inhibition by sulindac sulfide and exisulind in ER-positive and PR-positive human breast cancer cells may be mediated, in part, by inhibition of ER and PR signaling. Thus, these and related compounds may provide a novel approach to the prevention and treatment of human breast cancers, especially those that are ER positive.

Differential Requirement for DNA Methyltransferase 1 in Maintaining Human Cancer Cell Gene Promoter Hypermethylation

Previous work has shown that DNA hypermethylation of tumor suppressor genes in colorectal cancer cells may be maintained in the absence of the major mammalian methyltransferase, DNA methyltransferase 1 (DNMT1). In an effort to dissect the dependency on DNMT1 to maintain such hypermethylation in different cancer types, we performed a systematic analysis of depletion of DNMT1 in colorectal (SW48), bladder (T24), and breast (T47D) cancer cells by DNMT1-specific small hairpin RNA (shRNA) targeting. We show that although DNMT1-deficient SW48 and T24 cells exhibited no observable growth defects and were able to maintain promoter hypermethylation, DNMT1-deficient T47D breast cells failed to form comparable numbers of colonies when stably selected for the incorporation of the DNMT1-specific shRNA expression vector, suggesting a growth defect with reduced levels of DNMT1. Further treatment of T47D cells with transient transfection of small interfering RNA targeting DNMT1 revealed that severely DNMT1-deficient T47D cells could not fully maintain promoter hypermethylation, and gene silencing was partially reversed at two of the three assayed loci. These observations suggest that human cancer cells may differ in their reliance on DNMT1 for maintaining DNA methylation.

Discovery and Structure−Activity Relationship of 3-Aryl-5-aryl-1,2,4-oxadiazoles as a New Series of Apoptosis Inducers and Potential Anticancer Agents

We have identified 5-(3-chlorothiophen-2-yl)-3-(4-trifluoromethylphenyl)-1,2,4-oxadiazole (1d) as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 1d has good activity against several breast and colorectal cancer cell lines but is inactive against several other cancer cell lines. In a flow cytometry assay, treatment of T47D cells with 1d resulted in arrest of cells in the G(1) phase, followed by induction of apoptosis. SAR studies of 1d showed that the 3-phenyl group can be replaced by a pyridyl group, and a substituted five-member ring in the 5-position is important for activity. 5-(3-Chlorothiophen-2-yl)-3-(5-chloropyridin-2-yl)-1,2,4-oxadiazole (4l) has been found to have in vivo activity in a MX-1 tumor model. Using a photoaffinity agent, the molecular target has been identified as TIP47, an IGF II receptor binding protein. Therefore, our cell-based chemical genetics approach for the discovery of apoptosis inducers can identify potential anticancer agents as well as their molecular targets.