Human Breast Cell Line MCF-7 Research Articles

Heterobimetallic Fe(II)–Cu(I) Complexes Supported with 1,1′-Bis(diphenylphosphino)ferrocene and Chelating Sulfur Donor Ligands: Structural Characterization, DFT Analysis, Anti-tyrosinase Assay and Biological Studies

Heterobimetallic complexes [(dppf)CuSP(S)(OiPr)2] (1), [(dppf)CuSP(S)(OEt)2] (2), [(dppf)CuSP(S)(OCy)2] (3) and [(dppf)CuS2H2B(mt)2] (4), supported with sulfur donors from the family of dialkyldithiophosphate, SP(S)(OR)2 (R = iPr, Et, Cy) and borate ligands, (S)2H2B(mt)2, were synthesized in one pot by the reaction of 1,1′-Bis(diphenylphosphino)ferrocene (dppf) with [Cu(CH3CN)4]BF4 and subsequent addition of the chelating sulfur donor ligands at room temperature in an overall moderate yield. 1-4 were characterized using a combination of 1H, 13C{1H}, 31P{1H} NMR, IR spectroscopy techniques, and single crystal X-ray diffraction. Crystal structure analysis revealed that Cu(I) centers in 1-4 adopt a distorted tetrahedral geometry. The borate ligand in 4 adopts a κ3-S,S,H mode in the solid state. A density functional theory geometry optimization calculation was used to extract further structural and electronic information about 1-4. To investigate the possible cytotoxicity of 1-4, in vitro cellular tests were carried out on the human cancerous breast cell line MCF-7. The antibacterial activities of complexes 1 and 4 were screened against gram-positive Staphylococcus aureus PTCC 1112 and gram-negative Escherichia coli PTCC 1330.

Heteroleptic Silver(I) and Gold(I) N-Heterocyclic Carbene Complexes: Structural Characterization, Computational Analysis, Tyrosinase Inhibitory, and Biological Effects.

Derivatization of (NHC)M-Cl (M = Ag, Au) with selected sulfur donors from the family of dialkyldithiophosphates and bis(2-mercapto-1-methylimidazolyl)borate ligands gave a series of heteroleptic mononuclear complexes. In single-crystal X-ray diffraction analysis, Ag(I) complexes adopted a trigonal planar geometry, while Au(I) complexes are near-linear. TD-DFT and hole-electron analyses of the selected complexes gave insight into the electronic features of the metal complexes. In vitro cellular tests were conducted on the human cancerous breast cell line MCF-7 using 2 and 8. The antibacterial activities of complexes 1, 2, 3, 7, 8, and IPr-Ag-Cl were also screened against Gram-positive (Staphylococcus aureus PTCC 1112) and Gram-negative (Escherichia coli PTCC 1330) bacteria. Antityrosinase and hemolytic effects of the selected compounds were also determined.

Homobimetallic Au(I)-Au(I) and Heterotrimetallic Au(I)-Fe(II)-Au(I) Complexes with Dialkyldithiophosphates and Phosphine Ligands: Structural Characterization, DFT Analysis, and Tyrosinase Inhibitory and Biological Effects.

The role of bridging and terminal ligand electronic and steric properties on the structure and antiproliferative activity of two-coordinated gold(I) complexes was investigated on seven novel binuclear and trinuclear gold(I) complexes synthesized by the reaction of either Au2(dppm)Cl2, Au2(dppe)Cl2, or Au2(dppf)Cl2 with potassium diisopropyldithiophosphate, K[(S-OiPr)2], potassium dicyclohexyldithiophosphate, K[(S-OCy)2], or sodium bis(methimazolyl)borate, Na(S-Mt)2, which afforded air-stable gold(I) complexes. In 1-7, the gold(I) centers adopt a two-coordinated linear geometry and are structurally similar. However, their structural features and antiproliferative properties highly depend upon subtle ligand substituent changes. All complexes were validated by 1H, 13C{1H}, 31P NMR, and IR spectroscopy. The solid-state structures of 1, 2, 3, 6, and 7 were confirmed using single-crystal X-ray diffraction. A density functional theory geometry optimization calculation was used to extract further structural and electronic information. To investigate the possible cytotoxicities of 2, 3, and 7, in vitro cellular tests were carried out on the human cancerous breast cell line MCF-7. 2 and 7 show promising cytotoxicity.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

C-Myc induced the regulation of long non-coding RNA RHPN1-AS1 on breast cancer cell proliferation via inhibiting P53.

The global burden of breast cancer has been increasing, the mechanism of which is related to multifactorial accumulation of gene mutations. Dysregulation of long noncoding RNA (LncRNA) has been linked to multiple kinds of tumorigenesis. We aimed to identify functionally relevant targets of RHPN1 Antisense RNA 1 (RHPN1-AS1) in breast cancer using breast cancer cell line-based model. Quantitative RT-PCR revealed higher expression levels of RHPN1-AS1 in human breast cancer tissues and cell line MCF-7. RHPN1-AS1 was also located in MCF-7 cells by fluorescence in situ hybridization and western blot assays. Knockdown of RHPN1-AS1 delivered by lentivirus system inhibited MCF-7 cell proliferation indicated by the cell proliferation and colony formation assays, and the knockdown of RHPN1-AS1 enhanced P53 protein expression in MCF-7 and MDA-MB-231 cells. In addition, luciferase reporter assay validated that RHPN1-AS1 is a molecular sponge of miR-4261, and direct transcriptional target of c-Myc. RHPN1-AS1 exerts tumorigenesis by regulating P53 expression via MDM2 gene. These findings provide insights into the role and mechanism of action of lncRNA RHPN-AS1 in breast cancer.

Assessment of the cytotoxicity and genotoxicity of homosalate in MCF-7.

UV filters should be determined a strict toxicological safety prior to approval. Homosalate (HMS) is one of the most widely used organic UV filter. HMS accumulates in aquatic biota ecosystems and humans through the food chain; therefore, it is important to consider the effects on health and know its mechanisms of action. The study aimed to evaluate the cytotoxic and genotoxic effects of HMS in MCF-7 cell line. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) and cell membrane integrity by the lactate dehydrogenase release assays (LDH), and genotoxicity by using the micronucleus test at 250, 500, 750, 1000, 1500, and 2000µM concentrations with the human breast cell line MCF-7. Homosalate affected the cell viability dose-dependently at a concentrations of above 1000µM. Micronucleus formation was significantly induced at 750 and 1000µM within 24hours due to an increase in cytostatic effect, the cell viability of HMS decreased to 57% at a concentration of 2000µM, and a sufficient number of binucleated cells could not be obtained to count. Homosalate was also clastogenic when the cells were incubated at cytotoxic concentrations. These results suggest that homosalate can be considered as a cytotoxic and genotoxic substance.

Synthesis and anticancer activities of novel (tetrahydrobenzo [4,5] thieno [2,3-d] pyrimidine-4-yl)-pyrolidine-2-carboxylic acid derivatives

Cancer chemotherapy has been one of the major medical advances in the last few decades. The medications involved in treatment have a narrow therapeutic index, and often the responses produced are only just palliative as well as unpredictable. In contrast, targeted therapy such as Tyrosine kinases inhibitors, that has been introduced in recent years is directed against cancer-specific molecules and signaling pathways with some side effects Therefore, we studied these class of molecules to obtain new candidates of remarkable activities and minimal side effects. We herein reported SAR driven synthesis of 16 novel molecules and their anticancer profiles in specifically targeted human Breast cell line MCF-7 and Human Colon Cancer Cell Line HCT-15. Compounds 7e and 8f showed descent antitumor activities when compared to Doxorubicine (GI50 < 0.1) for human Breast cell line MCF-7. The synthesized analogs were ensured by spectral and elemental analysis.

Design, Synthesis and Biological Evaluation of Novel Class Diindolyl Methanes (DIMs) Derived from Naturally Occurring Phenolic Monoterpenoids

Several Diindolyl alkanes and their derivatives have been isolated from plant and marine sources. Among the various derivatives of indoles, Diindolyl methanes have wide medicinal applications such as to induce apoptosis in human cancer cells, antibacterial, Anti-inflammatory, antiviral and hormonal control activities. Therefore, they play essential role in marine as well as terrestrial living systems. In present studies we report novel class of Diindolyl methanes prepared from natural phenolic monoterpenoids, via ortho formylation of phenolic monoterpenoids (Carvacrol, Thymol and Eugenol), followed by synthesis, characterization, anticancer, antioxidant and α-amylase inhibitory activities. All the synthesized derivatives show moderate anticancer activities against human breast cell line MCF7, good antioxidant and α-amylase inhibitory activities using DPPH and α-amylase assay respectively.

Analogue-based design, synthesis and biological evaluation of 3-substituted-(methylenehydrazono)indolin-2-ones as anticancer agents

The docking studies on CDK2 and GSK-3β inspired us to synthesis a series of indoline-2,3-dione hydrazones 10a–l. Treatment of indoline-2,3-dione derivatives 7a–d with hydrazine gave 3-hydrazonoindolin-2-ones 8a–d which were reacted with the appropriate aldehydes 9a–c to yield 3-substituted-(methylenehydrazono)indolin-2-ones 10a–l. Compounds 10a–l showed a significant anticancer activity against human breast cell line MCF-7. Compounds 10c, f, i exhibited the highest activity almost the same of doxorubicin (IC50 = 6.10 μM) with IC50 = 7.75, 6.75, 6.25 μM, respectively.

Abstract P5-08-09: Synergistic cytotoxicity of digoxin and 5-fluorouracil in doxorubicin-resistant breast cancer cell lines

Abstract BACKGROUND: Metastatic breast cancer is the leading cause of cancer death for women in the US. Cytotoxic chemotherapy remains one of the most important systemic treatment options for breast cancer patients. To enhance the tumor response to chemotherapy, attention has been focused on agents that reverse multidrug resistance (MDR) and increase the sensitivity of tumor cells to chemical drugs. Although many reversal drugs have been identified in vitro, their clinical application has been limited due to their own toxicity. It was reported that 5 years after mastectomy, the recurrence rate of breast cancer among patients on cardiac glycoside therapy, namely digoxin or digitoxin, was almost 10 times lower as compared with those who were not on cardiac glycoside therapy. Digoxin has also been shown to inhibit hypoxia-inducible factors (HIF) and block lung metastasis in a breast cancer model. Interestingly, digoxin was demonstrated to stimulate cell death in various cell lines including breast cancer. Therefore the aim of this study was to examine the reversal effect of combined digoxin and 5-fluorouracil (5-FU) on MDR resistance in human breast cell lines MCF-7 and MDA-MB-231 in vitro, as well as its mechanism of action. METHODS: Wild-type and doxorubicin-resistant ER-positive (MCF-7 and MCF-7/DoxR) and triple negative (MDA-MB-231 and MDA-231/DoxR) breast cancer lines were used for this study. We have previously shown that both doxorubicin-resistant cell lines are also cross-resistant to 5-FU. Cell viability of both resistant cancer cell lines to 5-FU, digoxin, and both 5-FU with digoxin were compared to control at 96 hours of incubation both in normoxia and hypoxia using MTT assay. Western blot was used to quantify the level of HIF-1α and p-glycoprotein (P-gP). RESULTS: In both doxorubicin-resistant cell lines, sub-IC50 concentration of digoxin together with 5-FU significantly decreased the cell viabilities at 96 hours compared to single agent digoxin and 5-FU. Under both normoxic and hypoxic conditions, the same synergistic cytotoxic effects were demonstrated. Western blot revealed that HIF-1α and P-gP were decreased in both doxorubicin-resistant breast cancer cells treated with the digoxin and 5-FU combination compared to single agent digoxin or 5-FU. CONCLUSION: The combination of digoxin and 5-FU demonstrates a synergistic cytotoxic effect in doxorubicin-resistant breast cancer cell lines that is maintained under hypoxic conditions. This is, at least in part, via the inhibitory effects on both HIF-1α and P-gP. The combination of digoxin and 5-FU could be an effective clinical treatment strategy to overcome MDR in breast cancer. A phase II clinical trial is ongoing to test this hypothesis in patients with doxorubicin-resistant metastatic breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-09.

Abstract 4778: Computer-assisted molecular design use for the discovery and optimization of rationally-designed anticancer natural products. Discovery of olive oil-derived oleocanthal as a c-Met inhibitor and optimization of Veratrum alkaloids as Hedgehog signaling inhibitors

Abstract Dysregulation of the proto-oncogene receptor tyrosine kinase c-Met, which encodes the high-affinity receptor for hepatocyte growth factor (HGF) and the Hedgehog (Hh) pathway aberrantly associated with numerous malignancies including basal cell carcinoma, medulloblastoma, prostate, pancreatic and breast cancers. The Mediterranean diet is associated with lower incidences of cardiovascular disease, age-related cognitive disease and cancer. (-)-Oleocanthal is a naturally occurring minor secoiridoid from extra-virgin olive oil with potent anti-inflammatory activity via its ability to inhibit COX-1 and COX-2 and anti-Alzheimer's activity. Cyclopamine and its 11-oxo derivative jervine are C-nor-D-homosteroidal alkaloids with known potent teratogenic effects that induce cyclopic malformations in sheep. The jervane Veratrum alkaloids cyclopamine and jervine and the veratrane alkaloid veratramine were among the early known natural products that blocked the expression of the Hh pathway targets PTCH1 and GLI1, lowering Bcl2 levels and inducing apoptosis in many tumors. Computer-assisted molecular design (CAMD) identified oleocanthal as a potential virtual c-Met inhibitor hit. Oleocanthal inhibited the proliferation, migration, and invasion of the epithelial human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively. Oleocanthal demonstrated anti-angiogenic activity and inhibited the phosphorylation of c-Met kinase in vitro. CAMD study of our library of natural, biocatalytic, and semisynthetic jervane and veratrane alkaloids suggested that compounds with C-3-keto functionality with α4,5 or β1,2 and γ4,5 exhibited high virtual Hh binding affinity scores and possible Hh pathway activity. These compounds showed significant ability to inhibit the growth, proliferation, and migration of the prostate metastatic cancer cell line PC-3, which aided the establishment of a preliminary structure-activity relationship. (-)-Oleocanthal and Veratrum alkaloids can have potential therapeutic application for the control of c-Met- and Hh-dependent malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4778. doi:1538-7445.AM2012-4778

The effects of PXD101 on proliferation and apoptosis of human breast cell line MCF-7 and its mechanism

Objective:This work aims to investigate the effect of PXDIOI,a novel potent histone deacetylase inhibitor,on the cell proliferation,cycle arrest and apoptosis of human breast cancer cell line MCF-7 and to preliminarily explore its molecular mechanism. Methods:MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10%fetal bovin serum and were treated with PXDIOI at varying concentrations.The methyl thiazolyl tetrazolium(MTT ) assay and clonogenic assay were used to measure cell proliferation. Morphological changes of cells were observed by fluorescent microscope after staining by Hoechst33342.Flow cytometer was used to analyze the cell cycle arrest rates(PI staining ) and the cell apoptotic rates(AnnexinV-FITC/PI double-staining ).The protein expressions of p21,CyclinB1,PARP,Bcl-2 and Bax were detected by Western blot.Results:PXD101 was used to inhibit the proliferation of the MCF-7 cell line in a dose and time-dependent manner.Fluorescence microscope showed there were nuclear fragmentation and apoptosis bodies in the cells.Flow cytometric analysis indicated that PXD101 induced MCF-7 cells in G_2/M phase were significantly increased.After MCF-7 cells exposed to different concentrations of PXD101,i.e.,0,0.1,1 and 10μmol/L,for 24 h,the ratio of G_2/ M-phase cells was(12.66±1.55 )%,(20.63±1.32 )%,(23.20±1.82 )%and(32.19±2.37 )%respectively(P 0.05 ).The rates of apoptotic cells were also significantly increased,compared with the control group(P 0.05 ).PXDIOI could up-regulate the protein expression of p21 and down-regulate the expression of CyclinBl.The cleavage of PARP and the expression of pro-apoptosis protein Bax were increased while the anti-apoptosis protein Bcl-2 was decreased.Conclusion:PXD101 in vitro can significantly inhibit the proliferation and can induce cell cycle arrest and apoptosis on human breast cancer MCF-7 cell line in a dose-dependent manner.PXD101 may become a new anti-tumor drug for human breast cancer.

Illumina whole-genome complementary DNA–mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern–related genes in oral squamous cell carcinoma

High-throughput gene expression profiling from formalin-fixed, paraffin-embedded tissues has become a reality, and several methods are now commercially available. The Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay (Illumina, Inc) is a full-transcriptome version of the original 512-gene complementary DNA-mediated annealing, selection, extension and ligation assay, allowing high-throughput profiling of 24,526 annotated genes from degraded and formalin-fixed, paraffin-embedded RNA. This assay has the potential to allow identification of novel gene signatures associated with clinical outcome using banked archival pathology specimen resources. We tested the reproducibility of the whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay and its sensitivity for detecting differentially expressed genes in RNA extracted from matched fresh and formalin-fixed, paraffin-embedded cells, after 1 and 13 months of storage, using the human breast cell lines MCF7 and MCF10A. Then, using tumor worst pattern of invasion as a classifier, 1 component of the "risk model," we selected 12 formalin-fixed, paraffin-embedded oral squamous cell carcinomas for whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay analysis. We profiled 5 tumors with nonaggressive, nondispersed pattern of invasion, and 7 tumors with aggressive dispersed pattern of invasion and satellites scattered at least 1 mm apart. To minimize variability, the formalin-fixed, paraffin-embedded specimens were prepared from snap-frozen tissues, and RNA was obtained within 24 hours of fixation. One hundred four down-regulated genes and 72 up-regulated genes in tumors with aggressive dispersed pattern of invasion were identified. We performed quantitative reverse transcriptase polymerase chain reaction validation of 4 genes using Taqman assays and in situ protein detection of 1 gene by immunohistochemistry. Functional cluster analysis of genes up-regulated in tumors with aggressive pattern of invasion suggests presence of genes involved in cellular cytoarchitecture, some of which already associated with tumor invasion. Identification of these genes provides biologic rationale for our histologic classification, with regard to tumor invasion, and demonstrates that the whole-genome complementary DNA-mediated annealing, selection, extension and ligation assay is a powerful assay for profiling degraded RNA from archived specimens when combined with quantitative reverse transcriptase polymerase chain reaction validation.

(−)-Oleocanthal as a c-Met Inhibitor for the Control of Metastatic Breast and Prostate Cancers

The proto-oncogene receptor tyrosine kinase c-Met encodes the high-affinity receptor for hepatocyte growth factor (HGF). Dysregulation of the HGF-c-Met pathway plays a significant oncogenic role in many tumors. Overexpression of c-Met is a prognostic indicator for some transitional cell carcinomas. Extra-virgin olive oil (EVOO) provides a variety of minor phenolic compounds with beneficial properties. (-)-Oleocanthal (1) is a naturally occurring minor secoiridoid isolated from EVOO, which showed potent anti-inflammatory activity via its ability to inhibit COX-1 and COX-2. It altered the structure of neurotoxic proteins believed to contribute to the debilitating effects of Alzheimer's disease. Computer-Assisted Molecular Design (CAMD) identified 1 as a potential virtual c-Met inhibitor hit. Oleocanthal inhibited the proliferation, migration, and invasion of the epithelial human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively, with an IC (50) range of 10-20 µM, and demonstrated anti-angiogenic activity via downregulating the expression of the microvessel density marker CD31 in endothelial colony forming cells with an IC (50) of 4.4 µM. It inhibited the phosphorylation of c-Met kinase IN VITRO in the Z'-LYTE™ assay, with an IC (50) value of 4.8 µM. (-)-Oleocanthal and EVOO can have potential therapeutic use for the control of c-Met-dependent malignancies.

DNA methyltransferase inhibitor CDA-2 synergizes with high-dose thiotepa and paclitaxel in killing breast cancer stem cells

It has been suggested that breast cancer stem cells (CSCs), which characterized by CD44(+)CD24(-/low), may result in treatment failure in patients with breast cancer. It is possible therefore that that inhibiting such subpopulation might subsequently improve clinical outcome. In the present study, we found that the CD44(+)/CD24(-/low) CSCs, isolated from both human breast cell line MCF-7 and MDA-MB-231, were more resistant to thiotepa, paclitaxel and anthracycline, when compared with the non-breast cancer stem cell subset from the same cell lines, whereas the chemosensitivities were remarkably reversed by higher concentration of thiotepa and paclitaxel except for adriamycin. The percentage of CSCs was significantly decreased with an addition of DNA methyltransferase inhibitor CDA-2 and the expression of Smo, Shh, and Gli-1 of Hedgehog signaling pathway in CSCs was decreased. Of important findings, combination of thiotepa or paclitaxel with CDA-2 could significantly inhibit the proliferation of CSCs regardless of their dosages. These results unveiled that the selection of cytotoxic agents and increasing their dosage might be of great importance in the respect of eliminating CSCs. DNA methyltransferase inhibitor CDA-2 exhibited a synergistic effect with cytotoxic drugs, which might provide a conceptually new therapeutic strategy.

Novel polyazaheterocyclic systems: Synthesis, antitumor, and antimicrobial activities

A series of new polyazaheterocyclic ring systems was synthesized via the reaction of enaminone 5 with active methylene compounds, hydrazine hydrate, hydroxylamine, and heterocyclic amines. The structures of the newly synthesized compounds were substantiated on the basis of spectral data and elemental analyses. The antitumor activity of the enaminone 5 against the human breast cell line MCF-7, the liver carcinoma cell line HEPG2-1, and HELA cells was determined. In addition, the antimicrobial activity of some selected products was evaluated.

Raloxifene-stimulated experimental breast cancer with the paradoxical actions of estrogen to promote or prevent tumor growth: A unifying concept in anti-hormone resistance

We have previously demonstrated that prolonged treatments with raloxifene (RAL) in vitro will result in phase II RAL resistance and RAL-induced tumor growth. Clinical interest prompted us to re-examine RAL resistance in vivo, particularly the effects of long-term treatments (a decade or more) on the evolution of RAL resistance. In this study, we have addressed the question of this being a reproducible phenomenon in wild-type estrogen receptor (ER)-positive human breast cell line MCF-7. MCF-7 cells cultured under estrogen-deprived conditions in the presence of 1 microM RAL for more than a year develop RAL resistance resulting in an independent cell line, MCF7-RAL. The MCF7-RAL cells grow in response to both estradiol E2 and RAL. Fulvestrant (FUL) blocks RAL and E2-mediated growth. Transplantation of MCF7-RAL cells into athymic ovariectomized mice and treatment with physiologic doses of E2 causes early E2-stimulated tumor growth. In contrast, continuous treatment of implanted animals with daily oral RAL (1.5 mg daily) causes growth of small tumors within 15 weeks. Continuous re-transplantation of the tumors growing in RAL-treated mice indicated that RAL stimulated tumor growth. Tumors in the untreated mice did not grow. Bi-transplantation of MCF7-E2 and MCF7-RAL tumors into the opposing mammary fat pads of the same ovariectomized animal demonstrated that MCF7-E2 grew with E2 stimulation and not with RAL. Conversely, MCF7-RAL tumors grew with RAL and not E2, a characteristic of phase II resistance. Established phase II resistance of MCF7-RAL tumors was confirmed following up to 7 years of serial transplantation in RAL-treated athymic mice. The ERalpha was retained in these tumors. The cyclical nature of RAL resistance was confirmed and extended during a 2-year evolution of the resistant phases of the MCF7-RAL tumors. The MCF7-RAL tumors that initially were inhibited by E2 grew in the presence of E2 and subsequently grew with either RAL or E2. RAL remained the major grow stimulus and RAL enhanced E2-stimulated growth. Subsequent transplantation of E2 stimulated tumors and evaluations of the actions of RAL, demonstrated robust E2-stimulated growth that was blocked by RAL. These are the characteristics of the anti-estrogenic actions of RAL on E2-stimulated breast cancer growth with a minor component of phase I RAL resistance. Continuous transplantation of the phase I RAL-stimulated tumors for >8 months causes reversion to phase II resistance. These data and literature reports of the cyclical nature of anti-androgen/androgen responsiveness of prostate cancer growth, illustrate the generality of the evolution of anti-hormonal resistance in sex steroid-sensitive target tissues.

Reversal effect of mifepristone on adriamycin resistance in human breast cancer cell line MCF-7/ADM in vitro and in vivo

To explore the reversal effect of mifepristone(MIF) on adriamycin(ADM) resistance in human breast cell line MCF-7/ADM in vitro and in vivo. The transplantable models of MCF-7 cells resisting against adriamycin were established in nude mice by subcutaneous implantation to observe the reversal effect of MIF in vivo. The mice were randomly divided into 4 groups: a control group(treated with saline water 0.2 mL intraperitoneally and edible oil 0.5 mL orally), an MIF group (treated with mifepristone 30 mg/kg orally and saline water 0.2 mL intraperitoneally), an ADM group (treated with adriamycin 5 mg/kg intraperitoneally and edible oil 0.5 mL orally) and an ADM+MIF group (treated with ADM 5mg/kg intraperitoneally and mifepristone 30 mg/kg orally every 3 days). Tumor changes were investigated after different drug treatments. The reversal effect of 5 micromol/L MIF in vitro on the ADM resistance cell line MCF-7/ADM and non ADM resistance cell line MCF-7 was determined by 4,5-dimethylthiazol-2-yl (MTT) assay. (1) The inhibitory rate of 5 micromol/L of MIF for both cell lines MCF-7 and MCF-7/ADM was less than 5%, and it had no statistical difference compared with the group that was not treated with MIF(P > 0.05). (2) ADM could inhibit the growth of both MCF-7 and MCF-7/ADM,but the inhibition concentration 50 (IC(50)) of MCF-7 (0.42 mg/L) was obviously less than that of MCF-7/ADM(17.21 mg/L) (P < 0.05). (3) IC(50) of MCF-7/ADM of MIF+ADM group was 1.96 mg/L in vitro, which was significantly less than that in ADM alone group(17.21 mg/L) (P < 0.05), and 5 micromol/L of MIF reversed ADM resistance with fold-reversal of 8.78. (4) MIF had some effect on the inhibition of MCF-7/ADM cell growth in vivo, the xenograft volume in the MIF+ADM group [(232.5149 +/- 309.2377) mm(3)] was significantly smaller than that in the control group[(962.2309 +/- 261.1313) mm(3) ] after the 4 week treatment(P<0.05), and also smaller than that in the MIF group [(778.2846 +/- 42.6919) mm(3)] and in the ADM group [(508.9648 +/- 16.2609) mm(3)](P < 0.05). There was significant inhibition on xenograft weight after MIF combined with ADM treatment in vivo, and the inhibitory rate was 78.0%. MIF can effectively reverse ADM resistance in human breast cancer cell line MCF-7/ADM both in vitro and in vivo.

Synthesis, reactions and antitumor activity of new β-aminovinyl 3-pyrazolyl ketones

A new series of enaminones 4a-e was prepared and their reaction with the hydrazonoyl chloride 2c gave 3,4'-bis(pyrazolyl)ketones 8a-d. Hydrazinolysis of 8 proved to be site selective as it yielded the respective 4-(pyrazol-3-yl)-2H-pyrazolo(3,4-d)pyridazines 9. The results of screening of the antitumor activity of enaminones 4a-e against human breast cell line MCF-7 revealed that all such compounds exhibited lower activity in relation to the reference drug Doxorubicin and the activity of the enaminone having electron-accepting substituent is more than that having electron donating substituent.

Abstract B48: Xanthohumol from hops (Humulus lupulus L.) reactivates epigentically silenced genes by potential DNA methyltransferase inhibition

Abstract Epigenetic silencing of tumor suppressor genes by DNA hypermethylation is now recognized as a hallmark of cancer. Thus, reversal of DNA hypermethylation by demethylating agents and DNA methyltranserase (Dnmt) inhibitors has been extensively investigated. Recently, several studies have shown that natural dietary compounds have the potential to reactivate epigenetically silenced genes by various mechanisms, which might have an impact on chemoprevention strategies. Xanthohumol (XN), a prenylated chalcone from hops, exerts a broad spectrum of chemopreventive actions and inhibits mammary carcinogenesis in vivo. In this study, we report for the first time that epigenetic deregulation of gene expression during carcinogenesis is a novel target of XN action. We demonstrated that XN reactivates a series of candidate genes silenced by DNA promoter hypermethylation in human cancer cell lines. By treating human breast and prostate cancer cell lines MCF-7 and LNCaP with subtoxic XN-concentrations for 48 and 72h, respectively, we observed significant dose-dependent re-expression of glutathione-S-transferase Π (GSTPi), Cyclin D2 and RassF1A mRNA, measured by quantitative RT-PCR. The demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) was used as positive control. To address if the XN-mediated mechanism of gene re-expression involved inhibition of Dnmt activity, we tested XN in an in vitro system using the bacterial methlytransferase M.SssI as an enzyme source. XN at concentrations ranging from 3.13–50 µM dose-dependently inhibited M.SssI activity with a halfmaximal inhibitory concentration of 35.2 µM (p&amp;lt;0.05). Computational modelling of XN docking to Dnmt1 indicates steric interaction with the active site. In addition, in MCF-7 and LNCaP cells treated with XN, expression of Dnmt1, 3a and 3b was inhibited by XN-treatment in a dose-dependent manner, both at the transcriptional (p&amp;lt;0.05) and protein level. Next, demethylating potential of XN was investigated. DNA of XN-treated MCF-7 and LNCaP cells was subjected to quantitative methylation analyis using the Sequenom Massarray platform. Whereas treatment for 72h with 1µM of 5-aza-dC significantly reduced DNA hypermethylation of promoter CpG islands of GSTPi, Cyclin D2 and RassF1A by 10–20% (p&amp;lt;0.05), XN-treatment with 5–10µM did not show general DNA demethylation. However, individual CpG sites in the analyzed regions were strongly demethylated upon XN-treatment. Ongoing studies investigate whether demethylation of these single CpG sites is crucial for active transcription, and whether this can be correlated to induced gene-expression. In conclusion, we demonstrated that XN reactivates the expression of hypermethlyated genes by inhibition of Dnmt activity, expression and demethylation of selected CpG sites, which represents a novel chemopreventive mechanism by XN. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B48.