BACKGROUNDCancer stem cells (CSCs) are the cause of cancer recurrence because they are resistant to conventional therapy and contribute to cancer growth and metastasis. Endocrinotherapy is the most common breast cancer therapy and acquired tamoxifen (TAM) resistance is the main reason for endocrinotherapy failure during such therapy. Although acquired resistance to endocrine treatment has been extensively studied, the underlying mechanisms are unclear. We hypothesized that breast CSCs played an important role in TAM-induced resistance during breast cancer therapy. Therefore, we investigated the biological characteristics of TAM-resistant (TAM-R) breast cancer cells.

METHODSMammosphere formation and tumorigenicity of wild-type (WT) and TAM-R MCF7 cells were tested by a mammosphere assay and mouse tumor xenografts respectively. Stem-cell markers (SOX-2, OCT-4, and CD133) and epithelial-mesenchymal transition (EMT) markers were tested by quantitative real-time (qRT)-PCR. Morphological observation was performed to characterize EMT.

RESULTSAfter induction of TAM resistance, TAM-R MCF7 cells exhibited increased proliferation in the presence of TAM compared to that of WT MCF7 cells (P < 0.05), indicating enhanced TAM resistance of TAM-R MCF7 cells compared to that of WT MCF7 cells. TAM-R MCF7 cells showed enhanced mammosphere formation and tumorigenicity in nude mice compared to that of WT MCF7 cells (P < 0.01), demonstrating the elevated CSC properties of TAM-R MCF7 cells. Consistently, qRT-PCR revealed that TAM-R MCF7 cells expressed increased mRNA levels of stem cell markers including SOX-2, OCT-4, and CD133, compared to those of WT MCF7 cells (P < 0.05). Morphologically, TAM-R MCF7 cells showed a fibroblastic phenotype, but WT MCF7 cells were epithelial-like. After induction of TAM resistance, qRT-PCR indicated that MCF7 cells expressed increased mRNA levels of Snail, vimentin, and N-cadherin and decreased levels of E-cadherin, which are considered as EMT characteristics (P < 0.05).

CONCLUSIONTAM-R MCF7 cells possess CSC characteristics and may be responsible for TAM resistance during breast cancer therapy.

Animals ; Antineoplastic Agents, Hormonal ; pharmacology ; Breast Neoplasms ; drug therapy ; pathology ; Drug Resistance, Neoplasm ; Epithelial-Mesenchymal Transition ; Female ; Humans ; MCF-7 Cells ; Mice ; Neoplastic Stem Cells ; drug effects ; Tamoxifen ; pharmacology

OBJECTIVETo study the demethylation effect of arsenic trioxide (As2O3) on ERα-negative human breast cancer MDA-MB-435s cells and its possible mechanisms, and to observe its treatment efficacy in combination with tamoxifen (TAM) after ERα re-expression.

METHODSMTT assay was used to examine the inhibitory effect of As2O3 treatment alone or in combination with TAM on cell proliferation. A nude mouse xenograft model was used to further examine the treatment efficacy in vivo. MSP was used to detect the methylation status of ERα gene after treated with As2O3 in MDA-MB-435s cells and the transplanted tumor tissues. RT-PCR was used to detect the mRNA expression of DNMT1 and Erα. Western bolt was used to detect the DNMT1 and ERα protein expression. The diameter of xenograft tumors was measured weekly, and the tumor growth curve was drawn.

RESULTSThe level of proliferation of the MDA-MB-435s cells was significantly suppressed after treatment with different concentration of As2O3 alone or As2O3 combined with TAM, and the 4 µmol/L As2O3 + TAM treatment for 72 h showed the highest inhibition rate (62.6%). 1, 2, 4 µmol/L As2O3 had demethylation effect on MDA-MB-435s cells, and the DNMT1 mRNA and protein expression was inhibited and accompanied by ERα mRNA and protein re-expression. The unmethylation specific bands of ERα gene were enhanced after treated by As2O3 alone or As2O3 combined with TAM in the xenograft tumors. The expression of DNMT1 mRNA and protein was inhibited, and accompanied by ERα mRNA and protein re-expression. An significant decrease of volume and weight of the xenograft tumors in the As2O3 treated alone or combined with TAM groups was observed compared with those of the normal saline group or TAM alone group (P < 0.05), and the 4 mg/kg As2O3 + TAM group had the highest inhibition rate of tumor weight (79.5%) and volume (76.4%).

CONCLUSIONSERα can be re-expressed in ERα-negative breast cancer MDA-MB-435s cells after treated with As2O3 by inhibiting the DNMT1 activity. MDA-MB-435s cells are re-sensitized to endocrine therapy after ERα re-expression. As2O3 combined with TAM may provide a new therapeutic approach for patients with ERα-negative breast cancer in the clinic.

Animals ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Antineoplastic Agents, Hormonal ; administration & dosage ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Arsenicals ; administration & dosage ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; DNA Methylation ; Dose-Response Relationship, Drug ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Oxides ; administration & dosage ; pharmacology ; RNA, Messenger ; metabolism ; Tamoxifen ; administration & dosage ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.
Adolescent ; Antineoplastic Agents, Hormonal ; pharmacology ; Apoptosis ; drug effects ; Branched DNA Signal Amplification Assay ; methods ; Cell Line, Tumor ; Child ; Dexamethasone ; pharmacology ; Drug Resistance, Neoplasm ; Exons ; Glucocorticoids ; pharmacology ; Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; Receptors, Glucocorticoid ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Transcription, Genetic ; drug effects ; Up-Regulation

OBJECTIVETo explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene.

METHODSTwo recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines. Four kinds of BCRP expressing cell lines of MCF-7/Promoter-BCRP, MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP were established in which BCRP was promoted by the BCRP promoter and a CMV promoter as control, respectively. The drug resistant cells were treated with toremifene. Then RT-PCR, Western blot, mitoxantrone efflux assays and cytotoxicity assay were performed to detect the reversal function of BCRP by toremifene on the drug resistance cell lines.

RESULTSToremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERα-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP cells, toremifene at the dose of 0.1, 1 and 10 µmol/L decreased BCRP mRNA expression by 29.5% (P < 0.05), 68.1% (P < 0.01) and 97.4% (P < 0.01), respectively. After being treated with toremifene and 17β-estradiol, the BCRP mRNA level in MCF-7/Promoter-BCRP cells was 64.2% ± 1.3%, significantly higher than that of toremifene treatment control cells (3.8% ± 0.2%,P < 0.01). Furthermore, the effect of toremifene on BCRP protein is similar in BCRP mRNA. Toremifene obviously increased the mitoxantrone fluorescence intensity and decreased the efflux activity by 47.3% (P < 0.05) in MCF-7/promoter-BCRP cells when compared with the untreated control, whereas intracellular accumulation of mitoxantrone obviously decreased and the efflux activity increased by 61.5% were observed in combination with 17β-estradiol when compared with toremifene treatment alone. The results therefore suggested that toremifene reversed mitoxantrone resistance in MCF-7/Promoter-BCRP cells. However, in MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP cells, toremifene or in combination with 17β-estradiol did not affect intracellular mitoxantrone uptake.

CONCLUSIONTaken together, our findings indicate that expression of BCRP is downregulated by toremifene, via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through ER-mediated to inactivate the transcription of BCRP gene.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Hormonal ; administration & dosage ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cytomegalovirus ; genetics ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mitoxantrone ; pharmacology ; Neoplasm Proteins ; genetics ; metabolism ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Response Elements ; genetics ; Toremifene ; administration & dosage ; pharmacology

OBJECTIVEThe aim of this study was to investigate the changes in mTOR activity and survivin expression in liver cancer cell line HepG2 cells treated with tamoxifen.

METHODSSurvivin transcription level and p70S6K was demonstrated by PCR, dual-luciferase reporter assay and Western blot analysis, respectively, and the apoptosis in the HepG2 cells was detected by flow cytometry.

RESULTSTamoxifen leads to apoptosis of the cells and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6K. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduced the survivin protein level but not affected the survivin transcription, indicating that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells.

CONCLUSIONSOur results suggest that tamoxifen down-regulates survivin expression in HepG2 cells and it is mediated by transcriptional and post-transcriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents, Hormonal ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Drug Synergism ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Tamoxifen ; pharmacology

The in vitro antitumor activity of bakuchiol was exploited, compared with tamoxifen. The result of biological activities showed that bakuchiol could inhibit human breast cancer and the IC50 values were 2.89 x 10(-5) mol L(-1) and 8.29 x 10(-3) mol L(-1) against the cells line T-47D and MDA-MB-231 respectively. On the other hand, the key intermediate to synthesize bakuchiol was obtained by the method of Ireland-Claisen rearrangement. Comparing with traditional Claisen rearrangement, the reaction conditions are milder and the reaction reagents are safer.
Antineoplastic Agents, Hormonal ; pharmacology ; Antineoplastic Agents, Phytogenic ; chemical synthesis ; isolation & purification ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Humans ; Inhibitory Concentration 50 ; Phenols ; chemical synthesis ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Psoralea ; chemistry ; Tamoxifen ; pharmacology

Antineoplastic Agents, Hormonal ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; metabolism ; Tamoxifen ; pharmacology

AIMTo explore the effect of tamoxifen on voltage-dependent sodium channels in SHG-44 glioma cell line.

METHODSWhole-cell patch clamp technique was used to record the Na currents in SHG-44 cell line and to investigate the effect of tamoxifen of different concentration on this channel currents.

RESULTSThis channel activated and inactivated quickly. Tamoxifen could significantly decrease the amplitude of Na currents of SHG-44 cell line. This block effect was dose dependent and voltage dependent. When the holding potential was 0 mV, 8 micromol/L tamoxifen could block this currents 69%. The half inhibition concentration (IC50) was 5.54 micromol/L.

CONCLUSIONTamoxifen could significantly block the voltage dependent sodium channel in malignant glioma cell line SHG-44. It might be one of the mechanisms that tamoxifen inhibit glioma proliferation. clamp technique was used to record the Na currents in SHG-44 cell line and to investigate the effect of tamoxifen of different concentration on this channel currents.

RESULTSThis channel activated and inactivated quickly. Tamoxifen could significantly decrease the amplitude of Na currents of SHG-44 cell line. This block effect was dose dependent and voltage dependent. When the holding potential was 0 mV, 8 micromol/L tamoxifen could block this currents 69%. The half inhibition concentration (IC50) was 5.54 micromol/L.

CONCLUSIONTamoxifen could signifi-cantly block the voltage dependent sodium channel in malignant glioma cell line SHG-44. It might be one of the mechanisms that tamoxifen inhibit glioma proliferation.

Antineoplastic Agents, Hormonal ; pharmacology ; Brain Neoplasms ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Glioma ; pathology ; Humans ; Patch-Clamp Techniques ; Sodium Channel Blockers ; pharmacology ; Tamoxifen ; pharmacology

This study is to investigate the effect of dexamethasone on cell apoptosis of murine MIN6 pancreatic beta-cells, and to investigate the mechanism of dexamethasone-dependent cell apoptosis. The cell apoptosis model was established by choosing the murine MIN6 pancreatic beta-cells, which was cultured in vitro and induced by dexamethasone. The morphology of the cell apoptosis was observed through fluorescence microscopic analysis after Hochest/PI staining and flow cytometric assay after Annexin-V/PI staining. The expression of caspase-3 was detected with caspase-3 activity assay kit. The expressions of Cyt-c, Bcl-2, Bax, AKT and p-AKT were observed with Western blotting. The results indicated that after exposure to dexamethasone at a concentration ranging from 50-800 nmol x L(-1) for 48 h, the percentage of cell apoptosis was significantly increased with the concentration over 100 nmol x L(-1) of dexamethasone; after exposure to dexamethasone (100 nmol x L(-1)) for 72 h, the activity of caspase-3 increased significantly; after exposure to dexamethasone at a concentration ranging from 50-800 nmol x L(-1) for 48 h, the expression of Cyt-c increased, Bcl-2 and AKT phosphorylation decreased while Bax and T-AKT remained unchanged. It could be concluded that the effect of dexamethasone on murine MIN6 pancreatic beta-cells apoptosis is significant. The mechanism of dexamethasone-dependent cell apoptosis is probably related to down regulation of the Bcl-2 expression and reduction of AKT phosphorylation.
Animals ; Antineoplastic Agents, Hormonal ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; Cytochromes c ; metabolism ; Dexamethasone ; pharmacology ; Down-Regulation ; Insulin-Secreting Cells ; metabolism ; pathology ; Mice ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

AIMTo determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth.

METHODSThe cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor (GR) antagonist RU486. Reverse transcription-polymerase chain reaction verified the expression of GR mRNA in DU145 cells.

RESULTSDexamethasone significantly inhibited DU145 cell proliferation at the G(0)/G(1) phase. Western blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade.

CONCLUSIONDexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Antineoplastic Agents, Hormonal ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin D1 ; drug effects ; metabolism ; Dexamethasone ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Male ; Mitogen-Activated Protein Kinase 1 ; drug effects ; metabolism ; Mitogen-Activated Protein Kinase 3 ; drug effects ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Receptors, Glucocorticoid ; metabolism ; Signal Transduction ; drug effects