Proliferation Of MCF-7 Breast Cancer Cells Research Articles

Abstract 4771: Improved antitumor efficacy of an acid-sensitive doxorubicin derivative delivered by thermally responsive elastin-like polypeptides

Abstract Doxorubicin is a widely used anticancer drug used in the treatment of various cancers. It acts as an intercalator of DNA and inhibits topoisomerase II thus preventing cell replication. Its side effects include nausea, myelosuppression, alopecia, and dose-limiting cardiotoxicity. Dox is nonspecific, and high doses are needed to treat cancer cells. These high doses, however, are also harmful to normal cells. Because of this toxicity to normal cells, there is a great need for a delivery system that will target dox to the tumor site. In our research, we have developed a tumor targeted drug delivery system comprising the elastin-like polypeptide (ELP) which is a thermally responsive macromolecule that remains soluble in physiologic temperature but aggregates upon heating at 42 °C in the tumor. The N-terminus of ELP was modified with the cell-penetrating peptide SynB1. The SynB1-ELP construct was further modified by the addition of an acid-sensitive hydrazone derivative of doxorubicin (DOXO), by conjugation with a 6-maleimidocaproyl moiety on the drug to a terminal cysteine residue on ELP. SynB1-ELP-DOXO was found to have a thermal transition temperature of 41 °C. This construct was 2-fold more effective in inhibiting MCF-7 breast cancer cell proliferation at 42 °C than at 37 °C, indicating that the hyperthermia-induced phase transition of ELP increases the local concentration of the drug. This inhibition may be due to apoptosis since apoptosis was enhanced 2.3-fold in MCF-7 cells when SynB1-ELP-DOXO aggregation was induced by hyperthermia. SynB1-ELP-DOXO had a similar accumulation inside the cell as free Dox as shown by confocal fluorescence imaging, and the uptake into the cell was increased by hyperthermia. In the in vivo studies, a marked reduction in tumor size was seen with SynB1-ELP-DOXO exposed to hyperthermia in the E0771 syngeneic breast cancer model in C57BL/6 mice. The efficacy of the construct was further proved by the animal's body weight increasing over time as compared to treatment with free Dox and a decreasing body weight. An ongoing study uses the combination of free Dox and SynB1-ELP-DOXO at lower doses to reduce tumor size. The ELP macromolecule can also be applied to other small molecule therapeutics opening a potential platform technology for this thermally responsive biopolymer. 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 4771. doi:1538-7445.AM2012-4771

Kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii Korth. inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathway

Plants consumed by non-human primates represent potential drug sources for human disease management. In this study, we isolated kaempferol-3-O-rhamnoside as an active compound from the leaves of Schima wallichii Korth., a plant commonly consumed by non-human primates. Its anti-cancer activities, including its ability to induce apoptotic mechanisms, were investigated in MCF-7 breast cancer cells. Results showed that in MCF-7 cells, kaempferol-3-O-rhamnoside inhibits cell proliferation in a dose-dependent manner and promotes apoptosis via the activation of the caspase signaling cascade, which includes caspase-9, caspase-3 and PARP. Our results provide a basis for further exploration of kaempferol-3-O-rhamnoside as an active compound for potential anti-cancer therapeutics.

Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells

Ethnopharmacological relevanceZiziphus extracts have been used in Traditional Chinese Medicine for the treatment of cancer. Aim of the studyIn the present study we have investigated the effects of Ziziphus jujube extracts (ZEs) on breast cancer. Materials and methodsWe evaluated the effects of increasing concentrations of ZEs on ERα positive MCF-7 and ERα negative SKBR3 breast cancer cell proliferation using MTT assays. Apoptosis was analyzed by evaluating the involvement of some pro-apoptotic proteins, including Bax, Bad, Bid and PARP cleavage by immunoblotting analysis. Moreover, the effects of ZEs treatment on apoptosis were tested by both DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. By using chromatographic techniques, we identified the constituents of the effective extracts. ResultsZE1, ZE2, and ZE4 exerted significant antiproliferative effects on estrogen receptor alpha (ERα) positive MCF-7 (IC50 values of 14.42, 7.64, 1.69μg/mL) and ERα negative SKBR3 (IC50 values of 14.06, 6.21, 3.70μg/mL) human breast cancer cells. Remarkably, ZEs did not affect cell viability of both normal human fibroblasts BJ1-hTERT and nonmalignant breast epithelial MCF-10A cells. Treatment with ZEs induced cell death by apoptosis in both malignant breast cells. We found that the most effective extracts ZE2 and ZE4 shared a number of triterpenic acids, already known for their anticancer activities. ConclusionsOur data provide a rational base for the use of Ziziphus extracts in the treatment of breast cancer in Traditional Chinese Medicine.

MicroRNA let-7a suppresses breast cancer cell migration and invasion through downregulation of C-C chemokine receptor type 7

IntroductionC-C chemokine receptor type 7 (CCR7) plays an important role in chemotactic and metastatic responses in various cancers, including breast cancer. In the present study, the authors demonstrated that microRNA (miRNA) let-7a downregulates CCR7 expression and directly influences the migration and invasion of breast cancer cells.MethodsThe expression of CCR7, its ligand CCL21, and let-7a was detected in breast cancer cell lines and in breast cancer patient tissues. Synthetic let-7a and an inhibitor of let-7a were transfected into MDA-MB-231 and MCF-7 breast cancer cells, respectively, and cell proliferation, cell migration, and invasion assays were performed. To confirm the fact that 3'UTR of CCR7 is a direct target of let-7a, a luciferase assay for the reporter gene expressing the let-7a binding sites of CCR7 3'UTR was used. An in vivo invasion animal model system using transparent zebrafish embryos was also established to determine the let-7a effect on breast cancer cell invasion.ResultsFirst, a higher expression of both CCR7 and CCL21 in malignant tissues than in their normal counterparts from breast cancer patients was observed. In addition, a reverse correlation in the expression of CCR7 and let-7a in breast cancer cell lines and breast cancer patient tissues was detected. Synthetic let-7a decreased breast cancer cell proliferation, migration, and invasion, as well as CCR7 protein expression in MDA-MB-231 cells. The let-7a inhibitor reversed the let-7a effects on the MCF-7 cells. The 3'UTR of CCR7 was confirmed as a direct target of let-7a by using the luciferase assay for the reporter gene expressing let-7a CCR7 3'UTR binding sites. Notably, when analyzing in vivo invasion, MDA-MB 231 cells after synthetic let-7a transfection were unable to invade the vessels in zebrafish embryos.ConclusionsThe results from the present study suggest that targeting of CCL21-CCR7 signaling is a valid approach for breast cancer therapy and that let-7a directly binds to the 3'UTR of CCR7 and blocks its protein expression, thereby suppressing migration and invasion of human breast cancer cells. Furthermore, the present study underscores the therapeutic potential of let-7a as an antitumor and antimetastatic manager in breast cancer patients.

Mitochondria-targeted nitroxides exacerbate fluvastatin-mediated cytostatic and cytotoxic effects in breast cancer cells

Mito-CP11, a mitochondria-targeted nitroxide formed by conjugating a triphenylphosphonium cation to a five-membered nitroxide, carboxy-proxyl (CP), has been used as a superoxide dismutase (SOD) mimetic. In this study, we investigated the antiproliferative and cytotoxic properties of submicromolar levels of Mito-CP11 alone and in combination with fluvastatin, a well known cholesterol lowering drug, in breast cancer cells. Mito-CP11, but not CP or CP plus the cationic ligand, methyl triphenylphosphonium (Me-TPP+), inhibited MCF-7 breast cancer cell proliferation. Mito-CP11 had only minimal effect on MCF-10A, non-tumorigenic mammary epithelial cells. Mito-CP11, however, significantly enhanced fluvastatin-mediated cytotoxicity in MCF-7 cells. Mito-CP11 alone and in combination with fluvastatin inhibited nuclear factor kappa-B activity mainly in MCF-7 cells. We conclude that mitochondria-targeted nitroxide antioxidant molecules (such as Mito-CP11) that are non-toxic to non-tumorigenic cells could enhance the cytostatic and cytotoxic effects of statins in breast cancer cells. This strategy of combining mitochondria-targeted non-toxic molecules with cytotoxic chemotherapeutic drugs may be successfully used to enhance the efficacy of antitumor therapies in breast cancer treatment.

Design, synthesis, and biological evaluation of callophycin A and analogues as potential chemopreventive and anticancer agents

Callophycin A was originally isolated from the red algae Callophycusoppositifolius and shown to mediate anticancer and cytotoxic effects. In our collaborative effort to identify potential chemopreventive and anticancer agents with enhanced potency and selectivity, we employed a tetrahydro-β-carboline-based template inspired by callophycin A for production of a chemical library. Utilizing a parallel synthetic approach, 50 various functionalized tetrahydro-β-carboline derivatives were prepared and assessed for activities related to cancer chemoprevention and cancer treatment: induction of quinone reductase 1 (QR1) and inhibition of aromatase, nitric oxide (NO) production, tumor necrosis factor (TNF)-α-induced NFκB activity, and MCF7 breast cancer cell proliferation. Biological results showed that the n-pentyl urea S-isomer 6a was the strongest inducer of QR1 with an induction ratio (IR) value of 4.9 at 50μM [the concentration to double the activity (CD)=3.8μM] and its corresponding R-isomer 6f had an IR value of 4.3 (CD=0.2μM). The isobutyl carbamate derivative 3d with R stereochemistry demonstrated the most potent inhibitory activity of NFκB, with the half maximal inhibitory concentration (IC50) value of 4.8μM, and also showed over 60% inhibition at 50μM of NO production (IC50=2.8μM). The R-isomer urea derivative 6j, having an appended adamantyl group, exhibited the most potent MCF7 cell proliferation inhibitory activity (IC50=14.7μM). The S-isomer 12a of callophycin A showed the most potent activity in aromatase inhibition (IC50=10.5μM).

Domain and Functional Analysis of a Novel Breast Tumor Suppressor Protein, SCUBE2

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.

Antioxidant and Antiproliferative Activities of Loach (Misgurnus anguillicaudatus) Peptides Prepared by Papain Digestion

Loach protein hydrolysates (LPH) prepared by papain digestion were fractionated into four fractions, LPH-I (MW > 10 kDa), LPH-II (MW = 5-10 kDa), LPH-III (MW = 3-5 kDa), LPH-IV (MW < 3 kDa), and the in vitro antioxidant and antiproliferative (anticancer) activities of all fractions were determined. LPH-IV showed the lowest IC(50) value (16.9 ± 0.21 mg/mL) for hydroxyl radical scavenging activity and the highest oxygen radical scavenging capacity (ORAC) value (reaching 215 ± 5.9 mM Trolox/100 g loach peptide when the concentration was 60 μg/mL). Compared with other fractions, LPH-IV also exhibited stronger antiproliferative activity for human liver (HepG2), breast (MCF-7), and colon (Caco-2) cancer cell lines in a dose-dependent manner. When the protein concentration was 40 mg/mL, the HepG2 and MCF-7 cell proliferation of LPH-IV reached 7 and 4%, respectively, with no significant difference from those of LPH (8 and 7%, p > 0.05), with significantly less growth than those of LPH-I, LPH-II, and LPH-III, respectively (p < 0.05). The Caco-2 colon cell proliferation of LPH-IV was 12.8- and 8.7-fold smaller than those of LPH-I and LPH-II, respectively (p < 0.05). All of the fractions had a greater ability to inhibit Caco-2 colon cancer cell proliferation than to inhibit HepG2 liver cancer and MCF-7 breast cancer cell proliferation. The ORAC values of most of the fractions correlated (R(2) > 0.86, p < 0.01) with the antiproliferative activity of the three cancer cell lines, suggesting that higher antioxidant activity leads to better antiproliferative activity. However, further mechanistic and human clinical studies of the anticancer activity of loach protein hydrolysate fractions are needed.

Formulation of cashew nut shell liquid (CSNL) nanoemulsion, a potent inhibitor of human MCF-7 breast cancer cell proliferation

Cashew nut shell liquid (CSNL) nanoemulsion has been formulated and its cytotoxic activity was determined using human breast cancer cells (MCF-7) adopting MTT assay and established staining techniques. MCF-7 cells treated with CSNL nanoemulsion suffered loss of viability and death mostly through apoptosis and/or necrosis as evidenced by the changes in the nuclear morphology and cytoplasmic features. The IC50 value of 48-h treatment groups fall in the range of 88 ± 14.2 μl/ml. In conclusion, the antiproliferative and morphological effects of CSNL nanoemulsion appear to be target-specific killing of cells which gives further lead to study apoptotic pathways as target for breast cancer therapy. This study has provided a scientific basis for the anti-tumor property of CSNL nanoemulsion with respect to breast cancer.

Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies

BackgroundEpithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety.MethodsWe recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation.ResultsING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells.ConclusionA moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.

Abstract LB-135: The transcriptome of p300 loss in MCF-7 cells

Abstract Introduction: p300 is a transcriptional co-activator that promotes gene transcription by acetylating DNA-binding factors and bridging them to the basal transcription machinery. Recent studies have revealed decreased levels of p300 in breast cancer cells, suggesting that p300 may be involved in the control of breast cancer growth and progression. Preliminary data from our laboratory also indicates that decreased p300 function promotes MCF-7 breast cancer cell proliferation in vitro and tumorigenesis in vivo. Methodology: In order to identify the effects of p300 loss on gene expression in breast cancer cells, microarray analysis was performed on total RNA from MCF-7 cells, stably transduced with lentiviruses expressing one of 3 p300 shRNAs or a non-silencing sequence. Silencing of p300 was &amp;gt; 50% in response to each of the shRNAs. Endogenous RNA was converted to cDNA using random primers and then to biotinylated cRNA for hybridization to human exonic evidence-based oligonucleotide microarrays (Ocean Ridge Biosciences, Palm Beach Gardens, FL). 877 genes were found to be differentially expressed in p300 knockdown MCF-7 cells compared to non-silenced controls. Differentially regulated genes were ranked according to the significance of change (p value &amp;lt; 0.0005) and compared to previously reported data in the Oncomine database, for both directionality (up versus down-regulation) and positive correlation with decreased p300 levels. As an additional strategy we ranked the differentially expressed genes from our microarray with good level of significance (p&amp;lt;0.05) by fold change and repeated the comparative analysis with Oncomine as described above. Results: In our analysis, 877 genes were found to be differentially expressed out of which 27 genes were either significantly up-regulated or down-regulated. The rest of the gene expression levels found in the microarray analysis were not as highly significant as the top 27 genes. Among those 27 genes, four (SPAG7, OAS2, RARRES3, SERPINA6) showed highly significant (p&amp;lt;0.0005) increase in their expression and five genes (DHX9, EIF4G2, POLR2D, MCM6, CYP4F2) showed highly significant decrease. Among the genes with the largest fold changes, 10 (IFI27, IFI6, ATF-3, PIK3IP1, TNFAIP3, P8, RARRES3, FOS, IRF9, and RNF39) showed significantly increased expression and 8 were decreased (WNT3A, CAMK1, CENPH, DCTN1, CASC5, HSPA4, CDC25A, and CDKN2C). The second set of genes showed correlations with p300 levels as indicated in analyses of Oncomine data. Some of these genes may be contributing to epithelial to mesenchymal- transition (EMT) under p300 knock-down conditions. Conclusions: Our microarray analysis in MCF-7 cells with partial p300 loss has identified several key genes that are related to breast cancer growth. These genes may be important in mediating the function of p300 loss in tumorigenicity, and future studies will focus on the use of p300 or its downstream signaling components as therapeutic targets in breast cancer treatment. 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-135.

Abstract 5339: Inhibition of breast cancer cell proliferation and unique epitope recognition by EpCAM-specific human monoclonal antibody adecatumumab

Abstract Epithelial cell adhesion molecule (EpCAM; CD326) is frequently and highly expressed on a wide variety of human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients for safety and anti-tumor activity. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. We have recombinantly produced all antibodies but murine edrecolomab in Chinese hamster ovary cells and investigated all five antibodies for their binding affinity, EpCAM epitope recognition, antibody-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC), and inhibition of breast cancer cell proliferation. ING-1 and 3622W94 bound to recombinant EpCAM with much higher affinity than adecatumumab and edrecolomab, which was largely determined by slower off-rates. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded, N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing human cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities for recombinant EpCAM. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC with human effector cells than the murine IgG2a version of the antibody. Of the five antibodies, only adecatumumab showed a significant inhibition of MCF7 breast cancer cell proliferation in the absence of complement and immune cells. A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis. Adecatumumab emerges as an anti-EpCAM antibody with unique characteristics and biological activity. 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 5339.

Abstract 2653: Thermally targeted delivery of an acid-labile derivative of doxorubicin by elastin-like polypeptide

Abstract Doxorubicin (Dox) is an anthracycline used in the treatment of many types of cancer. It intercalates into the strands of DNA to inhibit the progression of topoisomerase II, stopping the cell from replicating. The side effects, however, are severe and include neutropenia, alopecia, nausea, myelosuppression, and cardiotoxicity. The drug is nonspecific; as a result, high doses are needed to treat cancer cells. These high doses are inadvertently harmful to normal cells. Therefore, there is a need for an efficient drug delivery system in order to deliver doxorubicin to the tumor site. Our lab has developed a macromolecular carrier that can provide this targeted delivery. This elastin-like polypeptide (ELP) is thermally responsive as it remains in solution at physiological temperature and aggregates upon heating at 42 °C. The N-terminus of ELP was modified with three different cell penetrating peptides (CPPs) - Bac, Tat, and SynB1. These peptides are derived from the bactenecin family of antimicrobial peptides, truncated version of the HIV-1 Tat protein, and porcine leukocytes, respectively. This study compared the efficiency of these three CPP-ELP carriers to deliver doxorubicin to the cells. ELP was attached to a hydrazone derivative of doxorubicin (DOXO) by conjugation of a 6-maleimidocaproyl moiety on the drug with a terminal cysteine residue on ELP. This linker allows the drug to be cleaved at acidic pH in the cell. The three CPP-ELP-DOXO conjugates had transition temperatures within the range of 38-42°C. All three carriers of DOXO inhibited MCF-7 breast cancer cell proliferation 2-fold more at 42 °C than at 37 °C, indicating that the hyperthermia induced phase transition of ELP does increase the local concentration of the drug. Inhibition of cell proliferation may be due to apoptosis, since apoptosis was enhanced 2.3-fold in MCF-7 cells when SynB1-ELP-DOXO aggregation was induced with hyperthermia. CPP-ELP-DOXO accumulated in cells to the same level as free Dox. Confocal imaging showed that when cells were treated with any of the three CPP-ELP-DOXO derivatives, Dox localized in the nucleus, similar to the free Dox. The results suggest that CPP-ELP-DOXO conjugates may provide a means to thermally target solid tumors. 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 2653.

Effect of androgen on the proliferation and apoptosis of MCF-7 breast cancer cells

Objective To observe the effect of testosterone on the proliferation, apoptosis of MCF-7 breast cancer cells and investigate the mechanism. Methods MCF-7 cells were treated with testosterone at concentrations of 1×10~(-5),1×10~(-7),1 ×10~(-9),1×10~(-11) mol/L for 24,48 and 72 h respectively. Cell via-bility and growth inhibition rate of MCF-7 cells were assayed by MTr methods. Cell cycle, apoptosis rate and the expression of Cyclin D1, androgen receptor (AR) were detected by flow cytometry. Results High concentration testosterone could inhibit the proliferation of MCF-7 cells. After MCF-7 cells were treated with 1×10~(-5) mol/L testosterone for 48 h ,the cell growth inhibition rate was 22.21% ,and the apoptosis rate was (58.60±5.41 )%. High concentration testosterone could accelerate MCF-7 cell cycle progression from G_1 to S phases by increasing the expression of Cyclin D1 and decreasing the expression of AR. Low concentra-tion testosterone (1×10~(-9) mol/L) could increase the expression of AR, but had no effect on Cyclin D1, and it could promote MCF-7 cells proliferation in short time (24 h). Conclusion High concentration tes-tosterone can increase the expression of Cyclin D1 of MCF-7 cells and accelerate their cell cycle from G_1 to S phases,and then induce them to apoptosis in vitro. Low concentration testosterone can promote MCF-7 cells proliferation in short time. Key words: Androgen; Breast carcinoma; Proliferation; Apoptosis

Abstract B131: Enhanced therapeutic effect of combined treatments with h7C10, a humanized antibody against insulin-like growth factor-1 receptor, and chemotherapeutic agents: In vitro results on MCF-7 cell line

Abstract IGF-1 receptor (IGF-1R) plays a key role in the development of numerous tumors. Blockade of IGF-1R axis using monoclonal antibodies constitutes an interesting approach to inhibit tumor growth. We have previously described h7C10 (also named MK-0646), a humanized anti-IGF-1R Mab, that exhibits a potent anti-tumor activity both in vitro and in vivo. It blocks IGF-1 and IGF-2 induced signaling and blocks in vivo tumor growth of IGF-IR expressing tumors. In this report, we examined the in vitro effects of this selective IGF-1R Mab in combination with a panel of chemotherapeutic agents. The effect of drugs and h7C10 on the growth of human breast cancer cell line MCF-7 was determined by 3H-thymidine incorporation in DNA. The combined effect of drugs and h7C10 on MCF-7 cell line was evaluated by the combination index (CI) analysis method described by Chou and Talalay. We showed that the combination of h7C10 with a panel of drugs namely, paclitaxel, docetaxel, doxorubicin and gemcitabine induced synergistic inhibitory effects on MCF-7 breast cancer cell proliferation in vitro. These findings demonstrated that h7C10 combined with established cancer therapeutics is highly effective against cancer cells that expressed IGF-1R and support clinical evaluation of h7C10 in combination with these drugs in the treatment of human cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B131.

Role of PDZ Domain-Containing 1 (PDZK1) in MCF-7 Breast Cancer Cells.

Breast cancer is the most common cancer and second leading cause of cancer death in women in the U.S. Approximately 70% of the breast cancers are estrogen receptor (ER) positive at the time of presentation. Estrogen induced genes are either considered as markers for diagnosis or candidate oncogenes of breast cancer. PDZ domain-containing 1 (PDZK1) is expressed in ER positive primary breast cancers and PDZK1 expression is induced by estrogen in ER positive MCF-7 breast cancer cells. Chromosome 1 was reported to be involved in the anomalies of 50-60% of breast cancer. PDZK1 gene is localized in chromosome 1q21 and shows significant overexpression correlated to copy number increase in breast cancer cells. PDZK1 is involved in the maintenance of endothelial cell monolayer integrity, stabilization of scavenger receptor class B, type I (SR-BI) in the liver, and lipid metabolism. SR-BI is a physiological receptor for high density lipoprotein (HDL) and HDL was reported to increase MCF-7 breast cancer cell proliferation. However, the role of PDZK1, its signaling pathway after estrogen stimulation, and its interaction with SR-BI in breast cancer cells are not known. The objectives of this study were to determine whether 1) estrogen induced PDZK1 expression is mediated through ER, 2) PDZK1 binds to Src kinase, 3) PDZK1 expression affects cholesterol ester uptake from HDL by its interaction with SR-BI, and 4) PDZK1 binding proteins are induced by estrogen in the MCF-7 breast cancer cells. MCF-7 breast cancer cells were cultured with 1 nM estradiol-17β (E2). PDZK1 mRNA and protein expression was determined by real-time PCR and western blotting, respectively. Knockdown of ERα and ERβ was performed by specific siRNA. PDZK1 binding to Src kinase was determined by immunoprecipitation followed by western blotting. Uptake of cholesterol ester was determined by flowcytometer and by fluorescence microscope after incubating cells with fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled HDL. Expression of PDZK1 binding proteins of ATP-binding cassette, subfamily C, member 2 (ABCC2) and PDZK1 interacting protein 1 (PDZK1-IP1) mRNAs was determined by real-time PCR. PDZK1 mRNA and protein expression was detectable after 12h and 24h of 1 nM E2 treatment, respectively. Knockdown of ERα by siRNA reduced E2 stimulated PDZK1 expression to the basal level. Knockdown of ERβ by siRNA reduced PDZK1 expression less than that of ERα. Immunoprecipitation by Src antibody showed that Src binds to PDZK1 and ERα. Fluorescence detected after incubation with DiI-HDL in the presence of E2 stimulation was reduced by PDZK1 or SR-BI siRNA. Though E2 induced PDZK1 expression, but it did not stimulate the expression of ABCC2 or PDZK1-IP1 mRNA. In conclusion, estrogen induced PDZK1 expression is mediated by ER in ER positive MCF-7 breast cancer cells. PDZK1 may bind phosphorylated Src after estrogen stimulated ER binds to Src, since PDZK1 protein was induced 24h after estrogen stimulation. This reaction may occur on the cell membrane. Estrogen treatment may enhance cholesterol ester uptake from circulating HDL by SR-BI and it may stimulate cell proliferation. Since PDZK1 binding proteins of ABCC2 and PDZK1-IP1 were not induced by estrogen and therefore may not participate as scaffolding proteins in the signaling of PDZK1. This pathway may be targeted as a new therapeutic intervention blocking the effect of estrogen on cell proliferation. (poster)

SCUBE2 Suppresses Breast Tumor Cell Proliferation and Confers a Favorable Prognosis in Invasive Breast Cancer

Signal peptide-CUB-epidermal growth factor-like domain-containing protein 2 (SCUBE2), originally identified from the endothelium and several nonendothelial primary cell types, was recently shown to be expressed in invasive breast carcinomas. However, the protein localization and biological significance of SCUBE2 in breast cancer are unknown. In this report, we show by anti-SCUBE2 immunostaining that SCUBE2 is mainly expressed in vascular endothelial and mammary ductal epithelial cells in normal breast tissue. In addition, we observed positive staining for SCUBE2 in 55% (86 of 156) of primary breast tumors. Patients with positive SCUBE2 protein-expressing tumors had better prognosis than those with negative SCUBE2 protein-expressing tumors in terms of disease-free survival. Multivariate analysis confirmed SCUBE2 protein expression as an independent prognostic factor for disease-free survival. Furthermore, overexpression of ectopic SCUBE2 protein resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH terminal region of SCUBE2 directly bound to and antagonized bone morphogenetic protein activity. Together, our results show for the first time that altered SCUBE2 expression is important in breast cancer progression and SCUBE2 may serve as a useful prognostic marker.

Effect of ursolic add on the growth of MCF-7 breast cancer cells

Objective To investigate the effect of ursolic acid on proliferation,apoptosis,invasion of MCF-7 breast cancer cells and MCF-7 breast tumor growth in nude mice. Methods In the ursolic acid group, MCF-7 cells were treated with ursolic acid at concentrations of 1, 10,or 100 μmol/L for 12,24 and 48 h respectively. Cell viability of MCF-7 cells and growth inhibition rate were assayed by MTT methods. Cell cycle, apoptosis rate and cell invasiveness were assayed by flow cytometry and transwell chamber methods. Nude mice transplanted with MCF-7 cells were divided into saline control group,low-dose ursolic plantation in the nude mice were detected. Chnages in hver and renal function,and routine blood at 15th days were tested. Results With the increase of ursolic acid concentration and the prolongation of action time,MCF-7 cell growth and apoptosis were affected significantly. After MCF-7 cells were treated with 100 μmol/L ursolic acid for 48 h,the cell growth inhibition rate was 46.0%, the growth cycle was arrested in G0/G1 phase,the apoptosis rate was (16.48±2.46)%, and the cell invasiveness decreased significantly. Tumor size in the ursolic acid group was significantly smaller than in the control group. In the high-dose ursolic acid group at 15th day,the tumor size was (323.5±33.5) mm3,the rumor growth inhibit rate was 50.9%,and the liver and renal function,routine blood had no significant changes. Conclusion Ursolic acid can inhibit proliferation and invasiveness of MCF-7 breast cancer cells, and induce apoptosis rate in vitro. Ursolic acid can significantly inhibit the growth of breast cancer in nude mice. Key words: Breast carcinoma; Ursolic acid; Proliferation

Bioassays for Estrogenic Activity: Development and Validation of Estrogen Receptor (ERα/ERβ) and Breast Cancer Proliferation Bioassays to Measure Serum Estrogenic Activity in Clinical Studies

Standard estrogenic prodrugs such as estradiol valerate (E2V) and increasingly popular phytoestrogen formulations are commonly prescribed to improve menopausal health. These drugs are metabolized to numerous bioactive compounds, known or unknown, which may exert combinatorial estrogenic effects in vivo. The aim of this study is to develop and validate estrogen receptor (ER) alpha/ERbeta reporter gene and MCF-7 breast cancer cell proliferation bioassays to quantify serum estrogenic activities in a clinical trial setting. We measured changes in serum estrogenicity following ingestion of E2V and compared this to mass spectrometric measurements of its bioactive metabolites, estrone and 17beta-stradiol. ERalpha bioactivity of the 192 serum samples correlated well (R = 79%) with 17beta-estradiol levels, and adding estrone improved R to 0.83 (likelihood ratio test, P < 0.0001), suggesting that the ERalpha assay reflects summated activity of compounds in serum. ERbeta correlated moderately (R = 0.52) with estrone and 17beta-estradiol, with an estrone/17beta-estradiol coefficient ratio that was twice that of ERalpha, indicating estrone was more active on a molar basis in the ERbeta assay. Unlike the ERalpha and ERbeta bioassays, MCF-7 cell proliferation was driven by 17beta-estradiol, and addition of estrone did not increase the predictive value of the model, suggesting that the driver or drivers for breast cancer cell proliferation were not the same as for ERalpha and ERbeta transactivation. In contrast, a decoction of the traditional Chinese medicinal herb Epimedium pubescens did not induce significant changes in estrogenic bioactivity over baseline. These data indicate that ERalpha/ERbeta reporter gene and MCF-7 breast cancer cell proliferation bioassays reflect different aspects of estrogenic activity and that these assays suggest that the Epimedium formulation tested is unlikely to exert significant estrogenic effects in humans.

8-Prenylnaringenin inhibits epidermal growth factor-induced MCF-7 breast cancer cell proliferation by targeting phosphatidylinositol-3-OH kinase activity

8-Prenylnaringenin (8PN), one of the strongest plant-derived oestrogen receptors (ERs) ligand, has been suggested to have potential cancer chemo-preventive activities and anti-angiogenic properties. Because published data suggest that ERs serve as nodal point that allows interactions between hormones and growth factors mediated pathways, we decided to investigate the effects exerted by 8PN on Epidermal growth factor (EGF)-elicited pathways in breast cancer cells. Here we show that in ER positive MCF-7 cells, 8PN interferes with EGF induced cell proliferation by strongly inhibiting activation of PI(3)K/Akt pathway, without affecting EGFR expression or tyrosine phosphorylation, and exerting a synergistic activation of Erk1/2 phosphorylation. Moreover, we demonstrate that 8PN is a direct inhibitor of PI(3)K activity as it is shown by in vitro experiments with the purified enzyme and by its inability to impair serine phosphorylation of a constitutive active form of Akt. These findings suggest that inhibition of PI(3)K is a novel mechanism which contributes to 8PN activity to inhibit cancer cell survival and EGF induced proliferation.