MCF-7 Xenograft Tumors Research Articles

IR-780 dye loaded tumor targeting theranostic nanoparticles for NIR imaging and photothermal therapy

IR-780 iodide is a near-infrared (NIR) fluorescence dye with higher and more stable fluorescence intensity than clinically applied dye indocyanine green (ICG). Meanwhile, IR-780 can be utilized in photothermal therapy with laser irradiation. IR-780 is an important theranostic agent but its lipophilicity limited its application. In this paper, we synthesize multifunctional heparin–folic acid-IR-780 nanoparticles (HF-IR-780 NPs) by self-assembly of the heparin–folic acid conjugate and IR-780 through ultrasonic sound method. The HF-IR-780 NPs exhibit good monodispersity, significant stability, and excellent molecular targeting to folate receptor over-expressing MCF-7 cells. Furthermore, the in vivo biodistribution experiments show that the HF-IR-780 NPs are specifically targeted to the tumor and can be used for tumor imaging. The in vitro cell viability assays and in vivo photothermal therapy experiments indicate that MCF-7 cells or MCF-7 xenograft tumors could be ablated by combining HF-IR-780 NPs with irradiation of an 808 nm laser. The photothermal therapy in vivo with a single-dose treatment has not caused significant adverse effect. The resulted HF-IR-780 NPs are a potential theranostic agent for imaging-guided cancer therapy.

Abstract 3920: In vivo tyrosinase reporter gene imaging with multispectral photoacoustic technology.

Abstract Photoacoustic imaging is a powerful tool for assessing tumor vasculature and oxygen saturation as well as detecting contrast agents for molecular imaging. Another exciting possibility is to visualize gene expression by using a reporter which produces a photoacoustic contrast agent. Tyrosinase is such a reporter in that its expression results in the production of melanin, which gives a strong photoacoustic signal. Here we describe the use of a commercially available photoacoustic (PA) imaging system (Vevo LAZR, VisualSonics, Toronto) to image inducible gene expression in subcutaneous xenograft tumors using tyrosinase as a reporter gene. The photoacoustic imaging system generated light from a tunable laser (680 - 970 nm) which was delivered through fiber optic bundles integrated into a linear array transducer (LZ-250, fc = 21 MHz), mounted to a linear stepper motor for 3D imaging. Animals (n=3) having MCF-7 xenograft tumors on either flank transfected with (+TYR) or without (-TYR) doxycycline-regulated tyrosinase were imaged before and one week after the induction of tyrosinase expression. 3D images of the tumors were acquired using multiple wavelengths (680, 750, 800, 850, 900 and 950nm) and 2D images were acquired across the entire wavelength range of the laser to generate absorption curves for blood and melanin. To confirm the presence of melanin and distinguish it from the endogenous blood signal, one animal was exsanguinated and the tumor was imaged again. Approximately 1mm-thick slices of the tumors were taken and photographed for visual confirmation of the presence of melanin. Comparison of pre- and post-doxycycline images of +TYR tumors clearly showed enhanced contrast in the tumor which closely matched the absorption spectrum of melanin. This signal persisted upon exsanguination. -TYR tumors showed signal which corresponded with the spectra of oxy and deoxy hemoglobin and changed little over the course of the experiment. Visual inspection of the excised sliced tumors revealed pigmentation in the +TYR tumors which was absent in the -TYR tumors. Here we have shown the ability of the Vevo LAZR photoacoustic imaging system to visualize inducible reporter gene expression in vivo. This has implications for assessing genetically controlled cellular processes and their response to anti-cancer drugs but also for monitoring gene therapy for cancer treatment non-invasively. Citation Format: Andrew Heinmiller, Minalini Lakshman, Dave Bates, Andrew Needles, Catherine Theodoropoulos, Robert J. Paproski, Roger J. Zemp. In vivo tyrosinase reporter gene imaging with multispectral photoacoustic technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3920. doi:10.1158/1538-7445.AM2013-3920

Abstract 1327: IGF-I and insulin signaling enhances levels of choline kinase in breast cancer cells: Implications for developing noninvasive magnetic resonance spectroscopy of choline containing compounds as a biomarker of response to IGF1R targeted therapy.

Abstract Insulin-like growth factor I (IGF-I) and insulin signal through the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively. Multiple drugs targeting IGF1R and IGF1R/IR are being tested in clinical trials. Biomarkers that enable the selection of patients with IGF driven tumors or the monitoring of response to this therapy have not yet been identified. Previously, we found that intratumoral total choline (tCho) metabolites decreased in MCF-7 xenograft tumors in mice twenty-four hours following administration of an inhibitory antibody against IGF1R. The tCho was measured by 1H magnetic resonance spectroscopy (MRS) and included three species of choline containing compounds, choline, phosphocholine (pCho), and glycerophosphocholine. These data suggested that noninvasive measurement of tCho may be a useful surrogate marker of response to IGF1R targeted therapy. The objective of the current study is to investigate whether there is a mechanistic link between IGF-I and insulin signaling and choline phospholipid metabolism to understand if decreased tCho levels could be a specific marker of inhibition of IGF signaling in cancer cells. In this study, we examined the effect of IGF-I and insulin signaling on levels of choline kinase (CK). CK is the enzyme that generates pCho, one of the species measured by MRS in our prior in vivo study. Estrogen receptor positive (ER+) human breast cancer cell lines (MCF-7, T47-D, ZR-75-1) and triple negative cells (MDA-MB-231, MDA-231-LM2 and MDA-MB-435A/LCC6) were treated with IGF-I or insulin for 10 minutes, 4h or 24h and levels of CK were examined. IGF-I and insulin signaling upregulated CK levels in ER+ breast cancer cells (MCF-7, T47-D, and ZR-75-1) but not in triple negative breast cancer cells (MDA-MB-231, MDA-231-LM2 and MDA-MB-435A/LCC6). In ER+ breast cancer cells, IGF-I and insulin activate the mitogen-activated protein kinase (MAPK) pathway. However, in triple negative breast cancer cells phosphorylation of p44/p42 MAPK are unaffected by IGF-I and insulin signaling or IGF1R inhibition. Inhibition of the MAPK pathway with U0126 blocked IGF-I and insulin induced upregulation of CK. These data suggest that IGF-I and insulin induce upregulation of CK via a MAPK dependent mechanism. We next investigated the effect of EM164, an inhibitory antibody against IGF1R, on IGF-I and insulin induced upregulation of CK. EM164 blocked IGF-I induced upregulation of CK but did not affect insulin induced upregulation of CK. These results provide molecular evidence for the observed decrease in intratumoral tCho after IGF1R inhibition in xenograft tumors. Our study is clinically significant as it provides evidence for developing decreased tCho, following IGF1R targeted drugs, as a specific biomarker for inhibition of IGF signaling. Citation Format: Huy T. Donguyen, Joseph C. Weber, Deepali Sachdev. IGF-I and insulin signaling enhances levels of choline kinase in breast cancer cells: Implications for developing noninvasive magnetic resonance spectroscopy of choline containing compounds as a biomarker of response to IGF1R targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1327. doi:10.1158/1538-7445.AM2013-1327

Abstract 3801A: Reoxygenation and repopulation of hypoxic cells in a solid tumor after chemotherapy: a cause of treatment failure.

Abstract Background: Hypoxia occurs in solid tumors: chronic diffusion-limited hypoxia occurs distal from functional blood vessels, while acute hypoxia may occur because of interruptions in blood flow. Well-nourished tumor cells close to blood vessels tend to be rapidly proliferating but hypoxic cells located farther away are slowly-proliferating. Chemotherapy may spare hypoxic cells because of poor drug distribution to them and because most drugs are selectively toxic to proliferating cells. Intervals between administration of chemotherapy allow for recovery of normal tissues (e.g. repopulation of bone marrow) but might allow re-oxygenation and resumed proliferation of formerly hypoxic cells due to better supply of nutrients to them, as is observed during radiotherapy. Here we evaluate these processes in human tumor xenografts Methods: Two specific markers of hypoxic cells (pimonidazole [pimo] and EF5) were injected into mice bearing MCF7 tumor xenografts, and tumor cells labeled with one or both markers were recognized in tumor sections (in relation to DioC7+ve functional tumor blood vessels) using appropriate fluorescence-labeled antibodies and imunohistochemistry. Proliferating cells were identified by an antibody to Ki67. Mice were treated with pimo and then either doxorubicin or saline one hour later; EF5 was given after a variable interval of 24, 48, 72, 96 or 120 hours; mice were killed two hours after the second injection. Changes in the location, proliferation and oxygen status of formerly hypoxic (pimo+ve) cells were quantified by their distance from hypoxia, Ki67 status and uptake of EF5 as a function of time. Results: Following treatment with doxorubicin, the proportion of hypoxic cells in the entire tumor decreased from 1.5% (pimo+ve) prior to injection to 0.7% (EF5+ve) at 24 hours. The percentage of pimo+ve formerly hypoxic cells that are no longer hypoxic (i.e. EF5 - ve) at 24 hours was 90% after docxorubicin compared to 27% in controls, indicating rescue of previous hypoxic cells that would have died in the absence of treatment. Proliferation of these pimo+ve cells that were cycling (Ki67+ve) increased from 7% to 15.0% at 24 Hours and then slowly decreased. Conclusions: Formerly hypoxic cells in MCF7 xenografts undergo re-oxygenation and increase their proliferation following treatment with doxorubicin. Originally hypoxic cells (that may have died in the absence of treatment) can move closer to blood vessels, re-oxygenate and repopulate a tumor. Treatment directed to killing hypoxic cells (e.g. with hypoxia activated pro-drugs) has potential to improve the outcome of chemotherapy. Supported by a research grant from the Canadian Institutes for Health Research. Citation Format: Jasdeep K. Saggar, Ian F. Tannock. Reoxygenation and repopulation of hypoxic cells in a solid tumor after chemotherapy: a cause of treatment failure. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3801A. doi:10.1158/1538-7445.AM2013-3801A

BMS-345541 Sensitizes MCF-7 Breast Cancer Cells to Ionizing Radiation by Selective Inhibition of Homologous Recombinational Repair of DNA Double-Strand Breaks

Our study was to elucidate the mechanisms whereby BMS-345541 (BMS, a specific IκB kinase β inhibitor) inhibits the repair of DNA double-strand breaks (DSBs) and evaluate whether BMS can sensitize MCF-7 breast cancer cells (MCF-7 cells) to ionizing radiation (IR) in an apoptosis-independent manner. In this study, MCF-7 cells were exposed to IR in vitro and in vivo with or without pretreatment of BMS. The effects of BMS on the repair of IR-induced DSBs by homologous recombination (HR) and non-homologous end-joining (NHEJ) were analyzed by the DR-GFP and EJ5-GFP reporter assays and IR-induced γ-H2AX, 53BP1, Brca1 and Rad51 foci assays. The mechanisms by which BMS inhibits HR were examined by microarray analysis and quantitative reverse transcription PCR. The effects of BMS on the sensitivity of MCF-7 cells to IR were determined by MTT and clonogenic assays in vitro and tumor growth inhibition in vivo in a xenograft mouse model. The results showed that BMS selectively inhibited HR repair of DSBs in MCF-7 cells, most likely by down-regulation of several genes that participate in HR. This resulted in a significant increase in the DNA damage response that sensitizes MCF-7 cells to IR-induced cell death in an apoptosis-independent manner. Furthermore, BMS treatment sensitized MCF-7 xenograft tumors to radiation therapy in vivo in an association with a significant delay in the repair of IR-induced DSBs. These data suggest that BMS is a novel HR inhibitor that has the potential to be used as a radiosensitizer to increase the responsiveness of cancer to radiotherapy.

Abstract P2-14-02: Preclinical Evaluation of Enzalutamide in Breast Cancer Models

Abstract Background: The vast majority of breast cancers express the androgen receptor (AR) with ∼80% of ER+ and 30% to50% of ER− tumors being positive for AR by immunohistochemistry (IHC). Approximately 30% of breast cancer patients with estrogen receptor positive (ER+) tumors do not respond to tamoxifen or aromatase inhibitors and AR signaling has been implicated as a possible mechanism of this insensitivity. AR overexpression has also been associated with resistance to either tamoxifen or aromatase inhibitors in cell line models. Hypothesis: ER+ breast cancers can switch from estrogen to androgen-dependent growth as they become resistant to traditional endocrine therapies and AR can serve as a therapeutic target in AR+ breast cancers, irrespective of ER status. Methods: The proliferative effect of dihydrotestosterone (DHT) and estradiol (E2) in the presence enzalutamide (formerly MDV3100) was examined in vitro and in xenograft studies using models of both ER+ and ER− breast cancer that express AR. Furthermore, AR and ER were examined by IHC in clinical specimens from breast cancer patients treated with neoadjuvant exemestane and adjuvant tamoxifen. Tumor tissue was compared to adjacent normal breast epithelium. Results: In ER+ models, bicalutamide and enzalutamide both inhibited DHT-mediated proliferation, while enzalutamide uniquely inhibited E2-mediated proliferation. In vivo, enzalutamide significantly reduced estrogen- and androgen-mediated growth of MCF7 (ER+/AR+) xenograft tumors. Remarkably, enzalutamide demonstrated an anti-proliferative effect comparable to tamoxifen. in vitro, enzalutamide inhibits E2-stimulated tumor cell proliferation and E2 bound ER activation of genes such as PR and SDF-1, despite no observed binding of enzalutamide to ER alpha or beta. In ER−/AR+ MDA-MB-453 xenografts, enzalutamide treatment resulted in decreased nuclear AR localization, increased apoptosis and tumor growth inhibition. Conclusions: Enzalutamide demonstrated significant anti-tumor activity in preclinical models of breast cancers that express AR, regardless of ER status. When enzalutamide opposes DHT, it functions by excluding AR from the nucleus, thereby reversing the anti-apoptotic effect of AR. In contrast, when opposing E2, it causes a decrease in tumor cell proliferation. Profiling of ER+ xenografts is underway to further elucidate the mechanism by which this occurs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-14-02.

HER2-Associated Radioresistance of Breast Cancer Stem Cells Isolated from HER2-Negative Breast Cancer Cells

To understand the role of HER2-associated signaling network in breast cancer stem cells (BCSC) using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2(-/low) breast cancers. HER2-expressing BCSCs (HER2(+)/CD44(+)/CD24(-/low)) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated signaling network was generated by MetaCore program. Compared with HER2-negative BCSCs, HER2(+)/CD44(+)/CD24(-/low) cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2(+)/CD44(+)/CD24(-/low) cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/MS-MS of HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs reported a unique HER2-associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function, and DNA repair. A specific feature of HER2-STAT3 network was identified. This study provides the evidence that HER2-mediated prosurvival signaling network is responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant HER2(-/low) breast cancer.

TIGAR induces p53-mediated cell-cycle arrest by regulation of RB–E2F1 complex

Background:p53 induces cell-cycle arrest and apoptosis in cancer cells and negatively regulates glycolysis via TIGAR. Glycolysis is crucial for cancer progression although TIGAR provides protection from reactive oxygen species and apoptosis. The relation between TIGAR-mediated inhibition of glycolysis and p53 tumour-suppressor activity is unknown.Methods:RT–PCR, western blot, luciferase and chromatin immunoprecipitation assays were used to study TIGAR gene regulation. Co-IPP was used to determine the role of TIGAR protein in regulating the protein–protein interaction between retinoblastoma (RB) and E2F1. MCF-7 tumour xenografts were utilised to study the role of TIGAR in tumour regression.Results:Our study shows that TIGAR promotes p21-independent, p53-mediated G1-phase arrest in cancer cells. p53 activates the TIGAR promoter only in cells exposed to repairable doses of stress. TIGAR regulates the expression of genes involved in cell-cycle progression; suppresses synthesis of CDK-2, CDK-4, CDK-6, Cyclin D, Cyclin E and promotes de-phosphorylation of RB protein. RB de-phosphorylation stabilises the complex between RB and E2F1 thus inhibiting the entry of cell cycle from G1 phase to S phase.Conclusion:TIGAR mediates de-phosphorylation of RB and stabilisation of RB–E2F1 complex thus delaying the entry of cells in S phase of the cell cycle. Thus, TIGAR inhibits proliferation of cancer cells and increases drug-mediated tumour regression by promoting p53-mediated cell-cycle arrest.

Human serum albumin-coated lipid nanoparticles for delivery of siRNA to breast cancer

Human serum albumin (HSA)-coated lipid nanoparticles (HSA-LNPs) loaded with phrGFP-targeted siRNA (HSA-LNPs-siRNA) were prepared and evaluated for gene downregulation effect in phrGFP-transfected breast cancer cells and the corresponding xenograft tumor model. HSA-LNPs-siRNA were successfully prepared with a particle size of 79.5±5.5 nm. In phrGFP-transfected MCF-7 cells, HSA-LNPs-siRNA significantly decreased cell fluorescence even in the presence of fetal bovine serum (FBS). Moreover, cell fluorescence and phrGFP mRNA expression were significantly downregulated by HSA-LNPs-siRNA in phrGFP-transfected MCF-7, MDA-MB-231, and SK-BR-3 cells in comparison with control or HSA-LNPs-siRNA (scrambled). In phrGFP-transfected MCF-7 xenograft tumor model, tumor fluorescence was significantly decreased after three IV administrations of HSA-LNPs-siRNA at a dose of 3 mg/kg in comparison with siRNA alone. HSA-LNPs-siRNA demonstrated a superior pharmacokinetic profile in comparison with siRNA at a dose of 1mg/kg. These results show that the novel nonviral carrier, HSA-LNPs, may be used for the delivery of siRNA to breast cancer cells. From the Clinical EditorTargeted delivery of siRNA to cancer cells may be a viable anti-cancer strategy with low toxicity. In this study the novel nonviral carrier, human serum albumin-coated lipid nanoparticles (HSA-LNP) were demonstrated as an efficient delivery agent of siRNA to breast cancer cells.

Abstract 5247: Use of two markers of hypoxia to study migration, re-oxygenation and repopulation of originally hypoxic cells in MCF-7 tumor xenografts following chemotherapy

Abstract Background: Hypoxia occurs in solid tumors: chronic diffusion-limited hypoxia occurs distal from functional blood vessels, while acute hypoxia may occur because of interruptions in blood flow. Well-nourished tumor cells close to blood vessels tend to be rapidly proliferating but hypoxic cells located farther away are slowly-proliferating. Chemotherapy may spare hypoxic cells because of poor drug distribution to them and because most drugs are selectively toxic to proliferating cells. Intervals between administration of chemotherapy allow for recovery of normal tissues (e.g. repopulation of bone marrow) but might allow re-oxygenation and resumed proliferation of formerly hypoxic cells due to better supply of nutrients to them, as is observed during radiotherapy. Therefore, it is important to establish a technique whereby hypoxia can be studied allowing for the assessment of different hypoxia-modulating therapies that can be used to prevent tumor relapse. Methods: Two specific markers of hypoxic cells (pimonidazole [pimo] and EF5) were injected into mice bearing MCF7 tumor xenografts, and tumor cells labeled with one or both markers were recognized in tumor sections (in relation to DioC7+ve functional tumor blood vessels) using appropriate fluorescence-labeled antibodies and imunohistochemistry. Proliferating cells were identified by an antibody to Ki67. Mice were treated with pimo and then either doxorubicin or saline one hour later; EF5 was given after a variable interval of 24, 48, 72, 96 or 120 hours; mice were killed two hours after the second injection. Changes in the location, proliferation and oxygen status of formerly hypoxic (pimo+ve) cells were quantified by their distance from blood vessels, Ki67 status and uptake of EF5 as a function of time. Results: Following treatment with doxorubicin, the proportion of hypoxic cells in the entire tumor decreased from1.5% (pimo+ve) prior to injection to 0.7% (EF5+ve) at 24 hours. The proportion of pimo+ve formerly hypoxic cells that are no longer hypoxic (i.e. EF5 - ve) at 24 hours was 75% after treatment compared to 18% in controls indicating rescue of previous hypoxic cells that would have died in the absence of treatment. The proportion of these pimo+ve cells that were cycling (Ki67+ve) increased from 4.7% to 15.0% at 24 Hours and then slowly decreased. Conclusions: There is a decrease in the proportion of hypoxic cells in MCF7 xenografts following treatment with doxorubicin. Originally hypoxic cells (that may have died in the absence of treatment) can move closer to blood vessels, re-oxygenate, and repopulate the tumor. Treatment directed to killing hypoxic cells (e.g. with hypoxia activated pro-drugs) has the potential to improve the outcome of chemotherapy by inhibiting tumor cell repopulation. Supported by are search grant from the Canadian Institutes for Health Research. 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 5247. doi:1538-7445.AM2012-5247

Re-oxygenation causes hypoxic tumor regression through restoration of p53 wild-type conformation and post-translational modifications

Hypoxic tumors are resistant to conventional therapies through indirect mechanisms such as the selection of resistant phenotype under chronic hypoxia. Hyperbaric oxygen (HBO) therapy has been shown to increase oxygen level and induce apoptosis in hypoxic tumor. However, it could produce significant adverse effects including oxygen toxic seizures and severe radiation tissue injury due to high pressure. We have shown that repeated oxygenation at 30% O2 (1 atmospheres absolute) results in significant regression of MCF-7 tumor xenografts without any adverse effect. In MCF-7 cells, re-oxygenation showed an eightfold increase in cellular apoptosis. Both in hypoxic tumor and in hypoxic cells, that exclusively favor p53 to exist in mutant conformation, re-oxygenation restores p53 wild-type conformation. The oxygen-mediated rescue of mutant p53 followed by its trans-activation is responsible for the induction of p53-downstream apoptotic, cell-cycle arrest and DNA-repair genes. Further, p53 trans-activation may thus be due to its post-translational modifications as a result of re-oxygenation. We have thus concluded that oxygen therapy without pressure, as opposed to HBO therapy, may be ideal for hypoxic tumor regression, which functions through oxygen-mediated rescue of mutant p53 followed by induction of apoptosis.

Furanodiene, a Natural Product, Inhibits Breast Cancer Growth Both in vitro and in vivo

Purpose: Previous studies have reported that the Curcuma wenyujin Y.H. Chen et C. Ling extract, which has a high furanodiene content, showed anti-cancer effects in breast cancer cells in vitro. The present study was designed to evaluate the in vitro and in vivo anti-cancer activity of furanodiene. Methods: The in vitro effects of furanodiene were examined on two human breast cancer cell lines, MCF-7 and MDA-MB-231 cells. Assays of proliferation, LDH release, mitochondrial membrane potential (△Ψm), cell cycle distribution, apoptosis and relevant signaling pathways were performed. The in vivo effect was determined with MCF7 tumor xenograft model in nude mice. Results: Furanodiene significantly inhibited the proliferation and increased the LDH release in both cell lines in a dose-dependent manner. △Ψm depolarization, chromatin condensation, and DNA fragmentation were also observed after furanodiene treatment. Furanodiene dose-dependently induced cell cycle arrest at the G0/G1 phase. The protein expressions of p-cyclin D1, total cyclin D1, p-CDK2, total CDK2, p-Rb, total Rb, Bcl-xL, and Akt were significantly inhibited by furanodiene, whereas the protein expressions of Bad and Bax, and the proteolytic cleavage of caspase-9, caspase-7, and poly-ADP-ribose polymerase (PARP) were dramatically increased. Furthermore, the z-VAD-fmk markedly reversed the furanodiene-induced cell cytotoxicity, the proteolytic cleavage of caspase-9, and DNA fragmentation but did not affect the proteolytic cleavage of PARP, whereas the Akt inhibitor VIII increased the furanodiene-induced cytotoxicity and PARP cleavage. In addition, furanodiene dose-dependently suppressed the tumor growth in vivo, achieving 32% and 54% inhibition rates after intraperitoneal injection of 15 mg/kg and 30 mg/kg, respectively. Conclusions: Taken together, we concluded that furanodiene suppresses breast cancer cell growth both in vitro and in vivo and could be a new lead compound for breast cancer chemotherapy.

Nanoformulation ofd-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancer therapy

The purpose of this research was to develop formulation of docetaxel-loaded biodegradable TPGS-b-(PCL-ran-PGA) nanoparticles for breast cancer chemotherapy. A novel diblock copolymer, d-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) [TPGS-b-(PCL-ran-PGA)], was synthesized from ε-caprolactone, glycolide and d-α-tocopheryl polyethylene glycol 1000 succinate by ring-opening polymerization using stannous octoate as catalyst. The obtained copolymers were characterized by (1)H NMR, GPC and TGA. The docetaxel-loaded TPGS-b-(PCL-ran-PGA) nanoparticles were prepared and characterized. The data showed that the fluorescence TPGS-b-(PCL-ran-PGA) nanoparticles could be internalized by MCF-7 cells. The TPGS-b-(PCL-ran-PGA) nanoparticles achieved significantly higher level of cytotoxicity than commercial Taxotere®. MCF-7 xenograft tumor model on SCID mice showed that docetaxel formulated in the TPGS-b-(PCL-ran-PGA) nanoparticles could effectively inhibit the growth of tumor over a longer period of time than Taxotere® at the same dose. In conclusion, the TPGS-b-(PCL-ran-PGA) copolymer could be acted as a novel and potential biologically active polymeric material for nanoformulation in breast cancer chemotherapy.

Molecular Characterization of the Tumor-Suppressive Function of Nischarin in Breast Cancer

Nischarin (encoded by NISCH), an α5 integrin-binding protein, has been identified as a regulator of breast cancer cell invasion. We hypothesized that it might be a tumor suppressor and were interested in its regulation. We examined nischarin expression in approximately 300 human breast cancer and normal tissues using quantitative polymerase chain reaction and immunohistochemistry. Loss of heterozygosity analysis was performed by examining three microsatellite markers located near the NISCH locus in normal and tumor tissues. We generated derivatives of MDA-MB-231 human metastatic breast cancer cells that overexpressed nischarin and measured tumor growth from these cells as xenografts in mice; metastasis by these cells after tail vein injection; and α5 integrin expression, Rac, and focal adhesion kinase (FAK) signaling using western blotting. We also generated clones of MCF-7 human breast cancer cells in which nischarin expression was silenced and measured tumor growth in mouse xenograft models (n = 5 for all mouse experiments). P values were from two-sided Student t tests in pairwise comparisons. Normal human breast tissue samples had statistically significantly higher expression of nischarin mRNA compared with tumor tissue samples (mean level in normal breast = 50.7 [arbitrary units], in breast tumor = 16.49 [arbitrary units], difference = 34.21, 95% confidence interval [CI] = 11.63 to 56.79, P = .003), and loss of heterozygosity was associated with loss of nischarin expression. MDA-MB-231 cells in which nischarin was overexpressed had statistically significantly reduced tumor growth and metastasis compared with parental MDA-MB-231 cells (mean volume at day 40, control vs nischarin-expressing tumors, 1977 vs 42.27 mm(3), difference = 1935 mm(3), 95% CI = 395 to 3475 mm(3), P = .025). Moreover, MCF-7 tumor xenografts in which nischarin expression was silenced grew statistically significantly faster than parental cells (mean volume at day 63, tumors with scrambled short hairpin RNA [shRNA] vs with nischarin shRNA, 224 vs 1262 mm(3), difference = 1038 mm(3), 95% CI = 899.6 to 1176 mm(3), P < .001). Overexpression of nischarin was associated with decreased α5 integrin expression, FAK phosphorylation, and Rac activation. Nischarin may be a novel tumor suppressor that limits breast cancer progression by regulating α5 integrin expression and subsequently α5 integrin-, FAK-, and Rac-mediated signaling.

Annonacin induces cell cycle-dependent growth arrest and apoptosis in estrogen receptor-α-related pathways in MCF-7 cells

Ethnopharmacological relevanceTamoxifen resistance is common in estrogen receptor-α (ERα)-positive breast cancers. Pawpaw and soursop are anticancer annonaceous plants in complementary medicine. Thus, we studied the effects of annonacin, an annonaceous acetogenin, in breast cancer cells. Materials and MethodsCell growth and ERα-related pathways were studied. The effects of annonacin were tested in MCF-7 xenografts in nude mice. ResultsIn ERα-positive MCF-7 cells, annonacin (half-effective dose ED50=0.31μM) and 4-hydroxytamoxifen (ED50=1.13μM) decreased cell survival whereas annonacin (0.5-1μM) increased cell death at 48h. Annonacin and 4-hydroxytamoxifen were additive in inhibiting cell survival. Annonacin (0.1μM) induced G0/G1 growth arrest while increasing p21WAF1 and p27kip1 and decreasing cyclin D1 protein expression. Annonacin (0.1μM) decreased cyclin D1 protein expression more than 4-hydroxytamoxifen (1μM). Annonacin (0.1μM) increased apoptosis while decreasing Bcl-2 protein expression. The combination of annonacin (0.1μM) and 4-hydroxytamoxifen (1μM) decreased Bcl-2 protein expression and ERα transcriptional activity more than annonacin (0.1μM) did alone. Annonacin, but not 4-hydroxytamoxifen, decreased ERα protein expression. Moreover, annonacin decreased phosphorylation of ERK1/2, JNK and STAT3. In nude mice, annonacin decreased MCF-7 xenograft tumor size at 7–22 days. Moreover, annonacin decreased ERα, cyclin D1 and Bcl-2 protein expression in the xenograft at 22 days. ConclusionsAnnonacin induced growth arrest and apoptosis in ERα-related pathways in MCF-7 cells. Annonacin and 4-hydroxytamoxifen were additive in inhibiting cell survival and ERα transcriptional activity. Moreover, annonacin attenuated MCF-7 xenograft tumor growth while inhibiting ERα, cyclin D1 and Bcl-2 protein expressions in nude mice.

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.

In Vivo and in Vitro Evidence That PPARγ Ligands Are Antagonists of Leptin Signaling in Breast Cancer

Obesity is a major risk factor for the development and progression of breast cancer. Leptin, a cytokine mainly produced by adipocytes, plays a crucial role in mammary carcinogenesis and is elevated in hyperinsulinemia and insulin resistance. The antidiabetic thiazolidinediones inhibit leptin gene expression through ligand activation of the peroxisome proliferator-activated receptor-γ (PPARγ) and exert antiproliferative and apoptotic effects on breast carcinoma. In this study, we investigated the ability of PPARγ ligands to counteract leptin stimulatory effects on breast cancer growth in either in vivo or in vitro models. The results show that activation of PPARγ prevented the development of leptin-induced MCF-7 tumor xenografts and inhibited the increased cell-cell aggregation and proliferation observed on leptin exposure. PPARγ ligands abrogated the leptin-induced up-regulation of leptin gene expression and its receptors in breast cancer. PPARγ-mediated repression of leptin gene involved the recruitment of nuclear receptor corepressor protein and silencing mediator of retinoid and thyroid hormone receptors corepressors on the glucocorticoid responsive element site in the leptin gene expression regulatory region in the presence of glucocorticoid receptor and PPARγ. In addition, PPARγ ligands inhibited leptin signaling mediated by MAPK/STAT3/Akt phosphorylation and counteracted leptin stimulatory effect on estrogen signaling. These findings suggest that PPARγ ligands may have potential therapeutic benefits in the treatment of breast cancer.

KX-01, a novel Src kinase inhibitor directed toward the peptide substrate site, synergizes with tamoxifen in estrogen receptor α positive breast cancer

KX-01 is the first clinical Src inhibitor of the novel peptidomimetic class that targets the peptide substrate site of Src providing more specificity toward Src kinase. The present study was designed to evaluate the effects of KX-01 as a single agent and in combination with tamoxifen (TAM) on cell growth and apoptosis of ERα positive breast cancer in vitro and in vivo. Flow cytometry demonstrated that KX-01 induced cell cycle arrest in G2/M phase. Immunofluorescent staining for mitotic phase markers and TUNEL staining indicated that cells had arrested in the mitotic phase and mitotic arrested cells were undergoing apoptosis. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2, and Cdc25C that is required for progression past the G2/M checkpoint. Apoptosis resulted from activation of caspases 6, 7, 8, and 9. Combinational index analysis revealed that combinations of KX-01 with TAM resulted in synergistic growth inhibition of breast cancer cell lines. KX-01 combined with TAM resulted in decreased ERα phosphorylation at Src-regulated phosphorylation sites serines 118 and 167 that were associated with reduced ERα transcriptional activity. Orally administered KX-01 resulted in a dose dependent growth inhibition of MCF-7 tumor xenografts, and in combination with TAM exhibited synergistic growth inhibition. Immunohistochemical analysis revealed that combinational treatment reduced angiogenesis, and ERα signaling in tumors compared to either drug alone that may underlie the synergistic tumor growth inhibition. Combinations of KX-01 with endocrine therapy present a promising new strategy for clinical management of ERα positive breast cancer.

Abstract LB-12: Estrogen-induced redox signaling via NRF-1 is involved in the development of breast cancer

Abstract The purpose of this study was to determine the role of estrogen-induced reactive oxygen species (ROS) signaling pathways in the development of breast cancer. Here we tested the postulate that activation of a redox sensitive transcription factor, nuclear respiratory factor-1 (NRF-1) signaling is essential for 4-hydroxyestradiol (4-OH-E2)-induced tumorigenesis. We observed that (i) 4-OH-E2 treatment of MCF-10A or MCF-7 cells increased the formation of ROS, (ii) Estrogen-induced ROS activated redox sensitive transcription factor NRF-1, (iii) 4-OH-E2 through activating a redox sensitive serine-threonine kinase AKT phosphorylated NRF-1, (iv) Overexpression of catalase and MnSOD, as well as silencing of NRF-1 prevented 4-OH-E2-induced anchorage-independent growth of MCF-10A and MCF-7 cells and xenograft tumor formation, and (v) 4-OH-E2-induced ROS through stimulating AKT kinase and histone acetyltransferase of CREB binding protein-associated factor (P/CAF), respectively, controlled phosphorylation and acetylation of NRF-1. The results also showed that 4-OH-E2-induced post-translational changes lead to NRF-1 activation, and silencing of NRF-1 and overexpression of MnSOD or catalase prevented 4-OH-E2-induced tumor formation. Taken together, these findings suggest a new paradigm for estrogen-induced breast carcinogenesis that deregulation of phosphorylation and aceytylation of NRF-1 may represent a major mechanism of 4-OH-E2-induced neoplastic transformation and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-12. doi:10.1158/1538-7445.AM2011-LB-12

Abstract 1146: Multiple HDAC inhibitors (HDACi) reduce the expression of the oncogenic miR-17∼92 cluster; a pan HDACi class effect

Abstract miR-17∼92 is an oncogenic polycistronic cluster encoding six mature miRNAs (MIR-17, MIR-20A, MIR-18A, MIR-19A, MIR-19B, and MIR-92A). This oncogenic cluster is driven by MYC activity and is highly-expressed in many tumor types where it has been shown to promote tumorigenesis through the suppression of oncogene-induced senescence. We recently observed reduced expression of the miR-17∼92 miRNA cluster in a miRNA microarray analysis of MCF-7 and MDA-MB-231 breast cancer cells treated with the pan-HDAC inhibitors (HDACi's) Vorinostat, MS-275, and LAQ824. Treatment with all three HDACi's, at concentrations shown to induce the expression of p21 and acetylated histone-H3, demonstrated this effect which was further validated by qRT-PCR. Furthermore, significant cell cycle arrest and induction of apoptosis and/or senescence were shown with treatment of both MCF-7 and MDA-MB-231 cells with the HDACi's. There was also significant reduction of MYC expression upon treatment with HDACi's which is consistent with studies showing that the miR-17∼92 cluster is driven by MYC. The miR-17∼92 cluster has also been shown to suppress the expression of several cellular targets important in tumor suppression including p21, PTEN, E2F1, and BIM. The expression of these proteins will also be assessed in MCF-7 and MDA-MB-231 cells after treatment with HDACi. The modulation of MYC, the miR-17∼92 cluster and its oncogenic targets will also be confirmed in MDA-MB-231 and MCF-7 tumor xenograft mice treated with the HDACi's. Lastly, antagomirs will be used to inhibit the expression of the miR-17∼92 cluster in MCF-7 and MDA-MB-231 cells in order to compare the functional (apoptosis, senescence, and cell cycle arrest) and cellular (repression of target proteins) effects elicited by HDACi treatment. Together, we have shown that pan-HDAC inhibitors inhibit the expression of MYC and the oncogenic miR-17-92 cluster. This finding could have important utility in the clinical development of pan-HDACi's by identifying MYC and/or the miR-17-92 cluster as potential biomarkers to select patients more likely to respond to pan-HDACi treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1146. doi:10.1158/1538-7445.AM2011-1146