MCF-7 Cell Death Research Articles

Abstract LB-58: Production of Thr-58-O-GlcNAcylated c-myc antibodies and detection of increased O-GlcNAcylated c-myc levels in MCF-7 cells after ketoconazole treatment.

Abstract Protein O-GlcNAcylation (N-acetylglucosamine modification) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. Most serine (Ser) and threonine (Thr) sites for O-GlcNAcylation are on or near to phosphorylation sites, which creates a system of mutual elimination. Thr-58 of c-myc, a mutational hot spot in lymphomas, is a site for both phosphorylation (primed by Ser-62 phosphorylation) and O-GlcNAcylation. Whereas Thr-58 O-GlcNAcylation induces ubiquitin-dependent c-myc degradation, Thr-58/Ser-62 phosphorylation increases c-myc stability and invasiveness and tumorigenesis of MCF-7 human breast cancer cells. Thr-58 site-specific anti-peptide antibodies have been produced by immunization of rabbits with KLH conjugated with c-myc peptides with and without Thr-58 O-GlcNAcylation. Whereas the antibodies for O-GlcNAcylated c-myc cross-reacted with BSA conjugated with Thr-58-O-GlcNAcylated c-myc peptides in ELISA and Western blot analysis, the antibodies did not cross-react with BSA conjugated with c-myc peptides without O-GlcNAcylation. Anti-peptide antibodies produced for the unmodified c-myc peptides did not cross-react with BSA conjugated with Thr-58-O-GlcNAcylated c-myc peptides. Using the antibodies for Thr-58-O-GlcNAcylated c-myc, Western blot analysis was carried out with nuclear lysates and cytosol-containing supernatant obtained from MCF-7 cells and it was found that Thr-58-O-GlcNAcylated c-myc proteins were minimally expressed in the cytosolic fraction but highly expressed in the nuclear fraction. Whereas c-myc proteins without Thr-58-O-GlcNAcylation in the nuclear fraction were detected primarily as 65 kDa proteins in Western blot analyses, Thr-58-O-GlcNAcylated c-myc proteins in the nuclear fraction were detected primarily as 68 kDa proteins. MCF-7 cells were treated for 4 hr with 1% DMSO and 2 mM β-N-acetylglucosaminidase (O-GlcNAcase) inhibitors (buspirone or ketoconazole) dissolved in DMSO and levels of Thr-58-O-GlcNAcylated and unmodified c-myc were determined by Western blot analysis using antibodies for the Thr-58-O-GlcNAcylated or unmodified c-myc. Whereas the O-GlcNAcylated c-myc level did not increase after buspirone treatment, the level dramatically increased after ketoconazole treatment. The Thr-58-unmodified c-myc protein level failed to increase after 2 mM ketoconazole treatment. The ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells as evidenced by lactate dehydrogenase (LDH) cytotoxicity assays. Our results showed that ketoconazole treatment increased Thr-58-O-GlcNAcylated c-myc level and induced cell death in MCF-7 cells, which may decrease invasiveness of the breast cancer cells. Supported by NCI SBIR Phase I Contract No. HHSN261201100073C. Citation Format: Hyesook Kim, Sohee Kim, Aby Joiakim, David Kaplan, David Putt. Production of Thr-58-O-GlcNAcylated c-myc antibodies and detection of increased O-GlcNAcylated c-myc levels in MCF-7 cells after ketoconazole treatment. [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 LB-58. doi:10.1158/1538-7445.AM2013-LB-58 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

Assessment of Cytotoxic Properties of Safranal and Nanoliposomal Safranal in Various Cancer Cell Lines

Saffron (Crocus sativus) is a widely used food additive used for its color and taste. It has been reported that saffron possesses significant in vivo and in vitro anti-tumor activity. In the present study, anti-tumor effects of safranal, the major aromatic compound in saffron, and its liposomal form were investigated. The role of apoptosis has also been explored in this toxicity. HeLa, MCF7 and L929 cell lines were cultured and exposed to safranal (0.01-3 mM) or liposomal safranal (0.04-0.32 mM). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay was performed to assess cytotoxicity. Apoptosis was evaluated by staining cells with propidium iodide and quantifying sub-Gl peak by flow cytometry. MTT assay revealed a significant and concentration-dependent cytotoxic effect of safranal on HeLa and MCF7 cell lines. Liposomal safranal showed enhanced effect compared to the safranal solution, as compared by their IC50 concentrations. Flow cytometry results revealed induction of apoptosis by safranal. It might be concluded that safranal could be involved in saffron-induced cell death in HeLa and MCF7 cells. Liposome encapsulation improved anti-tumor effect of safranal. Safranal and particularly its liposomal form could be investigated as promising chemotherapeutic agents in cancer treatment.

Enhancement of docetaxel-treated MCF-7 cell death by 900-MHz radiation

AbstractThe aim of the study was to investigate the effect of high-frequency electromagnetic field of 900 MHz at 8 W input power on metabolic activity of human breast adenocarcinoma MCF-7 cells. With the aid of the colorimetric MTT assay, it was shown that there is significant change in cell culture survival exposed to docetaxel in field-free conditions in comparison with cells treated with docetaxel simultaneously exposed to high-frequency electromagnetic field.

NP-1250, an ABCG2 inhibitor, induces apoptotic cell death in mitoxantrone-resistant breast carcinoma MCF7 cells via a caspase-independent pathway

Chemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with multidrug resistance (MDR). One of the most studied mechanisms of MDR is the high expression of ATP-binding cassette (ABC) transporters. Here, we demonstrated that NP-1250, an ABCG2 inhibitor, induced apoptotic cell death in ABCG2-overexpressing multidrug-resistant MCF7/mitoxantrone-resistant (MX) human breast carcinoma cells via a caspase-independent pathway. Incubation of MCF7/MX cells with NP-1250 significantly reduced cell viability, while NP-1250 had little effect on the viability of drug-sensitive MCF7/wild-type cells. Although the target molecules of NP-1250 in cell death remain unknown, investigation of NP-1250 will aid in the elucidation of the molecular mechanism of drug resistance and NP-1250 may become a new therapy for MDR cancers.

Vav1 Fine Tunes p53 Control of Apoptosis versus Proliferation in Breast Cancer

Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.

Synthesis and characterization of low-toxicity N-caprinoyl-N-trimethyl chitosan as self-assembled micelles carriers for osthole

Novel amphiphilic chitosan derivatives (N-caprinoyl-N-trimethyl chitosan [CA-TMC]) were synthesized by grafting the hydrophobic moiety caprinoyl (CA) and hydrophilic moiety trimethyl chitosan to prepare carriers with good compatibility for poorly soluble drugs. Based on self-assembly, CA-TMC can form micelles with sizes ranging from 136 nm to 212 nm. The critical aggregation concentration increased from 0.6 mg • L−1 to 88 mg • L−1 with decrease in the degree of CA substitution. Osthole (OST) could be easily encapsulated into the CA-TMC micelles. The highest entrapment efficiency and drug loading of OST-loaded CA-TMC micelles(OST/CA-TMC) were 79.1% and 19.1%, respectively. The antitumor efficacy results show that OST/CA-TMC micelles have significant antitumor activity on Hela and MCF-7 cells, with a 50% of cell growth inhibition (IC50) of 35.8 and 46.7 μg. mL−1, respectively. Cell apoptosis was the main effect on cell death of Hela and MCF-7 cells after OST administration. The blank micelles did not affect apoptosis or cell death of Hela and MCF-7 cells. The fluorescence imaging results indicated that OST/CA-TMC micelles could be easily uptaken by Hela and MCF-7 cells and could localize in the cell nuclei. These findings suggest that CA-TMC micelles are promising carriers for OST delivery in cancer therapy.

The effect of CTB on P53 protein acetylation and consequence apoptosis on MCF-7 and MRC-5 cell lines

Background:P300 is a member of the mammalian histone acetyl transferase (HAT) family, an enzyme that acetylates histones and several non-histone proteins including P53 (the most important tumor suppressor gene) during stress, which plays an important role in the apoptosis of tumor cells. Hereby, this study describes the potency of CTB (Cholera Toxin B subunit) as a P300 activator to induce apoptosis in a breast cancer cell line (MCF-7) and a lung fibroblast cell line (MRC-5) as a non-tumorigenic control sample.Materials and Methods:MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with or without CTB at a concentration of 85.43 μmol/L, based on half-maximal inhibitory concentration (IC50) index at different times (24, 48 and 72 h). The percentage of apoptotic cells were measured by flow cytometry. Real-time quantitative RT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with CTB at different times. ELISA and Bradford protein techniques were used to detect levels of total and acetylated P53 protein generated in MCF-7 and MRC-5.Results:Our findings indicated that CTB could effectively induce apoptosis in MCF-7 significantly higher than MRC-5. We showed that expression of P300 was up-regulated by increasing time of CTB treatment in MCF-7 but not in MRC-5 and the acetylated and total P53 protein levels were increased more in MCF-7 cells than MRC-5.Conclusion:CTB could induce acetylation of P53 protein through increasing expression of P300 and consequently induce the significant cell death in MCF-7 but it could be well tolerated in MRC-5. Therefore, CTB could be used as an anti-cancer agent.

The Acetone Extract of Sclerocarya birrea (Anacardiaceae) Possesses Antiproliferative and Apoptotic Potential against Human Breast Cancer Cell Lines (MCF‐7)

Interesting antimicrobial data from the stem bark of Sclerocarya birrea, which support its use in traditional medicine for the treatment of many diseases, have been delineated. The current study was aimed to further study some pharmacological and toxicological properties of the plant to scientifically justify its use. Anticancer activity of water and acetone extracts of S. birrea was evaluated on three different cell lines, HT-29, HeLa, and MCF-7 using the cell titre blue viability assay in 96-well plates. Apoptosis was evaluated using the acridine orange and propidium iodide staining method, while morphological structure of treated cells was examined using SEM. The acetone extract exhibited remarkable antiproliferative activities on MCF-7 cell lines at dose- and time-dependent manners (24 h and 48 h of incubation). The extract also exerted apoptotic programmed cell death in MCF-7 cells with significant effect on the DNA. Morphological examination also displayed apoptotic characteristics in the treated cells, including clumping, condensation, and culminating to budding of the cells to produce membrane-bound fragmentation, as well as formation of apoptotic bodies. The acetone extract of S. birrea possesses antiproliferative and apoptotic potential against MCF-7-treated cells and could be further exploited as a potential lead in anticancer therapy.

Honokiol synergizes chemotherapy drugs in multidrug resistant breast cancer cells via enhanced apoptosis and additional programmed necrotic death

Multidrug resistance (MDR) is a major challenge in cancer therapy. Apoptosis tolerance is one of the key mechanisms of MDR. Honokiol, a small-molecule pharmacologically active component, exhibits competent cytotoxicity in a variety of human cancer cells through apoptosis and other forms of programmed cell death (such as programmed necrosis). Although much work has been done on its antitumor effects, little attention has been paid on systemic evaluation of efficacy of honokiol combined with other chemotherapeutic agents, especially in drug‑resistant cell lines. Here, we systematically and quantitatively assess its combinational effect with different chemotherapeutic agents using the combination index (CI) equation. We found that honokiol synergized with chemotherapeutic agents both in sensitive and resistant, solid and non-solid (MCF-7, HL-60, MCF-7/ADR and HL-60/ADR) cell lines. Honokiol (40 µg/ml) induced necrotic cell death in MCF-7/ADR cells with characterized morphological and biochemical features. Co-incubation with honokiol and etoposide (VP-16) activated a complex death modality, which was composed of necrotic cell death and apoptosis. This dual-death pathway was shut down when pretreated with pan-caspase inhibitor (z-VAD-fmk) and cyclophilin D inhibitor (cyclosporin A). Western blot analysis results proved that honokiol also enhanced VP-16-induced apoptosis potentially via blocking nuclear factor‑κB (NF-κB) activation. Our data for the first time quantitatively demonstrate that honokiol synergizes frequently-used chemotherapeutic agents via enhanced apoptosis and additional programmed necrotic death. These findings indicate a promising way to circumvent MDR and apoptosis tolerance.

Negative regulation of ERα by a novel protein CAC1 through association with histone demethylase LSD1

ERα, a critical transcriptional factor for breast cancer proliferation, is regulated by a complex binding repertoire that includes coactivators and corepressors. Here, we identified a novel class of ERα coregulator called CAC1. The CoRNR box of CAC1 was required for the binding to and inactivation of ERα. CAC1 also associated with histone demethylase LSD1 and suppressed LSD1-enhanced ERα activity. CAC1 impaired recruitment of ERα and LSD1 to the ERα-responsive promoter, leading to greater H3K9me3 accumulation. This effect was reversed by CAC1 depletion. Finally, CAC1 increased paclitaxel-induced cell death in ERα-positive MCF7 cells, which are paclitaxel-resistant. Overall, our study provides the first evidence that CAC1, associated with LSD1, functions as an ERα corepressor, implicating a potential antitumor target in ERα-positive breast cancer. Structured summary of protein interactionsER-alphaphysically interacts with CAC1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)LSD1physically interacts with CAC1 by anti tag coimmunoprecipitation (View interaction)CAC1binds to ER-alpha by pull down (View interaction)CAC1 and ER-alphacolocalizebyfluorescence microscopy (View interaction)

8-Amino-Adenosine Activates p53-Independent Cell Death of Metastatic Breast Cancers

8-Amino-adenosine (8-NH(2)-Ado) is a ribose sugar nucleoside analogue that reduces cellular ATP levels and inhibits mRNA synthesis. Estrogen receptor-negative (ER-) metastatic breast cancers often contain mutant p53; therefore, we asked if 8-NH(2)-Ado could kill breast cancer cells without activating the p53-pathway. Regardless of the breast cancer subtype tested or the p53 status of the cells, 8-NH(2)-Ado was more cytotoxic than either gemcitabine or etoposide. 8-NH(2)-Ado treatment inhibited cell proliferation, activated cell death, and did not activate transcription of the p53 target gene p21 or increase protein levels of either p53 or p21. This occurred in the estrogen receptor-positive (ER+) MCF-7 cells that express wild-type p53, the ER+ T47-D cells that express mutant p53, and the ER- MDA-MB-468 cells or MDA-MB-231 cells that both express mutant p53. 8-NH(2)-Ado induced apoptotic death of MCF-7 cells and apoptosis was not inhibited by knockdown of functional p53. Moreover, the pan-caspase inhibitor Z-VAD blocked the 8-NH(2)-Ado-induced MCF-7 cell death. Interestingly, 8-NH(2)-Ado caused the MDA-MB-231 cells to detach from the plate with only limited evidence of apoptotic cell death markers and the cell death was not inhibited by Z-VAD. Inhibition of MDA-MB-231 cell autophagy, by reduction of ATG7 or 3-methyladenine treatment, did not block this 8-NH(2)-Ado-mediated cytotoxicity. Importantly 8-NH(2)-Ado was highly cytotoxic to triple-negative breast cancer cells and worked through a pathway that did not require wild-type p53 for cytoxicity. Therefore, 8-NH(2)-Ado should be considered for the treatment of triple-negative breast cancers that are chemotherapy resistant.

Ethanol Extract of Brazilian Red Propolis Induces Apoptosis in Human Breast Cancer MCF-7 Cells through Endoplasmic Reticulum Stress

Propolis, a natural product collected from plants by honey bees, is commonly used in folk medicines. Endoplasmic reticulum (ER) stress is known to induce apoptosis through the induction of CCAAT/enhancer-binding protein homologous protein (CHOP). Here, we investigated whether ethanol extracts of propolis and caffeic acid phenethyl ester (CAPE) induce apoptosis, mitochondrial dysfunction, and ER stress in human breast cancer MCF-7 cells and human fibroblasts. Among several ethanol extracts of propolis and CAPE, Brazilian red propolis (BRP) significantly reduced MCF-7 cell viability through the induction of mitochondrial dysfunction, caspase-3 activity, and DNA fragmentation but did not affect those of fibroblasts. Moreover, treatment with BRP significantly induced CHOP expression in MCF-7 cells compared to fibroblasts. Further, pretreatment with a chemical chaperone, 4-phenylbutyric acid, suppressed BRP-triggered MCF-7 cell death. Overall, we revealed that an ethanol extract of BRP induces MCF-7 cell apoptosis through, at least in part, ER stress-related signaling.

The role of ginseng total saponin in transient receptor potential melastatin type 7 channels

Although ginsenosides have a variety of physiologic or pharmacologic functions in various regions, there are only a few reports on the effects of transient receptor potential melastatin 7 (TRPM7) channels. Here, we showed evidence suggesting that TRPM7 channels play an important role in ginseng total saponin (GTS)-mediated cellular injury. The combination techniques of electrophysiology, pharmacological analysis, small interfering RNA (siRNA) method and cell death assays were used. GTS depolarized the resting membrane potentials and decreased the amplitude of pacemaker potentials in cultured interstitial cells of Cajal (ICCs) in gastrointestinal (GI) tract. The TRPM7-like currents in single ICCs and the overexpressing TRPM7 in HEK293 cells were inhibited by GTS. However, GTS had no effect on Ca2 +-activated Cl− conductance. GTS inhibited the survival of human gastric (AGS) and breast (MCF-7) adenocarcinoma cells. Also, GTS inhibited the TRPM7-like currents in AGS and MCF-7 cells. The GTS-mediated cytotoxicity was inhibited by TRPM7-specific siRNA. In addition, we showed that overexpression of TRPM7 channels in HEK293 cells was inhibited by GTS. Thus, TRPM7 channels are involved in GTS-mediated cell death in AGS and MCF-7 cells, and these channels may represent a novel target for physiological disorders where GTS plays an important role.

Involvement of Transient Receptor Potential Melastatin 7 Channels in Sophorae Radix-induced Apoptosis in Cancer Cells: Sophorae Radix and TRPM7.

Sophorae Radix (SR) plays a role in a number of physiologic and pharmacologic functions in many organs.Objective:The aim of this study was to clarify the potential role for transient receptor potential melastatin 7 (TRPM7) channels in SR-inhibited growth and survival of AGS and MCF-7 cells, the most common human gastric and breast adenocarcinoma cell lines.Methods:The AGS and the MCF-7 cells were treated with varying concentrations of SR. Analyses of the caspase-3 and - 9 activity, the mitochondrial depolarization and the poly (ADPribose) polymerase (PARP) cleavage were conducted to determine if AGS and MCF-7 cell death occured by apoptosis. TRPM7 channel blockers (Gd3+ or 2-APB) and small interfering RNA (siRNA) were used in this study to confirm the role of TRPM7 channels. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in AGS and MCF-7 cell growth and survival.Results:The addition of SR to a culture medium inhibited AGS and MCF-7 cell growth and survival. Experimental results showed that the caspase-3 and -9 activity, the mitochondrial depolarization, and the degree of PARP cleavage was increased. TRPM7 channel blockade, either by Gd3+ or 2-APB or by suppressing TRPM7 expression with small interfering RNA, blocked the SR-induced inhibition of cell growth and survival. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SR-induced cell death.Conclusions:These findings indicate that SR inhibits the growth and survival of gastric and breast cancer cells due to a blockade of the TRPM7 channel activity. Therefore, TRPM7 channels may play an important role in the survival of patients with gastric and breast cancer.

Wortmannin induces MCF-7 breast cancer cell death via the apoptotic pathway, involving chromatin condensation, generation of reactive oxygen species, and membrane blebbing

Apoptosis can be used as a reliable marker for evaluating potential chemotherapeutic agents. Because wortmannin is a microbial steroidal metabolite, it specifically inhibits the phosphatidyl inositol 3-kinase pathway, and could be used as a promising apoptosis-based therapeutic agent in the treatment of cancer. The objective of this study was to investigate the biomolecular mechanisms involved in wortmannin-induced cell death of breast cancer-derived MCF-7 cells. Our experimental results demonstrate that wortmannin has strong apoptotic effects through a combination of different actions, including reduction of cell viability in a dose-dependent manner, inhibition of proliferation, and enhanced generation of intracellular reactive oxygen species. Our findings suggest that wortmannin induces MCF-7 cell death via a programmed pathway showing chromatin condensation, nuclear fragmentation, reactive oxygen species, and membrane blebbing, which are characteristics typical of apoptosis.

JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells

N-Benzoyl-O-(N′-(1-benzyloxycarbonyl-4-piperidiylcarbonyl) -D-phenylalanyl)-D-phenylalaninol (BBP), a novel synthesized asperphenamate derivative with the increased solubility, showed growth inhibitory effect on human breast carcinoma MCF-7 cells in a time- and concentration-dependent manner. The growth inhibitory effect of BBP was associated with induction of autophagy, which was demonstrated by the development of acidic vesicular organelles, cleavage of LC3 and upregulation of Atg4 in BBP-treated MCF-7 cells. Since the application of Atg4 siRNA totally blocked the cleavage of LC3, we demonstrated a central role of Atg4 in BBP-induced autophagy. The further studies showed that BBP increased the levels of reactive oxygen species (ROS), and pretreatment with NAC effectively blocked the accumulation of ROS, autophagy and growth inhibition triggered by BBP. Moreover, BBP induced the activation of JNK, and JNK inhibitor SP600125 reversed autophagy, the increase of Atg4 levels, conversion of LC3 and growth inhibition induced by BBP. Knockdown of JNK by siRNA efficiently inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by BBP, indicating that JNK activation may be a upstream signaling of ROS and should be a core component in BBP-induced autophagic signaling pathway. These results suggest that BBP produces its growth inhibitory effect through induction of the autophagic cell death in MCF-7 cells, which is modulated by a JNK-dependent Atg4 upregulation involving ROS production.

The effect of cholesterol depletion in resistant breast cancer cells.

e11031 Background: A leading cause of breast cancer chemotherapeutic treatment failure is resistance of cancerous cells towards current anti-estrogen agents. This project investigated the relationship between inhibition of cholesterol synthesis and its effect on resistant breast cancer cells. Methods: MCF-7 cells were maintained in phenol red free media and pretreated with charcoal stripped media for 24 hours prior to the experiments (ensure all exogenous estrogen was taken out). Cells were plated in 96 well plates at 10,000 to 15,000 cells/well. MCF-7 cells were tested for their sensitivity to BIBB515 and whether the proliferation of cells with estrogen could be blocked with BIBB515. The cells were grown for 6 days, on day 7 the cell proliferation was determined by adding MTT and read at 570nM. Results: Estrogen, tamoxifen and anastrazole induced cell proliferation that was controlled by 10µM BIBB515. Inhibiting the cholesterol pathway in combination with tamoxifen induced cell death in MCF-7 cells (cell proliferation of 0.13 ±0.15) compared to tamoxifen alone (0.57±0.041) p<0.001 by one way ANNOVA and multiple comparison by DUNN test. Anastrozole alone induced statistically significant cell proliferation 0.403 ±0.03 compared to combination treatment with BIBB515 (0.05 ± 0.007 p<0.001 by one way ANNOVA and multiple comparison by DUNN test). BIBB515 treatment could also inhibit the estrogen induced cell proliferation in MCF-7 cells. These results indicate that cholesterol inhibitors can be developed to fill a niche area in breast cancer disease management where clinicians would prolong treatment on first-line anti-hormone therapies of tamoxifen and anastrazole for advanced disease patients. Conclusions: Results show that inhibition of cholesterol sensitizes resistant cells to current anti-estrogen chemotherapeutic agents. This finding is significant because it could potentially translate into reduce rates of breast cancer relapse due to regiment failure. Exploiting this mechanism could alter the current treatment regimens, leading to control of the diseases in advanced stages by either inducing tumors to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

92P Tff1 Trefoil Protein Involves in Resistance to Doxorubicin-Induced Apoptosis in Breast Cancer Cell

ABSTRACT TFF1 trefoil protein is a small secretory protein expressed in a various type of carcinomas including breast cancer. TFF1 gene contained estrogen responsive element and its expression could be regulated by estrogen. In addition, estrogen has been demonstrated its ability to promote resistance to doxorubicin in estrogen receptor positive MCF-7 breast cancer cell line. Moreover, it has been reported that TFF1 can protect conjunctival cells from UV-induced apoptosis. In this study, we demonstrated the role of TFF1 in estrogen-promoted resistance to doxorubicin-induced apoptosis using MCF-7 model. Permanent knockdown of TFF1 gene in MCF-7 cell had been generated and used to test the sensitivity to doxorubicin treatment compared to parental cell in the conditions present or absent of 17b-estradiol, a potent estrogenic agent. The apoptosis cells were measured by fluorescence staining and flow cytometry method. The results showed that among the stimulation of apoptosis by doxorubicin, 17b-estradiol could suppress this process in parental MCF-7 cell but not in TFF1 knockdown MCF-7 cell. Moreover, by trypan blue staining method, it has been shown that anti-TFF1 antibody could reverse the anti-apoptotic effect of estrogen in parental MCF-7 cell and recombinant TFF1 could recover TFF1 knockdown MCF-7 cell death induced by doxorubicin. However, this process could not be inhibited by fulvestrant, an estrogen antagonist. Apoptosis protein array experiment reflected the role of the anti-oxidative enzymes catalase and heme oxygenase 1 in estrogen and TFF1 modulated apoptosis. These phenomena determine the role of TFF1 in estrogen-promoted resistance to apoptosis induced by doxorubicin in MCF-7 breast cancer cell. TFF1 gene may be a target for enhancing of sensitivity to chemotherapy in breast cancer treatment. Disclosure All authors have declared no conflicts of interest.

Abstract 956: The liver-X-receptor-α is involved in the induction by Tamoxifen of breast cancer cell differentiation and death

Abstract Hormonotherapy with Tamoxifen (Tam) is one of the main strategies used world-wide for the treatment of estrogen receptor positive breast cancer. However, partial responses, tumor recurrence and acquired resistance to Tam may involve mechanisms unrelated to Estrogen Receptors (ER). Studies on non-ER related mechanisms of action of Tam led us to find that it induced breast cancer cell differentiation and death through the modulation of sterol metabolism and the stimulation of sterol oxidation (de Medina et al, Cell Death Differ, 2009), and that Tam inhibited cholesterol epoxide hydrolase (ChEH). ChEH catalyzes the trans hydration of cholesterol-5,6-epoxides (CE) and this activity is a property of the microsomal antiestrogen binding site (AEBS) (de Medina et al, PNAS, 2010). In the present study we report that Tam and AEBS ligands stimulated the oxidation of cholesterol into CE and induced the accumulation of CE in MCF-7 cells through the inhibition of ChEH at therapeutic concentrations. Since CE has been reported to be a modulator of Liver-X-Receptors (LXR), we investigated the impact of Tam and other AEBS ligands on LXR-dependent gene expression: Tam and AEBS ligands modulated the expression LXR-dependent genes in MCF-7 cells. This effect had a 24 to 48 hour lag time while the effect of direct LXR modulators was observable after 6 hours, suggesting an indirect action of AEBS ligands. The involvement of CE in the control of transcription by Tam and AEBS ligands was supported by the observation that vitamin E, which inhibited the formation and the accumulation of CE, totally blocked this transcriptional modulation. We found that knocking down LXR-α using interfering RNAs in MCF-7 cells blocked the transcriptional modulation of LXR-controlled genes induced by CE, Tam and AEBS ligands. Finally, we found that the knock down of LXR-α reproduced the vitamin E inhibition of the induction of differentiation and death of MCF-7 cells. Altogether, these data showed that LXR-α is implicated in the anticancer action of Tam and identified a new signaling pathway that could explain sensitivity and resistance to Tam. 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 956. doi:1538-7445.AM2012-956