MCF-7 Cell Death Research Articles

Synthesis, structure information, DNA/BSA binding affinity and in vitro cytotoxic studies of mixed ligand copper(II) complexes containing a phenylalanine derivative and diimine co-ligands

Binary [Cu(PAIC)(H2O)2]·H2O (1) and mixed ligand [Cu(PAIC)(L)]·2H2O complexes, where PAIC=phenylalanine imidazole carboxylic acid, L=diimine coligands [2,2′-bipyridine (bpy) (2) and 1,10-phenanthroline (phen) (3)] have been synthesized and fully characterized by analytical and spectral techniques. The X-ray structure of [Cu(PAIC)(phen)]·2H2O (3) shows a N4O coordination with square pyramidal geometry around the copper (II) atom. The spin Hamiltonian parameters calculated for the complexes account for the distorted square planar structure and rules out the possibility of a trigonal bipyramidal structure. Interaction of the complexes (1–3) with calf thymus DNA (CT DNA) was studied by using different techniques (absorption titration, fluorescence quenching and thermal melting) and the studies suggest that these complexes bind to CT DNA through intercalation. The DNA-binding affinity of the complexes has further been explained by DFT computational results. Binding activity of Bovine serum albumin (BSA) reveals that the complexes can strongly quench the intrinsic fluorescence of BSA through a static quenching mechanism. DNA cleavage experiments using plasmid DNA pUC 19 show that the complexes exhibit efficient chemical nuclease activity even in the absence of any external additives. The cytotoxicity of the complexes against human normal cell line (HBL 100) and human breast cancer cell line (MCF-7) shows that metal complexation of the ligands results in a significant enhancement in the cell death of MCF-7. Finally, docking studies on DNA and protein binding interactions were performed.

Selective cytotoxicity and modulation of apoptotic signature of breast cancer cells by Pithecellobium dulce leaf extracts.

We report the potent and selective cytotoxicity of the crude aqueous leaf extract from the medicinal plant, Pithecellobium dulce toward the human breast cancer cells (MCF-7), but not the normal cells (MCF-10A). The cytotoxicity was found to be dose and time dependent, as 300 µg/mL of the extract decreased the cell viability to 50% (IC50 ) in 48 h. The induction of apoptosis in the breast cancer cells after treatment was confirmed by significant percentage (24.7%), of early apoptotic cells (AnnexinV (+) Propidium Iodide(_) ) in treated cells as compared to control cells (3.5%). We observed a significant upregulation in the mRNA expression of various pro-apoptotic gene such as Bax (21.1 folds), p21(14.4 folds), p53 (11.7 folds), TNF (10.2 folds) and fas (6.3 folds) after treatment as compared to untreated cells. On the other hand, the relative mRNA expression of anti-apoptotic genes such as Bcl-2, NF-KB and Cdk was reduced. The selective upregulation of pro-apoptotic gene and down regulation of specific anti-apoptotic genes could be the inducing factor for apoptotic cell death in MCF-7 cells after treatment with the herbal extract. We believe that our findings provide a foundation for further studies on this formulation as a potential therapeutic candidate for breast cancer. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:756-766, 2016.

Molecular Mechanism of Crocin Induced Caspase Mediated MCF-7 Cell Death: In Vivo Toxicity Profiling and Ex Vivo Macrophage Activation.

Crocus sativus and its major constituent crocin are well established to have anti-cancer properties in breast cancer cells (MCF-7). However the role of C. sativus extract (CSE) and crocin on caspase signaling mediated MCF-7 cell death at molecular level is remains unclear. In this study, we tried to unravel role of CSE and crocin on caspase mediated MCF-7 cells death and their in vivo preclinical toxicity profiling and immune stimulatory effect. CSE extract was fractionated by HPLC and crocin was isolated and characterized by NMR, IR, and MS. MCF-7 cells were treated with both CSE and crocin and expression of Bcl-2 and Bax was assessed after 24 and 36 hours. Furthermore, caspase 3, caspase 8 and caspase 9 expression was determined by Western blotting after 24 hours of treatment. DNA fragmentation analysis was performed for genotoxicity of CSE and crocin in MCF-7 cells. The in vivo toxicity profile of CSE (300 mg/kg of b.wt) was investigated in normal Swiss albino mice. In addition, peritoneal macrophages were collected from crocin (1, 1.5 and 2 mg/kg body weight) treated mice and analyzed for ex vivo yeast phagocytosis. Immunoblot analysis revealed that there was time dependent decline in anti-apoptotic Bcl-2 with simultaneous upregulation of Bax in CSE and crocin treated MCF-7 cells. Further CSE and crocin treatment downregulated caspase 8 and 9 and cleaved the caspase 3 after 24 hours. Both CSE and crocin elicited considerable DNA damage in MCF-7 cells at each concentration tested. In vivo toxicity profile by histological studies revealed no observable histopathologic differences in the liver, kidney, spleen, lungs and heart in CSE treated and untreated groups. Crocin treatment elicited significant dose and time dependent ex vivo yeast phagocytosis by peritoneal macrophages. Our study delineated involvement of pro-apoptotic and caspase mediated MCF-7 cell death by CSE and crocin at the molecular level accompanied with extensive DNA damage. Further we found that normal swiss albino mice can tolerate the maximum dose of CSE. Crocin enhanced ex vivo macrophage yeast phagocytic ability.

Abstract P4-07-09: MYB is involved in the DNA damage response in human ER+ breast cancer cells

Abstract Aims: Over 70% of human breast cancer cells are oestrogen receptor positive (ER+) and express MYB. MYB expression is necessary for the proliferation of ER+ breast cancer cells in vitro and for tumour development in vivo. Our previous studies found that shRNA-mediated MYB knock-down greatly sensitised breast cancer cells to chemically-induced apoptosis by down-regulating the BCL2 (a MYB target gene) (Drabsch et al., 2010). Furthermore, several published studies (Taha et al., 2004; Thomadaki et al., 2006) indicated that actinomycin D and etoposide treatment could induce DNA damage in human ER+ breast cancer (MCF-7) cells. Moreover, silencing MYB increased DNA damage-induced cell death in castration resistant prostate cancer cells by down-regulating DNA damage response genes (Li et al., 2014). However, there is very little information on MYB function in the DNA damage response of ER+ breast cancer cells. Therefore, the aim of this study was to investigate whether silencing MYB affected the DNA damage repair and resulting in cell death in MCF-7 cells. Methods: To achieve down-regulation of MYB expression in human ER+ breast cancer, we used doxycycline inducible shRNA lentiviral vectors (pLV711 (Drabsch et al., 2007; Brown et al., 2010)) in human MCF-7 breast cancer cells. We used PI staining plus FACS analysis for cell death assessment. We also used γ-H2AX protein expression and γ-H2AX foci counting to assess DNA damage. Results: We found that silencing MYB alone did not result in cell death, as reported previously (Drabsch et al., 2010). However, silencing MYB significantly increased actinomycin D-induced cell death. This similar result was also found on etoposide-induced cell death. Furthermore, we found that silencing MYB significantly increased actinomycin D or etoposide-induced γ-H2AX expression. Moreover, anti-apoptotic BCL2 expression, measured by western blotting, was dramatically reduced after the combination of MYB knock down and actinomycin D or etoposide, compared to wild type and nonsilencing controls. Conclusions: Silencing MYB significantly increased DNA damage-induced cell death and D???damage. This may result from down-regulation of BCL2 expression, an effect on DNA damage response genes, or both. This requires further investigation. For example, Rad51 and γ-H2AX colocalization and 53BP1 expression in response to DNA damage will be presented.

Cytotoxic Effects of Phosphonate-Functionalized Mesoporous Silica Nanoparticles.

In this work, we synthesized pristine mesoporous silica nanoparticles (MSN) and functionalized these with phosphonate groups (MSN-Phos). We report, for the first time, cell death in MCF-7 cells (human breast adenocarcinoma cell line) when exposed to the empty MSN and MSN-Phos nanoparticles. In comparison, the same nanoparticles were found to elicit few deleterious effects on normal human foreskin fibroblast cells (BJ cells). MCF-7 cells were found to exhibit a concentration-dependent uptake, whereas no detectable nanoparticle uptake was observed in the BJ cells, irrespective of treatment dosage. A disruption of the cell cycle in the MCF-7 cells was determined to be the cause of cell death from the nanoparticle exposure, thereby suggesting the role of nondrug loaded MSN and MSN-Phos as effective anticancer drugs.

Crude Extracts of Marine-derived and Soil Fungi of the Genus Neosartorya Exhibit Selective Anticancer Activity by Inducing Cell Death in Colon, Breast and Skin Cancer Cell Lines.

Background:The crude ethyl acetate extracts of marine-derived fungi Neosartorya tsunodae KUFC 9213 (E1) and N. laciniosa KUFC 7896 (E2), and soil fungus N. fischeri KUFC 6344 (E3) were evaluated for their in vitro anticancer activities on a panel of seven human cancer cell lines.Materials and Methods:The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, after 48 h treatments with different concentrations of extracts, to determine their concentration of the extract or Dox that inhibits cell viability by 50% for each cell line. The effects of the crude extracts on DNA damage, clonogenic potential and their ability to induce cell death were also assessed.Results:E1 was found to the void of anti-proliferative effects. E2 was shown to decrease the clonogenic potential in human colorectal carcinoma cell line (HCT116), human malignant melanoma cell line (A375), human breast adenocarcinoma cell line (MCF7), and human caucasian colon adenocarcinoma Grade II cell line (HT29) cells, whereas E3 showed such effect only in HCT116 and MCF7 cells. Both extracts were found to increase DNA damage in some cell lines. E2 was found to induce cell death in HT29, HCT116, MCF7, and A375 cells while extract E3 increased cell death in MCF7 and HCT116 cell lines.Conclusion:The results reveal that E2 and E3 possess anticancer activities in human colon carcinoma, breast adenocarcinoma, and melanoma cells, validating the interest for an identification of molecular targets involved in the anticancer activity.SUMMARY The crude ethyl acetate extract of N. tsunodae (E1) did not decrease cell viability in any of the tested cell linesThe crude ethyl acetate extracts of N. laciniosa (E2) and N. fischeri (E3) decreased cell proliferation in some human cancer cell lines tested at both short- and long-termN. laciniosa (E2) induced a significant increase in the number of cell death, in part, due to the induction of DNA damageN. fischeri (E3) induce cell death but in some cell lines without induction of DNA damage detected by comet assayCrude ethyl extracts of N. laciniosa (E2) and N. fischeri (E3) exert an anticancer activity in human colon carcinoma, breast adenocarcinoma, and malignant melanoma cells. Abbreviations Used: A375: Human malignant melanoma cell line; A549: Human non-small lung cancer cell line; DAPI: 4,6-diamidino-2-phenylindole; DMEM: Dulbecco's Modified Eagle Medium; DMSO: Dimethylsulfoxide; Dox: Doxorubicin; E1: Neosartorya tsunodae KUFC 9213; E2: Neosartorya laciniosa KUFC 7896; E3: Neosartorya fischeri KUFC 6344; FBS: Fetal bovine serum; HCT116: Human colorectal carcinoma cell line; HEPES: (N-[2-hydroxyethyl] piperazine-N’-[2-ethane-sulfonic acid]); HepG2: Human hepatocellular carcinoma cell line; HT29: Human caucasian colon adenocarcinoma Grade II cell line; IC50: Concentration of the extract or Dox that inhibits cell viability by 50%; MCF7: Human breast adenocarcinoma cell line; MEM: Minimum Essential Medium Eagle; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCI-H460: Human non-small lung cancer cell line; PBS: Phosphate buffered saline; PE: Plating efficiency; RPMI: Roswell park memorial institute medium; SF: Surviving fraction; U-251: Human malignant glioblastoma cell line.

Anticancer Effects of a New SIRT Inhibitor, MHY2256, against Human Breast Cancer MCF-7 Cells via Regulation of MDM2-p53 Binding.

The sirtuins (SIRTs), a family of NAD+-dependent class III histone deacetylase, are involved in various biological processes including cell survival, division, senescence, and metabolism via activation of the stress-response pathway. Recently, inhibition of SIRTs has been considered a promising anticancer strategy, but their precise mechanisms of action are not well understood. In particular, the relevance of p53 to SIRT-induced effects has not been fully elucidated. We investigated the anticancer effects of a novel SIRT inhibitor, MHY2256, and its efficacy was compared to that of salermide in MCF-7 (wild-type p53) and SKOV-3 (null-type p53) cells. Cell viability, SIRT1 enzyme activity, cell cycle regulation, apoptosis, and autophagic cell death were measured. We compared sensitivity to cytotoxicity in MCF-7 and SKOV-3 cells. MHY2256 significantly decreased the viability of MCF-7 (IC50, 4.8 μM) and SKOV-3 (IC50, 5.6 μM) cells after a 48 h treatment period. MHY2256 showed potent inhibition (IC50, 0.27 mM) against SIRT1 enzyme activity compared with nicotinamide (IC50, >1 mM). Moreover, expression of SIRT (1, 2, or 3) protein levels was significantly reduced by MHY2256 treatment in both MCF-7 and SKOV-3 cells. Flow cytometry analysis revealed that MHY2256 significantly induced cell cycle arrest in the G1 phase, leading to an effective increase in apoptotic cell death in MCF-7 and SKOV-3 cells. A significant increase in acetylated p53, a target protein of SIRT, was observed in MCF-7 cells after MHY2256 treatment. MHY2256 up-regulated LC3-II and induced autophagic cell death in MCF-7 cells. Furthermore, MHY2256 markedly inhibited tumor growth in a tumor xenograft model of MCF-7 cells. These results suggest that a new SIRT inhibitor, MHY2256, has anticancer activity through p53 acetylation in MCF-7 human breast cancer cells.

Chinese Herbal Mixture, Tien-Hsien Liquid, Induces G2/M Cycle Arrest and Radiosensitivity in MCF-7 Human Breast Cancer Cells through Mechanisms Involving DNMT1 and Rad51 Downregulation.

The Chinese herbal mixture, Tien-Hsien Liquid (THL), has been proven to suppress the growth and invasiveness of cancer cells and is currently regarded as a complementary medicine for the treatment of cancer. Our previous study using acute promyelocytic leukemia cells uncovered its effect on the downregulation of DNA methyltransferase 1 (DNMT1) which is often overexpressed in cancer cells resulting in the repression of tumor suppressors via hypermethylation. Herein, we explored the effects of THL in MCF-7 breast cancer cells that also demonstrate elevated DNMT1. The results show that THL dose-dependently downregulated DNMT1 accompanied by the induction of tumor suppressors such as p21 and p15. THL arrested cell cycle in G2/M phase and decreased the protein levels of cyclin A, cyclin B1, phospho-pRb, and AKT. DNMT1 inhibition was previously reported to exert a radiosensitizing effect in cancer cells through the repression of DNA repair. We found that THL enhanced radiation-induced clonogenic cell death in MCF-7 cells and decreased the level of DNA double-strand break repair protein, Rad51. Our observations may be the result of DNMT1 downregulation. Due to the fact that DNMT1 inhibition is now a mainstream strategy for anticancer therapy, further clinical trials of THL to confirm its clinical efficacy are warranted.

Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1) Mutation and TRAP1 Inhibitor Gamitrinib-triphenylphosphonium (G-TPP) Induce a Forkhead Box O (FOXO)-dependent Cell Protective Signal from Mitochondria

TRAP1 (tumor necrosis factor receptor-associated protein 1), a mitochondrial Hsp90 family chaperone, has been identified as a critical regulator of cell survival and bioenergetics in tumor cells. To discover novel signaling networks regulated by TRAP1, we generated Drosophila TRAP1 mutants. The mutants successfully developed into adults and produced fertile progeny, showing that TRAP1 is dispensable in development and reproduction. Surprisingly, mutation or knockdown of TRAP1 markedly enhanced Drosophila survival under oxidative stress. Moreover, TRAP1 mutation ameliorated mitochondrial dysfunction and dopaminergic (DA) neuron loss induced by deletion of a familial Parkinson disease gene PINK1 (Pten-induced kinase 1) in Drosophila. Gamitrinib-triphenylphosphonium, a mitochondria-targeted Hsp90 inhibitor that increases cell death in HeLa and MCF7 cells, consistently inhibited cell death induced by oxidative stress and mitochondrial dysfunction induced by PINK1 mutation in mouse embryonic fibroblast cells and DA cell models such as SH-SY5Y and SN4741 cells. Additionally, gamitrinib-triphenylphosphonium also suppressed the defective locomotive activity and DA neuron loss in Drosophila PINK1 null mutants. In further genetic analyses, we showed enhanced expression of Thor, a downstream target gene of transcription factor FOXO, in TRAP1 mutants. Furthermore, deletion of FOXO almost nullified the protective roles of TRAP1 mutation against oxidative stress and PINK1 mutation. These results strongly suggest that inhibition of the mitochondrial chaperone TRAP1 generates a retrograde cell protective signal from mitochondria to the nucleus in a FOXO-dependent manner.

Evaluation of gamma radiation-induced cytotoxicity of breast cancer cells: Is there a time-dependent dose with high efficiency?

Radiotherapy is one of the important treatment modalities in the management of breast cancer. The aim of this study is to study the efficient treatment of breast cancer as related to the dose delivery. The human breast cancer cell lines (MCF-7) cells were cultured and exposed by 1, 2, 4, 6, 8, 10, and 20 Gy of γ-rays. Radiation-induced cell death was detected and evaluated, using three assay methods: Cell viability, clonogenic cell survival assay and induction of apoptosis. The cell viability was determined using trypan blue staining, 24 and 72 h post-irradiation. The survival fraction (SF) was determined by colony counting, 14 days after exposure and the apoptotic cell death was determined using the TUNEL assay. One- or two-way analysis of variance was deemed as appropriate, followed by relevant post t-test to determine P values. The difference of MCF-7 cell death through increasing post-radiation time from 24 to 72 h following the dose of 1, 6 and 10 Gy was found to be 2%, 9.6% and 7.14%, respectively. D0of MCF-7 was 220 cGy and the SF in the cells irradiated by 1 Gy and 10 Gy doses were 0.8 and 0.0001, respectively. The estimated variances were 2%, 11.1% and 8.4%, between 24 h and 72 h post-radiation apoptosis death for 1, 6, and 10 Gy, respectively. The dose and time dependence inducing apoptotic death was significant (P = 0.001). The delayed mortality and apoptosis was observed in MCF-7 cell, but the variance of total cell death and apoptosis in 24 and 72 h post-radiation with 6 Gy was obviously more than that with other doses.

The Roles of 4β-Hydroxywithanolide E from Physalis peruviana on the Nrf2-Anti-Oxidant System and the Cell Cycle in Breast Cancer Cells.

4[Formula: see text]-Hydroxywithanolide E is an active component of the extract of Physalis peruviana that has been reported to exhibit antitumor effects. Although the involvement of reactive oxygen species (ROS) production and the ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway in 4[Formula: see text]-hydroxywithanolide E-induced apoptosis of breast cancer MCF-7 cells was demonstrated in our previous study, the relationship between ROS production and the cellular defense system response in 4[Formula: see text]-hydroxywithanolide E-induced cell death requires further verification. The present study suggests that ROS play an important role in 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in which anti-oxidants, such as glutathione or N-acetylcysteine, can resist the 4[Formula: see text]-hydroxywithanolide E-induced accumulation of ROS and cell death. Furthermore, N-acetylcysteine or glutathione can reverse the 4[Formula: see text]-hydroxywithanolide E-induced changes in the cell cycle distribution and the expression of cell cycle regulators. We found that the 4[Formula: see text]-hydroxywithanolide E-induced ROS accumulation was correlated with the upregulation of Nrf2 and Nrf2-downstream genes, such as antioxidative defense enzymes. In general, the activity of Nrf2 is regulated by the Ras signalling pathway. However, we demonstrated that Nrf2 was activated during 4[Formula: see text]-hydroxywithanolide E-induced MCF-7 cell death in spite of the 4[Formula: see text]-hydroxywithanolide E-induced inhibition of the Ras/Raf/ERK pathway. The activity and protein expression of superoxide dismutase and catalase were involved in the 4[Formula: see text]-hydroxywithanolide E-induced ROS production in MCF-7 cells. Furthermore, 4[Formula: see text]-hydroxywithanolide E was demonstrated to significantly reduce the sizes of the tumor nodules in the human breast cancer MDA-MB231 xenograft tumor model.

In Vitro Apoptotic Activity of Endophytic Fungal Lectin Isolated from Endophyte, Aspergillus flavus of Viscum album on Human Breast Adenocarcinoma Cell Line (MCF-7

Lectin was isolated and purified from the endophytic fungi Aspergillus flavus of Viscum album and it was a D-galactose and N-acetylgalactoseamine specific lectin capable of agglutinating A+ve blood group tested in hemeagglutination method. The lectin was confirmed a glycoprotein PAS staining on NATIVE-PAGE and it is having single band of 64 kDa protein (in SDS-PAGE) similar to plant lectin. The peptide sequences were identified from purified lectin 64 kDa protein band is shown. NVRFDLSGATSSSYK similar to Cucurbia pepo protein peptide sequence and it is a ribosome inactivating protein. Apoptotic activity was tested on human breast adenocarcinoma cancer (MCF-7) cell line and it is a time and dose dependent manner. Dose dependent inhibition of cancer cells was observed with IC50 values of 0.02 μg/ ml from Aspergillus flavus. MCF-7 cell death elicited by the lectin extract was found to be apoptotic in nature based the indication of nucleus condensation, shrinkage of nucleus membrane; membrane bebbling and also DNA fragmentation are the hallmark of apoptosis was observed in MCF-7 cells after staining with Hoechst 33342. Caspase mediated apoptosis was observed in lectin treated MCF-7 cells by cleaving fluorogenic peptide substrates, Ac-DEVD-AMC. The caspase-7 was observed as an effector in cleaving other caspases, further inhibition of caspase with an inhibitor provide clear evidence of caspase-7 mediated apoptosis in MCF-7 treated cells.

Synthesis of platinum complexes with 2-(5-perfluoroalkyl-1,2,4-oxadiazol-3yl)-pyridine and 2-(3-perfluoroalkyl-1-methyl-1,2,4-triazole-5yl)-pyridine ligands and their in vitro antitumor activity

Five new mononuclear Pt(II) complexes with 5-perfluoroalkyl-1,2,4-oxadiazolyl-pyridine and 3-perfluoroalkyl-1,2,4-triazolyl-pyridine ligands are reported. The ligands 2-(5-perfluoroheptyl-1,2,4-oxadiazole-3yl)-pyridine (pfhop), 2-(5-perfluoropropyl)-1,2,4-oxadiazole-3yl)-pyridine (pfpop), 2-(3-perfluoroheptyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfhtp), 2-(3-perfluoropropyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfptp) and their complexes [PtCl2(pfhop)2]·1.5 DMSO (2a), [PtCl2(pfpop)2]·1.5 DMSO (3a), [PtCl2(pfhtp)2]·1.5 DMSO (4a), PtCl2(pfhtp) (4b), [PtCl2(pfptp)2]·1.5 DMSO (5a) have been synthesized and structurally characterized. The complexes 2a, 3a, 4a and 5a have the same chemical environment of Pt(II) where PtCl2 moieties coordinate two molecules of ligand via N1 atom of pyridine in the case of pfhop and pfpop, and N2 atom of 1,2,4-triazole in the case of pfhtp and pfptp. For 4b, pfhtp behaves as bidentate ligand, coordinating Pt(II) ion via N4 atom of triazole and N1 atom of pyridine. All complexes have been tested in vitro by 3-(4,5-dimethyl-2-thiazolyl)bromide-2,5-diphenyl-2H-tetrazolium (MTT) test on four tumor cell lines MCF-7 (human breast cancer), HepG2 (human hepatocellular carcinoma), HCT116 (human colorectal carcinoma). Compounds 2a and 4b showed a dose-dependent anti-proliferative effect against the three tumor cell lines whereas did not affect viability of intestinal normal-like differentiated Caco-2 cells. The cell death of HepG2, MCF-7 and HCT116 induced by the compounds, was considered to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and observing the typical apoptotic morphological change by acridine orange (AO)/ethidium bromide (EB) staining.

Synergic Effects of Doxorubicin and Melatonin on Apoptosis and Mitochondrial Oxidative Stress in MCF-7 Breast Cancer Cells: Involvement of TRPV1 Channels.

Transient receptor transient receptor potential vanilloid 1 (TRPV1) is a Ca(2+)-permeable channel gated by oxidative stress and capsaicin (CAP) and modulated by melatonin (MEL) and capsazepine (CPZ). A combination of doxorubicin (DOX) and MEL may offer a potential therapy for breast cancer by exerting antitumor and anti-apoptotic effects and modulating Ca(2+) influx and TRPV1 activity. We aimed to investigate the effects of MEL and DOX on the oxidative toxicity of MCF-7 human breast cancer cells, in addition to the activity of the TRPV1 channel and apoptosis. The MCF-7 cells were divided into the following six treatment groups: control, incubated with MEL (0.3mM), incubated with 0.5μM DOX, incubated with 1μM DOX, incubated with MEL+0.5μM DOX, or incubated with MEL+1μM DOX. The intracellular free Ca(2+) concentration was higher in the DOX groups than in the control, and the concentration was decreased by MEL. The intracellular free Ca(2+) concentration was further increased by treatment with the TRPV1 channel activator CAP (0.01mM), and it was decreased by the CPZ (0.1mM). The intracellular production of reactive oxygen species, mitochondrial membrane depolarization, apoptosis level, procaspase 9 and PARP activities, and caspase 3 and caspase 9 activities were higher in the DOX and MEL groups than in the control. Apoptosis and the activity of caspase 9 were further increased in the DOX plus MEL groups. Taken together, the findings indicate that MEL supported the effects of DOX by activation of TRPV1 and apoptosis, as well as by inducing MCF-7 cell death. As the apoptosis and caspase activity of cancer cells increase because of their elevated metabolism, MEL may be useful in supporting their apoptotic capacity.

Dynamin-related protein 1 is involved in micheliolide-induced breast cancer cell death.

Dynamin-related protein 1 (Drp1) is a newly discovered therapeutic target for tumor initiation, migration, proliferation, and chemosensitivity. Thus, therapeutic strategies that focus on targeting Drp1 and its related signaling pathway pave a new way to address the ineffectiveness of traditional cancer therapies. Micheliolide (MCL), a guaianolide sesquiterpene lactone, can selectively eradicate acute myeloid leukemia stem or progenitor cells. But the effect of MCL on the mitochondrial dynamics of cancer cells is still not well demonstrated. In this study, we show that MCL inhibited the growth of MCF-7 human breast cancer cells, accompanied by increased mitochondrial fission and upregulation of Drp1. The results obtained from overexpression experiments of wild or dominant-negative mutant type of Drp1 demonstrate that Drp1 is both necessary and sufficient to induce MDA-MB-231 and MCF-7 cell death. Furthermore, mitochondrial membrane potential decreased, whereas reactive oxygen species (ROS) generation, cytochrome c release, and PARP cleavage were enhanced after overexpression of Drp1 wild type. On the other hand, overexpression of Drp1-K38A (a dominant-negative mutant of Drp1) rescued cells from increased apoptosis, confirming the role of MCL-induced Drp1 in the observed apoptosis. Finally, MCL-induced Drp1-mediated cell death could be reversed by N-acetyl-L-cysteine (the ROS scavenger) in breast cancer cells. Taken together, the present study shows a novel role for Drp1 in MCL-induced breast cancer cell death, potentially through regulation of ROS–mitochondrial apoptotic pathway.

Abstract A26: Effects of O-GlcNAcase inhibitors on O-GlcNAcylated c-myc expression in MCF-7 cells

Abstract Protein N-acetylglucosamine modification (O-GlcNAcylation) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. 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, which are conserved among human rat and mouse. Whereas Thr-58O-GlcNAcylation induces ubiquitin-dependent c-myc degradation, Thr-58/Ser-62 phosphorylation increases c-myc stability and thus induces invasiveness and tumorigenesis of MCF-7 human breast cancer cells. c-Myc antibodies specific for (a) Thr-58-O-GlcNAcylation, (b) Thr-58-unmodification and (c) Thr-58/Ser-62 phosphorylation were produced and specificities of the antibodies have been characterized by Western blot analyses using BSA conjugated with synthetic peptides containing the O-GlcNAcylated, unmodified and phosphorylated sites of c-myc. Western blot analysis of MCF-7 cells revealed that, whereas ~68 kDa Thr-58-O-GlcNAcylated c-myc proteins were primarily detected in the nuclear fraction, the 65-68 kDa Thr-58/Ser-62 phosphorylated and 65 kDa and 40 kDa Thr-58 unmodified c-myc proteins were expressed in both nuclear and cytosolic fractions. When MCF-7 cells were treated with 1% DMSO, 2 mM ketoconazole, a medicine-like human and bacterial β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, or 2 mM streptozotocin (STZ), an irreversible inhibitor of O-GlcNAcase, dissolved in DMSO (final concentration, 1%) for 4 hr, Thr-58-O-GlcNAcylated c-myc protein levels in nuclear and total cell lysates increased after 2 mM ketoconazole treatment but not with STZ treatment. Thr-58/Ser-62 phosphorylated protein levels did not change after either ketoconazole or STZ treatments. Treatment of the cells with 1% DMSO, 2 mM ketoconazole, buspirone or acetazolamide dissolved in DMSO (final concentration, 1%) or N6-methyladenosine 5'-monophosphate (dissolved in media), a medicine-like O-GlcNAcase inhibitor, for 4 hr revealed that Thr-58-O-GlcNAcylated c-myc protein levels in nuclear lysates increased only when the cells were treated with 2 mM ketoconazole. None of the O-GlcNAcase inhibitors including ketoconazole treatments changed levels of c-myc proteins unmodified at the Thr-58 site or phosphorylated at the Thr-58/Ser-62 sites. Whereas treatment of the MCF-7 cells with 2 mM STZ and N6-methyladenosine 5'-monophosphate failed to induce cell death and treatment with 2 mM buspirone and acetazolamide induced minimal cell death, 2 mM ketoconazole treatment, which dramatically increased Thr-58-O-GlcNAcylated c-myc protein levels, induced severe cell death. Subsequent lactate dehydrogenase (LDH) cytotoxicity assays demonstrated that the ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells by lactate dehydrogenase (LDH) cytotoxicity assays. Dose-dependent cell proliferation assays were carried out by treatment of the cells with and without 10, 20, 50 or 100 µM ketoconazole dissolved in DMSO for 72 hr, staining the cells with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). The ketoconazole treatment inhibited cell proliferation of the MCF-7 cells by 60% and 90% at 50 and 100 µM, respectively (p < 0.05). These results suggest that, in MCF-7 cells, ketoconazole, a medicine-like O-GlcNAcase inhibitor, dramatically increased Thr-58-O-GlcNAcylated c-myc proteins, which coincided with increased cell death and inhibited cell proliferation. Supported by NCI SBIR Phases I and II Contracts N261201100073C and N261201300058C. Citation Format: Hyesook Kim, So Hee Kim, Aby Joiakim, David Kaplan, David Putt. Effects of O-GlcNAcase inhibitors on O-GlcNAcylated c-myc expression in MCF-7 cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A26.

SZC017, a novel oleanolic acid derivative, induces apoptosis and autophagy in human breast cancer cells.

Oleanolic acid (OA) and its derivatives such as 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), CDDO-Me, and CDDO-Im show potent anticancer function. In this study, we elucidated the anticancer effect of SZC017, a novel OA derivative and identified the mechanisms by which SZC017 induces MCF-7 cell death. We found that SZC017 effectively decreased the cell viability of these breast cancer cells, but was less toxic to MCF10A mammary epithelial cells. Mechanisms underlying the inhibition of cell viability are apoptosis, autophagy induction, and G0/G1 phase arrest. SZC017 treatment suppressed the levels of Akt, phosphorylated-Akt (p-Akt), p-IκBα, total p65, and total p-p65, in addition to p-p65 in both the cytoplasm and nucleus. Furthermore, the inhibition of p65 nuclear translocation was confirmed by immunofluorescence staining. Cell viability was increased after pretreatment with chloroquine, an inhibitor of autophagy, whereas the level of procaspase-3 was significantly decreased. A concentration-dependent increase in the intracellular reactive oxygen species (ROS) level was observed in both MCF-7 and MDA-MB-231 cells. Additionally, pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, increased cell viability and the expression of Akt and procaspase-3, but decreased the ratio of LC3-II/I. These data show that SZC017 is an effectively selective anticancer agent against breast cancer cells, highlighting the potential use of this derivative as a breast cancer therapeutic agent.

Cytoprotective effects of ferritin on doxorubicin-induced breast cancer cell death

Ferritin is a major iron storage protein and essential for iron homeostasis. It has a wide range of functions in the body including iron delivery, immunosuppression, angiogenesis, and cell proliferation. Ferritin is overexpressed in many cancer cells, but its precise role in cancer is unclear. In the present study, we examined the functional roles of ferritin in protecting the MCF-7 breast cancer cell line against treatment with the chemotherapeutic agent doxorubicin. The effects of ferritin (human liver ferritin) and doxorubicin on the human MCF-7 breast cancer cell line were evaluated using the cell viability assay. The impact of decreasing ferritin light chain (FTL) and ferritin heavy chain (FTH) expression on doxorubicin sensitivity was assessed using siRNA. Reactive oxygen species (ROS) was also measured using the fluorescence probe CM-H2DCFDA. The mechanism of modulated chemosensitivity was evaluated by western blot analysis. Ferritin treatment activated MCF-7 cell proliferation in a concentration- and time-dependent manner. While treatment with doxorubicin alone significantly increased intracelullar ROS production, the addition of ferritin decreased this ROS formation, thereby reducing doxorubicin-induced MCF-7 cell death. The inhibition of FTL and FTH by siRNA sensitized cells to doxorubicin. Treatment with doxorubicin alone significantly induced the cell cycle-dependent kinase inhibitor protein p21, whereas ferritin reduced p21 expression. Thus, ferritin plays a critical role in protecting MCF-7 cells against the chemotherapeutic drug doxorubicin. A targeted reduction of ferritin may be a useful strategy for overcoming chemoresistance in breast cancer.

Mitogen-activated protein kinase phosphatase 1 is involved in tamoxifen resistance in MCF7cells.

Tamoxifen resistance is a major clinical problem for ER-positive breast cancer, but the underlying mechanism is not completely elucidated. In the present study, we reported that mitogen-activated protein kinase (MAPK) phosphatase1 (MKP-1), a member of the family of MKPs, is involved in tamoxifen resistance. We found that MKP1 expression increased in tamoxifen resistant MCF7cells. To explore the possible role of MKP1 in tamoxifen resistance, siRNA targeting MKP1 was transfected into tamoxifen resistant MCF7cells. To our surprise, knockdown of MKP-1 promoted cell death induced by tamoxifen. On the other hand, the MKP1 overexpressed MCF7 cell clone was established and MKP1 overexpression effectively attenuated MCF7 cell death induced by tamoxifen. In addition, we revealed that MKP1 inhibited tamoxifen‑mediated JNK activation in tamoxifen resistant MCF7 and MCF7cells, and by this mechanism MKP1 was able to inhibit tamoxifen-induced cell death. We also showed that combined appliaction of MKP1 inhibitor triptolide and tamoxifen can effectively increase tamoxifen sensitivity in tamoxifen resistant MCF7cells. Collectively, our results indicated that MKP-1 can attenuate tamoxifen-induced cell death through inhibiting the JNK signal pathway, which represents a novel mechanism of tamoxifen resistance in MCF7cells.

Abstract 1230: Detection of increased O-GlcNAcylated p53 levels in MCF-7 cells after O-GlcNAcase inhibitor treatment using antibodies specific for Ser-149-O-GlcNAcylated p53 proteins

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. O-GlcNAcylation of p53, a tumor suppressor protein, at Ser-149 prevents p53 degradation and stabilizes p53 proteins expressed in MCF-7 human breast cancer cells whereas phosphorylation of the p53 at Thr-155 induces ubiquitin-dependent degradation, which decreases p53 levels. Accumulation of the p53 protein in cancer cells induces arrest of cell growth and apoptosis in response to DNA damage. Ser-149 site-specific anti-peptide antibodies have been produced using KLH conjugated with p53 peptides with and without O-GlcNAcylation at Ser-149 by immunization of rabbits for polyclonal and mice for monoclonal antibodies. Whereas polyclonal antibodies and hybridoma media for O-GlcNAcylated p53 cross-reacted with BSA conjugated with Ser-149-O-GlcNAcylated p53 peptides in ELISA or Western blot analysis, the antibodies did not cross-react with BSA conjugated with p53 peptides without O-GlcNAcylation. Anti-peptide polyclonal antibodies produced for the unmodified p53 peptides did not cross-react with BSA conjugated with Ser-149 O-GlcNAcylated p53 peptides. Using the hybridoma media for Ser-149-O-GlcNAcylated p53, Western blot analysis was carried out with nuclear lysates and cytosol-containing supernatant obtained from MCF10A (normal breast cancer cell line) and MCF-7 cells. It was found that, in MCF-10A cells, Ser-149-O-GlcNAcylated p53 proteins (∼55 kDa) were minimally expressed in the nuclear fraction but highly expressed in the cytosolic fraction whereas, in MCF-7 cells, the level of cytosolic Ser-149-O-GlcNAcylated p53 protein was low compared with the level in MCF-10A cells. MCF-7 cells were treated for 24 hr with 1% DMSO or 2 mM β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, acetazolamide, dissolved in DMSO and levels of Ser-149-O-GlcNAcylated p53 were determined by Western blot analysis using antibodies for the Ser-149-O-GlcNAcylated p53. Whereas the level of O-GlcNAcylated p53 did not increase after 1% DMSO treatment, the level dramatically increased after treatment of acetazolamide in 1% DMSO and induced cell death. Our results showed that acetazolamide treatment increased Ser-149-O-GlcNAcylated p53 level and induced cell death in MCF-7 cells, which may decrease invasiveness of the breast cancer cells. Supported by NCI SBIR Phases I and II Contracts No. N261201100073C and N261201300058C. Citation Format: Hyesook Kim, So Hee KIm, Aby Joiakim, David Kaplan, Sung Hun Bae, David Putt. Detection of increased O-GlcNAcylated p53 levels in MCF-7 cells after O-GlcNAcase inhibitor treatment using antibodies specific for Ser-149-O-GlcNAcylated p53 proteins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1230. doi:10.1158/1538-7445.AM2015-1230