MCF-7 Cell Death Research Articles

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

Micheliolide overcomes KLF4-mediated cisplatin resistance in breast cancer cells by downregulating glutathione.

Micheliolide (MCL) is a promising novel compound with broad-spectrum anticancer activity. However, little is known regarding its action and mechanism in breast cancer. To explore the potential therapeutic application of MCL as a chemosensitivity modulator, this study investigated the effects of MCL on cisplatin sensitivity in breast cancer and the underlying mechanisms. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay and a xenograft tumor model, MCL enhanced the cisplatin sensitivity of the breast cancer cell line MCF-7 both in vitro and in vivo. Treatment of MCF-7 cells with low-dose cisplatin (10 µM) was sufficient to enrich the proportion of ALDH+ cells and upregulate Krüppel-like factor 4 (KLF4) expression. The results obtained from knockdown and overexpression experiments demonstrate that KLF4 is both necessary and sufficient to induce a cisplatin resistance phenotype in breast cancer cells. Furthermore, the glutathione (GSH) content was elevated in MCF-7 cells after overexpression of KLF4. KLF4-mediated resistance to cisplatin was found to be abrogated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis. MCL induced GSH depletion and severe cell death in KLF4-overexpressing MCF-7 cells following exposure to cisplatin. Therefore, these results suggest that MCL-mediated direct depletion of GSH represents a major mechanism in reversing KLF4-induced cisplatin resistance in MCF-7 cells.

The interactions between SATB1 and F-actin are important for mechanisms of active cell death.

The direct involvement of nuclear actin filaments in gene transcription and remodeling of chromatin is still debatable. However, nuclear localization of F-actin and its interactions with other nuclear matrix proteins have been reported. The aim of the study was to estimate the interactions between nuclear F-actin and one of the matrix proteins, special AT-rich sequence-binding protein 1 (SATB1), during active cell death induced in vitro by geldanamycin (GA). The expression of SATB1 was modified by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The amount and localization of F-actin were altered by changes of cofilin-1 (CFL1) expression in MCF-7 cells. The association between SATB1 and F-actin during GA-induced cell death was analyzed using confocal and transmission electron microscopy. Our studies revealed the colocalization between nuclear F-actin and SATB1 protein, during GA-induced death of breast cancer MCF-7 cells. The colocalization was enhanced in cells with overexpressed SATB1 and cofilin-1. At the ultrastructural level the SATB1 and F-actin complexes were seen at the border of condensed and decondensed chromatin. The presence of SATB1/F-actin molecular complexes was confirmed by magnetic separation of F-actin and interacting proteins. We suggest that the molecular interactions between SATB1 and F-actin are necessary for active cell death to occur.

Synthesis of cobalt ferrite nanoparticles using spermine and their effect on death in human breast cancer cells under an alternating magnetic field

To compare heat generation under an AC magnetic field for inducing cancer cell death, CoFe2O4 and Fe3O4 nanoparticles with a diameter of ∼10nm were synthesized by hydrolysis in an aqueous solution containing the metal chlorides iron(III) chloride and cobalt(II) or iron(II) chloride, with spermine or N,N'-bis(3-aminopropyl)butane-1,4-diamine, as a base and a protective reagent. Both CoFe2O4 and Fe3O4 nanoparticles were incorporated into human breast cancer MCF-7 cells; the nanoparticles exhibited little toxicity on the cells. When the cells containing nanoparticles were subjected to an AC magnetic field, MCF-7 cell death was induced by heat generated from ∼10-nm CoFe2O4 nanoparticles but not from ∼10-nm Fe3O4 nanoparticles. An increase in coercivity derived from the substitution of Fe2+ with Co2+ facilitated the ability of the nanoparticles to induce cell death in human breast cancer cells under an AC magnetic field.

NJK14013, a novel synthetic estrogen receptor-α agonist, exhibits estrogen receptor-independent, tumor cell-specific cytotoxicity.

Estrogens act through interactions with estrogen receptors (ERs) to play diverse roles in various pathophysiological conditions. A number of synthetic selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, have been developed and used to treat ER-related diseases, including breast cancer and osteoporosis. Here, we identified a novel compound, bis(4-hydroxyphenyl)methanone-O-isopentyl oxime, designated NJK14013, as an ER agonist. NJK14013 activated ER-dependent transcription in a concentration-dependent manner, while suppressing androgen receptor-dependent transcriptional activity. It induced the activation-related phosphorylation of ER and enhanced the transcription of growth regulation by estrogen in breast cancer 1 (GREB1), further supporting its ER-stimulating activity. NJK14013 exerted anti-proliferative effects on various cancer cell lines, including an ER-negative breast cancer cell line, suggesting that it is capable of suppressing the growth of cancer cells independent of its ER-modulating activity. In addition, NJK14013 treatment resulted in significant apoptotic death of MCF7 and Ishikawa cancer cells, but did not induce apoptosis in non-cancer human umbilical vein endothelial cells. Collectively, our findings demonstrate that NJK14013 is a novel SERM that can activate ER-mediated transcription in MCF7 cells and suppress the proliferation of various cancer cells, including breast cancer cells and endometrial cancer cells. These results suggest that NJK14013 has potential as a novel SERM for anticancer or hormone-replacement therapy with reduced risk of carcinogenesis.

Quercetin protects necrotic insult and promotes apoptosis by attenuating the expression of RAGE and its ligand HMGB1 in human breast adenocarcinoma cells.

The receptor for advanced glycation end-products (RAGE) is a multiligand member of the immunoglobulin superfamily, which plays an important role in maintaining cellular homeostasis. It is normally expressed on immune cells, including macrophages, monocytes, dendritic cells and T cells to maintain homeostasis, but highly upregulated at sites of vascular pathology. Accumulating evidence suggest that the elevated expression of RAGE and its ligand HMGB-1 was found in various types of cancer. The accumulation of RAGE and its ligand high-mobility group box proteins-1 (HMGB1) activates complex signaling network for cell survival and evades apoptosis. Therefore, targeting the RAGE-mediated signaling could be the promising strategies for the therapeutic potential of cancer. This study was aimed to examine the biological potential of quercetin on the regulation of RAGE- and HMGB1-mediated activation of NF-κB and induction of apoptotic cell death in MCF-7 cells. Our findings demonstrate that quercetin inhibits the expression of RAGE and HMGB1 in MCF-7 cells. In addition, quercetin protects necrotic insult and augments apoptosis in MCF-7 cells. Taken together, these results suggest that quercetin plays an important role in modulating RAGE and HMGB1 signaling and induces apoptotic cell death in MCF-7 cells.

Abstract P6-14-07: High miR-19a serum levels correlate with favorable prognosis in patients with metastatic HER2+ inflammatory breast cancer and may result from an effective antibody-dependent cell-mediated cytotoxicity induced by trastuzumab

Abstract Background: IBC is a rare but highly aggressive form of locally advanced breast cancer (5-year OS rate: 40.5% IBC vs 85% non-IBC patients) accounting for 10% of all breast cancer deaths. To date, no unique molecular diagnostic or prognostic biomarker has been identified for IBC. Increasing evidence supports the potential value of miRNA as prognostic and predictive serum biomarker in cancer. We found that IBC cells expressed high levels of miR-19a and patients with metastatic IBC HER2+ (MIBC HER2+) and high miR-19a serum levels had better prognosis than patients with MIBC HER2+ and low miR-19a serum levels. As one of the mechanisms of action of trastuzumab is the induction of antibody-dependent cell-mediated cytotoxicity (ADCC), we hypothesized that the increased miR-19a serum levels in MIBC HER2+ patients with favorable clinical outcome could result from an effective ADCC and be used as biomarker to monitor the response to trastuzumab. Methods: Total RNA was isolated using the Total RNA Purification Kit (Invitrogen, Norgen Biotek). MiR-19a levels in tumor tissue, serum, cell lines and supernatants were evaluated by qRT-PCR (Applied Biosystems). ADCC was evaluated by Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen). Results: Microarray was performed in IBC (n=23) and non-IBC (n=24) tumors and normal tissue (n=12). Microarray showed higher miR-19a expression in IBC compared with non-IBC (p=0.028) and normal tissue (p=0.0002). The two IBC cell lines SUM149 (triple receptor-negative) and KPL-4 (HER2-amplified) expressed higher levels of miR-19a compared with the non-IBC cell lines MDA-231 (triple receptor-negative), SKBR3 (HER2-amplified), and MCF-7 (HER2-non-amplified) (p<0.001, p<0.001, p<0.001 and p<0.05, p<0.05, p<0.01 respectively). To assess whether miR-19a could be released from IBC cells upon NK cell-mediated ADCC, we performed a NK cytotoxicity test using the NK-resistant KPL-4 cells and the NK-sensitive SKBR3 and the NK-sensitive MCF-7 cells as control. Co-incubation with trastuzumab induced increased MCF-7, SKBR3 and KPL-4 cell death (2.1-fold, 2.4-fold and 3.5-fold respectively) and accordingly increased miR-19a levels in their supernatants (MCF-7: 3-fold, p=0.017; SKBR3: 6-fold, p=0.005 and KPL-4: 8-fold, p=0.0001). The pattern of miR-19a levels in the supernatants correlated with that expressed at cellular levels (MCF-7<SKBR3<KPL-4). We measured miR-19a serum levels in patients with MIBC HER2+ (n=27) and metastatic nonIBC HER2+ (n=24). Patients with MIBC HER2+ and high miR-19a level had longer PFS (10.3 vs 3.2 months, p=0.022) and OS (median not reached vs 11.2 months, p=0.003) than patients with low miR-19a levels. Patients with metastatic nonIBC HER2+ and high miR-19a levels had longer OS (32.9 vs 13.3 months; p=0.015) than patients with low miR-19a levels (32.9 vs 13.3 months; p=0.015). Conclusion: High miR-19a serum levels are associated with favorable prognosis in patients with MIBC HER2+ and could result from an effective NK cell-mediated ADCC. MiR-19a may represent a novel serum biomarker to monitor the response to trastuzumab therapy and predict clinical outcome in patients with MIBC HER2+. Citation Format: Simone Anfossi, Antonio Giordano, Lei Huo, Ricardo H Alvarez, Vicente Valero, Gabriel N Hortobagyi, Wendy A Woodward, Naoto T Ueno, George A Calin, James M Reuben. High miR-19a serum levels correlate with favorable prognosis in patients with metastatic HER2+ inflammatory breast cancer and may result from an effective antibody-dependent cell-mediated cytotoxicity induced by trastuzumab [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-07.

Cytotoxicity of Vitex trifolia leaf extracts on MCF-7 and Vero cell lines

Pharmacological and preventive properties of Vitex trifolia leaf extracts are well known, but the anticancer activity of methanol and petroleum ether extracts of Vitex trifolia leafs on human breast cancer cells have not been explored so far. Therefore, the present study was designed to investigate the cytotoxic activities of these extracts against MCF-7 and Vero cell line. Cells were exposed to 125 to 500 μg/ml of the extracts of Vitex trifolia for 72 h. Post-treatment, percent cell viability was studied by 3-(4, 5-dimethylthiazol-2yl)-2, 5-biphenyl tetrazolium bromide (MTT) assays. The results showed that petroleum ether and methanol extracts significantly reduced cell viability of MCF-7 cells in a concentration dependent manner. Concentrations of 125 μg/ml and above of petroleum ether and 500 μg/ml of methanol extract were found to be cytotoxic in MCF-7 cells. Cell mortality at 125, 250 and 500 μg/ml of petroleum ether extract was recorded as 98.80%, 93.40% and 87.75% respectively, whereas at 125, 250 and 500 of methanol extract values were 79.98%, 75.70% and 70.25%, respectively by MTT assay. MCF-7 and Vero cells exposed to 125, 250 and 500 μg/ml. The data revealed that the treatment with petroleum ether and methanol of Vitex trifolia leaf extract induced cell death in MCF-7 cells. Meanwhile the same extract showed a moderate inhibition against Vero cell lines. It may be concluded that Vitex trifolia can cause cell death in MCF-7 cancer cells, which can be considered as a promising chemotherapeutic agent in breast cancer treatment.

The cytotoxic nature of Acanthopanax sessiliflorus stem bark extracts in human breast cancer cells

Acanthopanax sessiliflorus, a small woody shrub has traditionally been referred to have anticancer activity, but it has not been scientifically explored so far. Therefore, to investigate the anticancer effects of A. sessiliflorus stem bark extracts (ASSBE), MDA-MB-231 and MCF-7 human breast cancer cells were treated with one of its bioactive fractions, n-hexane (ASSBE-nHF). Cytotoxicity (24h) was determined by MTT assay and antiproliferative effect was assessed by counting cell numbers after 72h treatment using hemocytometer. The role of ASSBE-nHF on apoptosis was analysed by annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation pattern and immunoblotting of apoptosis markers. For the assay of enhanced production of ROS and mitochondrial membrane depolarization, specific stains such as DCFH-DA and JC-1 were used, respectively. To understand the mode of action of ASSBE-nHF on MCF-7 cells, cells were pre-treated with antioxidant, n-acetylcysteine. The hexane fraction of ASSBE showed maximum activity towards human breast cancer cells compared to other two fractions at a minimal concentration of 50μg/ml. The annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation and immunoblotting assays showed that ASSBE-nHF induces non-apoptotic cell death in MCF-7 and MDA-MB-231 cells. ASSBE-nHF significantly increased the production of ROS and decreased the mitochondrial membrane potential (MMP) in MCF-7 cells. Similarly, it decreased the MMP in MDA-MB-231 cells, but had no effect on ROS production. Further, the cytotoxic effect of ASSBE-nHF in MCF-7 cells was not significantly reversed even in the presence of n-acetylcysteine, an antioxidant. These findings revealed that ASSBE-nHF induces non-apoptotic cell death via mitochondria associated with both ROS dependent and independent pathways in human breast cancer cells.

Participation of non-neuronal muscarinic receptors in the effect of carbachol with paclitaxel on human breast adenocarcinoma cells. Roles of nitric oxide synthase and arginase

Breast cancer is the most common type of cancer in women and represents a major issue in public health. The most frequent methods to treat these tumors are surgery and/or chemotherapy. The latter can exert not only beneficial effects by reducing tumor growth and metastasis, but also toxic actions on normal tissues. Metronomic therapy involves the use of low doses of cytotoxic drugs alone or in combination to improve efficacy and to reduce adverse effects. We have previously reported that breast tumors highly express functional muscarinic acetylcholine receptors (mAChRs) that regulate tumor progression. For this reason, mAChRs could be considered as therapeutic targets in breast cancer. In this paper, we investigated the ability of a combination of the cytotoxic drug paclitaxel plus carbachol, a cholinergic agonist, at low doses, to induce death in breast tumor MCF-7 cells, via mAChR activation, and the role of nitric oxide synthase (NOS) and arginase in this effect. We observed that the combination of carbachol plus paclitaxel at subthreshold doses significantly increased cytotoxicity in tumor cells without affecting MCF-10A cells, derived from human normal mammary gland. This effect was reduced in the presence of the muscarinic antagonist atropine. The combination also increased nitric oxide production by NOS1 and NOS3 via mAChR activation, concomitantly with an up-regulation of NOS3 expression. The latter effects were accompanied by a reduction in arginase II activity. In conclusion, our work demonstrates that mAChRs expressed in breast tumor cells could be considered as candidates to become targets for metronomic therapy in cancer treatment.

Changes in the response of MCF-7 cells to ionizing radiation after the combination of ATM and DNA-PK inhibition.

The aim of the present study is to evaluate the role of ATM (KU55933) and DNA-PK (NU7441) inhibitors in the repair of double-strand breaks and downstream signaling of DNA damage introduced by ionizing radiation. The irradiation of MCF-7 cells alone increased the proportion of cells in the G1 phase in comparison with mock-treated cells. After ATM inhibitor pretreatment, the cells were more accumulated in the G2 phase, whereas DNA-PK inhibitor application increased the percentage of cells in the G1 phase. ATM and DNA-PK inhibitor application alone increased the sensitivity of MCF-7 cells to ionizing radiation; however, combining both inhibitors together resulted in a further enhancement of cell death. Unexpectedly, combining both inhibitors decreased the percentage of senescent cells and increased G2 cell cycle arrest 3 days after treatment. After irradiation, the p21 protein was increased and Chk1 and Chk2 were activated. These proteins were not increased in cells pretreated with the ATM inhibitor prior to ionizing radiation exposure, albeit DNA-PK inhibitor application did not affect the amount of proteins detected. Formation of γH2AX was found to be ATM and DNA-PK dependent, application of the ATM inhibitor suppressed incidence of γH2AX, whereas DNA-PK caused persistence of γH2AX. Our results suggest that the further investigation of the ATM inhibitor in combination with the DNA-PK inhibitor as sensitizers preventing cell senescence and promoting cell death in breast carcinoma MCF-7 cells is warranted.

ZNF32 inhibits autophagy through the mTOR pathway and protects MCF-7 cells from stimulus-induced cell death.

ZNF32 is a recently identified zinc finger protein and its functions remain largely unknown. Autophagy has been shown to affect cell proliferation and survival. Here, we innovatively show the effect of ZNF32 on cell autophagy and autophagy-associated cell death in breast carcinoma cells and also elucidate its underlying mechanisms. We examined the autophagic activity and LC3 II expression in human carcinoma cell lines with increased or decreased ZNF32 expression. Pharmacological inhibition (rapamycin) or activation (EGF) assays were used to investigate the function of the AKT/mTOR pathway during this process. H2O2- and diamide-induced MCF-7 cell death models were used to elucidate the role of ZNF32-associated autophagy in breast carcinoma cell death. Our results show that increasing ZNF32 expression in MCF-7 cells inhibits autophagy initiation by activating the AKT/mTOR pathway, and further reduced autophagy-associated cell death and maintained MCF-7 cell survival. Conversely, impairing ZNF32 expression by transfecting ZNF32 siRNA strongly promoted autophagy, further augmenting autophagy-associated cell death. Furthermore, correlations between ZNF32 and autophagy were observed in both MCF-7 xenograft tumors and in breast cancer patients. In conclusion, ZNF32 acts as an effective autophagy inhibitor to protect breast cancer cells from excessive stimulus-autophagy-induced cell death.

Anticancer effect of black tea extract in human cancer cell lines.

In this study we investigated effects of natural extract from the black tea Camellia sinensis (BTE) against human colon carcinoma cell line HT-29, human breast carcinoma cell line MCF-7, human alveolar carcinoma cell line A549 and healthy cell line NIH-3T3. We identified concentration range for cytotoxic/antiproliferative effects using MTT assay and the trypan blue assay, gel electrophoresis we employed to determine the type of cell death induced by BTE and DNA damage we determined by comet assay. Different concentrations of the extract (0.00078 - 5 μg/mL) we added to the cultured cells and incubated for 216 h. BTE showed cytotoxic effects against all carcinoma cell lines, however HT-29 and MCF-7 cells were more sensitive than A549. BTE showed no antiproliferative effect against healthy cells NIH-3T3 at tested concentrations. We found no apoptotic cell death in HT-29 and MCF-7 cells after 72 h of incubation in case of single administration of BTE but in case of repetitive administration of BTE (BTE was added to the cells each day) we found apoptotic cell death in HT-29 after 72 h incubation. BTE induced also DNA strand breaks and oxidative damage to DNA in carcinoma cells HT-29 and MCF-7.

Synthesis and biological evaluation of novel tyrosyl-DNA phosphodiesterase 1 inhibitors with a benzopentathiepine moiety

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a promising target for antitumor therapy based on Top1 poison-mediated DNA damage. Several novel benzopentathiepines were synthesized and tested as inhibitors of TDP1 using a new oligonucleotide-based fluorescence assay. The benzopentathiepines have IC50 values in the range of 0.2–6.0μM. According to the molecular modeling, the conformational flexibility of the dibutylamine group of the most effective inhibitor (3d) allows it to occupy an advantageous position for effective binding compared to its cyclic counterparts. The study of cytotoxicity of these compounds revealed that all compounds cause an apoptotic cell death in MCF-7 and Hep G2 cells. Therefore the new class of very effective inhibitors of TDP1 was elaborated.

Induction of DNA damage by the leaves and rhizomes of Curcuma amada Roxb in breast cancer cell lines

ObjectiveTo evaluate DNA damage inducing effect of the methanolic extract of both the leaves and the rhizomes of Curcuma amada (C. amada) against breast cancer cell lines MCF-7 and MDA MB 231 and analyze the active components present in the methanolic extract of the leaves and the rhizomes. MethodsThe DNA damage induced in yeast was assessed using diphenylamine method. The DNA damage induced by the extracts in cell lines was assessed using single cell gel electrophoresis (Comet assay). Various phytochemicals present in the leaves and the rhizomes were analysed using various chromatographic and spectral studies. A normal non-cancer cell line HBL-100 and an eukaryotic model organism yeast was also used for comparison. ResultsThe results indicated that the methanolic extract of both the leaves and the rhizomes of C. amada induced cell death in the breast cancer cell lines MCF-7 and MDA MB 231. The extracts showed less DNA damage in yeast and HBL-100 cells. The phytochemical investigation revealed the presence of more amounts of terpenoids and steroids in both the leaves and rhizomes. ConclusionsThe results indicated that the methanolic extract of leaves of rhizomes of C. amada possess genotoxic and cytotoxic activity against the breast cancer cell lines.

DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) inhibits chemotherapeutic drug-induced autophagy

Our previous work found that DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl]quinoline) was a novel autophagy inhibitor. Here, we aimed to investigate the effects of DMH1 on chemotherapeutic drug-induced autophagy as well as the efficacy of chemotherapeutic drugs in different cancer cells. We found that DMH1 inhibited tamoxifen- and cispcis-diaminedichloroplatinum (II) (CDDP)-induced autophagy responses in MCF-7 and HeLa cells, and potentiated the anti-tumor activity of tamoxifen and CDDP for both cells. DMH1 inhibited 5-fluorouracil (5-FU)-induced autophagy responses in MCF-7 and HeLa cells, but did not affect the anti-tumor activity of 5-FU for these two cell lines. DMH1 itself did not induce cell death in MCF-7 and HeLa cells, but inhibited the proliferation of these cells. In conclusion, DMH1 inhibits chemotherapeutic drug-induced autophagy response and the enhancement of efficacy of chemotherapeutic drugs by DMH1 is dependent on the cell sensitivity to drugs.

Phosphorylation-independent mTORC1 inhibition by the autophagy inducer Rottlerin

We recently found that Rottlerin not only inhibits proliferation but also causes Bcl-2- and Beclin 1-independent autophagic death in apoptosis-resistant breast adenocarcinoma MCF-7 cells. Having excluded a role for canonical signaling pathways, the current study was aimed to investigate the contribution of the AMPK/mTOR axis in autophagy induction and to search for the upstream signaling molecules potentially targeted by Rottlerin. Using several enzyme inhibitors, Western blotting analysis, mTOR siRNA and pull down assay, we demonstrate that the Rottlerin-triggered autophagy is mediated by inhibition of mTORC1 activity through a novel AMPK and mTORC1 phosphorylation-independent mechanism, likely mediated by the direct interaction between Rottlerin and mTOR.

ROS-induced toxicity: exposure of 3T3, RAW264.7, and MCF7 cells to superparamagnetic iron oxide nanoparticles results in cell death by mitochondria-dependent apoptosis

Superparamagnetic nanoparticles (Fe3O4, SPIO) have been used as magnetic resonance imaging enhancers for years. However, bio-safety issues concerning nanoparticles remain largely unexplored. Of particular concern is the possible cellular impact of nanoparticles during SPIO uptake and subsequent oxidative stress. SPIO causes cell death by apoptosis via a little understood mitochondrial pathway. To more closely examine this process, three kinds of cells—3T3, RAW264.7, and MCF7—were treated with SPIO coated with polyethylene glycol (SPIO-PEG) and monitored by transmission electron microscopy (TEM), using cytotoxicity evaluation, mitochondrial activity, reactive oxygen species (ROS) generation, and Annexin V assay. TEM revealed that SPIO-PEG nanoparticles surrounded the cellular endosome membrane, creating a bulge in the endosome. Compared to 3T3 cells, greater numbers of SPIO-PEG nanoparticles infiltrated the mitochondria of RAW264.7 and MCF7 cells. SPIO-PEG residency is associated with boosted ROS, with elevated levels of mitochondrial activity, and advancement of cell apoptosis. Furthermore, correlation analysis showed that a polynomial model demonstrates a better fit than a linear model in MCF7, implying that cytotoxicity may have alternative impacts on cell death at different concentrations. Thus, we believe that MCF7 cell death results from the apoptosis pathway triggered by mitochondria, and we find lower cytotoxicity in 3T3. We propose that optimal levels of SPIO-PEG nanoparticles lead to increased levels of ROS and a resulting oxidative stress environment which will kill only cancer cells while sparing normal cells. This finding has great potential for use in cancer therapies in the future.

Interaction of Aβ peptide with tubulin causes an inhibition of tubulin polymerization and the apoptotic death of cancer cells.

We report in this work that the Aβ peptide directly interacts with tubulin close to the vinblastine and GTP/GDP binding site, inhibits the tubulin polymerization rate, induces tubulin aggregation, causes cell shrinking, enhances Mad2, BubR1, p53, and p21 activation in MCF7 cells and induces the apoptotic death of A549, HeLa and MCF7 cells.

Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.