Phytochemical-capped silver nanoparticles from the medicinal plant Embelia laeta disrupting 3D tumorspheres and inducing ROS-mediated apoptosis in MCF-7 breast cancer cells

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.

We previously reported that expression of NRIF3 (nuclear receptor interacting factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (also known as interferon regulatory factor-2 binding protein 2 [IRF-2BP2]), the cellular target of NRIF3, was identified as a transcriptional repressor, and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF-2BP1 and EAP1 (enhanced at puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF-2BP1, and EAP1 occurs through the conserved C4 zinc fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the death domain (DD1) region of NRIF3. The DIF-1 complex was found to repress FASTKD2, a putative proapoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown, while expression of FASTKD2 leads to apoptosis of both breast and nonbreast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer.

Survivin expression has been shown to be associated with cancer progression, poor prognosis, and drug resistance. The aim of this study was to examine whether survivin knock-down could enhance paclitaxel-induced apoptosis in breast cancer cells in vitro. MCF-7 cells were infected with an siRNA-expressing adenovirus vector against survivin (Adv-siSurv) or Renilla luciferase as a control (Adv-siRL). After treatment with paclitaxel, cells were analyzed by apoptotic, cell cycle and immunoblotting assays. Of cells treated with paclitaxel alone, only 20.2±2.08% showed apoptotic features. An increase in the paclitaxel dose was associated with increased survivin expression. In contrast, Adv-siSurv infection resulted in a marked increase in apoptotic cell death in paclitaxel-treated MCF-7 cells (49.9±7.70%). The percentage of cells in the G2M phase was lower (23.9±1.64%) in Adv-siSurv-infected cells than that of Adv-siRL-treated cells (40.0±2.43%). Adv-siSurv infection reduced survivin, procaspase-9, and procaspase-3 levels in paclitaxel-treated MCF-7 cells. Loss of survivin expression enhanced paclitaxel-induced apoptosis in MCF-7 breast cancer cells in vitro.

Objective To explore the mechanism of endoplasmic reticulum (ER) stress inducing autophagy and apoptosis of human breast cancer cells by inhibiting c-Jun N-terminal kinases(JNK) -c-Jun signaling pathway. Methods The optimal concentration of tunicamycin was determined based on its effect at different concentration and time on human breast cancer cells. Cells were divided into ER activation group, ER activation+ SP600125 (JNK depressor) group and control group(routindely cultured cells without specific procedure). Methyl thiazolyl tetrazolium assay, dansylcadaverine assay and flow cytometry were used to detect viability, autophagy and apoptosis of cells. Western blot was applied to analyze ER stress-related proteins 78 kDa glucose regulated protein (GRP78) and CCAAT-enhancer-binding protein homolgous protein (CHOP), JNK-c-Jun signaling pathway-related proteins JNK and c-Jun, autophagy-related proteins Beclin1, and apoptosis-related proteins Bax, caspase-3 and caspase-12 expression levels. Results The optimum concentration of tunicamycin on human breast cancer cells was 100 ng/ml, and the action time was 72 h. Under these conditions, the expression levels of GRP78 and CHOP were significantly higher than those before treatment (all P <0.05). The expression levels of JNK and c-Jun in ER activation group were significantly higher than those in ER activation+ SP600125 group and control group (all P <0.05). Compared with the control group and ER activation+ SP600125 group, the proliferation activity of cells in ER activation group was significantly inhibited, while the fluorescence intensity of MDC and the apoptotic rate were significantly increased (all P <0.05). Meanwhile, the expression levels of key apoptotic factors (Bax, Caspase-3 and Caspase-12) in ER activation group, were significantly higher than those in ER activation+ SP600125 group and control group (all P <0.05). Conclusions ER stress may inhibit the cell growth, further induce autophagy and apoptosis of breast cancer cells by activating JNK-c-Jun signaling pathways. Key words: Endoplasmic reticulum; Stress; JNK/c-Jun signaling pathway; Cell autophagy; Cell apoptosis

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.

Objective To investigate the expression of thyroid cancer-1 (TC-1) in breast cancer and its influence on the proliferation and apoptosis of breast cancer cells. Methods Reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the expression of TC-1 in breast cancer tissues and corresponding paracancerous tissues. The breast cancer cell MCF-7 were transfected with TC-1 siRNA and siRNA control, it was named as interference group and negative group, the cells without treatment were used as control group. Cell proliferation was detected by methyl thiazol tetrazolium (MTT), apoptosis was detected by flow cytometry, the expressions of β-catenin, c-Myc, Cleaved Caspase-3 were detected by Western blotting. Results The level of TC-1 in breast cancer tissues was higher than that in paracancerous tissues (t=17.746, P=0.000; t=12.705, P=0.000). The apoptosis rate, survival rate, TC-1, β-catenin, c-Myc, Cleaved Caspase-3 in the control group and the negative group did not change significantly. The rate of apoptosis in the interference group was higher than that in the control group, the cell survival rate was lower than that of the control group, and the levels of β-catenin and c-Myc were lower than those of the control group, the levels of Cleaved Caspase-3 was higher than those of the control group (t=13.986, P=0.000; t=7.015, P=0.002; t=10.457, P=0.001; t=27.886, P=0.000; t=7.426, P=0.002). Conclusion The expression of TC-1 was up-regulated in breast cancer tissues, interference with TC-1 can inhibit proliferation of breast cancer cells, promoting apoptosis of breast cancer cells, the Wnt signaling pathway may be one of its mechanisms. Key words: Breast cancer; Proliferation; Apoptosis; Thyroid cancer-1

To investigate the effects of adenovirus melanoma differentiation associated gene-7 (mda-7)/IL-24 on the growth and apoptosis of human breast cancer cells. Human breast cancer cells of the lines MCF-7 and MD-MBA-453 were cultured and transfected with purified adenovirus mda-7/IL-24. 48 h later, Western blotting was used to detect the protein expression of mda-7/IL-24 in the MCF-7 cells. The growth of Ad-mda-7/IL-24 in the MCF-7 cells was assayed by crystal violet staining and MTT method. The apoptotic effect of Ad-mda-7/IL-24 in the MCF-7 and MDA-MB-453 cells was detected by Hoechst staining and annexin V method. Nude mice were inoculated with MCF-7 cells and randomly divided into 2 groups to be injected with Ad-mda-7/IL-24 of PBS. The size of tumor was observed. Western blotting showed that mda-7/IL-24 was expressed in the transfected MCF-7 cells. Crystal violet staining and MTT method showed that Ad-mda-7/IL-24 dose and time-dependently inhibited the growth of the MCF-7 and MDA-MB-453 cells. Hochst staining and annexin V test indicated obvious apoptosis of the breast cancer cells. The size of tumor of the nude mice injected with Ad-mda-7/IL-24 was significantly smaller than that of the control mice. Ad-mda-7/IL-24 effectively inhibits the growth of breast cancer cells and induces its apoptosis.

We investigated the effect of high mobility group protein N2 (HMGN2) on the proliferation and apoptosis of the human MCF-7 breast cancer cell line, and its effect on tumor growth in a subcutaneous heterotopic transplantation tumor model of breast cancer. The cell viability assay was used to verify the effect of the recombinant human HMGN2 on MCF-7 cell proliferation. The Transwell chamber assay was used to verify the effect of HMGN2 on MCF-7 cell migration. Flow cytometry and Hoechst staining were used to detect the effect of HMGN2 on MCF-7 cell apoptosis. MCF-7 was injected to establish a subcutaneous heterotopic transplantation tumor model of breast cancer in nude mice. The size, weight and volume of tumor in each group were compared after the administration of different concentrations of HMGN2 solution around the tumor tissue at day 1, 3, 5 and 7. The tumor tissue was removed and cut into sections, and the apoptotic cells in tumors of nude mice were detected by a TUNEL kit. The CCK-8 assay showed that HMGN2 at different concentrations inhibited the proliferation of the MCF-7 breast cancer cells, and the proliferation of MCF-7 cells were significantly inhibited when the concentration of HMGN2 reached 3 µg/ml (P<0.01). The Transwell chamber assay showed that 3 µg/ml of HMGN2 significantly decreased the migration capacity of MCF-7 cells (P<0.01). Flow cytometry and Hoechst staining showed that 3 µg/ml of HMGN2 significantly increased apoptosis of MCF-7 cells (P<0.01). After the nude mouse model of breast cancer was established, HMGN2 at different concentrations was injected around the tumor tissue at day 1, 3, 5 and 7. We demonstrated that the growth of breast cancer was significantly inhibited when the concentration of HMGN2 reached 15 µg/ml. TUNEL staining showed that the number of apoptotic cells in the 15 µg/ml dose group was significantly higher than that in the control group (P<0.01). Therefore, in vitro and in vivo experiments proved that recombinant human HMGN2 could significantly inhibit the proliferation and migration of breast cancer cells, which increased the apoptosis of breast cancer cells and exerted anti-breast cancer effects, which enriched our understanding of the biological roles of HMGN2.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR also known as fenretinide) is a potent inducer of apoptosis in breast cancer cells. We observed a 4.5-fold reduction in 4-HPR-mediated apoptosis in MCF-7 breast cancer cells transfected with HER2/neu (MCF-7/HER2) as compared with the parental MCF-7 (MCF-7/WT) cells. Blocking HER2/neu with trastuzumab (Herceptin) led to a six-fold increase in 4-HPR-induced apoptosis in HER2/neu-overexpressing cells. These data indicate that HER2/neu reduces the sensitivity of breast cancer cells to 4-HPR. We showed previously that nitric oxide (NO) is essential for 4-HPR to induce apoptosis in breast cancer cells. The inhibitory effects of the 4-HPR and trastuzumab combination correlated with the amount of NO produced in HER2/neu-overexpressing cells. When a NO synthase (NOS) inhibitor was used to block NO production, decreased apoptosis by the 4-HPR and trastuzumab combination was observed. Furthermore, 4-HPR-mediated NOSII expression was lower in MCF-7/HER2 than MCF-7/WT cells, but was increased by trastuzumab in HER2/neu-overexpressing cells. Here we report the novel findings that HER2/neu reduces the ability of 4-HPR to induce apoptosis in breast cancer cells, and that one mechanism by which HER2/neu increases the resistance of breast cancer cells to 4-HPR is by decreasing NOSII-mediated NO production.

Quercetin is a dietary flavonoid which exerts anti-oxidant, anti-inflammatory and anti-cancer properties. In this study, we investigated the anti-proliferative effect of quercetin in two breast cancer cell lines (MCF-7 and MDA-MB-231), which differed in hormone receptor. IC50 value (37μM) of quercetin showed significant cytotoxicity in MCF-7 cells, which was not observed in MDA-MB-231 cells even at 100μM of quercetin treatment. To study the response of cancer cells to quercetin, with respect to different hormone receptors, both the cell lines were treated with a fixed concentration (40μM) of quercetin. MCF-7 cells on quercetin treatment showed more apoptotic cells with G1 phase arrest. In addition, quercetin effectively suppressed the expression of CyclinD1, p21, Twist and phospho p38MAPK, which was not observed in MDA-MB-231 cells. To analyse the molecular mechanism of quercetin in exerting an apoptotic effect in MCF-7 cells, Twist was over-expressed and the molecular changes were observed after quercetin administration. Quercetin effectively regulated the expression of Twist, in turn p16 and p21 which induced apoptosis in MCF-7 cells. In conclusion, quercetin induces apoptosis in breast cancer cells through suppression of Twist via p38MAPK pathway.

Benzyl isothiocyanate (BITC), a dietary cancer chemopreventive agent, causes apoptosis in MDA-MB-231 and MCF-7 human breast cancer cells, but the mechanism of cell death is not fully understood. We now demonstrate that the BITC-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) due to inhibition of complex III of the mitochondrial respiratory chain. The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase and pharmacological inhibition of the mitochondrial respiratory chain. The mitochondrial DNA-deficient Rho-0 variant of MDA-MB-231 cells was nearly completely resistant to BITC-mediated ROS generation and apoptosis. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of Bax. Biochemical assays revealed inhibition of complex III activity in BITC-treated MDA-MB-231 cells as early as at 1 h of treatment. The BITC treatment caused activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which function upstream of Bax activation in apoptotic response to various stimuli. Pharmacological inhibition of both JNK and p38 MAPK conferred partial yet significant protection against BITC-induced apoptosis. Activation of JNK and p38 MAPK resulting from BITC exposure was abolished by overexpression of catalase. The BITC-mediated conformational change of Bax was markedly suppressed by ectopic expression of catalytically inactive mutant of JNK kinase 2 (JNKK2(AA)). Interestingly, a normal human mammary epithelial cell line was resistant to BITC-mediated ROS generation, JNK/p38 MAPK activation, and apoptosis. In conclusion, the present study indicates that the BITC-induced apoptosis in human breast cancer cells is initiated by mitochondria-derived ROS.

Some experimental and clinical studies have been conducted for the usage of chemotherapeutic drugs encapsulated into nanoparticles (NPs). However, no study has been conducted so far on the co-encapsulation of doxorubicin (Dox) and epoxomicin (Epo) into NPs as biocompatible drug delivery carriers. Therefore, we investigated if co-encapsulation of doxorubicin (Dox) and/or epoxomicin (Epo) into NPs enhance their anticancer efficiency and prevent drug resistance and toxicity to normal cells. We synthesized Dox and/or Epo loaded poly (lactic-co-glycolic acid) (PLGA) NPs using a multiple emulsion solvent evaporation technique and characterized them in terms of their particle size and stability, surface, molecular, thermal, encapsulation efficiency and in vitro release properties. We studied the effects of drug encapsulated NPs on cellular accumulation, intracellular drug levels, oxidative stress status, cellular viability, drug resistance, 20S proteasome activity, cytosolic Nuclear Factor Kappa B (NF-κB-p65), and apoptosis in breast cancer and normal cells. Our results proved that the nanoparticles we synthesized were thermally stable possessing higher encapsulation efficiency and particle stability. Thermal, morphological and molecular analyses demonstrated the presence of Dox and/or Epo within NPs, indicating that they were successfully loaded. Cell line assays proved that Dox and Epo loaded NPs were less cytotoxic to single-layer normal HUVECs than free Dox and Epo, suggesting that the NPs would be biocompatible drug delivery carriers. The apoptotic index of free Dox and Epo increased 50% through their encapsulation into NPs, proving combination strategy to enhance apoptosis in breast cancer cells. Our results demonstrated that the co-encapsulation of Dox and Epo within NPs would be a promising treatment strategy to overcome multidrug resistance and toxicity to normal tissues that can be studied in further in vivo and clinical studies in breast cancer.

The sensitivity of cells to chemotherapeutic agents has a major effect on disease outcome in breast cancer patients. Unfortunately, there are numerous factors involved in the regulation of chemosensitivity, and the mechanisms need to be further investigated. Autophagy/Beclin 1 regulator 1 (Ambra1) is a key protein in the crosstalk between autophagy and apoptosis. It controls the switch between these two processes, which determines whether cells survive or die. Induction of apoptosis is the primary mechanism by which most chemotherapeutic drugs eliminate cancer cells. Recently, Ambra1 has been shown to modulate paclitaxel-induced apoptosis in breast cancer cells via the Bim/mitochondrial pathway, thereby modifying the sensitivity of cells to paclitaxel. However, how Ambra1 regulates Bim expression remains unclear. Here, we further confirmed that Bim plays an indispensable role in Ambra1's regulation of apoptosis and chemosensitivity in breast cancer cells. Furthermore, Ambra1 was found to regulate Bim expression at the transcriptional level through the Akt-FoxO1 pathway. Therefore, we propose a novel pathway, Ambra1-Akt-FoxO1-Bim, which regulates apoptosis and chemosensitivity in breast cancer cells. Thus, Ambra1 may represent a potential target for breast cancer treatment.

Breast cancer is one of the most common cancers in women all over the world and new treatment options are urgent. ER stress in cancer cells results in apoptotic cell death, and it is being proposed as a new therapeutic target. SH003, a newly developed herbal medicine, has been reported to have anti-cancer effects. However, its molecular mechanism is not yet clearly defined. Microarray was performed to check the differential gene expression patterns in various breast cancer cell lines. Cell viability was measured by MTT assays to detect cytotoxic effects. Annexin V-FITC and 7AAD staining, TUNEL assay and DCF-DA staining were analyzed by flow cytometry to evaluate apoptosis and ROS levels, respectively. Protein expression was examined in SH003-breast cancer cells using immunoblotting assays. The expression of C/EBP Homologous Protein (CHOP) mRNA was measured by real-time PCR. The effects of CHOP by SH003 treatment were investigated using transfection method. Herein, we investigated the molecular mechanisms through which SH003 causes apoptosis of human breast cancer cells. Both cell viability and apoptosis assays confirmed the SH003-induced apoptosis of breast cancer cells. Meanwhile, SH003 altered the expression patterns of several genes in a variety of breast cancer cell lines. More specifically, it upregulated gene sets including the response to unfolded proteins, independently of the breast cancer cell subtype. In addition, SH003-induced apoptosis was due to an increase in ROS production and an activation of the ER stress-signaling pathway. Moreover, CHOP gene silencing blocked SH003-induced apoptosis. SH003 causes apoptosis of breast cancer cells by upregulating ROS production and activating the ER stress-mediated pathway. Thus, our findings suggest that SH003 can be a potential therapeutic agent for breast cancer.

SZC015, a synthetic oleanolic acid derivative, induces both apoptosis and autophagy in MCF-7 breast cancer cells