Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

Inhibiting ROS-TFE3-dependent autophagy enhances the therapeutic response to metformin in breast cancer

Autophagy modulation is a potential therapeutic strategy for breast cancer, and a previous study indicated that metformin exhibits significant anti-carcinogenic activity. However, the ability of metformin to induce autophagy and its role in breast cancer cell death remains unclear. In this study, we exposed MCF-7 cells to different concentrations of metformin (2.5, 5, and 10?mM) for 48?h, and metformin-induced significant apoptosis in the MCF-7 cells. The expression levels of CL-PARP (poly(ADP-ribose) polymerase 1) and the ratio of BAX to BCL-2 were significantly increased. In addition to apoptosis, we showed that metformin increased autophagic flux in MCF-7 cells, as evidenced by the upregulation of LC3-II and downregulation of P62/SQSTM1. Moreover, pharmacological or genetic blocking of autophagy increased metformin-induced apoptosis, indicating a cytoprotective role of autophagy in metformin-treated MCF-7 cells. Mechanistically, metformin-induced TFE3^(Ser321) dephosphorylation activated TFE3 nuclear translocation and increased of TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes and, subsequently, initiated autophagy in MCF-7 cells. Importantly, we found that metformin triggered the generation of reactive oxygen species (ROS) in MCF-7 cells. Furthermore, N-acetyl-l-cysteine (NAC), a ROS scavenger, abrogated the effects of metformin on TFE3-dependent autophagy. Notably, TFE3 expression positively correlated with breast cancer development and poor prognosis in patients. Taken together, these data demonstrate that blocking ROS-TFE3-dependent autophagy to enhance the activity of metformin warrants further attention as a treatment strategy for breast cancer.     

Seleno-short-chain chitosan induces apoptosis in human breast cancer cells through mitochondrial apoptosis pathway in vitro

Seleno-short-chain chitosan (SSCC) was a synthesized chitosan derivative with the molecular weight of 4826.986 Da. The study is aimed to investigate cytotoxicity of SSCC on human breast cancer MCF-7 and BT-20 cells and explore apoptosis-related mechanism in vitro. The MTT (3- [4,5-Dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide) assay showed that SSCC exhibited significantly cytotoxic effects on MCF-7 and BT-20 cells in a dose- and time-dependent manner, and the effective inhibitory concentration was 100 μg/ml and 200 μg/ml, respectively. Apoptosis assay of these two kinds of cells was determined by Hoechst 33,342/PI and Annexin V-FITC/PI double staining. The cell cycle assay showed that SSCC triggered S and G2/M phase cell cycle arrest in MCF-7 cells and S phase cell cycle arrest in BT-20 cells in a time-dependent manner. Further studies demonstrated that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) in these two kinds of cells. N- acetyl-L cysteine (NAC), as a radical scavenger, significantly inhibited the generation of ROS and decreased the apoptosis of MCF-7 and BT-20 cells. Moreover, the expression of mitochondrial apoptosis-related proteins was detected by western blot assay. SSCC up-regulated the expression of Bax, down-regulated the expression of Bcl-2, subsequently increased the release of cytochrome c from mitochondria to cytoplasm, and activated the cleavage of caspase-9 and ?3, which finally induced apoptosis in MCF-7 and BT-20 cells in vitro. Consequently, these data indicated that SSCC could induce apoptosis of MCF-7and BT-20 cells in vitro by mitochondrial pathway.     

Chronic Oxidative Stress Increases Growth and Tumorigenic Potential of MCF-7 Breast Cancer Cells

Accumulating evidence suggests that exposures to elevated levels of either endogenous estrogen or environmental estrogenic chemicals are associated with breast cancer development and progression. These natural or synthetic estrogens are known to produce reactive oxygen species (ROS) and increased ROS has been implicated in both cellular apoptosis and carcinogenesis. Though there are several studies on direct involvement of ROS in cellular apoptosis using short-term exposure model, there is no experimental evidence to directly implicate chronic exposure to ROS in increased growth and tumorigenicity of breast cancer cells. Therefore, the objective of this study was to evaluate the effects of chronic oxidative stress on growth, survival and tumorigenic potential of MCF-7 breast cancer cells. MCF-7 cells were exposed to exogenous hydrogen peroxide (H₂O₂) as a source of ROS at doses of 25 µM and 250 µM for acute (24 hours) and chronic period (3 months) and their effects on cell growth/survival and tumorigenic potential were evaluated. The results of cell count, MTT and cell cycle analysis showed that while acute exposure inhibits the growth of MCF-7 cells in a dose-dependent manner, the chronic exposure to H₂O₂-induced ROS leads to increased cell growth and survival of MCF-7 cells. This was further confirmed by gene expression analysis of cell cycle and cell survival related genes. Significant increase in number of soft agar colonies, up-regulation of pro-metastatic genes VEGF, WNT1 and CD44, whereas down-regulation of anti-metastatic gene E-Cadherin in H₂O₂ treated MCF-7 cells observed in this study further suggests that persistent exposure to oxidative stress increases tumorigenic and metastatic potential of MCF-7 cells. Since many chemotherapeutic drugs are known to induce their cytotoxicity by increasing ROS levels, the results of this study are also highly significant in understanding the mechanism for adaptation to ROS-induced toxicity leading to acquired chemotherapeutic resistance in breast cancer cells.     

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H₂O₂ levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.     

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 (24 h) was determined by MTT assay and antiproliferative effect was assessed by counting cell numbers after 72 h 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 50 μg/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.     

Antiproliferative action of menadione and 1,25(OH)2D3 on breast cancer cells

Calcitriol or 1,25(OH)₂D₃ is a negative growth regulator of MCF-7 breast cancer cells. The growth arrest is due to apoptosis activation, which involves mitochondrial disruption. This effect is blunted in vitamin D resistant cells (MCF-7^DRes cells). Menadione (MEN), a glutathione (GSH)-depleting compound, may potentiate antitumoral effects of anticancer drugs. The aim of this study was to investigate whether MEN enhances cellular responsiveness of MCF-7 cells to 1,25(OH)₂D₃. Cells were cultured and treated with different concentrations of 1,25(OH)₂D₃ ± MEN or vehicle for 96 h. GSH levels and the activity of antioxidant enzymes were determined by spectrophotometry and ROS production by flow cytometry. Both drugs decreased growth and enhanced ROS in MCF-7 cells, obtaining the maximal effects when 1,25(OH)₂D₃ was combined with MEN (P < 0.01 vs. Control and vs. each compound alone). MCF-7^DRes cells were not responsive to 1,25(OH)₂D₃, but the cell proliferation was slightly inhibited by the combined treatment. Calcitriol and MEN separately enhanced antioxidant enzyme activities, but when they were used in combination, the effect was more pronounced (P < 0.05 vs. Control and vs. each compound alone). MEN, calcitriol and the combined treatment decreased GSH levels (P < 0.05 vs. Control). The data indicate that MEN potentiates the effect of 1,25(OH)₂D₃ on growth arrest in MCF-7 cells by oxidative stress and increases the activities of antioxidant enzymes, probably as a compensatory mechanism.     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.     

Regulatory effects of resveratrol on antioxidant enzymes: A mechanism of growth inhibition and apoptosis induction in cancer cells

Resveratrol (RSV) is a natural polyphenol that is known as a powerful chemopreventive and chemotherapeutic anticancer molecule. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells. Prostate cancer PC-3 cells, hepatic cancer HepG2 cells, breast cancer MCF-7 cells and the non-cancerous HEK293T kidney epithelial cells were treated with a wide range of RSV concentrations (10-100 μM) for 24–72 h. Cell growth was estimated by trypan blue staining, activities of the antioxidant enzymes were measured spectrophotometrically, expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots, and the percentage of apoptotic cells was determined by flow cytometry. Treatment with a low concentration of RSV (25 μM) significantly increased superoxide dismutase (SOD) activity in PC-3, HepG2 and MCF-7 cells, but not in HEK293T cells. Catalase (CAT) activity was increased in HepG2 cells, but no effect was found on glutathione peroxidase (GPX) upon RSV treatment. RSV-induced SOD2 expression was observed in cancer cells, although the expression of SOD1, CAT and GPX1 was unaffected. Apoptosis increased upon RSV treatment of cancer cells, especially in PC-3 and HepG2 cells. Together, our data demonstrated that RSV inhibits cancer cell growth with minimal effects on non-cancerous cells. We postulate that the disproportional up-regulation of SOD, CAT and GPX expression and enzymatic activity in cancer cells results in the mitochondrial accumulation of H₂O₂, which in turn induces cancer cell apoptosis.     

The presence of Estrogen Receptor β modulates the response of breast cancer cells to therapeutic agents

Breast cancer is a leading cause of death for women. The estrogen receptors (ERs) ratio is important in the maintenance of mitochondrial redox status, and higher levels of ERβ increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERβ ratio and the response to cytotoxic treatments such as cisplatin (CDDP), paclitaxel (PTX) and tamoxifen (TAM). Cell viability, apoptosis, autophagy, ROS production, mitochondrial membrane potential, mitochondrial mass and mitochondrial functionality were analyzed in MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) breast cancer cell lines. Cell viability decreased more in MCF-7 when treated with CDDP and PTX. Apoptosis was less activated after cytotoxic treatments in T47D than in MCF-7 cells. Nevertheless, autophagy was increased more in CDDP-treated MCF-7, but less in TAM-treated cells than in T47D. CDDP treatment produced a raise in mitochondrial mass in MCF-7, as well as the citochrome c oxidase (COX) and ATP synthase protein levels, however significantly reduced COX activity. In CDDP-treated cells, the overexpression of ERβ in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERβ in T47D cells promoted the opposite effects. In TAM-treated cells, ERβ-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERβ in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERβ expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.     

Proteomic Comparison of MCF-7 Tumoursphere and Monolayer Cultures

Breast cancer is a heterogenous disease, composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently, a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells, or alternatively, in the case of breast cancer, breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in “tumoursphere” culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence, proteomic analysis of tumourspheres may find use in identifying novel targets for future therapy. The therapeutic targeting of breast cancer stem cells, a highly clinically relevant sub-population of tumour cells, has the potential to eliminate residual disease and may become an important component of a multi-modality treatment of cancer.     

Ascorbate promotes the cellular accumulation of doxorubicin and reverses the multidrug resistance in breast cancer cells by inducing ROS-dependent ATP depletion

Chemotherapy is the most effective strategy for the treatment of metastatic breast cancer. However, P-glycoprotein (P-gp)-mediated multidrug resistance severely limits the efficacy of chemotherapy and is a major cause of the failure during chemotherapeutic treatment. In this study, we investigated the anticancer effects of combining chemotherapeutic drugs with ascorbate (AA) in human breast cancer cells. We found that combined administration of AA can improve the sensitivity of both MCF-7 and doxorubicin (Dox)-resistant MCF-7/Adr cells to Dox in vitro and in vivo by a reactive oxygen species (ROS)-dependent mechanism. Further studies proved that AA can promote the accumulation of Dox in MCF-7/Adr cells when combined with doxorubicin. AA had no effect on the expression of P-gp at the mRNA and protein levels, but could decrease its activity as demonstrated by an obvious inhibition of efflux of P-gp substrate Rh 123. AA reduced ATP levels in both MCF-7 and MCF-7/Adr cells, and pretreating AA-stimulating cells with catalase completely rescued ATP levels. With ATP reduction, we observed an increased cellular calcium and the appearance of vacuoles and micropores on the cell surface, indicating the increased cell membrane permeability in AA-treated MCF-7/Adr cells. The above results suggest that AA could promote the cellular accumulation of doxorubicin by inducing ROS-dependent ATP depletion. Clinically, a combination of AA with doxorubicin would be a novel strategy for reversal of the multidrug resistance in human breast cancer cells during chemotherapy.     

Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.     

Dryofragin, a phloroglucinol derivative, induces apoptosis in human breast cancer MCF-7 cells through ROS-mediated mitochondrial pathway

Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. In this study, the anticancer activity of dryofragin on human breast cancer MCF-7 cells was investigated. Dryofragin inhibited the growth of MCF-7 cells in a time and concentration-dependent manner. The cell viability was measured using MTT assay. After treatment with dryofragin for 72, 48 and 24h, the IC(50) values were 27.26, 37.51 and 76.10μM, respectively. Further analyses of DNA fragmentation and Annexin V-PI double-labeling indicated an induction of apoptosis. Dryofragin-treatment MCF-7 cells had a significantly accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. These phenomena were blocked by pretreatment for 2h of MCF-7 cells with the antioxidant compound N-acetyl-l-cysteine (NAC, 5mM). These results speak for the involvement of a ROS-mediated mitochondria-dependent pathway in dryofragin-induced apoptosis. Western blot results showed that dryofragin inhibited Bcl-2 and induced Bax expression which led to an activation of caspases-9 and -3 in the cytosol, and further cleavage of poly ADP-ribose polymerase (PARP) in the nucleus, then induced cell apoptosis. In conclusion, the present study provides evidence that dryofragin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway.     

Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

Functional analysis of Csk and CHK kinases in breast cancer cells.

In this report, we analyzed the expression and kinase activities of Csk and CHK kinases in normal breast tissues and breast tumors and their involvement in HRG-mediated signaling in breast cancer cells. Csk expression and kinase activity were abundant in normal human breast tissues, breast carcinomas, and breast cancer cell lines, whereas CHK expression was negative in normal breast tissues and low in some breast tumors and in the MCF-7 breast cancer cell line. CHK kinase activity was not detected in human breast carcinoma tissues (12 of 12) or in the MCF-7 breast cancer cell line (due to the low level of CHK protein expression), but was significantly induced upon heregulin (HRG) stimulation. We have previously shown that CHK associates with the ErbB-2/neu receptor upon HRG stimulation via its SH2 domain and that it down-regulates the ErbB-2/neu-activated Src kinases. Our new findings demonstrate that Csk has no effect on ErbB-2/neu-activated Src kinases upon HRG treatment and that its kinase activity is not modulated by HRG. CHK significantly inhibited in vitro cell growth, transformation, and invasion induced upon HRG stimulation. In addition, tumor growth of wt CHK-transfected MCF-7 cells was significantly inhibited in nude mice. Furthermore, CHK down-regulated c-Src and Lyn protein expression and kinase activity, and the entry into mitosis was delayed in the wt CHK-transfected MCF-7 cells upon HRG treatment. These results indicate that CHK, but not Csk, is involved in HRG-mediated signaling pathways, down-regulates ErbB-2/neu-activated Src kinases, and inhibits invasion and transformation of breast cancer cells upon HRG stimulation. These findings strongly suggest that CHK is a novel negative growth regulator of HRG-mediated ErbB-2/neu and Src family kinase signaling pathways in breast cancer cells.     

Antiproliferative and apoptosis-induction studies of 5-hydroxy 3′,4′,7-trimethoxyflavone in human breast cancer cells MCF-7: an in vitro and in silico approach

The aim of this study was to find the efficacy of 5-hydroxy 3′,4′,7-trimethoxyflavone (HTMF), a flavonoid compound isolated from the medicinal plant Lippia nodiflora, in inhibiting the proliferation and inducing apoptosis in human breast cancer cell line MCF-7. The anti-proliferative effect of the compound HTMF was confirmed using MTT cytotoxicity assay. Increased apoptotic induction by HTMF was demonstrated by acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33258 staining studies. The phosphatidylserine translocation, an early feature of apoptosis and DNA damage were revealed through AnnexinV-Cy3 staining and comet assay. Moreover, the significant elevation of cellular ROS was observed in the treated cells, as measured by 2,7-diacetyl dichlorofluorescein (DCFH-DA). The mRNA expression studies also supported the effectiveness of HTMF by shifting the Bax:Bcl-2 ratio. The treatment of MCF-7 cells with HTMF encouraged apoptosis through the modulation of apoptotic markers, such as p53, Bcl-2, Bax, and cleaved PARP. In silico molecular docking and dynamics studies with MDM2-p53 protein revealed that HTMF was more potent compound that could inhibit the binding of MDM2 with p53 and, therefore, could trigger apoptosis in cancer cell. Overall, this study brings up scientific evidence for the efficacy of HTMF against MCF-7 breast cancer cells.