10H‐3,6‐Diazaphenothiazines triggered the mitochondrial‐dependent and cell death receptor‐dependent apoptosis pathways and further increased the chemosensitivity of MCF‐7 breast cancer cells via inhibition of AKT1 pathways

Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H‐3,6‐diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF‐7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC₅₀ = 0.895 µM) toward MCF‐7 cells. Further, cell cycle analysis illustrated that the S‐phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF‐10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT² PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria‐dependent and cell death receptor‐dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF‐7 cells, thus suggesting its potential as a chemotherapeutic drug.     

PA‐MSHA inhibits proliferation and induces apoptosis through the up‐regulation and activation of caspases in the human breast cancer cell lines

To investigate the effects of PA‐MSHA (Pseudomonas aeruginosa‐mannose sensitive hemagglutinin) on inhibiting proliferation of breast cancer cell lines and to explore its mechanisms of action in human breast cancer cells. MCF‐10A, MCF‐7, MDA‐MB‐468, and MDA‐MB‐231HM cells were treated with PA‐MSHA or PA (Heat‐killed P. aeruginosa) at different concentrations and different times. Changes of cell super‐microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA‐MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V‐FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis‐related molecules. A time‐dependent and concentration‐dependent cytotoxic effect of PA‐MSHA was observed in MDA‐MB‐468 and MDA‐MB‐231HM cells but not in MCF‐10A or MCF‐7 cells. The advent of PA‐MSHA changed cell morphology, that is to say, increases in autophagosomes, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V‐FITC and Hoechst33258 staining showed that the different concentrations of PA‐MSHA could all induce the apoptosis and G₀–G₁ cell cycle arrest of breast cancer cells. Cleaved caspase 3, 8, 9, and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA‐MSHA but not of PA. Completely different from PA, PA‐MSHA may impart antiproliferative effects against breast cancer cells by inducing apoptosis mediated by at least a death receptor‐related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. J. Cell. Biochem. 108: 195–206, 2009. © 2009 Wiley‐Liss, Inc.     

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase‐2

The apoptotic initiator caspase‐2 has been implicated in oocyte death, in DNA damage‐ and heat shock‐induced death, and in mitotic catastrophe. We show here that the mitosis‐promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase‐2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase‐2 interdomain, prevents caspase‐2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase‐2 detected during interphase was lost in mitosis. Expression of S340A non‐phosphorylatable caspase‐2 abrogated mitotic suppression of caspase‐2 and apoptosis in various settings, including oocytes induced to undergo cdk1‐dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase‐2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase‐2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase‐2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur.     

Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti‐tumour properties. The underlying mechanisms by which statins‐induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti‐tumour mechanisms of a lipophilic statin, lovastatin, in MCF‐7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF‐7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1‐AMPK signalling blockade abrogated lovastatin‐induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1‐AMPK‐p38MAPK‐p53‐survivin cascade to cause MCF‐7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.     

Design,Synthesis, and Biological Evaluation of 2‐(2‐Bromo‐3‐nitrophenyl)‐5‐phenyl‐1,3,4‐oxadiazole Derivatives as Possible Anti‐Breast Cancer Agents

Breast Cancer (BCa) is the most often diagnosed cancer among women who were in the late 1940’s. Breast cancer growth is largely dependent on the expression of estrogen and progesterone receptor. Breast cancer cells may have one, both, or none of these receptors. The treatment for breast cancer may involve surgery, hormonal therapy (Tamoxifen, an aromatase inhibitor, etc.) and oral chemotherapeutic drugs. The molecular docking technique reported the findings on the potential binding modes of the 2‐(2‐bromo‐3‐nitrophenyl)‐5‐phenyl‐1,3,4‐oxadiazole derivatives with the estrogen receptor (PDB ID: 3ERT). The 1,3,4‐oxadiazole derivatives 4a – 4j have been synthesized and described by spectroscopic method. 2‐(2‐Bromo‐6‐nitrophenyl)‐5‐(4‐bromophenyl)‐1,3,4‐oxadiazole ( 4c ) was reconfirmed by single‐crystal XRD. All the compounds have been tested in combination with generic Imatinib pharmaceutical drug against breast cancer cell lines isolated from Caucasian woman MCF‐7, MDA‐MB‐453 and MCF‐10A non‐cancer cell lines. The compounds with the methoxy (in 4c ) and methyl (in 4j ) substitution were shown to have significant cytotoxicity, with 4c showing dose‐dependent activation and decreased cell viability. The mechanism of action was reported by induced apoptosis and tested by a DNA enzyme inhibitor experiment (ELISA) for Methyl Transferase. Molecular dynamics simulations were made for hit molecule 4c to study the stability and interaction of the protein?ligand complex. The toxicity properties of ADME were calculated for all the compounds. All these results provide essential information for further clinical trials.     

NF‐κB/NKILA signaling modulates the anti‐cancerous effects of EZH2 inhibition

A wealth of evidence supports the broad therapeutic potential of NF‐κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF‐κB and the NF‐κB interacting long non‐coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non‐canonical actions of EZH2 on the regulation of NF‐κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non‐transformed breast epithelial cells, triple negative MDA‐MB‐231 cells and hormone responsive MCF‐7 cells, and measured changes in EZH2/NF‐κB/NKILA levels to confirm their interdependence. We demonstrate cell line‐specific fluctuations in these factors that functionally contribute to epithelial‐to‐mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA‐MB‐231 cell motility and CDK4‐mediated MCF‐7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non‐transformed cells. Notably, these events are mediated by a cell‐context dependent gain or loss of NKILA and NF‐κB. Depletion of NF‐κB in non‐transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF‐κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.     

DNA damage during mitosis invokes a JNK‐mediated stress response that leads to cell death

Mitotic catastrophe is a phenomenon displayed by cells undergoing aberrant mitosis to eliminate cells that fail to repair the errors. Why and how mitotic catastrophe would lead to cell death remains to be resolved and the answer will prove valuable in design of better therapeutic agents that specifically target such cells in mitosis. The antibiotic actinomycin D has been shown to induce chromosomal lesions in lower order organisms as well as in human interphase cells. Relatively few studies have been conducted to elucidate molecular events in the context of mitotic DNA damage. We have previously established a model of mitotic catastrophe in human HeLa cells induced by actinomycin D. Here, we show that actinomycin D induce cellular stress via DNA damage during mitosis. The higher order packing of chromosomes during mitosis might impede efficient DNA repair. γH2AX serves as a marker for DNA repair and active JNK interacts with γH2AX in actinomycin D‐treated mitotic extracts. We believe JNK might be in part, responsible for the phosphorylation of H2AX and thereby, facilitate the propagation of a positive signal for cell death, when repair is not achieved. The mitotic cell activates JNK‐mediated cell death response that progresses through a caspase cascade downstream of the mitochondria. In the mean time, remaining checkpoint signals may be sufficient to put a restraining hand on entry into anaphase and the cell eventually dies in mitosis. J. Cell. Biochem. 110: 725–731, 2010. © 2010 Wiley‐Liss, Inc.     

In vitro effects of 2‐methoxyestradiol‐bis‐sulphamate on the non‐tumorigenic MCF‐12A cell line

A priority in recent anti‐cancer drug development has been attaining better side‐effect profiles for potential compounds. To produce highly specific cancer therapies it is necessary to understand both the effects of the proposed compound on cancer and on normal cells comprising the rest of the human body. Thus in vitro evaluation of these compounds against non‐carcinogenic cell lines is of critical importance. One of the most recent developments in experimental anti‐cancer agents is 2‐methoxyestradiol‐bis‐sulphamate (2ME‐BM), a sulphamoylated derivative of 2‐methoxyestradiol. The aim of this study was to evaluate the in vitro effects of 2ME‐BM on cell proliferation, morphology and mechanisms of cell death in the non‐carcinogenic MCF‐12A breast epithelial cell line. The study revealed changes in proliferative capacity, morphology and cell death induction in response to 2ME‐BM exposure (24 h at 0.4 µM). Microscopy showed decreased cell density and cell death‐associated morphology (increased apoptotic characteristics), a slight increase in acidic intracellular vesicles and insignificant ultra‐structural aberrations. Mitotic indices revealed a G₂M‐phase cell cycle block. This was confirmed by flow cytometry, where an increased fraction of abnormal cells and a decrease in cyclin B1 levels were observed. These results evidently demonstrate that the non‐carcinogenic MCF‐12A cell line is less susceptible when compared to 2ME‐BM‐exposed cancer cell lines previously tested. Further in vitro research into the mechanism of this potentially useful compound is warranted. Copyright © 2010 John Wiley & Sons, Ltd.     

Mitotic Catastrophe Results in Cell Death by Caspase-Dependentand Caspase-Independent Mechanisms

Exposure of MDA-MB-231 and MCF-7/VP human breast carcinoma cells to theanthracyclines doxorubicin and WP631 induced polyploidy, formation of multinucleated cellsand cell death by mitotic catastrophe through caspase-dependent and caspase-independentmechanisms. In both cell lines, the antiproliferative effect of WP631 was higher than that ofdoxorubicin and a transient halt in G2/M was observed without cell senescence, while p53-dependent apoptosis did not occur in these cells. Mitotic catastrophe was linked to necrosis, butalso to apoptosis-like death, estimated by differential cell staining with Annexin-V-fluoresceinand propidium iodide. Drug-induced changes in the expression of c-myc and p21WAF1, and in theirrespective protein levels, were observed. They depended on the cell line, the anthracycline usedand its concentration, and they were consistent with the cell cycle progression through G2 tomitosis. Significant activation of caspase-2 and caspase-3 was only observed in MDA-MB-231cells treated with doxorubicin but not with WP631, indicating that caspases may be notmandatory for the occurrence of cell death through mitotic catastrophe. In MCF-7/VP cells,which do not express functional caspase-3, mitotic catastrophe was also induced.     

miR‐10b expression in breast cancer stem cells supports self‐renewal through negative PTEN regulation and sustained AKT activation

Cancer stem cells (CSCs) are linked to metastasis. Moreover, a discrete group of miRNAs (metastamiRs) has been shown to promote metastasis. Accordingly, we propose that miRNAs that function as metastatic promoters may influence the CSC phenotype. To study this issue, we compared the expression of 353 miRNAs in CSCs enriched from breast cancer cell lines using qRT–PCR analysis. One of the most altered miRNAs was miR‐10b, which is a reported promoter of metastasis and migration. Stable overexpression of miR‐10b in MCF‐7 cells (miR‐10b‐OE cells) promoted higher self‐renewal and expression of stemness and epithelial–mesenchymal transition (EMT) markers. In agreement with these results, inhibiting miR‐10b expression using synthetic antisense RNAs resulted in a decrease in CSCs self‐renewal. Bioinformatics analyses identified several potential miR‐10b mRNA targets, including phosphatase and tensin homolog (PTEN), a key regulator of the PI3K/AKT pathway involved in metastasis, cell survival, and self‐renewal. The targeting of PTEN by miR‐10b was confirmed using a luciferase reporter, qRT–PCR, and Western blot analyses. Lower PTEN levels were observed in CSCs, and miR‐10b depletion not only increased PTEN mRNA and protein expression but also decreased the activity of AKT, a downstream PTEN target kinase. Correspondingly, PTEN knockdown increased stem cell markers, whereas AKT inhibitors compromised the self‐renewal ability of CSCs and breast cancer cell lines overexpressing miR‐10b. In conclusion, miR‐10b regulates the self‐renewal of the breast CSC phenotype by inhibiting PTEN and maintaining AKT pathway activation.     

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.     

Dominant-negative polo-like kinase 1 induces mitotic catastrophe independent of cdc25C function.

Polo-like kinase 1 (PLK1), which has been shown to have a critical role in mitosis, is one possible target for cancer therapeutic intervention. PLK1, at least in Xenopus, starts the mitotic cascade by phosphorylating and activating cdc25C phosphatase. Also, loss of PLK1 function has been shown to induce mitotic catastrophe in a HeLa cervical carcinoma cell line but not in normal Hs68 fibroblasts. We wanted to understand whether the selective mitotic catastrophe in HeLa cells could be extended to other tumor types, and, if so, whether it could be attributable to a tumor-specific loss of dependence on PLK1 for cdc25C activation. When PLK1 function was blocked through adenovirus delivery of a dominant-negative gene, we observed tumor-selective apoptosis in most tumor cell lines. In some lines, dominant-negative PLK1 induced a mitotic catastrophe similar to that published in HeLa cells (K. E. Mundt et al., Biochem. Biophys Res. Commun., 239: 377-385, 1997). Normal human mammary epithelial cells, although arrested in mitosis, appeared to escape the loss of centrosome maturation and mitotic catastrophe seen in tumor lines. Mitotic phosphorylation of cdc25C and activation of cdk1 was blocked by dominant-negative PLK1 in human mammary epithelial cells as well as in the tumor lines regardless of whether they underwent mitotic catastrophe. These data strongly argue that the mitotic catastrophe is not attributable to a lack of dependence for PLK1 in activating cdc25C.     

Securin enhances the anti-cancer effects of 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075) in human colorectal cancer cells

BPR0L075 [6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole] is a novel anti-microtubule drug with anti-tumor and anti-angiogenic activities in vitro and in vivo. Securin is required for genome stability, and is expressed abundantly in most cancer cells, promoting cell proliferation and tumorigenesis. In this study, we found that BPR0L075 efficiently induced cell death of HCT116 human colorectal cancer cells that have higher expression levels of securin. The cytotoxicity of BPR0L075 was attenuated in isogenic securin-null HCT116 cells. BPR0L075 induced DNA damage response, G(2)/M arrest, and activation of the spindle assembly checkpoint in HCT116 cells. Interestingly, BPR0L075 induced phosphorylation of securin. BPR0L075 withdrawal resulted in degradation of securin, mitotic exit, and mitotic catastrophe, which were attenuated in securin-null cells. Inhibition of cdc2 decreased securin phosphorylation, G(2)/M arrest and cell death induced by BPR0L075. Moreover, BPR0L075 caused cell death through a caspase-independent mechanism and activation of JNK and p38 MAPK pathways. These findings provided evidence for the first time that BPR0L075 treatment is beneficial for the treatment of human colorectal tumors with higher levels of securin. Thus, we suggest that the expression levels of securin may be a predictive factor for application in anti-cancer therapy with BPR0L075 in human cancer cells.     

MDM2 is implicated in high‐glucose‐induced podocyte mitotic catastrophe via Notch1 signalling

Podocyte injury and depletion are essential events involved in the pathogenesis of diabetic nephropathy (DN). As a terminally differentiated cell, podocyte is restricted in ‘post‐mitosis’ state and unable to regenerate. Re‐entering mitotic phase will cause podocyte disastrous death which is defined as mitotic catastrophe (MC). Murine double minute 2 (MDM2), a cell cycle regulator, is widely expressed in renal resident cells including podocytes. Here, we explore whether MDM2 is involved in podocyte MC during hyperglycaemia. We found aberrant mitotic podocytes with multi‐nucleation in DN patients. In vitro, cultured podocytes treated by high glucose (HG) also showed an up‐regulation of mitotic markers and abnormal mitotic status, accompanied by elevated expression of MDM2. HG exposure forced podocytes to enter into S phase and bypass G2/M checkpoint with enhanced expression of Ki67, cyclin B1, Aurora B and p‐H3. Genetic deletion of MDM2 partly reversed HG‐induced mitotic phase re‐entering of podocytes. Moreover, HG‐induced podocyte injury was alleviated by MDM2 knocking down but not by nutlin‐3a, an inhibitor of MDM2‐p53 interaction. Interestingly, knocking down MDM2 or MDM2 overexpression showed inhibition or activation of Notch1 signalling, respectively. In addition, genetic silencing of Notch1 prevented HG‐mediated podocyte MC. In conclusion, high glucose up‐regulates MDM2 expression and leads to podocyte MC. Notch1 signalling is an essential downstream pathway of MDM2 in mediating HG‐induced MC in podocytes.     

Inhibition of PARP activity by PJ‐34 leads to growth impairment and cell death associated with aberrant mitotic pattern and nucleolar actin accumulation in M14 melanoma cell line

The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo‐ and radio‐sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ‐34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time‐lapse videomicroscopy, we evidenced that 10 µM PJ‐34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ‐34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken toghether, our results demonstrate that, targeting PARP activity by PJ‐34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 µM PJ‐34 for 24 h to obtain the same citotoxic effect; (b) effects dependent on PJ‐34 treatment are reversible. Our data suggest that, to reduce the harm done to non‐tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ‐34 treatment. J. Cell. Physiol. 222: 401–410, 2010. © 2009 Wiley‐Liss, Inc.     

Iejimalides A and B inhibit lysosomal vacuolar H+‐ATPase (V‐ATPase) activity and induce S‐phase arrest and apoptosis in MCF‐7 cells

Iejimalides are novel macrolides that are cytostatic or cytotoxic against a wide range of cancer cells at low nanomolar concentrations. A recent study by our laboratory characterized the expression of genes and proteins that determine the downstream effects of iejimalide B. However, little is known about the cellular target(s) of iejimalide or downstream signaling that lead to cell‐cycle arrest and/or apoptosis. Iejimalides have been shown to inhibit the activity of vacuolar H⁺‐ATPase (V‐ATPase) in osteoclasts, but how this inhibition may lead to cell‐cycle arrest and/or apoptosis in epithelial cells is not known. In this study, MCF‐7 breast cancer cells were treated with iejimalide A or B and analyzed for changes in cell‐cycle dynamics, apoptosis, lysosomal pH, cytoplasmic pH, mitochondrial membrane potential, and generation of reactive oxygen species. Both iejimalides A and B sequentially neutralize the pH of lysosomes, induce S‐phase cell‐cycle arrest, and trigger apoptosis in MCF‐7 cells. Apoptosis occurs through a mechanism that involves oxidative stress and mitochondrial depolarization but not cytoplasmic acidification. These data confirm that iejimalides inhibit V‐ATPase activity in the context of epithelial tumor cells, and that this inhibition may lead to a lysosome‐initiated cell death process. J. Cell. Biochem. 109: 634–642, 2010. © 2009 Wiley‐Liss, Inc.     

Down‐regulation of Orai3 arrests cell‐cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells

Breast cancer (BC) is the leading cancer in the world in terms of incidence and mortality in women. However, the mechanism by which BC develops remains largely unknown. The increase in cytosolic free Ca²⁺ can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca²⁺ selective channels. In the present study, we analyzed Orai3 expression in normal and cancerous breast tissue samples, and its role in MCF‐7 BC and normal MCF‐10A mammary epithelial cell lines. We found that the expression of Orai3 mRNAs was higher in BC tissues and MCF‐7 cells than in normal tissues and MCF‐10A cells. Down‐regulation of Orai3 by siRNA inhibited MCF‐7 cell proliferation and arrested cell cycle at G1 phase. This phenomenon is associated with a reduction in CDKs 4/2 (cyclin‐dependent kinases) and cyclins E and D1 expression and an accumulation of p21^Waf1/Cip1 (a cyclin‐dependent kinase inhibitor) and p53 (a tumor‐suppressing protein). Orai3 was also involved in MCF‐7 cell survival. Furthermore, Orai3 mediated Ca²⁺ entry and contributed to intracellular calcium concentration ([Ca²⁺]_i). In MCF‐10A cells, silencing Orai3 failed to modify [Ca²⁺]_i, cell proliferation, cell‐cycle progression, cyclins (D1, E), CDKs (4, 2), and p21^Waf1/Cip1 expression. Our results provide strong evidence for a significant effect of Orai3 on BC cell growth in vitro and show that this effect is associated with the induction of cell cycle and apoptosis resistance. Our study highlights a possible role of Orai3 as therapeutic target in BC therapy. J. Cell. Physiol. 226: 542–551, 2011. © 2010 Wiley‐Liss, Inc.     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of tamoxifen and retinoic acid on bradykinin induced proliferation in MCF‐7 cells

Chemopreventive approaches for the treatment of breast cancer have been validated clinically and with in vitro studies. The combined action of tamoxifen/all‐trans retinoic acid was advantageous in MCF‐7 cells, reducing cell proliferation, Bcl‐2 and c‐Myc protein levels and increasing E‐Cadherin protein levels and Gap junctional Intercellular Communication. We further investigated their combined effect in the presence of bradykinin, a pro‐inflammatory agent, previously reported to contribute to the proliferation of breast cancer cells. Bradykinin increased MCF‐7 cell proliferation, c‐Myc levels and ERK1/2 activity. The co‐incubation of bradykinin‐MCF‐7 cells with tamoxifen/all‐trans retinoic acid reduced cell proliferation, ERK1/2 activity, as well as Bcl‐2, c‐Myc, and bradykinin receptor‐2 levels, without altering the enhanced E‐cadherin levels induced by tamoxifen/all‐trans retinoic acid. We showed that the anti‐tumoral effect of tamoxifen/all‐trans retinoic acid is beneficial in MCF‐7 breast cancer cells grown in a bradykinin‐pro‐mitogenic environment, an effect that might be, at least in part, through the MAPK pathway and B2‐bradykinin receptor inhibition. J. Cell. Biochem. 106: 473–481, 2009. © 2008 Wiley‐Liss, Inc.