rottlerin and Breast-Neoplasms

In the past few years, we focused the interest on rottlerin, an old/new natural substance that, over the time, has revealed a number of cellular and molecular targets, all potentially implicated in the fight against cancer. Past and recent literature well demonstrated that rottlerin is an inhibitor of enzymes, transcription factors and signaling molecules that control cancer cell life and death. Although the rottlerin anticancer activity has been mainly ascribed to apoptosis and/or autophagy induction, recent findings unveiled the existence of additional mechanisms of toxicity. The major novelties highlighted in this mini review are the ability to bind and inhibit key molecules, such as ERK and mTOR, directly, thus independently of upstream signaling cascades, and to cause a profound dysregulation of cap-dependent protein translation through the mTORC1/4EBP1/eIF4E axis and by inhibition of eIF2, an initiation factor of translation that is negatively regulated by endoplasmic reticulum (ER) stress. These last mechanisms, proved to be lethal in cancer cell lines derived from breast and skin, strongly enforce the potential of rottlerin as a promising natural lead compound for the development of novel therapeutic approaches.

Topics: Acetophenones; Antineoplastic Agents; Benzopyrans; Breast Neoplasms; Humans; Melanoma

Aurora kinases and protein kinase C (PKC) have been shown to be involved in different aspects of cancer progression. To date, no dual Aurora/PKC inhibitor with clinical efficacy and low toxicity is available. Here, we report the identification of compound

Topics: Antineoplastic Agents; Aurora Kinase A; Breast Neoplasms; Cell Line, Tumor; Drug Screening Assays, Antitumor; Female; Humans; Hydrogen Bonding; Isoenzymes; Molecular Docking Simulation; Protein Kinase C; Protein Kinase Inhibitors; Selenium Compounds; Small Molecule Libraries; Structure-Activity Relationship; Substrate Specificity; Tumor Stem Cell Assay

Besides the crucial role of hyperinsulinemia in the development of breast cancer with Type 2 diabetes mellitus (T2DM), it has been shown that hyperglycemia could contribute to promote cancer progression. A remarkable association within hyperglycemia, PKCδ and Ubiquitin-proteasome system (UPS) has been reported, suggesting that PKCδ may mediate high glucose-induced UPS activation in breast cancer cells. Although the independent effects of PKCδ or UPS on breast cancer and T2DM are increasingly supported by experimental evidence, the complex interactional link between PKCδ and UPS is still unclear. Hence, we focus on the relationship between PKCδ and UPS in breast cancer with T2DM. We hypothesize that PKCδ may have the function to regulate the activity of UPS. Further, we speculate that PKCδ combine with proteasome α2 promoter, that indicate PKCδ regulate the function of UPS by change the composition of proteasome. Therefore, we surmise that PKCδ mediated high glucose-induced UPS activation in breast cancer cells, and specific PKCδ inhibitor rottlerin significantly suppressed elevated glucose induced the activity of UPS. We hope that our paper will stimulate further studies the relationship between PKCδ and UPS, and a new targeted therapy and early medical intervention for PKCδ could be a useful option for breast cancer cases complicated with T2DM or hyperglycemia.

Topics: Acetophenones; Benzopyrans; Breast Neoplasms; Diabetes Mellitus, Type 2; Disease-Free Survival; Enzyme Activation; Enzyme Inhibitors; Female; Glucose; Humans; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Protein Binding; Protein Kinase C-delta; Ubiquitin

Studies have investigated the tumor suppressive role of rottlerin in carcinogenesis. However, the molecular mechanisms of rottlerin-induced anti-tumor activity are largely unclear. Skp2 (S-phase kinase associated protein 2) has been validated to play an oncogenic role in a variety of human malignancies. Therefore, inactivation of Skp2 could be helpful for the treatment of human cancers. In the current study, we explore whether rottlerin could inhibit Skp2 expression, leading to inhibition of cell growth, migration and invasion in breast cancer cells. We found that rottlerin treatment inhibited cell growth, induced apoptosis and cell cycle arrest. We also revealed that rottlerin suppressed cell migration and invasion in breast cancer cells. Mechanically, we observed that rottlerin significantly down-regulated the expression of Skp2 in breast cancer cells. Importantly, overexpression of Skp2 abrogated rottlerin-mediated tumor suppressive activity, whereas down-regulation of Skp2 enhanced rottlerin-triggered anti-tumor function. Strikingly, we identified that rottlerin exhibited its anti-tumor potential partly through inactivation of Skp2 in breast cancer. Our findings indicate that rottlerin could be a potential safe agent for the treatment of breast cancer.

Topics: Acetophenones; Angiogenesis Inhibitors; Antineoplastic Agents; Apoptosis; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Female; Humans; S-Phase Kinase-Associated Proteins

Dibenzoylmethane (DB), a minor constituent of the root extract of licorice, belongs to the flavonoid family. Hydroxydibenzoylmethane (HDB) and hydroxymethyldibenzoylmethane (HMDB) have an identical structure to DB, but also possess a hydroxyl group and a hydroxyl and methyl group bonded to aromatic rings, respectively. They inhibit cellular proliferation and induce apoptosis in a variety of types of cancer cell, however, the antimetastatic effects of DB, HDB and HMDB on human breast carcinoma cells remain to be elucidated. The present study aimed to clarify the molecular mechanisms underlying the effects of DB and its analogues on phorbol‑12‑myristate 13‑acetate (PMA)‑induced MCF‑7 cell metastasis. The results revealed that DB, HDB and HMDB inhibited cell migration and invasion. In addition, PMA‑mediated MCF‑7 cell invasion was inhibited by DB, HDB and HMDB by inhibiting the expression of matrix metalloproteinase (MMP)‑9. Rottlerin, a protein kinase C (PKC)δ inhibitor and LY294002, a phosphatidylinositide 3‑kinase (PI3K) inhibitor, reduced the PMA‑mediated expression of MMP‑9 and cell invasion. Furthermore, DB, HDB and HMDB prevented the activation of PKCδ and PI3K by inhibiting their phosphorylation. The present study was the first, to the best of our knowledge, to demonstrate the antimetastatic potential of DB, HDB and HDMB, which decreased cancer cell invasion through the PI3K/PKCδ‑mediated MMP‑9 pathway.

Topics: Acetophenones; Benzopyrans; Breast Neoplasms; Cell Movement; Chalcones; Chromones; Female; Humans; Ketones; Matrix Metalloproteinase 9; MCF-7 Cells; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Propane; Protein Kinase C-delta; Signal Transduction; Tetradecanoylphorbol Acetate

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.

Topics: Acetophenones; Adenocarcinoma; AMP-Activated Protein Kinases; Antineoplastic Agents; Autophagy; Benzopyrans; Breast Neoplasms; Female; Humans; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphorylation; Protein Kinase C-delta; Protein Kinase Inhibitors; RNA Interference; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transfection

Activation of Wnt/β-catenin signaling can result in up-regulation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. We found that rottlerin, a natural plant polyphenol, suppressed LRP6 expression and phosphorylation, and inhibited Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of rottlerin on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were confirmed in human prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. Mechanistically, rottlerin promoted LRP6 degradation, but had no effects on LRP6 transcriptional activity. In addition, rottlerin-mediated LRP6 down-regulation was unrelated to activation of 5'-AMP-activated protein kinase (AMPK). Importantly, we also found that rottlerin inhibited mTORC1 signaling in prostate and breast cancer cells. Finally, we demonstrated that rottlerin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity with IC(50) values between 0.7 and 1.7 μM for prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells. Our data indicate that rottlerin is a novel LRP6 inhibitor and suppresses both Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells, and that LRP6 represents a potential therapeutic target for cancers.

Topics: Acetophenones; Antineoplastic Agents; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; Female; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Inhibitory Concentration 50; Low Density Lipoprotein Receptor-Related Protein-6; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Promoter Regions, Genetic; Prostatic Neoplasms; Proteolysis; Survivin; TOR Serine-Threonine Kinases; Transcription, Genetic; Wnt Signaling Pathway; Wnt3A Protein

Protein tyrosine phosphatase alpha (PTPα) functions as an activator of Src by dephosphorylating Tyr527/530, a critical negative regulatory site. The increase of PTPα catalytic activity requires its phosphorylation at Ser180 and/or Ser204 and its dissociation from PTPα/Grb2 complex. Here, we show that epidermal growth factor (EGF) stimulation increases the ability of PTPα to activate Src by dephosphorylating Tyr530 in BT-20 and SKBR3 breast cancer cell lines. Treatment of these cells with EGF transiently decreased the association of PTPα with Grb2 and enhanced PTPα catalytic activity via Ser180 and Ser204 phosphorylation that was blocked by the protein kinase C delta (PKCδ) inhibitor rottlerin or knockdown of PKCδ by siRNA or by the overexpression of PTPαS180A/S204A mutant. PTPα siRNA blocked EGF-mediated Src activation in cancer cells and inhibited on colony formation, whereas control siRNA did not. These results suggested that PTPα links activation of epidermal growth factor receptor (EGFR) signaling with Src activation and may provide a novel therapeutic target for treatment of breast cancer.

Topics: Acetophenones; Benzopyrans; Biocatalysis; Breast Neoplasms; Cell Line, Tumor; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; GRB2 Adaptor Protein; Humans; Immunoblotting; Mutation; Phosphorylation; Protein Binding; Protein Kinase C-delta; Proto-Oncogene Proteins pp60(c-src); Receptor-Like Protein Tyrosine Phosphatases, Class 4; RNA Interference; Serine; Signal Transduction; Tyrosine

Type 2 diabetes mellitus (T2DM) has contributed to advanced breast cancer development over the past decades. However, the mechanism underlying this contribution is poorly understood. In this study, we determined that high glucose enhanced proteasome activity was accompanied by enhanced proliferation, migration and invasion, as well as suppressed apoptosis, in human breast cancer MCF-7 cells. Proteasome inhibitor bortezomib (BZM) pretreatment mitigated high glucose-induced MCF-7 cell growth and invasion. Furthermore, high glucose increased protein kinase C delta (PKC?)-phosphorylation. Administration of the specific PKC? inhibitor rottlerin attenuated high glucose-stimulated cancer cell growth and invasion. In addition, PKC? inhibition by both rottlerin and PKC? shRNA significantly suppressed high glucose-induced proteasome activity. Our results suggest that PKC?-dependent ubiquitin proteasome system activation plays an important role in high glucose- induced breast cancer cell growth and metastasis.

Topics: Acetophenones; Apoptosis; Benzopyrans; Boronic Acids; Bortezomib; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Glucose; Humans; MCF-7 Cells; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase C-delta; Pyrazines; Ubiquitin

Autophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles. Autophagy is associated with the several pathological processes, including cancer. Cancer stem cells (CSCs) play significant roles in cancer initiation, progression and drug resistance. Recent studies have demonstrated the antitumor activities of plant-derived chemopreventive agent rottlerin (Rott). However, the molecular mechanism by which Rott induces autophagy in breast CSCs has not been investigated.. The objectives of this study were to examine the molecular mechanism by which Rott induces autophagy which leads to apoptosis in breast CSCs. Treatment of breast CSCs with Rott for 24 h resulted in a concentration dependent induction of autophagy, followed by apoptosis as measured by flow cytometry. Electron microscopy confirmed the presence of autophagosomes in Rott treated breast CSCs. Western blot analysis showed that Rott treatment increased the expression of LC3, Beclin-1 and Atg12 that are accumulated during autophagy. Prolonged exposure of breast CSCs to Rott caused apoptosis which was associated with the suppression of phosphorylated Akt and mTOR, upregulation of phosphorylated AMPK, and downregulation of anti-apoptosis Bcl-2, Bcl-X(L), XIAP and cIAP-1. Knock-down of Atg7 or Beclin-1 by shRNA inhibited Rott-induced autophagy at 24 h. Our study also demonstrates that pre-treatment of breast CSCs with autophagosome inhibitors 3-methyladenine and Bafilomycin, as well as protein synthesis inhibitor cycloheximide inhibited Rott-induced autophagy and apoptosis. Rott induces autophagy via extensive cytoplasmic vacuolization in breast CSCs. Molecular docking results between C2-domain of protein kinase C-delta and Rott indicated that both hydrogen bonding and hydrophobic interactions contributed significantly for ligand binding with minimum binding affinity of ≈ 7.5 Kcal/mol. Although, autophagy inhibitors suppress the formation of cytoplasmic vacuolization and autophagy in breast CSCs, the potency of Rott to induce autophagy and apoptosis might be based on its capability to activate several pathways such as AMPK and proteasome inhibition.. A better understanding of the relationship between autophagy and apoptosis would eventually allow us to discover novel drugs for the treatment of breast cancer by eliminating CSCs.

Topics: Acetophenones; AMP-Activated Protein Kinases; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Benzopyrans; Breast Neoplasms; Cell Survival; Female; Humans; Microtubule-Associated Proteins; Molecular Docking Simulation; Neoplastic Stem Cells; Protein Kinase C-delta; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Vacuoles

This study was designed to investigate the role of protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in tamoxifen (TAM)-induced apoptosis and drug resistance in human breast cancer cells. Drug-sensitive, or estrogen receptor (ER)-positive human breast carcinoma cells (MCF-7) and the multi-drug-resistant variant (ER-negative) MCF-7/ADR cells were treated with doses of TAM for various periods of time. Cell viability and apoptosis were assessed using cell counting, DNA fragmentation and flow cytometric analysis. We found that TAM administration caused a significant increase in apoptosis of MCF-7 cells but not MCF-7/ADR cells. Western blot analysis revealed enhanced expression of PKCδ but decreased expression of PKCα in ER-positive MCF-7 cells; while ER-negative MCF-7/ADR cells had decreased levels of PKCδ and increased levels of PKCα. Interestingly, we observed that in MCF-7 cells, TAM stimulated apoptosis by promoting rapid activation of PKCδ, antagonizing downstream signaling of ERK phosphorylation; while in MCF-7/ADR cells, TAM upregulated PKCα, which promoted ERK phosphorylation. These results suggest that PKCδ enhances apoptosis in TAM-treated MCF-7 cells by antagonizing ERK phosphorylation; while the PKCα pathway plays an important role in TAM-induced drug resistance by activating ERK signaling in MCF-7/ADR cells. The combination of TAM with PKCα and ERK inhibitors could promote TAM-induced apoptosis in breast cancer cells.

Topics: Acetophenones; Antineoplastic Agents, Hormonal; Apoptosis; Benzopyrans; Breast Neoplasms; Carbazoles; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Humans; Protein Kinase C-alpha; Protein Kinase C-delta; Receptors, Estrogen; Signal Transduction; Tamoxifen

HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7).. We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. Doxorubicin-induced apoptosis was also correlated with the presence of HuR in the cytoplasm. Rottlerin, which was able to block HuR nuclear export, had correspondingly antagonistic effects with doxorubicin on cell toxicity. The proapoptotic activity of HuR was not due to cleavage to an active form, as was previously reported. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response.. The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin and that restoration of HuR expression in resistant cells resensitizes them to the action of this drug, thereby identifying HuR as a key protein in doxorubicin pharmacology.

Topics: Acetophenones; Apoptosis; Benzopyrans; Biological Transport; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; ELAV Proteins; Electrophoresis, Gel, Two-Dimensional; Female; Humans; Immunoprecipitation; Oligonucleotide Array Sequence Analysis; Phosphorylation

Cyclooxygenase-2 (COX-2) is an important enzyme in inflammation. In this study, we investigated the underlying molecular mechanism of the synergistic effect of rottlerin on interleukin1β (IL-1β)-induced COX-2 expression in MDA-MB-231 human breast cancer cell line. Treatment with rottlerin enhanced IL-1β-induced COX-2 expression at both the protein and mRNA levels. Combined treatment with rottlerin and IL-1β significantly induced COX-2 expression, at least in part, through the enhancement of COX-2 mRNA stability. In addition, rottlerin and IL-1β treatment drove sustained activation of p38 Mitogen-activated protein kinase (MAPK), which is involved in induced COX-2 expression. Also, a pharmacological inhibitor of p38 MAPK (SB 203580) and transient transfection with inactive p38 MAPK inhibited rottlerin and IL-1β-induced COX-2 upregulation. However, suppression of protein kinase C δ (PKC δ) expression by siRNA or overexpression of dominant-negative PKC δ (DN-PKC-δ) did not abrogate the rottlerin plus IL-1β-induced COX-2 expression. Furthermore, rottlerin also enhanced tumor necrosis factor-α (TNF-α), phorbol myristate acetate (PMA), and lipopolysaccharide (LPS)-induced COX-2 expression. Taken together, our results suggest that rottlerin causes IL-1β-induced COX-2 upregulation through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells.

Topics: Acetophenones; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Cyclooxygenase 2; Enzyme Activation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1beta; Mallotus Plant; MAP Kinase Signaling System; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Protein Kinase C-delta; Reactive Oxygen Species

Induction of matrix metalloproteinase (MMP)-9 is particularly important for the invasiveness of breast cancers. We investigated the inhibitory effect of kalopanaxsaponin A (KPS-A) on cell invasion and MMP-9 activation in phorbol 12-myristate 13-acetate (PMA)-treated MCF-7 human breast cancer cells. KPS-A inhibited PMA-induced cell proliferation and invasion. PMA-induced cell invasion was blocked in the presence of a primary antibody of MMP-9, and KPS-A suppressed the increased expression and/or secretion of MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1. Using specific inhibitors, we confirmed that PMA-induced cell invasion and MMP-9 expression is primarily regulated by nuclear factor-kappa B (NF-kappaB) activation via phosphatidylinositol 3-kinase (PI3K)/Akt and activator protein-1 (AP-1) activation via extracellular signal-regulated kinase (ERK)1/2. KPS-A decreased PMA-induced transcriptional activation of NF-kappaB and AP-1 and inhibited PMA-induced phosphorylation of ERK1/2 and Akt. Treatment with the protein kinase C (PKC)delta inhibitor rottlerin caused a marked decrease in PMA-induced MMP-9 secretion and cell invasion, as well as ERK/AP-1 activation, and KPS-A reduced PMA-induced membrane localization of PKCdelta. Furthermore, oral administration of KPS-A led to a substantial decrease in tumor volume and expression of proliferating cell nuclear antigen, MMP-9, TIMP-1 and PKCdelta in mice with MCF-7 breast cancer xenografts in the presence of 17beta-estradiol. These results suggest that KPS-A inhibits PMA-induced invasion by reducing MMP-9 activation, mainly via the PI3K/Akt/NF-kappaB and PKCdelta/ERK/AP-1 pathways in MCF-7 cells and blocks tumor growth and MMP-9-mediated invasiveness in mice with breast carcinoma. Therefore, KPS-A may be a promising anti-invasive agent with the advantage of oral dosing.

Topics: Acetophenones; Animals; Antineoplastic Agents, Phytogenic; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Transplantation; NF-kappa B; Oleanolic Acid; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C-delta; Proto-Oncogene Proteins c-akt; Saponins; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transplantation, Heterologous

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Topics: Acetophenones; Benzopyrans; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Indoles; Maleimides; NF-kappa B; Proto-Oncogene Proteins c-akt; Signal Transduction

We have reported that Erbin expression was down-regulated in the Jurkat leukaemia T lymphocytes treated with the recombinant soluble tumour necrosis factor-related apoptosis-inducing ligand (rsTRAIL). Herein, we studied the expression and the regulation of Erbin and its binding partner, ErbB2, in the MCF-7 breast cancer cell line. We showed that the expressions of Erbin and ErbB2 were modulated by PKCdelta inhibitor, rottlerin, in the TRAIL-resistant MCF-7 cell line. The affinity of Erbin-ErbB2 interaction was reduced by ErbB2 phosphorylation. Inhibiting the expression of Erbin facilitated the sensitivity of the MCF-7 cells to TRAIL via suppressing the ErbB2/AKT/NF-kappaB signalling pathway.

Topics: Acetophenones; Adaptor Proteins, Signal Transducing; Apoptosis; Benzopyrans; Blotting, Western; Breast Neoplasms; Caspases; Enzyme Inhibitors; Female; Humans; I-kappa B Kinase; Immunoblotting; Immunoprecipitation; NF-kappa B; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a number of tumorogenic or transformed cells, yet is relatively non-toxic to most normal cells, therefore, it is a promising agent for cancer therapy. However, some cancer cell lines were resistant to TRAIL cytoxicity, including MCF-7 breast cancer cells. The mechanism is not clear. Here, we report that protein kinase C delta (PKCdelta) protects MCF-7 cells from the recombinant soluble TRAIL (rsTRAIL)- mediated apoptosis. It was demonstrated that rottlerin, a PKCdelta inhibitor, sensitized MCF-7 cells to rsTRAIL cytoxicity. Combination of rottlerin and rsTRAIL inhibited PKCdelta translocation from the cytosol to membrane, and PKCdelta kinase activity on the cell membrane was kept pace with the change of PKCdelta expression. Moreover, inhibition of PKCdelta by interference RNA could facilitate apoptosis of MCF-7 cells induced by rsTRAIL. Further experiments on the signal machinery showed that rottlerin increased the sensitivity of MCF-7 cells to rsTRAIL by suppressing the transcription activity of NF-kappaB, and enhancing the caspase-processing to generate executive apoptotic signals. These findings indicate that PKCdelta functions as a survival factor protecting MCF-7 cells from the apoptosis induced by rsTRAIL.

Topics: Acetophenones; Apoptosis; Apoptosis Regulatory Proteins; Benzopyrans; Breast Neoplasms; Caspase 9; Caspases; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Membrane Glycoproteins; NF-kappa B; Protein Kinase C-delta; Recombinant Proteins; RNA Interference; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Protein kinase C (PKC) promotes cell survival in response to ionizing radiation in a variety of experimental models including human carcinoma, human glioblastoma, and transformed mouse embryo fibroblast cell lines. We have introduced specific antisense oligonucleotides into human mammary tumor cell lines in vitro to analyze the role of individual PKC isoforms in radiation-induced cell death in breast cancer. MDA-MB-231 and MCF-7 cells treated with oligonucleotide directed against the PKC delta isoform exhibited impaired survival in response to 5.6 Gy gamma-radiation as measured by mitochondrial metabolism of tetrazolium dye. The role of PKC delta in the breast tumor cell lines was of particular interest, because contradictory reports exist in the literature regarding the role of PKC delta in cell survival and apoptosis. A comparison of the effects of the PKC delta antisense oligonucleotide and a nucleotide scrambled version of this nucleotide revealed only the antisense oligonucleotide decreased cell survival. The PKC delta antisense oligonucleotide decreased cell survival after exposure to low (1.5 Gy) radiation doses and in the absence of radiation insult. We found 3 micro M rottlerin, a selective PKC delta inhibitor, to reduce MCF-7 and MDA-MB-231 cell survival. Furthermore, MCF-7 cells transformed to express a dominant-negative mutant of PKC delta exhibited reduced survival. Comet analysis showed that PKC delta oligonucleotide treatment caused an accumulation of cells containing damaged DNA similar to that seen in 1.5 Gy radiation-treated cells. We conclude that PKC delta acts as a prosurvival factor in human breast tumor cells in vitro.

Topics: Acetophenones; Benzopyrans; Blotting, Western; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Cell Survival; Comet Assay; DNA, Neoplasm; Down-Regulation; Enzyme Inhibitors; Gamma Rays; Genes, Dominant; Humans; Isoenzymes; Oligoribonucleotides, Antisense; Protein Kinase C; Protein Kinase C-delta; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

The goal of this study was to investigate the differential sensitivity of estrogen receptor (ER) positive MCF-7 and ER negative MDA-MB 231 breast cancer cells to phorbol myristate acetate (PMA)-dependent growth arrest. MCF-7 cells were growth arrested by 80% while MDA-MB 231 cells were arrested by 20% in response to seven days of treatment with 10 nM PMA. Coincident with the increased sensitivity of MCF-7 cells to be growth arrested by the protein kinase C (PKC) activator PMA, PMA induced 9-fold higher levels of the cyclin dependent kinase (Cdk) inhibitor p21(WAF1/GIP1) in MCF-7 compared to MDA-MB 231 cells. A comparison of the PKC isoforms expressed in MCF-7 versus MDA-MB 231 cells showed that only the PMA-sensitive PKC delta and eta isoforms were expressed at markedly (> or =10-fold) elevated levels in MCF7 versus MDA-MB 231 cells. These results suggested that the differential sensitivity to growth arrest and induction of p2l(WAFl/CIPl) could reflect, at least in part, increased expression of PMA-dependent PKC isoforms delta and/or eta. Direct evidence to support this hypothesis was provided by the ability of transient transfections into MCF-7 cells of constitutively active PKC delta but not of PKC's eta or alpha or epsilon to enhance p21(WAFl/CIP1) promoter activity. These results suggest that PKC delta plays a fundamental role in the regulation of growth in estrogen receptor positive breast cancer cells.

Topics: Acetophenones; Animals; Benzopyrans; Blotting, Western; Breast Neoplasms; Carcinogens; Cell Division; Cell Membrane; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytosol; Detergents; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Humans; Isoenzymes; Luciferases; Octoxynol; Plasmids; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Rats; Receptors, Estrogen; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured