Phosphomimetic Mutation at Ser165 of α-Tubulin Promotes the Persistence of GTP Caps in Microtubules.

Protein kinase C (PKC)-mediated phosphorylation of α-tubulin at Ser165 or expression of phosphomimetic (S165D)-α-tubulin stimulates microtubule (MT) polymerization (Cytoskeleton 2014, 71, 257-272). Ser165 lies near the interface between adjacent αß-tubulin heterodimers and helix H8, which contains Glu254, the catalytic residue in α-tubulin that hydrolyzes the exchangeable GTP in ß-tubulin (ß:GTP) and triggers MT depolymerization. It was hypothesized that S165D, a phosphomimetic variant of α-tubulin, perturbs the alignment of α:Glu254 with respect to ß:GTP, thereby impairing its hydrolysis. Molecular simulations were performed with cryoEM structures of MTs (PDB ID: 3J6E) in which phosphomimetic S165D or control S165N had been substituted. Unlike native and S165N structures, the distance between S165D and α:Glu254 increased by 0.6 Å, while the distance between α:Glu254 and ß:GTP decreased by 0.4 Å. Rotation of ß:GTP by 4 Å occurred in the S165D variant, whereas ß:GTP in the S165N control was unchanged from the native structure. Additionally, the S165D variant exhibited an altered pattern of H-bonding to ß:GTP, including the loss of three H-bonds. The significance of these findings to ß:GTP hydrolysis was analyzed in MCF-10A human breast cells treated with an antibody that detects GTP-bound tubulin. Compared with controls, GTP-tubulin signals were at higher levels in cells that ectopically expressed S165D-α-tubulin (TUBA1C) or had been treated with PKC activator DAG-lactone to induce phosphorylation of Ser165 in native α-tubulin. These findings support a model whereby conformational changes induced by Ser165 phosphorylation alter the spatial relationship between ß:GTP and α:Glu254, thereby slowing GTP hydrolysis and promoting GTP caps.

Electrochemical Resistive-Pulse Sensing of Extracellular Vesicles.

Extracellular vesicles (EVs) released from biological cells have attracted considerable interest due to their potential for cancer diagnostics and important role in cell signaling. Most previously reported studies have been concerned with the detection of EVs in biofluids and analysis of proteins and nucleic acids they contain. Electrochemical resistive-pulse (ERP) sensing enables direct detection of single EVs released from a specific cell and analysis of reactive oxygen and nitrogen species in such vesicles. Here, we demonstrate the applicability of ERP sensing to distinguish between nontransformed and cancerous breast cell lines as well as between breast cancer cell lines with different metastatic potential. Another application of ERP sensing is in real-time monitoring of changes in a single cell induced by a chemical agent. This approach is potentially useful for evaluating the efficacy of therapeutic agents, including those that trigger breast cancer cell death by inducing intense oxidative stress.

Resistive-Pulse Sensing Inside Single Living Cells.

Resistive-pulse sensing is a technique widely used to detect single nanoscopic entities such as nanoparticles and large molecules that can block the ion current flow through a nanopore or a nanopipette. Although the species of interest, e.g., antibodies, DNA, and biological vesicles, are typically produced by living cells, so far, they have only been detected in the bulk solution since no localized resistive-pulse sensing in biological systems has yet been reported. In this report, we used a nanopipette as a scanning ion conductance microscopy (SICM) tip to carry out resistive-pulse experiments both inside immobilized living cells and near their surfaces. The characteristic changes in the ion current that occur when the pipet punctures the cell membrane are used to monitor its insertion into the cell cytoplasm. Following the penetration, cellular vesicles (phagosomes, lysosomes, and/or phagolysosomes) were detected inside a RAW 264.7 macrophage. Much smaller pipettes were used to selectively detect 10 nm Au nanoparticles in the macrophage cytoplasm. The in situ resistive-pulse detection of extracellular vesicles released by metastatic human breast cells (MDA-MB-231) is also demonstrated. Electrochemical resistive-pulse experiments were carried out by inserting a conductive carbon nanopipette into a macrophage cell to sample single vesicles and measure reactive oxygen and nitrogen species (ROS/RNS) contained inside them.

Electrochemical Measurements of Reactive Oxygen and Nitrogen Species inside Single Phagolysosomes of Living Macrophages.

The release of reactive oxygen and nitrogen species (ROS/RNS) by macrophages undergoing phagocytosis is crucial for the efficiency of the immune system. In this work, platinized carbon nanoelectrodes were used to detect, characterize, and quantify for the first time the intracellular production rates of the four primary ROS/RNS (i.e., H2O2, ONOO-, NO•, and NO2-) inside single phagolysosomes of living RAW 264.7 murine macrophages stimulated by interferon-γ and lipopolysaccharide (IFN-γ/LPS) to mimic an in vivo inflammatory activation. The time-dependent concentrations of the four primary ROS/RNS in individual phagolysosomes monitored using a four-step chronoamperometric method evidenced a high variability of their production rates. This intrinsic variability unravels the complexity of phagocytosis.

Direct Electrochemical Measurements of Reactive Oxygen and Nitrogen Species in Nontransformed and Metastatic Human Breast Cells.

The production of reactive oxygen and nitrogen species (ROS and RNS) in human cells is implicated in various diseases, including cancer. Micrometer-sized electrodes coated with Pt black and platinized Pt nanoelectrodes have previously been used for the detection of primary ROS and RNS in biological systems. In this Article, we report the development of platinized carbon nanoelectrodes with well-characterized geometry and use them as scanning electrochemical microscopy (SECM) tips to measure ROS and RNS inside noncancerous and metastatic human breast cells. By performing time-dependent quantitative amperometric measurements at different potentials, the relative concentrations of four key ROS/RNS in the cell cytoplasm and their dynamics were determined and used to elucidate the chemical origins and production rates of ROS/RNS in nontransformed and metastatic human breast cells.

Phosphorylation of Cdc42 effector protein-4 (CEP4) by protein kinase C promotes motility of human breast cells.

Cdc42 effector protein-4 (CEP4) was recently identified by our laboratory to be a substrate of multiple PKC isoforms in non-transformed MCF-10A human breast cells. The significance of phosphorylated CEP4 to PKC-stimulated motility of MCF-10A cells was evaluated. Single site mutants at Ser residues embedded in potential PKC consensus sites (Ser(18), Ser(77), Ser(80), and Ser(86)) were individually replaced with Asp residues to simulate phosphorylation. Following expression in weakly motile MCF-10A cells, the S18D and S80D mutants each promoted increased motility, and the double mutant (S18D/S80D) produced a stronger effect. MS/MS analysis verified that Ser(18) and Ser(80) were directly phosphorylated by PKCα in vitro. Phosphorylation of CEP4 severely diminished its affinity for Cdc42 while promoting Rac activation and formation of filopodia (microspikes). In contrast, the phosphorylation-resistant double mutant S18A/S80A-CEP4 blocked CEP4 phosphorylation and inhibited motility of MCF-10A cells that had been stimulated with PKC activator diacylglycerol lactone. In view of the dissociation of phospho-CEP4 from Cdc42, intracellular binding partners were explored by expressing each CEP4 double mutant from a tandem affinity purification vector followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as tumor endothelial marker-4 (TEM4; ARHGEF17), a guanine nucleotide exchange factor that is involved in migration. In motile cells expressing S18D/S80D-CEP4, knockdown of TEM4 inhibited both Rac activation and motility. These findings support a model in which PKC-mediated phosphorylation of CEP4 at Ser(18) and Ser(80) causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodium formation, and cell motility.

Anti-tumor properties of cis-resveratrol methylated analogs in metastatic mouse melanoma cells.

Resveratrol (E-3,5,4'-trihydroxystilbene) is a polyphenol found in red wine that has been shown to have multiple anti-cancer properties. Although cis-(Z)- and trans-(E)-isomers of resveratrol occur in nature, the cis form is not biologically active. However, methylation at key positions of the cis form results in more potent anti-cancer properties. This study determined that synthetic cis-polymethoxystilbenes (methylated analogs of cis-resveratrol) inhibited cancer-related phenotypes of metastatic B16 F10 and non-metastatic B16 F1 mouse melanoma cells. In contrast with cis- or trans-resveratrol and trans-polymethoxystilbene which were ineffective at 10 µM, cis-polymethoxystilbenes inhibited motility and proliferation of melanoma cells with low micromolar specificity (IC50 < 10 µM). Inhibitory effects by cis-polymethoxystilbenes were significantly stronger with B16 F10 cells and were accompanied by decreased expression of ß-tubulin and pleckstrin homology domain-interacting protein, a marker of metastatic B16 cells. Thus, cis-polymethoxystilbenes have potential as chemotherapeutic agents for metastatic melanoma.

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins.

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.

Analysis of substrates of protein kinase C isoforms in human breast cells by the traceable kinase method.

A previous report [Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370] described the application of the traceable kinase method in identifying substrates of protein kinase Cα (PKC-α) in nontransformed human breast MCF-10A cells. Here, a nonradioactive variation of this method compared the phosphoprotein profiles of three traceable PKC isoforms (α, δ, and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high-affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N(6)-phenyl-ATP, and the resulting phosphoproteins were analyzed by Western blotting with an antibody that recognizes the phosphorylated PKC consensus site. Phosphoprotein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, -δ, and -ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (moles of phospho-CEP4 per mole of CEP4). Following knockdown with isoform-specific shRNA-encoding plasmids, the level of phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC-α, -δ, and -ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and -δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms.

Nanoelectrochemistry of mammalian cells.

There is a significant current interest in development of new techniques for direct characterization of the intracellular redox state and high-resolution imaging of living cells. We used nanometer-sized amperometric probes in combination with the scanning electrochemical microscope (SECM) to carry out spatially resolved electrochemical experiments in cultured human breast cells. With the tip radius approximately 1,000 times smaller than that of a cell, an electrochemical probe can penetrate a cell and travel inside it without apparent damage to the membrane. The data demonstrate the possibility of measuring the rate of transmembrane charge transport and membrane potential and probing redox properties at the subcellular level. The same experimental setup was used for nanoscale electrochemical imaging of the cell surface.

Phosphorylation state of Ser165 in α-tubulin is a toggle switch that controls proliferating human breast tumors.

Engineered overexpression of protein kinase Cα (PKCα) is known to phosphorylate Ser165 in α-tubulin resulting in stimulated microtubule dynamics and cell motility, and activation of an epithelial-mesenchymal transition (EMT) in non-transformed human breast cells. Here it is shown that endogenous phosphorylation of native α-tubulin in two metastatic breast cell lines, MDA-MB-231-LM2-4175 and MDA-MB-468 is detected at PKC phosphorylation sites. α-Tubulin mutants that simulated phosphorylated (S165D) or non-phosphorylated (S165 N) states were stably expressed in MDA-MB-231-LM2-4175 cells. The S165D-α-tubulin mutant engendered expression of the EMT biomarker N-cadherin, whereas S165 N-α-tubulin suppressed N-cadherin and induced E-cadherin expression, revealing a 'cadherin switch'. S165 N-α-tubulin engendered more rapid passage through the cell cycle, induced shorter spindle fibers and exhibited more rapid proliferation. In nude mice injected with MDA-MB-231-LM2-4175 cells, cells expressing S165 N-α-tubulin (but not the S165D mutant) produced hyper-proliferative lung tumors with increased tumor incidence and higher Ki67 expression. These results implicate the phosphorylation state of Ser165 in α-tubulin as a PKC-regulated molecular switch that causes breast cells to exhibit either EMT characteristics or hyper-proliferation. Evaluation of genomic databases of human tumors strengthens the clinical significance of these findings.

Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(µ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(µ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

Dequalinium blocks macrophage-induced metastasis following local radiation.

A major therapeutic obstacle in clinical oncology is intrinsic or acquired resistance to therapy, leading to subsequent relapse. We have previously shown that systemic administration of different cytotoxic drugs can induce a host response that contributes to tumor angiogenesis, regrowth and metastasis. Here we characterize the host response to a single dose of local radiation, and its contribution to tumor progression and metastasis. We show that plasma from locally irradiated mice increases the migratory and invasive properties of colon carcinoma cells. Furthermore, locally irradiated mice intravenously injected with CT26 colon carcinoma cells succumb to pulmonary metastasis earlier than their respective controls. Consequently, orthotopically implanted SW480 human colon carcinoma cells in mice that underwent radiation, exhibited increased metastasis to the lungs and liver compared to their control tumors. The irradiated tumors exhibited an increase in the colonization of macrophages compared to their respective controls; and macrophage depletion in irradiated tumor-bearing mice reduces the number of metastatic lesions. Finally, the anti-tumor agent, dequalinium-14, in addition to its anti-tumor effect, reduces macrophage motility, inhibits macrophage infiltration of irradiated tumors and reduces the extent of metastasis in locally irradiated mice. Overall, this study demonstrates the adverse effects of local radiation on the host that result in macrophage-induced metastasis.

Immunohistochemical analysis of advanced human breast carcinomas reveals downregulation of protein kinase C alpha.

Forty-six advanced-stage human breast carcinoma specimens were evaluated by immunohistochemistry for PKC alpha expression and compared with 25 samples of normal adjacent breast tissue. For normal tissue, the median staining of ductal epithelia was of moderate intensity. No staining was observed for 67% of tumor specimens, and only 4% showed intensities greater than the median observed in normal tissue. Faint to moderate PKC alpha staining was observed in the stroma, inflammatory cells, and fibroblasts of tumors but was absent in normal tissue. These findings demonstrate that downregulation of PKC alpha protein occurs in epithelial cells of advanced breast tumors (p<0.001).

Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells.

Protein kinase C (PKC) engenders motility through phosphorylation of α-tubulin at Ser-165 in nontransformed MCF-10A cells. Live cell imaging explored the impact of PKC-mediated phosphorylation on microtubule (MT) dynamics. MTs fluorescently labeled with GFP-α-tubulin were treated with diacylglycerol (DAG)-lactone (a membrane-permeable PKC activator), or cotransfected with a pseudophosphorylated S165D-α6-tubulin mutant. Each condition increased the dynamicity of MTs by stimulating the rate and duration of the growth phase and decreasing the frequency of catastrophe. In MDA-MB-231 metastatic breast cells where the intrinsic PKC activity is high, these MT growth parameters were also high but could be suppressed by expression of phosphorylation-resistant S165N-α6-tubulin or by treatment with a pan-PKC inhibitor (bis-indoleylmaleimide). Subcellular fractionation and immunofluorescence of MCF-10A cells showed that phosphorylation (via DAG-lactone) or pseudophosphorylation of α6-tubulin increased its partitioning into MTs as compared to controls, and produced longer, more stable MTs. Following expression of the plus-end binding protein GFP-EB1, DAG-lactone accelerated the formation and increased the number of nascent MTs. Expression of S165D-α6-tubulin promoted Rac1 activation and Rac1-dependent cell motility. These findings call attention to PKC-mediated phosphorylation of α-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement.

Export of a single drug molecule in two transport cycles by a multidrug efflux pump.

Secondary multidrug transporters use ion concentration gradients to energize the removal from cells of various antibiotics. The Escherichia coli multidrug transporter MdfA exchanges a single proton with a single monovalent cationic drug molecule. This stoichiometry renders the efflux of divalent cationic drugs energetically unfavourable, as it requires exchange with at least two protons. Here we show that surprisingly, MdfA catalyses efflux of divalent cations, provided that they have a unique architecture: the two charged moieties must be separated by a long linker. These drugs are exchanged for two protons despite the apparent inability of MdfA to exchange two protons for a single drug molecule. Our results suggest that these drugs are transported in two consecutive transport cycles, where each cationic moiety is transported as if it were a separate substrate. We propose that secondary transport can adopt a processive-like mode of action, thus expanding the substrate spectrum of multidrug transporters.

Development of a highly potent, selective, and cell-active inhibitor of cysteine cathepsin L-A hybrid design approach.

A hybrid-design approach is undertaken to develop a highly potent and selective inhibitor of human cathepsin L. Studies involving human breast carcinoma MDA-MB-231 cells establish that this inhibitor can successfully block intracellular cathepsin L activity, and retard the cell-migratory potential of these highly metastatic cells.

PhosphoMARCKS drives motility of mouse melanoma cells.

Phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by protein kinase C alpha (PKC alpha) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that once released into the cytoplasm, phosphoMARCKS directly promotes motility of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas in weakly motile B16 F1 cells it is undetectable. Following treatment with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibited a dramatic increase in phosphoMARCKS that was co-incident with a 5-fold increase in motility. Both MARCKS phosphorylation and motility were substantially decreased when prior to OA addition, MARCKS expression was knocked out by a MARCKS-specific shRNA, thereby implicating MARCKS as a major component of the motility pathway. Decreased motility and phosphoMARCKS levels in OA-treated cells were observed with a PKC inhibitor (calphostin C), thus indicating that PKC actively phosphorylates MARCKS in F1 cells but that this reaction is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility was further established with a pseudo-phosphorylated mutant of MARCKS-GFP in which Asp residues replaced Ser residues known to be phosphorylated by PKC alpha. This mutant localized to the cytoplasm and engendered three-fold higher motility in F1 cells. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localized to the cytoplasm, blocked motility by 40-50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These results demonstrate that phosphoMARCKS contributes to the metastatic potential of melanoma cells, and reveal a previously undocumented signaling role for this cytoplasmic phospho-protein.

Selective targeting of neuroblastoma tumour-initiating cells by compounds identified in stem cell-based small molecule screens.

Neuroblastoma (NB) is the most deadly extra-cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour-initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient-derived cancer stem cell-like tumour-initiating cells (TICs) while sparing normal paediatric stem cells (skin-derived precursors, SKPs) and characterize two therapeutic candidates. DECA-14 and rapamycin were identified as NB TIC-selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self-renewal or tumour-initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient-specific and potentially less toxic therapies.