kt-5720 and staurosporine-aglycone

The highly dynamic nature of voltage-gated Na+ channel (VGSC) expression and its controlling mechanism(s) are not well understood. In this study, we investigated the possible involvement of nerve growth factor (NGF) in regulating VGSC activity in the strongly metastatic Mat-LyLu cell model of rat prostate cancer (PCa). NGF increased peak VGSC current density in a time- and dose-dependent manner. NGF also shifted voltage to peak and the half-activation voltage to more positive potentials, and produced currents with faster kinetics of activation; sensitivity to the VGSC blocker tetrodotoxin (TTX) was not affected. The NGF-induced increase in peak VGSC current density was suppressed by both the pan-trk antagonist K252a, and the protein kinase A (PKA) inhibitor KT5720. NGF did not affect the Nav1.7 mRNA level, but the total VGSC alpha-subunit protein level was upregulated. NGF potentiated the cells' migration in Transwell assays, and this was not affected by TTX. We concluded that NGF upregulated functional VGSC expression in Mat-LyLu cells, with PKA as a signaling intermediate, but enhancement of migration by NGF was independent of VGSC activity.

Topics: Animals; Carbazoles; Cell Line, Tumor; Cell Movement; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Indole Alkaloids; Indoles; Male; NAV1.7 Voltage-Gated Sodium Channel; Nerve Growth Factor; Patch-Clamp Techniques; Prostatic Neoplasms; Pyrroles; Rats; Receptors, Nerve Growth Factor; RNA, Messenger; Sodium Channels; Time Factors; Up-Regulation

We have studied the interactions of five indolocarbazoles with N-[methyl-(3)H]scopolamine (NMS) and unlabeled acetylcholine at M(1)-M(4) muscarinic receptors, using equilibrium and nonequilibrium radioligand binding studies. The results are consistent with an allosteric model in which the primary and allosteric ligands bind simultaneously to the receptor and modify each other's affinities. The compounds were generally most active at M(1) receptors. [(3)H]NMS binding was enhanced by staurosporine, KT5720, and KT5823 at M(1) and M(2) receptors, and by K-252a at M(1) receptors. Gö 7874 reduced [(3)H]NMS affinity by up to threefold for all subtypes. A range of cooperative effects with acetylcholine was seen, and, at the M(1) receptor, KT5720 had a log affinity of 6.4 and enhanced acetylcholine affinity by 40%. The compounds inhibited the dissociation of [(3)H]NMS to different extents across the receptor subtypes, with the largest effects at M(1) receptors. In equilibrium binding studies the inhibitory potency of gallamine at M(1) receptors was not affected by KT5720, indicating that these agents bind to two distinct allosteric sites and have neutral cooperativity with each other. In contrast, gallamine and staurosporine had a negatively cooperative or competitive interaction at M(1) receptors. Similarly, the potency and relative effectiveness of KT5720 for inhibiting [(3)H]NMS dissociation from M(1) receptors were not affected by gallamine or brucine, but were affected in a complex manner by staurosporine. These results demonstrate that there are at least two distinct allosteric sites on the M(1) receptor, both of which can support positive cooperativity with acetylcholine.

Topics: Acetylcholine; Allosteric Regulation; Allosteric Site; Animals; Carbazoles; CHO Cells; Cricetinae; Enzyme Inhibitors; Indole Alkaloids; Indoles; N-Methylscopolamine; Pyrroles; Radioligand Assay; Receptors, Muscarinic; Staurosporine

The incubation of rat peritoneal macrophages in the presence of staurosporine, a non-specific protein kinase inhibitor, induced interleukin-6 (IL-6) production in a time- and concentration-dependent manner at 6.3-63 nM, but at 210 nM, the stimulant effect on IL-6 production was reduced. The levels of IL-6 mRNA as determined by a reverse transcription-polymerase chain reaction were also increased by staurosporine in parallel with the ability to induce IL-6 production. Compounds structurally related to staurosporine including K-252a (non-specific protein kinase inhibitor) and KT-5720 (inhibitor of cyclic AMP-dependent protein kinase, PKA), did not increase IL-6 production by peritoneal macrophages. Staurosporine-induced increases in IL-6 production and expression of IL-6 mRNA were decreased by the PKC inhibitors, H-7 (2.7-27 microM), Ro 31-8425 (1-10 microM) and calphostin C (0.3-3 microM) and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 (30-100 microM), but were further increased by the protein tyrosine kinase (PTK) inhibitor, genistein (12-37 microM). The staurosporine-induced increase in IL-6 production was not affected by the PKA inhibitor, H-89 (0.1-3 microM). These findings suggest that the induction of IL-6 production by staurosporine is secondary to elevation of IL-6 mRNA level, which, in turn, is positively regulated by the activation of PKC and PI 3-kinase and negatively regulated by the activation of PTK. PKA does not appear to play a significant role.

Topics: Animals; Carbazoles; Chromones; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; Indole Alkaloids; Indoles; Interleukin-6; Isoquinolines; Macrophages, Peritoneal; Male; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Pyrroles; Rats; Rats, Sprague-Dawley; RNA, Messenger; Staurosporine; Sulfonamides; Time Factors

We examined alterations in cell morphology and expression of adhesion molecules in response to a general protein kinase inhibitor K252a treatment of non-adherent colon adenocarcinoma Colo201 cells. K252a induced rapid cell adhesion and spreading with concomitant formation of actin stress fibers. A protein kinase A inhibitor KT5720 also induced cell adhesion, but the rate of spread was slower than that seen with K252a. These adhesions were mediated by integrin molecules since cell adhesion required Mg2+, Mn2+ or Ca2+, and was inhibited by monoclonal antibodies for integrins alpha2 and beta1. Indirect immunofluorescence microscopic observations revealed that integrin alpha2 and beta1 molecules in K252a-treated cells were concentrated at sites of focal adhesion, but expressions of integrin molecules were not modulated. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin increased during K252a- or KT5720-induced cell adhesion. Immunosuppressants FK506 and cyclosporin A suppressed the K252a-induced cell adhesion and abolished tyrosine phosphorylation of cellular proteins including FAK and paxillin. Furthermore, W7 and calmidazolium, inhibitors of calmodulin, also inhibited the cell adhesion. Based on findings that FK506 and cyclosporin A are inhibitors of the calcium calmodulin-dependent protein phosphatase, calcineurin, this phosphatase may regulate integrin-dependent cell adhesion and spread of Colo201 cells. This Colo201 cell model provides a pertinent system for studying molecules involved in signal transduction pathways and can shed light on mechanisms of metastasis and invasion of colon carcinoma cells.

Topics: Adenocarcinoma; Carbazoles; Cell Adhesion; Cell Adhesion Molecules; Colonic Neoplasms; Cyclosporine; Cytoskeletal Proteins; Enzyme Inhibitors; Extracellular Matrix; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Immunosuppressive Agents; Indole Alkaloids; Indoles; Integrins; Neoplasm Metastasis; Paxillin; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Pyrroles; Sulfonamides; Tacrolimus; Tumor Cells, Cultured; Tyrosine

The role of phosphorylation in modulating an inwardly rectifying ATP-regulated K+ channel with inward conductance of about 90 pS was examined using the patch-clamp technique on opossum kidney (OK) cells. The activity of the inwardly rectifying K+ channel observed in cell-attached patches rapidly declined (channel "rundown") upon excision of the membrane into inside-out patches in a control bath solution (3 mM Mg2+, ATP-free). The declined channel activity was partially restored by applying ATP to the bath, and the ATP-induced channel restoration reached the near maximal level at an ATP concentration of 3 mM. The channel activity maintained by 3 mM ATP in inside-out patches was inhibited by K-252a (10 microM), a nonspecific protein kinase inhibitor, or KT5720 (200 nm), a specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA), and was further stimulated by the addition of a catalytic subunit of PKA (20 nM). In cell-attached patches, the channel activity was also inhibited by K-252a (10 microM) or KT5720 (200 nM). The application of dibutyryl-cAMP (100 microM) alone failed to enhance channel activity, but significantly stimulated channel activity after the pretreatment of cells with Ro-20-1724 (100 microM), an inhibitor of cAMP-specific phosphodiesterase. These results suggest that maintenance of the activity of ATP-regulated K+ channels in OK cells requires protein kinase-mediated phosphorylation with ATP-hydrolysis, and that phosphorylation is mainly induced by PKA.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenosine Triphosphate; Animals; Bucladesine; Carbazoles; Cell Line; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Indole Alkaloids; Indoles; Kidney; Opossums; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Phosphorylation; Potassium Channels; Protein Kinase Inhibitors; Pyrroles

We have used paclitaxel-dependent Tax 2-4 cells to screen for compounds that have paclitaxel-like functional activity. The indolocarbazole serine/threonine kinase inhibitor K252a and analogues such as KT5926, KT5720, and K252b partially support the growth of the paclitaxel-dependent cells in the absence of paclitaxel. A novel kinase inhibitor of similar structure, U98017, supports the growth of the dependent cells to 48% of that seen with paclitaxel. Used in combination with paclitaxel, these compounds reduce the amount of paclitaxel required for maximum growth of the dependent cells. Isobologram analysis demonstrates that these compounds also act synergistically with paclitaxel to promote toxicity in wild-type Chinese hamster ovary cells. These selected indolocarbazoles may act at sites distinct from that of paclitaxel and may specifically inhibit kinases that contribute to the destabilization of microtubules. Other indolocarbazoles such as staurosporine and rebeccamycin do not support paclitaxel-dependent cell growth. Structurally unrelated serine/threonine kinase inhibitors such as H-9 and H-7 or tyrosine kinase inhibitors such as lavendustin do not support the growth of these cells. These results define a screen for functional paclitaxel analogues and suggest that it may be useful to investigate the possible synergy of selected indolocarbazoles and paclitaxel in vivo.

Topics: Alkaloids; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carbazoles; Cell Division; Cell Line; CHO Cells; Cricetinae; Drug Synergism; Indole Alkaloids; Indoles; Myosin-Light-Chain Kinase; Paclitaxel; Pyrroles; Staurosporine

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of 32P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723-20729, 1989; Mol Cell Biol 10:3020-3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca++, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr(R) and murine melanoma B16/Adr(R) cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.

Topics: Alkaloids; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Calcium; Carbazoles; Carrier Proteins; Clone Cells; Cyclic AMP; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Humans; Indole Alkaloids; Indoles; KB Cells; Melanoma, Experimental; Membrane Glycoproteins; Mice; Neoplasm Proteins; Phosphatidylserines; Phosphorylation; Phosphothreonine; Phosphotyrosine; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Pyrroles; Staurosporine; Transfection; Tumor Cells, Cultured; Tyrosine

We have shown that nontransformed mammalian cells arrest early in the G1 phase of the cell cycle when treated with exceedingly low concentrations of the nonspecific kinase inhibitor staurosporine, whereas transformed cells continue to progress through the cell cycle. We have now treated normal or transformed human skin fibroblasts with four other kinase inhibitors. Three of these inhibitors are highly specific: KT5720 inhibits cAMP-dependent protein kinase, KT5823 inhibits cGMP-dependent protein kinase, and KT5926 inhibits myosin light-chain kinase. The fourth inhibitor K252b has a moderate specificity for protein kinase C but also inhibits the three kinases just mentioned. We have found that these inhibitors reversibly arrest normal human skin fibroblasts at different times in the G1 phase but do not affect the cell cycle progression of transformed cells. The times of arrest within the G1 phase can be divided into two categories. Two of the inhibitors, KT5926 and K252b, act at an early time that is approximately 4 h after the transition from G0 to G1. The cAMP- and cGMP-dependent protein kinase inhibitors KT5720 and KT5823 arrest cells at a later time that is approximately 6 h after the G0/G1 boundary. These data indicate that there are multiple kinase-mediated phosphorylations of different substrates that are essential for the progression of normal cells, but not transformed cells, through the G1 phase. These inhibitors provide us with a set of biochemical probes that should be invaluable in the study of the function of kinases during G1 phase progression of normal cells.

Topics: Alkaloids; Carbazoles; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Humans; In Vitro Techniques; Indole Alkaloids; Indoles; Interphase; Protein Kinase Inhibitors; Protein Kinases; Pyrroles; Staurosporine

To clarify the role of protein kinase C and protein kinase A in cell proliferation and differentiation, the effects of K252a and its derivatives (K252b, KT5720), which have different inhibitory activity to these protein kinases, on the proliferation and differentiation of HL-60 cells were investigated. The proliferation and DNA synthesis of the HL-60 cells were inhibited by K252a in a dose dependent manner. However, K252b and KT5720 which are more specific inhibitors of protein kinase C or protein kinase A, respectively, had no observable effect on cell proliferation. K252a (40nM) enhanced the differentiation of HL-60 cells induced by 1,25(OH)2D3, retinoic acid and DMSO. K252b and KT5720 did not affect 1,25(OH)2D3-induced differentiation. K252a significantly inhibited the differentiation induced by PMA. These results demonstrate that K252a but not its derivatives can function as an antitumor drug and enhancer of the differentiation induced by various inducers.

Topics: Carbazoles; Cell Differentiation; Cell Division; DNA, Neoplasm; Humans; Indole Alkaloids; Indoles; Leukemia, Promyelocytic, Acute; Protein Kinase C; Protein Kinases; Pyrroles; Tumor Cells, Cultured

Possible involvement of protein kinases in the serotonin (5-HT) transport system in platelets and the inhibitory effect of concanavalin A (Con A) on platelet 5-HT uptake were investigated. Staurosporine and K-252a, highly active inhibitors of protein kinases, did not inhibit 5-HT transport, but they antagonized the inhibitory effect of Con A on 5-HT uptake. KT5720, a protein kinase A inhibitor that has no effect on protein kinase C, neither affected 5-HT transport nor antagonized the inhibitory effect of Con A on 5-HT uptake. The Con A effect on 5-HT uptake was also antagonized by LaCl3, a Ca++ entry blocker. When the activity of Ca++ transport into platelets was estimated, Con A was shown to have a stimulative effect, which was antagonized by alpha-methyl-D-mannoside, a specific antagonist of Con A binding to cell membrane glycoproteins. Furthermore, Con A was shown to stimulate the protein kinase C activity of platelets, which phosphorylates a 40-kDa platelet protein; the Con A effects were antagonized by alpha-methyl-D-mannoside, staurosporine and K-252a, but not by KT5720. We suggest that the activation of protein kinase C and phosphorylation of 40-kDa protein might be involved in the inhibitory effect of Con A on platelet 5-HT transport.

Topics: Alkaloids; Animals; Biological Transport, Active; Blood Platelets; Blood Proteins; Calcium; Calcium Radioisotopes; Carbazoles; Concanavalin A; In Vitro Techniques; Indole Alkaloids; Indoles; Lanthanum; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Pyrroles; Rabbits; Serotonin; Staurosporine