Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C.

Isolated protein kinase C (PKC) was irreversibly inactivated by a brief (min) incubation with calphostin C in the presence of light. This inactivation required Ca2+ either in a millimolar range in the absence of lipid activators or in a submicromolar range in the presence of lipid activators. In addition, an oxygen atmosphere was required suggesting the involvement of oxidation(s) in this inactivation process. Furthermore, PKC inactivation might involve a site-specific oxidative modification of the enzyme at the Ca(2+)-induced hydrophobic region. Physical quenchers of singlet oxygen such as lycopene, beta-carotene, and alpha-tocopherol all reduced the calphostin C-induced inactivation of PKC. In intact cells treated with calphostin C, the inactivation of PKC was rapid in the membrane fraction compared to cytosol. This intracellular PKC inactivation was also found to be irreversible. Therefore, calphostin C can bring prolonged effects for several hours in cells treated for a short time. Taken together these results suggest that the calphostin C-mediated inactivation of PKC involves a site-specific and a 'cage' type oxidative modification of PKC.     

Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells

Impaired function of apoptosis-related genes is deeply involved in oncogenesis and the progression of cancers, and caspase-3 plays a critical role as an executioner of apoptosis. We introduced the caspase-3 gene via an adenovirus (Adv) vector into Alexander hepatoma cells, MCF-7 breast cancer cells, and U251 and U-373MG glioma cells which have different endogenous levels of caspase-3 expression. None of the cell lines underwent apoptosis by overexpression of caspase-3, indicating that induction of caspase-3 alone is not applicable for cancer gene therapy. Next, we investigated whether overexpression of caspase-3 could enhance Fas ligand-mediated apoptosis in these four cell lines. In U-373MG cells, which showed the highest level of expression of surface Fas among the four cell lines, coinfection of the Adv for caspase-3 (Adv-caspase-3) and the Adv for Fas ligand (Adv-FL) induced a remarkably increased degree of apoptosis compared with that induced by the single infection of either Adv-caspase-3 or Adv-FL. Similar results were obtained by cotreatment with anti-Fas antibody in U-373MG cells. These data suggest that when strong proapoptotic upstream stimuli are induced, the level of caspase-3 expression determines the degree of apoptosis in cancer cell lines. In conclusion, overexpression of caspase-3 alone did not induce apoptosis in cancer cells. Both a strong proapoptotic signal and a high expression of caspase-3 were required to induce drastic apoptosis in cancers. This strategy would be highly beneficial for selected cancer patients.     

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells

In this study we investigated the responses of intracellular calcium ([Ca²⁺]_i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [³H]PDBu binding approximately 2–3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca²⁺]_i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca²⁺]_i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.Special issue dedicated to Dr. Leon S. Wolfe.     

The protein kinase C inhibitor, calphostin C, inhibits succinate-dependent mitochondrial reduction of MTT by a mechanism that does not involve protein kinase C.

The light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.     

Neobavaisoflavone sensitizes apoptosis via the inhibition of metastasis in TRAIL-resistant human glioma U373MG cells

Aims

Neobavaisoflavone (NBIF), an isoflavone isolated from Psoralea corylifolia (Leguminosae), has striking anti-inflammatory and anti-cancer effects. NBIF inhibits the proliferation of prostate cancer in vitro and in vivo.

Main methods

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a key endogenous molecule that selectively induces apoptosis in cancer cells with little or no toxicity in normal cells. However, some cancer cells, including U373MG cells, are resistant to TRAIL-mediated apoptosis. We demonstrated that the cell viability, migration and invasion assay were used in U373MG glioma cells.

Key findings

In this study, we found that NBIF sensitizes human U373MG glioma cells to TRAIL-mediated apoptosis. Co-treatment of TRAIL and NBIF effectively induced Bid cleavage and activated caspases 3, 8, and 9. Importantly, DR5 expression was upregulated by NBIF. We also observed that the combination NBIF and TRAIL increased expression of BAX. We further demonstrate that NBIF induced TRAIL-mediated apoptosis in human glioma cells by suppressing migration and invasion, and by inhibiting anoikis resistance.

Significance

Taken together, our results suggest that NBIF reduces the resistance of cancer cells to TRAIL and that the combination of NBIF and TRAIL may be a new therapeutic strategy for treating TRAIL-resistant glioma cells.     

Comparative effects of a highly specific protein kinase C inhibitor, calphostin C and calmodulin inhibitors on angiotensin-stimulated aldosterone secretion

We have examined the relative roles of the calcium-calmodulin system and protein kinase C in angiotensin-mediated aldosterone secretion. We used a highly specific protein kinase C inhibitor, calphostin C and two well-accepted calmodulin inhibitors, W-7 and calmidazolium. Although both types of inhibitors affected angiotensin-induced aldosterone secretion, as judged by the inhibitory doses of these compounds, angiotensin-evoked aldosterone secretion was more sensitive to calmodulin inhibition than protein kinase C inhibition. Manipulation of intracellular calcium by dantrolene and thapsigargin also modified aldosterone secretion significantly.     

Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells

Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells.     

Inhibition of protein kinase C by calphostin C is light-dependent.

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, inhibits protein kinase C by competing at the binding site for diacylglycerol and phorbol esters. Calphostin C is a polycyclic hydrocarbon with strong absorbance in the visible and ultraviolet ranges. In characterizing the activity of this compound, we unexpectedly found that the inhibition of [3H]phorbol dibutyrate binding was dependent on exposure to light. Ordinary fluorescent light was sufficient for full activation. The inhibition of protein kinase C activity in cell-free systems and intact cells also required light. Light-dependent cytotoxicity was seen at concentrations about 5-fold higher than those inhibiting protein kinase C.     

Mechanisms through which PDGF alters intracellular calcium levels in U-1242 MG human glioma cells

PDGF-BB induces a rapid, sustained increase in intracellular calcium levels in U-1242 MG cells. We used several calcium channel blockers to identify the types of channels involved. L channel blockers (verapamil, nimodipine, nicardipine, nitrendipine and taicatoxin) had no effect on PDGF-BB induced alterations in intracellular calcium. Blockers of P, Q and N channels (ω-agatoxin-IVA, ω-conotoxin MVIIC and ω-conotoxin GVIA) also had no effect. This indicates that these channels play an insignificant role in supplying the Ca²⁺ necessary for PDGF stimulated events in U-1242 MG cells. However, a T channel blocker (NDGA) and the non-specific (NS) calcium channel blockers (FFA and SK&F 9365) abolished PDGF-induced increases in intracellular calcium. This indicates that PDGF causes calcium influx through both non-specific cationic channels and T channels. To study the participation of intracellular calcium stores in this process, we used thapsigargin, caffeine and ryanodine, all of which cause depletion of intracellular calcium stores. The PDGF effect was abolished using both thapsigargin and caffeine but not ryanodine. Collectively, these data indicate that in these human glioma cells PDGF-BB induces release of intracellular calcium from caffeine- and thapsigargin-sensitive calcium stores which in turn lead to further calcium influx through both NS and T channels.     

Transfection of 2,6 and 2,3-sialyltransferase genes and GlcNAc-transferase genes into human glioma cell line U-373 MG affects glycoconjugate expression and enhances cell death

Human glioma cell line U-373 MG expresses CMP-NeuAc : Galbeta1,3GlcNAc alpha2,3-sialyltransferase [EC No. 2.4.99.6] (alpha2,3ST), UDP-GlcNAc : beta-d-mannoside beta1,6-N-acetylglucosaminyltransferase V [EC 2.4.1.155] (GnT-V) and UDP-GlcNAc3: beta-d-mannoside beta1,4-N-acetylglucosaminyltransferase III [EC 2.4.1.144] (GnT-III) but not CMP-NeuAc : Galbeta1,4GlcNAc alpha2,6-sialyltransferase [EC 2.4.99.1] (alpha2,6ST) under normal culture conditions. We have previously shown that transfection of the alpha2,6ST gene into U-373 cells replaced alpha2,3-linked sialic acids with alpha2,6 sialic acids, resulting in a marked inhibition of glioma cell invasivity and a significant reduction in adhesivity. We now show that U-373 cells, which are typically highly resistant to cell death induced by chemotherapeutic agents (< 10% death in 18 h), become more sensitive to apoptosis following overexpression of these four glycoprotein glycosyltransferases. U-373 cell viability showed a three-fold decrease (from 20 to 60% cell death) following treatment with staurosporine, C2-ceramide or etoposide, when either alpha2,6ST and GnT-V genes were stably overexpressed. Even glycosyltransferases typically raised in cancer cells, such as alpha2,3ST and GnT-III, were able to decrease viability two-fold (from 20 to 40% cell death) following stable overexpression. The increased susceptibility of glycosyltransferase-transfected U-373 cells to pro-apoptotic drugs was associated with increased ceramide levels in Rafts, increased caspase-3 activity and increased DNA fragmentation. In contrast, the same glycosyltransferase overexpression protected U-373 cells against a different class of apoptotic drugs, namely the phosphatidylinositol 3-kinase inhibitor LY294002. Thus altered surface protein glycosylation of a human glioblastoma cell line can lead to lowered resistance to chemotherapeutic agents.     

Calcium response after stimulation by substance P of U373 MG cells: inhibition of store-operated calcium entry by protein kinase C

In this paper we investigate the Ca2+ response after Substance P (SP) stimulation of U373 MG cells. SP is a tachykinin and physiologically acts as a neurotransmitter and neuromodulator in the nervous system, but pathologically triggers malignant glial cells, such as U373 MG, to release cytokines and increase proliferation rate.In this paper we show that SP increases the proliferation rate of U373 MG cells and the intracellular Ca2+ concentration by mobilizing Ca2+ only from thapsigargin-sensitive stores. In fact, Ca2+ entry through store-operated calcium entry (SOCE) channels, which was observed after thapsigargin treatment, was not detected after stimulation by SP. The inhibition of SOCE after SP stimulation must be mediated by protein kinase C (PKC), because it was not observed in the presence of calphostin C (an inhibitor of PKC). Moreover, stimulation by SP-induced membrane potential hyperpolarization. Our results are consistent with the following sequence of events: (i) SP interacts with NK(1) receptors; (ii) fast homologous receptor desensitization occurs; (iii) reuptake by endoplasmic reticulum Ca(2+)-ATPase quantitatively overwhelms the extrusion by plasma membrane Ca2+-ATPase. These results have two important consequences. In U373 MG cells the SOCE does not contribute to the Ca2+ response after SP, and is not necessarily involved in promoting cell proliferation.     

IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A(1), A(2a), A(2b) and A(3) adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA (N6-(R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A(2b) receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A(2b) dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.     

H7, a protein kinase C inhibitor, increases the glutathione content of neuroblastoma cells.

It is shown that the intracellular glutathione (GSH) concentration of neuroblastoma-2a cells in culture increases with a maximum at 24 h after starting treatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C (PKC). Other inhibitors of this and other protein kinases, e.g. sphingosine, staurosporine, and HA 1004, at the concentrations tested, had a less marked or negligible effect on intracellular GSH concentration. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was also tested and showed no significant effect 24 h after addition.     

A specific inhibitor of protein kinase C induces differentiation of neuroblastoma cells

Recent reports suggest that protein kinase C is involved in neural differentiation. We show that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), the more specific inhibitor of protein kinase C known, induces morphological and functional differentiation of neuro 2a cells, as indicated by the marked increase in the number of neurites/cell and in acetylcholinesterase activity. HA 1004 does not induce differentiation of neural cells. The induction of differentiation by H7 was very rapid; 3 h after addition of H7 the percentages of differentiated cells were 17, 33, 37, 55, and 75% for 17, 50, 85, 250, and 500 microM H7, respectively, while for controls it was 9%. When 500 microM H7 was added to the culture medium, protein kinase C was inhibited by 72 and 62% in cytosol and membrane, respectively. Also, acetylcholinesterase activity (a marker of functional differentiation) increased with time, reaching a 7-fold increase after 48 h.     

Stimulation of mitogen activated protein kinase by LDL and oxLDL in human U-937 macrophage-like cells

Mitogen activated protein kinase in extracts of U-937 macrophage-like cells was stimulated by LDL and oxLDL. A maximum value (161% of the basal phosphotransferase activity) was obtained after 6 min exposure to oxidized LDL (27 μ/ml) using APRTPGGRR peptide substrate. The activatory effect was more pronounced (LDL 181%, oxLDL 201%) when MAPK of stimulated cells was immunoprecipitated with anti-p42^MAPK antibodies and phosphotransferase activity was assayed in immune complexes. Stimulation produced by oxLDL was inhibited by poly I, fucoidan, dextran sulfate and by the MAPKK inhibitor PD 098059 but not by PMA-mediated depletion of PKC or by pre-treatment with chloroquine or with pertussis toxin. These results suggest a direct mitogenic effect of LDL which, in the case of oxLDL, is dependent on scavenger receptor ligation but not on G-protein mediated or PKC-dependent signal transduction.     

Gangliosides in the human glioma cell line U-118 MG grown in culture or as xenografts in nude rats

This study was undertaken to characterize gangliosides in the human glioma cell line U-118 MG. The cell line was grown both in cell culture and as xenografts in nude rats. A common finding in both culture and xenograft cells was the high proportion of the lactoseries ganglioside 3'-LM1, approximately one third of the total ganglioside sialic acid. Otherwise, there were marked differences between the two cell sources. The cells grown in culture had a more simple ganglioside pattern than those grown in xenografts. In the latter instance, more complex gangliosides of the lactoseries, including 3'8'-LD1, sialyllactonorhexaosylceramide and a branched structure with two terminal NeuAc alpha 2-3Gal beta 1- 4GlcNAc chains, and the gangliotetraose series were found. Another marked difference involved GM2, which in the cultured cells was a major fraction, indicating that the synthesis of the gangliotetraose series gangliosides in the former stopped at the level of GM2. These results show that the ganglioside composition of a glioma cell line is strongly influenced by environmental factors.     

Staurosporine, a protein kinase C inhibitor interferes with proliferation of arterial smooth muscle cells

Staurosporine (10 ng/ml and more), a protein kinase C inhibitor, inhibited the serum-stimulated growth of smooth muscle cells. This inhibitory effect proved to be linked to the inhibition of transition from the G0 to the S phase of the cell cycle, as measured by 5-bromodeoxyuridine incorporation into the nuclei. As this inhibitory effect of staurosporine was restricted to the first 10 hours of the stimulation, reactions which are essential for the signal transduction may be associated with actions of protein kinase C. The possibility that staurosporine may inhibit cell growth via inhibition of this step warrants further attention.     

Ultrastructural study of protein kinase C-betaII localization during the cell cycle of human glioma cells

Transmission electron microscopy and immunogold labeling were used to determine how PKC-betaII is localized at stages in the cell cycle of the human glioma cell line U-373MG. Results show that immunogold particles in both dimethylsulfoxide (DMSO) and calphostin C (0.5 microM)-treated cells were mainly located in the cytoplasm. The concentration of gold particles in the nucleus was relatively small and constant throughout the cell cycle of both DMSO and calphostin C treated cells. Micrographs revealed changes in PKC-betaII during the cell cycle. The concentration of gold particles in the DMSO-treated cells was constant until 8 h. Subsequently, cytoplasmic PKC-betaII oscillated with an increased at 10 h, a rapid decrease at 12 h, and a rise at 14 h. The concentration of the gold particles then gradually decreased. In contrast, immunogold labeling in calphostin C-treated cells increased gradually up to 10 h. Subsequently, the pattern of PKC-betaII labeling in calphostin C-treated cells recapitulated those of control cells as seen by the rapid decline of PKC-betaII labeling at 12 h and its re-accumulation at 14 h. Additionally, there was a rapid increase at 20 h. Western blots of PKC-betaII showed constant PKC-betaII immunoreactivity throughout the cell cycle. In comparison to Western blots, in-situ immunogold labeling revealed changes in PKC-II immunoreactivity at 10 h and 14 h. This technique may represent intracellular immunoreactivity of PKC-betaII. The results from the immunogold labeling technique suggest that binding of calphostin C to the regulatory domain of PKC-betaII provokes a conformation change in PKC-betaII, preventing its activation and degradation.