PKC 412 sensitizes U1810 non-small cell lung cancer cells to DNA damage

Non-small cell lung carcinoma (NSCLC) is characterized by resistance to drug-induced apoptosis, which might explain the survival of lung cancer cells following treatment. Recently we have shown that the broad-range kinase inhibitor staurosporine (STS) reactivates the apoptotic machinery in U1810 NSCLC cells [Joseph et al., Oncogene 21 (2002) 65]. Lately, several STS analogs that are more specific in kinase inhibition have been suggested for tumor treatment. In this study the apoptosis-inducing ability of the STS analogs PKC 412 and Ro 31-8220 used alone or in combination with DNA-damaging agents in U1810 cells was investigated. In these cells Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to etoposide treatment. PKC 412 as a single agent induced death of a small number of U1810 cells, whereas it efficiently triggered a dose- and time-dependent apoptosis in U1285 small cell lung carcinoma cells. In both cell types PKC 412 triggered release of mitochondrial proteins followed by caspase activation. However, concomitant activation of a caspase-independent pathway was essential to kill NSCLC cells. Importantly, PKC 412 was able to sensitize etoposide- and radiation-induced death of U1810 cells. The best sensitization was achieved when PKC 412 was administered 24 h after treatments. In U1810 cells, Ro 31-8220 decreased PMA-induced ERK phosphorylation as efficiently as PKC 412, indicating that the failure of Ro 31-8220 to induce apoptosis was not due to weaker inhibition of conventional and novel PKC isoforms. However, Ro 31-8220 increased the basal level of ERK and Akt phosphorylation in both cell lines, whereas Akt phosphorylation was suppressed in the U1810 cells, which might influence apoptosis. These results suggest that PKC 412 could be a useful tool in increasing the efficiency of therapy of NSCLC.     

新生牛肝中三种低分子化合物牛磺酸、鸟氨酸、肌肽对HL-60细胞凋亡的诱导作用

目的:研究三种新生牛肝源低分子化合物:牛磺酸、鸟氨酸、肌肽对HL-60白血病细胞增殖的抑制作用并探讨其调控机理.方法:用MTT法分别检测三种活性成分作用后HL-60细胞和正常人淋巴细胞的存活率.分别用琼脂糖凝胶电泳,顺磁共振ESR技术,免疫组化法测定其对HL-60细胞的核DNA、氧自由基活性和细胞周期蛋白水平的影响.结果:三种化合物能够有效地抑制HL-60细胞的增殖,而对正常的人淋巴细胞的生长没有抑制作用;三种化合物使HL-60细胞的核DNA产生30 kb片段,使HL-60细胞内的氧自由基活性降至痕量水平,并下调HL-60的p45/skp2的水平,而上调p27/kip的水平.结论:牛磺酸、鸟氨酸、肌肽能够通过调控细胞周期蛋白的水平而选择性的抑制肿瘤细胞的增殖.     

Proliferation of human melanoma cells is under tight control of protein kinase C alpha

Exponential proliferation of human melanoma cells has been associated with low levels of protein kinase C (PKC)-alpha. The aim of the present study was to investigate the functional relationship between PKC-alpha and melanoma cell proliferation. Treatment of human melanoma cells with the selective PKC inhibitor Ro-31-8220 resulted in a significant increase of cell proliferation as measured by (3)H-thymidine incorporation and a fluorometric microassay. In addition, phosphorothioate antisense-oligodeoxynucleotides (ODNs) to PKC-alpha enhanced DNA-synthesis of human melanoma cells. Furthermore, microinjection and transient transfection of melanoma cells with PKC-alpha decreased their proliferation, as shown by the reduction of nuclear staining with the proliferation marker Ki-67. The presented data demonstrate a cause-effect relationship between PKC-alpha and melanoma cell growth, whereby PKC-alpha reversely influences the rate of cell proliferation.     

The involvement of lipid activators of protein kinase C in the induction of ZIF268 in PC12 cells exposed to lead

Lead (Pb) induces the expression of immediate early genes (IEG) in PC12 cells by a mechanism that involves protein kinase C (PKC). To define the mechanisms, the involvement of two commonly observed lipid activators of PKC, diacylglycerols, and phosphatidylinositols, were examined. A dose-dependent increase in the expression of the IEG zif268 was observed in PC12 cells exposed to Pb. The PKC inhibitor Ro-31-8220 blocked the induction. An increase in levels of diacylglycerols was observed in PC12 cells exposed to Pb, but the increase was inhibited by Ro-31-8220. The phosphatidylinositol 3-kinase inhibitor Wortmannin, but not the inhibitor LY 294002, blocked the induction zif268 in Pb-exposed cells. Small increases in phosphatidylinositol 3-kinase activity were observed after exposure to Pb. In summary, diacylglycerols are elevated in PC12 cells exposed to Pb by a mechanism that requires PKC. It is possible that diacylglycerols contribute to the induction of zif268 by Pb by sustaining PKC activation.     

Hybrid molecules containing benzo[4,5]imidazo[1,2-d][1,2,4]thiadiazole and alpha-bromoacryloyl moieties as potent apoptosis inducers on human myeloid leukaemia cells

The synthesis and biological activity of a series of hybrids 1-5 prepared combining a benzo[4,5]imidazo[1,2-d][1,2,4]thiadiazole and different benzoheterocyclic alpha-bromoacryloyl amides have been described and their structure-activity relationships discussed. All these hetero-bifunctional compounds were highly cytotoxic against the human myeloid leukaemia cell lines HL-60 and U937 (IC(50) 0.24-1.72microM), significantly superior to that of both alkylating units alone. In human myeloid leukaemia HL-60 cells we observed that these compounds suppress survival and proliferation by triggering morphological changes and internucleosomal DNA fragmentation characteristic of apoptotic cell death. The apoptosis induced by these compounds is mediated by caspase-3 activation and is also associated to an early release of cytochrome c from the mitochondria.     

Dissociation of tyrosine phosphorylation and activation of phosphoinositide phospholipase C induced by the protein kinase C inhibitor Ro-31-8220 in Swiss 3T3 cells treated with platelet-derived growth factor

Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP₂) via phospholipase C-γ1 (PLC-γ1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC-γ1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC-γ1 translocation to the particulate fraction. Thus, although activation of PLC-γ1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC-γ1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC-γ1 induced by PDGF and slowed the translocation of PLC-γ1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP₂ in cells labeled with myo[³H]inositol. The increased synthesis of PtdInsP₂ could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC-γ1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC-γ1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC-γ1, and on PtdInsP₂ synthesis cannot be attributed to inhibition of PKC. © 1997 Elsevier Science B.V. All rights reserved.     

Studies with protein kinase C inhibitors presently available cannot elucidate the role of protein kinase C in the activation of NADPH oxidase

The effects of various protein kinase C (PKC) inhibitors on NADPH oxidase (NO) activation by the phorbol ester PMA and by the chemotactic peptide FMLP were studied. H-7 reduced the effects of both stimuli in human neutrophils (HN) and HL-60 cells by 13-63%. Polymyxin B did not inhibit NO activation by PMA and FMLP in HN and reduced the effects of both stimuli in HL-60 cells by 27-55%. Retinal and retinoic acid enhanced the effects of PMA and FMLP in HL-60 cells and of FMLP in HN up to 4.5-fold. In contrast, retinoic acid inhibited the effect of PMA in HN. In the presence of cytochalasin B, retinal inhibited the effect of FMLP in HN, whereas retinoic acid inhibited NO activation by FMLP in both cell types. The dual PKC/calmodulin inhibitors trifluoperazine and W-7 abolished NO activation by PMA and FMLP in HN and HL-60 cells. Thus, the effects of PKC inhibitors on NO activation exhibit (1) cell type specificity, (2) stimulus dependency and (3) no correlation with in vitro inhibition of PKC. Our results suggest that studies with PKC inhibitors presently available cannot clarify the role of PKC in NO activation.     

IL-1beta enhances beta2-adrenergic receptor expression in human airway epithelial cells by activating PKC

Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1beta upregulates beta2-adrenergic receptor (beta2-AR) density and beta2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1beta-induced beta-AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta-AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on beta-AR density, and 3) IL-1beta treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1beta (0.2 nM for 18 h) increased beta-AR density to 213% of control values (P < 0.001). PMA (1 microM for 18 h) increased beta-AR density to 225% of control values (P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1beta-induced upregulation of beta-AR in dose-dependent fashion. PKC isozymes detected by Western blotting included alpha, betaII, epsilon, mu, zeta, and lambda/iota. IL-1beta increased PKC-mu immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1beta-induced beta-AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-mu isozyme. We conclude that signal transduction pathways activated by PKC-mu upregulate beta2-AR expression in human airway epithelial cells.     

Up-regulation of p21 and TNF-α is mediated in lycorine-induced death of HL-60 cells

Background  

Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia (APL) is a common type of leukemia. Many natural compounds have already been found to exhibit significant anti-tumor effects. Lycorine, a natural alkaloid extracted from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. The survival rate of HL-60 cells exposed to lycorine was decreased, cell growth was slowed down, and cell regeneration potential was inhibited. HL-60 cells exhibited typical apoptotic characteristic. Lycorine can suppress leukemia growth and reduce cell survival and inducing apoptosis of tumor cells. The purpose of this work is to elucidate the mechanism by which lycorine induces APL cells.     

Intracellular Interaction of Interleukin (IL)-32α with Protein Kinase C? (PKC?) and STAT3 Protein Augments IL-6 Production in THP-1 Promonocytic Cells

IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCϵ inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCϵ, and this interaction was totally inhibited by the PKCϵ inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCϵ and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCϵ and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.     

ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells

In this study, we analyzed in ratmyometrial cells the signaling pathways involved in the endothelin(ET)-1-induced extracellular signal-regulated kinase (ERK) activationrequired for the induction of DNA synthesis. We found that inhibitionof protein kinase C (PKC) by Ro-31-8220 abolished ERK activation.Inhibition of phospholipase C (PLC) by U-73122 or of phosphoinositide(PI) 3-kinase by wortmannin partially reduced ERK activation. A similarpartial inhibition was observed after treatment with pertussis toxin orPKC downregulation by phorbol ester treatment. The effect of wortmanninwas additive with that produced by PKC downregulation but not with thatdue to pertussis toxin. These results suggest that bothdiacylglycerol-sensitive PKC, activated by PLC products, anddiacylglycerol-insensitive PKC, possibly activated by aG_i-PI 3-kinase-dependent process, are involved inET-1-induced ERK activation. These two pathways were found to beactivated mainly through the ET_A receptor subtype. ET-1 andphorbol ester stimulated Src activity in a PKC-dependent manner, bothresponses being abolished in the presence of Ro-31-8220. Inhibition of Src kinases by PP1 abrogated phorbol ester- and ET-1-induced ERK activation. Finally, ET-1 activated Ras in a PP1-and Ro-31-8220-sensitive manner. Altogether, our results indicatethat ET-1 induces ERK activation in rat myometrial cells through thesequential stimulation of PKC, Src, and Ras.

     

PKC phosphorylates MARCKS Ser159 not only directly but also through RhoA/ROCK

It is well recognized that phorbol 12,13-dibutyrate (PDBu)-activated PKC directly phosphorylates myristoylated alanine-rich C kinase substrate (MARCKS), whose phosphorylation is used as a marker of PKC activation. However, in SH-SY5Y neuroblastoma cells, Western blotting analyses revealed that Rho-associated coiled-coil kinase (ROCK)-specific inhibitor H-1152 inhibited PDBu-induced phosphorylation, and that a small G-protein inhibitor, toxin B, also inhibited MARCKS phosphorylation. Furthermore, in GST pull-down assays, PDBu induced RhoA activation in SH-SY5Y cells, and this activation was inhibited by PKC inhibitor Ro-31-8220. Finally, we showed that the transfection of a dominant negative form of RhoA inhibited PDBu-induced MARCKS phosphorylation in immunocytochemistries. These findings suggest that some PDBu-induced MARCKS phosphorylation includes the RhoA/ROCK pathway in SH-SY5Y cells.     

FAK overexpression upregulates cyclin D3 and enhances cell proliferation via the PKC and PI3-kinase-Akt pathways

We previously demonstrated that FAK-transfected HL-60 (HL-60/FAK) cells exhibit anti-apoptotic capacity. Here, we report that HL-60/FAK cells proliferate much faster than vector-transfected control (HL-60/Vect) cells with a 1.5-fold faster doubling time. This observation prompted us to investigate the mechanism of how HL-60/FAK cells augment cell proliferation. Since a protein kinase C (PKC) inhibitor, chelerythrine, or a PI3-kinase inhibitor, LY294002, suppressed cell proliferation effectively, both PKC and PI-3-kinase pathways are presumed to be involved in the cell proliferation. Among cyclins and CDKs, cyclin D3 expression was particularly prominent in the HL-60/FAK cells. Among PKC family, particularly PKCalpha, beta and eta isoforms were activated and directly associated with FAK in HL-60/FAK cells. We assumed that FAK activates PKC and PI3-kinase-Akt pathway, which resulted in marked induction of cyclin D3 expression and CDK activity.     

1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL-60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells).

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D(3)-26,23-lactone (1alpha,25-(OH)(2)D(3)-26,23-lactone) analogs on 1alpha,25(OH)(2)D(3)-induced differentiation of human leukemia HL-60 cells thought to be mediated by the genomic action of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non-genomic actions of 1alpha,25-(OH)(2)D(3). We found that the 1alpha,25-(OH)(2)D(3)-26,23-lactone analogs, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9648), inhibited differentiation of HL-60 cells induced by 1alpha,25-(OH)(2)D(3). However, 1beta-hydroxyl diastereomers of these analogs, i.e. (23S)-25-dehydro-1beta-hydroxyvitamin D(3)-26, 23-lactone (1beta-TEI-9647) and (23R)-25-dehydro-1beta-hydroxyvitamin D(3)-26,23-lactone (1beta-TEI-9648), did not inhibit differentiation of HL-60 cells caused by 1alpha,25-(OH)(2)D(3). A separate study showed that the nuclear vitamin D receptor (VDR) binding affinities of the 1-hydroxyl diastereomers were about 200 and 90 times weaker than that of 1alpha-hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In contrast, 1beta,25-dihydroxyvitamin D(3) (1beta,25-(OH)(2)D(3)) and 1beta,24R-dihydroxyvitamin D(3) (1beta,24R-(OH)(2)D(3)) inhibited NB4 cell differentiation but not HL-60 cell differentiation. Collectively, the results suggested that 1-hydroxyl lactone analogs, i.e. TEI-9647 and TEI-9648, are antagonists of 1alpha,25-(OH)(2)D(3), specifically for the nuclear VDR-mediated genomic actions, but not for non-genomic actions.     

Regulation of the human vitamin C transporters expressed in COS-1 cells by protein kinase C [corrected

Protein kinase C (PKC) regulation of l-ascorbic acid transport mediated by the Na+/ascorbic acid transporters, hSVCT1 and hSVCT2, expressed in COS-1 cells was studied using recombinant carboxyl-terminal V5 epitope-tagged forms of the transporters. The PKC activator phorbol 12-myristate 13-acetate (PMA) caused a time-dependent and concentration-dependent decrease (40-60%) in ascorbic acid transport activity. Effects of PMA were not observed with the inactive phorbol ester 4 alpha-phorbol and were reversed by treatment of the cells with the PKC-specific inhibitor Ro-31-8220. Kinetically, the reduction in hSVCT1 and hSVCT2 activity arose from a decrease in maximal velocity with no change in the apparent affinity. Western blot and confocal microscopy analyses indicated that the total pool of hSVCT1 or hSVCT2 proteins expressed in the transfected COS-1 cells remained unaffected by PMA treatment. For hSVCT1 the decrease in L-ascorbic acid correlated with a redistribution of the transporter from the cell surface to intracellular membranes. However, for hSVCT2 there was no apparent change in transporter distribution, suggesting that the PKC-dependent modulation of L-ascorbic acid transport mediated by hSVCT2 was the result of reduced catalytic transport efficiency.     

Possible role of duration of PKC-induced ERK activation in the effects of agonists and phorbol esters on DNA synthesis in Panc-1 cells

Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) have been implicated in the effects of regulatory peptides on proliferation. We studied how ERK was activated by PKC following regulatory peptide or phorbol ester stimulation and we also investigated the effect of ERK activation on proliferation in Panc-1 cells. Panc-1 cells transfected with CCK1 receptors were treated with cholecystokinin (CCK), neurotensin (NT), or phorbol 12-myristate 13-acetate (PMA). DNA synthesis was studied by measuring tritiated thymidine incorporation. PKC isoforms were selectively inhibited with G?6983 and 200 nM Ro-32-0432, their translocation was detected by confocal microscopy and by subcellular fractionation followed by immunoblotting. ERK cascade activation was detected with phosphoERK immunoblotting and inhibited with 20 microM PD98059. PMA and CCK inhibited, NT stimulated DNA synthesis. These effects were inhibited by Ro-32-0432 but not by G?6983 suggesting the involvement of PKCepsilon in proliferation control. Confocal microscopy and subcellular fractionation demonstrated that PMA, CCK, and NT caused cytosol to membrane translocation of PKCepsilon and ERK activation that was inhibited by Ro-32-0432 but not by G?6983. ERK activation was prolonged following PMA and CCK, but transient after NT treatment. PMA, CCK, and NT all activated cyclinD1, while p21CIP1 expression was increased by only PMA and CCK, but not by NT; each of these effects is inhibited by PD98059. In conclusion, our results provide evidence for PKCepsilon-mediated differential ERK activation and growth regulation in Panc-1C cells. Identification of the mechanisms by which these key signaling pathways are modulated could provide a basis for the development of novel therapeutic interventions to treat pancreatic cancer.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.