PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug — Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.     

Proliferation of human breast cancer cells and anti-cancer action of doxorubicin and vinblastine are independent of PKC-alpha

Protein kinase C (PKC) has been considered for a potential target of anticancer chemotherapy. PKC-alpha has been associated with growth and metastasis of some cancer cells. However, the role of PKC-alpha in human breast cancer cell proliferation and anticancer chemotherapy remains unclear. In this study, we examined whether alterations of PKC-alpha by phorbol esters and PKC inhibitors could affect proliferation of human breast cancer MCF-7 cells and the cytotoxic effect of chemotherapeutic agents. Exposure for 24 h to doxorubicin (DOX) and vinblastine (VIN) caused a concentration-dependent reduction in proliferation of MCF-7 cells. However, these two anticancer drugs altered cellular morphology and growth pattern in distinct manners. Phorbol 12,13-dibutyrate (PDBu, 100 nM), which enhanced activities of PKC-alpha, increased cancer cell proliferation and attenuated VIN (1 microM)-induced cytotoxicity. These effects were not affected in the presence of 10 nM staurosporine. Phorbol myristate acetate (PMA, 100 nM) that completely depleted PKC-alpha also enhanced cancer cell proliferation and attenuated VIN-induced cytotoxicity. Three potent PKC inhibitors, staurosporine (10 nM), chelerythrine (5 microM) and bisindolylmaleimide-I (100 nM), had no significant effect on MCF-7 cell proliferation; staurosporine and chelerythrine, but not bisindolylmaleimide-I, attenuated VIN-induced cytotoxicity. Moreover, neither phorbol esters nor PKC inhibitors had an effect on cytotoxic effects of DOX (1 microM) on MCF-7 cell proliferation. Thus, these data suggest that MCF-7 cell proliferation or the anti-cancer action of DOX and VIN on breast cancer cells is independent of PKC-alpha.     

Protein kinase C desensitization by phorbol esters and its impact on growth of human breast cancer cells

Active phorbol esters such as TPA (12-0-tetra-decanoylphorbol-13-acetate) inhibited growth of mammary carcinoma cells (MCF-7 greater than BT-20 greater than MDA-MB-231 greater than = ZR-75-1 greater than HBL-100) with the exception of T-47-D cells presumably by interacting with the phospholipid/Ca2+-dependent protein kinase (PKC). The nonresponsive T-47-D cells exhibited the lowest PKC activity. A rapid (30 min) TPA-dependent translocation of cytosolic PKC to membranes was found in the five TPA-sensitive cell without affecting cell growth. However, TPA-treatment of more than 10 hours inhibited reversibly the growth of TPA-responsive cells. This effect coincided with the complete loss of cellular PKC activity due to the proteolysis of the translocated membrane-bound PKC holoenzyme (75K) into 60K and 50K PKC fragments. Resumption of cell growth after TPA-removal was closely related to the specific reappearance of the PKC holoenzyme activity (75K) in the TPA-responsive human mammary tumor cell lines suggesting an involvement of PKC in growth regulation.     

Differential effects of protein kinase C-eta on apoptosis versus senescence

Protein kinase C-eta (PKCη) is considered an anti-apoptotic kinase, which promotes cell survival and chemoresistance in several cancers, including breast cancer. We have recently shown that PKCη positively regulates the anti-apoptotic protein Mcl-1 in breast cancer cells, and depletion of PKCη induced proteasomal degradation of Mcl-1. We therefore examined if depletion of PKCη would enhance cellular sensitivity to chemotherapeutic agents. Silencing of PKCη by siRNA attenuated apoptosis induced by doxorubicin and paclitaxel in both MCF-7 and T47D breast cancer cells. While silencing of Mcl-1 caused a substantial increase in apoptosis induced by doxorubicin, the combined knockdown of PKCη and Mcl-1 was less effective. Depletion of PKCη also caused an increase in the abundance of the cell cycle inhibitor p27 and a decrease in the clonogenic survival of MCF-7 and T47D cells. PKCη knockdown was associated with an increase in senescence-associated β-galactosidase (SA-β-gal) activity but this increase was attenuated by knockdown of p27. The suppression of doxorubicin-induced apoptosis by PKCη knockdown was partially relieved when p27 was depleted. Since loss of proliferative capacity during senescence could cause resistance to chemotherapeutic drugs, our results suggest that PKCη knockdown inhibits apoptosis by inducing p27-mediated senescence.     

Estrogenic and antiestrogenic activities of 16alpha- and 2-hydroxy metabolites of 17beta-estradiol in MCF-7 and T47D human breast cancer cells

The comparative mitogenic activities of 17beta-estradiol (E2) and four metabolites, 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 16alpha-hydroxyestradiol (16alpha-OHE2) and 16alpha-hydroxyestrone (16alpha-OHE1) were determined in estrogen receptor (ER)-positive MCF-7 and T47D human breast cancer cells. E2 (1 nM) induced a 7- to 13-fold increase in cell number in both cell lines compared to untreated cells and the mitogenic potencies of 16alpha-OHE1 or 16alpha-OHE2 were comparable to or greater than E2. In contrast, 2-OHE1 and 2-OHE2 were weak mitogens in both cell lines and in cells cotreated with 1 nM E2 and 100 or 1000 nM 2-OHE1 or 2-OHE2, there was a significant inhibition of hormone-induced cell proliferation. The comparative ER agonist/antagonist activities of E2 and the metabolites on transactivation were determined in T47D cells transiently transfected with constructs containing promoter inserts from the cathepsin D (pCD) and creatine kinase B (pCKB) genes. E2, 16alpha-OHE2 and 16alpha-OHE1 induced reporter gene activity in both MCF-7 or T47D cells transfected with pCKB or pCD. In contrast, 2-OHE1 and 2-OHE2 did not exhibit ER agonist activity for these transactivation assays, but in cells cotreated with E2 plus 2-OHE1 or 2-OHE2, there was a significant decrease in the hormone-induced response. These results demonstrate that 16alpha-OHE1/16alpha-OHE2 exhibit estrogenic activities similar to that observed for E2, whereas the 2-catecholestrogens are weak ER agonists (cell proliferation) or antagonists (cell proliferation and transactivation).     

The mitogenic signaling pathway but not the plasminogen activator- inducing pathway of basic fibroblast growth factor is mediated through protein kinase C in fetal bovine aortic endothelial cells

Basic fibroblast growth factor (bFGF) induces cell proliferation and plasminogen activator (PA) activity in transformed fetal bovine aortic endothelial (FBAE) GM 7373 cells. A similar response is observed after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In these cells, bFGF and TPA cause activation of protein kinase C (PKC), as demonstrated by the induction of the phosphorylation of an 87-kD PKC substrate in intact cells and by the increase in membrane-associated PKC activity. Activation of PKC by bFGF or TPA is inhibited in cells made PKC-deficient by pretreatment with high concentrations of TPA. The mitogenic activity of bFGF or of TPA is completely inhibited in PKC- deficient cells or in naive cells treated with the PKC inhibitor H-7. However, these cells proliferate in response to serum, epidermal growth factor, and dibutyryl cyclic AMP. Similar results are obtained in normal FBAE AG 7680 cells. These data indicate that activation of PKC is responsible for the mitogenic activity of bFGF in FBAE cells. On the contrary, the PA-inducing activity of bFGF is unaffected by down- regulation of PKC or by treatment with the PKC inhibitor H-7 in both transformed GM 7373 and normal AG 7680 cells. bFGF induces a rapid 45Ca influx in naive and in PKC-deprived GM 7373 cells. In these cells, addition of EGTA to the incubation medium prevents both the 45Ca influx and the increase in PA activity induced by bFGF, without affecting its mitogenic activity. Even though the involvement of PKC in the increase of cell-associated PA activity induced by bFGF can not be completely dismissed, the present results suggest a role of calcium entry in the modulation of the PA-inducing activity of bFGF.     

The role and proposed mechanism by which oestradiol 17β-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations

Synthesis of the biologically active oestrogen, oestradiol, within breast tumours makes an important contribution to the high concentrations of oestrogens which are present in malignant breast tissues. In breast tumours, oestrone is preferentially converted to oestradiol by the Type I oestradiol 17β-hydroxysteroid dehydrogenase (E2DH). Several growth factors, such as insulin-like growth factor Type I, and cytokines, such as Tumour Necrosis Factor (TNF), have been shown to stimulate E2DH activity in MCF-7 breast cancer cells. As little is known about the regulation of Type I E2DH expression and activity in other breast cancer cell lines, the expression and activity of this enzyme was examined in other oestrogen receptor positive and also oestrogen receptor negative breast cancer cell lines. As it is possible that E2DH activity may be limited by co-factor availability, the effects of exogenous co-factors on enzyme activity in these cell lines was also investigated. For T47D and BT20 breast cancer cells, the addition of exogenous co-factors was found to enhance enzyme activity. TNF, in addition to stimulating E2DH activity in MCF-7 cells, also increased activity in T47D and MDA-MB-231 cells, although to a lesser extent than in MCF-7 cells. An investigation of signalling pathways involved in the regulation of E2DH activity revealed that stimulation of both the protein kinase C (PKC) and PKA pathways may be involved in regulation of E2DH activity. As several growth factors and cytokines have now been found to be involved in regulating E2DH activity, the role that macrophages and lymphocytes have in supplying these factors and the mechanism by which these factors may stimulate tumour growth, is also reviewed.     

The increase in cytosolic levels of protein kinase C precedes the growth factor requirements during the transition stem cells to differentiated progeny in the MCF-7 breast tumor cells.

The expression of regulatory genes within the context of a differentiation program can have profound long-term consequences in tissues with permanent renewing populations. The breast tumor cell line MCF-7 retains in culture some of the characteristics of a unidirectional differentiation pathway. We show that the cytosolic activity of the regulatory enzyme protein kinase C (PKC) precedes and continues the sequence of maturation in pre-differentiated subpopulations derived from a stem cell fraction. However, the activity declines in the most differentiated, post-mitotic fraction. These results indicate that PKC may be considered among the regulatory genes in MCF-7 cells that specify maturation of the stem cell progeny.     

Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways

IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda.     

Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.     

Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells.

Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two molecules require 16 and 6 h, respectively, of activation of PKC to induce 50% of maximal cell proliferation. The requirement for a long-lasting activation of PKC appears to be a mechanism for the control of cell proliferation capable of discriminating among transient nonmitogenic stimuli and long-lasting mitogenic stimuli.     

Regulation of motility and protease expression in PKC-mediated induction of MCF-7 breast cancer cell invasiveness

We investigated a potentially central role of protein kinase C (PKC) in controlling multiple pathways in breast cancer cell invasiveness. To do this we evaluated the ability of pharmacologic agents that alter PKC activity to regulate the behavior of the poorly invasive human breast cancer cell line MCF-7. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) produced a dramatic induction of the invasiveness of these cells (18-fold), an effect that concurrent treatment with the PKC inhibitor Bryostatin-1 was able to block. To characterize alterations in the cellular properties that might be responsible for these effects we measured the impact of these two agents on a number of processes thought to be important for invasiveness. The motility of the cells was first examined; it was markedly increased by treatment with TPA (20-fold) and again, Bryostatin-1 inhibited this stimulation. We next examined the expression of MMP-1, 3, 9, 10, and 11 (matrix metalloproteinases), all of which have been shown to be PKC responsive in other systems. We found that the expression and secretion of MMP-9 were increased by at least 100-fold, though all of the enzyme secreted was in the latent form. Finally, the expression of both urokinase plasminogen activator (UPA) and its receptor (UPAR) were induced after TPA treatment by 8- and 7-fold, respectively. In conclusion, we have shown that stimulation of PKC activity markedly increases the invasiveness of MCF-7 cells, and that this change in behavior is correlated with a coordinated set of biochemical and cellular changes which are likely to contribute to this process. These data highlight the possible utility of PKC inhibitors such as Bryostatin-1 as anti-invasive and/or antimetastatic agents. Bryostatin-1 is currently in early clinical trials as an anticancer agent.     

Estrogenic constituents of the heartwood of Dalbergia parviflora

From the heartwood of Dalbergia parviflora, five compounds, dalparvin A (1), B (2), C (3), dalparvinol C (4), and neokhriol A (5), along with 11 known compounds, kenusanone G (6), cajanin (7), sophorol (8), alpinetin (9), hesperetin (10), 3'-O-methylorobol, odoratin, (2R)(3R)-2,3-trans 7-hydroxy-5-methoxydihydroflavonol, (6aR, 11aR)-3,8-dihydroxy-9-methoxypterocarpan, (6aR, 11aR)- vesticarpan, and methyl-3,4-dihydroxy-2-methoxybenzoate were isolated and characterized. Isolates were evaluated for their cell proliferation stimulatory activity against MCF-7, T-47D, and BT20 human breast cancer cell lines. Along with 7-10, two compounds 2 and 3 stimulated not only MCF-7, but also T-47D human breast cancer cell proliferation. Compound 6 had activity only against MCF-7 cells, and the activity of 7 was more than equivalent to that of daidzein. On the other hand, none of the isolates had any significant effects on BT20 cell proliferation, and these results indicated that the stimulative activity of these compounds was not general to any cell proliferations. Furthermore, these compounds were tested in the estrogen-responsive transient luciferase reporter assay.     

Induction of plasminogen activator activity by phorbol ester in transformed fetal bovine aortic endothelial cells. Possible independence from protein kinase C

12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol (diC8) activate protein kinase C (PKC) in transformed fetal bovine aortic endothelial GM 7373 cells. Both molecules cause a similar increase in membrane-associated PKC activity and in the phosphorylation of a PKC-specific endogenous 87-kDa substrate in intact cells. Even though both TPA and diC8 exert a mitogenic activity in GM 7373 cells, only TPA induces also an increase in cell-associated plasminogen activator (PA) activity. Down-regulation of PKC which follows TPA-pretreatment completely abolishes the mitogenic activity of diC8 and the mitogenic and PA-inducing activity of TPA. However, both the PKC inhibitor H-7 and the down-regulation of PKC which follows a prolonged stimulation with diC8 do not abolish the PA-inducing activity of TPA. The PA-inducing activity of TPA is instead inhibited in cultures incubated in the presence of 1 mM EGTA or in a calcium-free medium. The data indicate that TPA and diC8 induce a different pattern of cellular activation in GM 7373 cells and that the PA-inducing activity of TPA might not be mediated by PKC.     

Quantitative analysis of CD5 antigen modulation by 12-O-tetradecanoylphorbol-13-acetate in T-lymphoblastic leukemia cells: individual response patterns and their relationships with both maturation and protein kinase C content.

Modulation of CD5 expression by TPA was investigated on T-leukemic cell lines corresponding to different stages of ontogeny. These CD5 changes have been analyzed simultaneously with modifications of cell growth, cell cycle, cell surface phenotype, and PKC content. CD5 expression was found 6- to 17-fold increased by TPA in a dose-dependent manner on phenotypically mature T-cells (Jurkat, JM, and T-CLL) while T-cells from earlier stages of differentiation (CEM III, CEM 95, and CEM 44) were found unresponsive. CD5 upregulation on TPA-sensitive JM cells appears correlated with inhibition of cell growth, blockage in G1 phase, and phenotypic maturation (downregulation of CD7 and CD1 antigens) and seemed to be related to PKC activation since DiC8 (a PKC activator) mimicked this TPA effect and H7 (a PKC inhibitor) partially reduced it. On the other hand, on CEM III cells TPA induced no modulation of CD5 antigen, a less dramatic effect on cell growth and cell cycle, but a CD7 downregulation. TPA appeared fully effective in binding and translocating PKC in both CEM III and JM cells, although the PKC activity level was three times higher in the latter. Finally, our study suggests that CD5 expression is at least partially under control of PKC in phenotypically mature neoplastic T-cells while PKC could not be directly involved in the regulation of CD5 antigen in leukemic cells arrested at earlier stages of differentiation.     

Role of PTHrp and PTHrp-engaged pathways in MCF-7 cells migration/invasion.

In this paper the effect of N-terminal parathyroid hormone-related protein (PTHrp) and PTHrp-engaged pathways on MCF-7 breast cancer cell migration/invasivity and matrix metalloproteinases (MMPs) production were investigated. We found that: a) migration is not affected by PTHrp and Forskolin (FK)-activated PKA, while Phorbol Myristate Acetate (PMA)-activated PKC strongly stimulates MCF-7 cells motility. b) MMPs production was unaffected by PTHrp, but FK reduced membrane-type (MT)-1 MMP expression. Conversely, PMA induced a marked increase of MT1-MMP and MMP-9. c) Chemical activation of PKC is not sufficient, by itself, to confer invasive ability to MCF-7 cells, unless they were provided with additional factors, supplied by fibroblasts. d) Matrix invasion likely occurs through an activation cascade, involving at least three components: pro-MMP-9 and MT-1 MMP (supplied by PMA-stimulated MCF-7 cells) and pro MMP-2 (supplied by fibroblasts). e) The selective chemical inhibition of the adenylylciclase (AC)/PKA and phospholipase C (PLC)/PKC pathways confirmed that MCF-7 cells invasivity is not affected by exogenous PTHrp, which can only modulate their growth. However, the PTHrp responsibility in breast cancer invasion cannot be completely excluded. Indeed, fibroblasts are known to respond to PTHrp (which is a normal product of MCF-7 as well as other breast cancer cells) with enhanced release of MMP-2. On the basis of the documented requirement of fibroblast-derived MMP-2 for MCF-7 cell invasivity, a novel humoral fibroblast-breast cancer cell interaction, mediated by PTHrp, can be recognised.     

Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation.

CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26-specific mAb anti-Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF-HSB-2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co-immobilized mAb AC7 and mAb OKT3 (anti-CD3). Cultures of T cells growing in the presence of IL-2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells.