Involvement of nucleophosmin/B23 in TPA-induced megakaryocytic differentiation of K562 cells

Human myelogenous leukaemia K562 cells were induced to undergo megakaryocytic differentiation by treatment with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (20 nM, 24-72 h). The steady-state level of nucleophosmin/B23 mRNA decreased during the TPA-induced differentiation. There was also decrease in the level of cellular nucleophosmin/B23 protein and appearance of its degraded product (25 kDa) during the TPA-induced differentiation. Furthermore, K562/B23 (wild type), K562/D1 (delta280-294) and K562/D2 (delta263-294) cells were less, while K562/D3 (delta244-294) cells were more responsive to TPA-induced differentiation as compared to K562/vector or parental K562 cells. Activation of the ERK/MAPK was observed in parental K562 cells upon TPA treatment (5 nM, 5-30 min). As compared to K562/vector cells, less activation of ERK/MAPK was observed in K562/D2 cells, while ERK/MAPK was highly activated in K562/D3 cells upon TPA treatment. Our results indicate that nucleophosmin/B23 plays an important role in TPA-induced differentiation of K562 cells and the amino acids 244-294 at C-terminal of nucleophosmin/B23 could be an important site for regulation of cellular response to differentiation.     

Novel function of the chromosome 7 open reading frame 41 gene to promote leukemic megakaryocyte differentiation by modulating TPA-induced signaling

12-O-tetradecanoylphorbol-13-acetate (TPA) activates multiple signaling pathways, alters gene expression and causes leukemic cell differentiation. How TPA-induced genes contribute to leukemic cell differentiation remains elusive. We noticed that chromosome 7 open reading frame 41 (C7ORF41) was a TPA-responsive gene and its upregulation concurred with human megakaryocyte differentiation. In K562 cells, ectopic expression of C7ORF41 significantly increased CD61 expression, enhanced ERK and JNK signaling, and upregulated RUNX1 and FLI1, whereas C7ORF41 knockdown caused an opposite phenotype. These observations suggest that C7ORF41 may promote megakaryocyte differentiation partially through modulating ERK and JNK signaling that leads to upregulation of RUNX1 and FLI1. In supporting this, C7ORF41 overexpression rescued megakaryocyte differentiation blocked by ERK inhibition while JNK inhibition abrogated the upregulation of FLI1 by C7ORF41. Furthermore, we found that Y34F mutant C7ORF41 inhibited megakaryocyte differentiation. nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was the major activator of C7ORF41 that in turn repressed NF-κB activity by inhibiting its phosphorylation at serine 536, while MAPK/ERK was the potent repressor of C7ORF41. Finally, we showed that C7ORF41 knockdown in mouse fetal liver cells impaired megakaryocyte differentiation. Taken together, we have identified the function of a novel gene C7ORF41 that forms interplaying regulatory network in TPA-induced signaling and promotes leukemic and normal megakaryocyte differentiation.     

Mutant human tumor suppressor p53 modulates the activation of mitogen-activated protein kinase and nuclear factor-kappaB, but not c-Jun N-terminal kinase and activated protein-1

The roles of the mitogen-activated kinase protein (MAPK) pathway, nuclear factor-kappa B (NF-kappaB), and activator protein-1 (AP-1) in cellular responses to growth factors and mitogen are well established. However, the manner by which these proliferative pathways are affected by the tumor suppressor protein p53 is not fully understood. We report here the results of an investigation of the status of p53 on two human melanoma cell lines with wild-type p53 (SK-Mel-186) or mutant p53 (SK-Mel-110). The basal levels of the activated extracellular-signal regulated kinases 1 and 2 (ERK1/2) were high in cells with wild-type p53, but low in cells with mutant p53. The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of ERK1/2 through the phosphorylation of threonine and tyrosine at 202 and 204, respectively, was demonstrated in both cell lines, however, in a discrete manner. TPA-induced activation of ERK1/2 was sustained in wild-type p53 cells, while only a transient activation was seen in mutant p53 cells. Inhibition of MAPK kinase (MEK), an upstream kinase, by U0126, blocked TPA-induced activation of ERK1/2 in wild-type p53 cells and in mutant p53 cells. Treatment of wild-type p53 (SK-Mel 186) cells with small interfering RNA (siRNA) of p53 displayed a transient induction of activation of ERK1/2 following TPA treatment, indicating that p53 has a role in the regulation of the activation of ERK1/2. NF-kappaB activity decreased significantly in cells with wild-type p53, while enhanced NF-kappaB activity was evident in cells with mutant p53. The expression of either wild-type or mutant p53 had a similar effect on TPA-induced Jun N-terminal kinase (JNK) activation, indicating specificity for the ERK pathway. Similarly, AP-1 binding activity showed a transient variation in both cell lines after TPA treatment but with different kinetics. These observations suggest that both wild-type and mutant p53 can modulate the activation pathways for ERK1/2, and NF-kappaB distinctively, while modulating the pathways of JNK and AP-1 similarly. These differences may influence cellular processes such as proliferation, differentiation, and apoptosis.     

Regulation of hairy-cell survival through constitutive activation of mitogen-activated protein kinase pathways

The hairy cells (HCs) of hairy-cell leukemia are intrinsically activated mature clonal B cells. The aims of this study were to gain further insights into the nature of this activation and to assess its importance for the prolonged HC survival in this chronic disease. We show that HCs contain phosphorylated/activated p38 MAPK, JNK and ERK1/ERK2 (ERK1/2). PKC inhibitors increased the activation of p38 and JNK, but reduced the phosphorylation of ERK1/2. Moreover, PKC inhibition resulted in cell death; cell death was also observed when the activation of ERK1/2 in HCs was abrogated with an inhibitor of MEK1/2 activation. In addition to PKC, active Src kinase was also shown to be involved in the maintenance of Raf-independent ERK activation in HCs. During cell culture on a nonadherent surface, ERK phosphorylation was sustained, while phosphorylation of p38 and JNK decreased. This decrease was not observed in HCs cultured on vitronectin (VN), indicating that p38/JNK activation is probably a consequence of in vivo HC interaction with VN present in abundance in the red pulp of the spleen. Taken together, these results suggest that active p38/JNK make HCs susceptible to apoptosis, but the cells are effectively rescued by ERK activation involving constitutively active PKC and Src. These findings are relevant for the understanding of the prolonged cell survival of HCs and their selective sensitivity to some chemotherapeutic agents.     

Humulone inhibits phorbol ester-induced COX-2 expression in mouse skin by blocking activation of NF-kappaB and AP-1: IkappaB kinase and c-Jun-N-terminal kinase as respective potential upstream targets

Humulone, a bitter acid derived from hop (Humulus lupulus L.), possesses antioxidative, anti-inflammatory and other biologically active activities. Although humulone has been reported to inhibit chemically induced mouse skin tumor promotion, the underlying mechanisms are yet to be elucidated. Since an inappropriate over-expression of cyclooxygenase-2 (COX-2) is implicated in carcinogenesis, we investigated effects of humulone on COX-2 expression in mouse skin stimulated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical application of humulone (10 mumol) significantly inhibited TPA-induced epidermal COX-2 expression. Humulone also diminished TPA-induced DNA binding of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Pre-treatment with humulone attenuated TPA-induced phosphorylation of p65 and nuclear translocation of NF-kappaB subunit proteins. Humulone blunted TPA-induced activation of inhibitory kappaB (IkappaB) kinase (IKK) in mouse skin, which accounts for its suppression of phosphorylation and subsequent degradation of IkappaBalpha. An in vitro kinase assay revealed that humulone could directly inhibit the catalytic activity of IKKbeta. Humulone suppressed the activation of mitogen-activated protein kinases (MAPKs) in TPA-treated mouse skin. The roles of extracellular signal-regulated protein kinase-1/2 and p38 MAPK in TPA-induced activation of NF-kappaB in mouse skin had been defined in our previous studies. The present study revealed that topical application of SP600125, a pharmacological inhibitor of c-Jun-N-terminal kinase (JNK), abrogated the activation of AP-1 and the expression of COX-2 in TPA-treated mouse skin. Taken together, humulone suppressed TPA-induced activation of NF-kappaB and AP-1 and subsequent expression of COX-2 by blocking upstream kinases IKK and JNK, respectively, which may account for its antitumor-promoting effects on mouse skin carcinogenesis.     

Frondoside A has an anti-invasive effect by inhibiting TPA-induced MMP-9 activation via NF-κB and AP-1 signaling in human breast cancer cells

Metastasis and invasion are among the main causes of death in patients with malignant tumors. The aim of this study was to determine the anti-invasive activity of frondoside?A against human breast cancer cells. We investigated the inhibitory effect of frondoside?A on cell clonogenicity, invasion and migration in TPA-stimulated human breast cancer cells at non-cytotoxic concentrations. Frondoside?A significantly attenuated TPA-induced colony formation, invasion and migration in MBA-MB-231 human breast cancer cells. Induction of MMP-9 is especially important for the metastasis of many cancer tumor cell types. Additionally, we found that frondoside?A suppresses TPA-induced MMP-9 enzymatic activity, secretion and expression. This effect was associated with reduced activation of AP-1 and NF-κB, and correlated with enhanced expression of TIMP-1 and TIMP-2. Frondoside?A significantly inhibited the TPA-induced MMP-9 expression possibly via the suppression of AP-1 and NF-κB signaling pathways. Frondoside?A reduces the activation of the PI3K/Akt, ERK1/2 and p38 MAPK signals. These results suggest that the anti-metastatic effects of frondoside?A on human breast cancer cells might result from inhibited TPA activation of AP-1 and NF-κB and reduced TPA activation of PI3K/Akt, ERK1/2 and p38 MAPK signals, ultimately leading to downregulation of MMP-9 expression. These results indicate the role of frondoside?A in metastasis and its underlying molecular mechanisms, thus, suggesting frondoside A as a chemopreventive agent for metastatic breast cancer.     

Involvement of both extracellular signal-regulated kinase and c-jun N-terminal kinase pathways in the 12-O-tetradecanoylphorbol-13-acetate-induced upregulation of p21(Cip1) in colon cancer cells

Protein kinase C (PKC), a family of serine-threonine kinases, has been implicated in the regulation of colon tumorigenesis. However, the specific isoform of PKC involved in this process is not clear. In the present study, we found that treatment of the cultured human colon cancer cell line COLO-205 with a PKC agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in cell-cycle arrest at the G(0)/G(1) phase, decrease in cell number, PKCgamma isoform translocation, and upregulation of p21(Cip1) protein. Pretreatment of the cells with a PKC inhibitor, staurosporine, prevented the TPA-induced upregulation of p21(Cip1) protein. Based on the findings of the present study including that (a) both extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) were activated in the TPA-treated COLO-205 cells, (b) pretreatment with the mitogen-activated protein kinase kinase inhibitor PD98059 but not with the p38 mitogen-activated protein kinase inhibitor SB203580 blocked the TPA-induced p21(Cip1) in COLO-205 cells, and (c) transient transfection of the COLO-205 cells with dominant negative ERK or JNK plasmid significantly suppressed the TPA-induced p21(Cip1) protein induction, we conclude that both the ERK and JNK pathways are involved in the TPA-induced upregulation of p21(Cip1) protein in the COLO-205 cells.     

TPA-induced p21 expression augments G2/M arrest through a p53-independent mechanism in human breast cancer cells

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.     

Protein kinase C alpha trigger Ras and Raf-independent MEK/ERK activation for TPA-induced growth inhibition of human hepatoma cell HepG2

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a well-known activator of both protein kinase C (PKC) and mitogen activated protein kinase (MAPK) signal cascade triggering a lot of effects in many non-tumor and tumor cells. We have reported activation of PKCalpha isozyme was specifically required for TPA-induced ERK (MAPK) signaling that mediated gene expressions of the CDK inhibitors p15(INK4b) and p16 (INK4a) leading to growth inhibition of hepatoma cell HepG2. We further investigated the upstream signal molecule linking PKCalpha to ERK. In the Ras activation assay, HepG2 cell exhibited substantial amount of Ras activity. Treatment of the cell with 50nM TPA for 10min slightly inhibited Ras activity by about 10-20%. Pretreatment of the cell with 10microM manumycin A, which abolish basal Ras activity, did not prevent TPA-triggered ERK phosphorylation. Immunoprecipitation coupled with kinase assay demonstrated that MEK-1 activity was strongly induced by treatment of TPA for 5-30min in HepG2. In contrast, c-Raf activity was not significantly induced by TPA within 5-15min. Consistently, Western blot of Phospho(ser-218/222)-MEK demonstrated that phosphorylation of MEK-1 was greatly induced by 50nM TPA, which can be prevented by the PKC inhibitor Bisindolylmaleimides II. Moreover, pretreatment of the MEK1/2 inhibitor, but not c-Raf inhibitor prevented the TPA-induced ERK phosphorylation, gene expression of p15(INK4b) and p16 (INK4a) and growth inhibition of HepG2. In addition, transient expression of a dominant negative Raf mutant in HepG2 did not prevent these effects of TPA. Constitutive expression of an active PKCalpha mutant in HepG2 enhanced phosphorylation of both MEK and ERK accompanied with induction of gene expression of p16(INK4a) and growth inhibition of HepG2. In contrast, Ras and Raf activity were not increased by expression of active PKCalpha. Taken together, we conclude that PKCalpha may activate MEK, independently of Raf and Ras, to trigger sustained ERK (MAPK) signaling and cell cycle arrest of HepG2 induced by TPA.     

B7-H1 expression is regulated by MEK/ERK signaling pathway in anaplastic large cell lymphoma and Hodgkin lymphoma

B7-H1 is a member of the B7 family that inhibits the function of T-cells through its receptor programmed death-1 (PD-1). We examined B7-H1 expression in anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) and found that it was constitutively expressed in both clinical samples and cell lines. In anaplastic lymphoma kinase–positive (ALK⁺) ALCL cells, B7-H1 expression was suppressed by the blocking of extracellular signal-regulated kinase (ERK) signaling and upregulated by the augmentation of ERK activity by phorbol 13-myristate 12-acetate stimulation, suggesting that B7-H1 expression is regulated by ERK signaling pathway in ALCL. ERK is one of the downstream mediators of nucleophosmin (NPM)/ALK signaling in ALK⁺ALCL, and pharmacological inhibition of ALK was shown to dephosphorylate ERK and down-regulate B7-H1. The involvement of NPM/ALK in B7-H1 expression was also demonstrated by introducing the construct into human non-ALCL lymphoid cell lines, which resulted in B7-H1 expression. In the case of HL, B7-H1 expression was shown to be dependent on the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways. These results suggest that B7-H1 expression is controlled by common ERK signaling pathways in ALCL and HL cells. Our findings provide a potentially effective immunotherapeutic strategy for these B7-H1-expressing tumors. ( Cancer Sci 2009)     

The inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in chronic myelogenous leukemic K562 cells

In this study, the downstream signaling of Bcr-Abl tyrosine kinase responsible for apoptosis resistance was investigated. DNA fragmentation, a hallmark of apoptosis, was observed after 2 days of herbimycin A treatment with a peak on 3 day. During the apoptosis induced by the treatment of herbimycin A, stress-activated protein kinase (SAPK) and p38 kinase were activated time- and dose-dependently, while extracellular signal-regulated kinase (ERK) was inhibited. However, apoptosis was induced by the treatment of PD98059, a specific inhibitor of MEK (MAPK or ERK kinase), not by the treatment of sorbitol, a strong activator of SAPK and p38 kinase. Although K562 cells were very resistant to sorbitol-induced apoptosis, DNA fragmentation was induced rapidly in Jurkat, HL-60 and U937 cells after exposure to sorbitol, despite that these apoptosis-sensitive cells have similar or lower activities of JNK/SAPK and p38 kinase compared with K562 cells after treatment of sorbitol. K562 cells had a much higher basal activity of ERK/MAPK than other apoptosis-sensitive cell lines, which were very susceptible to apoptosis induced by low dose of PD98059 compared with K562 cells. In HL-60 cells, sorbitol-induced apoptosis was prevented by the treatment of phorbol myristate 13-acetate (PMA), which activates the ERK/MAPK pathway, and this was blocked by PD98059. From these results, it could be suggested that the inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in K562 cells.     

Involvement of c-Jun N-terminal kinase in G2/M arrest and FasL-mediated apoptosis induced by a novel indoloquinoline derivative, IQDMA, in K562 cells

N'-(11H-Indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (IQDMA), an indoloquinoline derivative, synthesized in our laboratory, has been demonstrated to be an effective anti-tumor agent in human leukemia cells. Treatment of K562 cells with IQDMA resulted in G2/M phase cell cycle arrest, presumably involving the concomitant up-regulation of p21 and apoptosis through up-regulation of FasL and sequential activation of caspase-8 and caspase-3. In contrast to the lack of appreciable effect on the phosphorylation of ERK and p38 MAPK, activation of JNK was noted when K562 cells were exposed to IQDMA. Moreover, IQDMA-mediated G2/M phase arrest and apoptosis were reversed after treatment with the JNK-specific inhibitors, SP600125 and JNK inhibitor 1. Further investigation showed that SP600125 reduced the activation of FasL, caspase-3, caspase-8, and led to a marked decline of p21. Taken together, our data show that JNK plays an important role in IQDMA-mediated G2/M arrest and apoptosis of K562 cancer cells.     

Immunocytochemical evidence for phorbol ester-induced directional translocations of protein kinase C in HL60, K562, CHO, and E7SKS cells: possible role in differentiation

The effects of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the directions of protein kinase C (PKC) translocation in two leukemic cell lines (HL60 and K562) and two fibroblastic cell lines (CHO and E7SKS), related to their susceptibility to the differentiating effect of TPA, were examined. Immunocytochemical evidence indicated that TPA induced a redistribution (outward) of PKC to the plasma membrane in TPA-sensitive HL60 cells, whereas it caused a translocation (inward) of the enzyme to the nucleus or the perinuclear region in K562, CHO, and E7SKS cells, which are resistant to TPA in terms of cell growth and differentiation. Immunoblot analysis of the nuclear proteins from K562 cells revealed that TPA induced an increase in the amount of immunoreactive proteins. TPA, however, did not increase the amount of these immunoreactive species in nuclei isolated from CHO and E7SKS cells, indicating that the translocated PKC was associated only with perinuclear structures of the TPA-treated cells. It is suggested that directional redistribution of PKC to the plasma membrane, as opposed to the nuclear and perinuclear region, might represent an early event required for the TPA-induced differentiation and maturation of HL60 cells.     

Coadministration of UCN-01 with MEK1/2 inhibitors potently induces apoptosis in BCR/ABL+ leukemia cells sensitive and resistant to ST1571

Interactions between the PKC and Chk1 inhibitor UCN-01 and pharmacologic MEK1/2 inhibitors (e.g., U0126, PD184352) were examined in Bcr/Abl(+) = human leukemia cells (K562, LAMA 84) sensitive and resistant to the Bcr/Abl kinase inhibitor STI571. Coexposure of K562 cells to UCN-01 (e.g., 100 nM) or U0126 (30 microM) resulted in a marked increase in mitochondrial injury (e.g., release of cytochrome c; loss of deltapsi(m)) and apoptosis. Similar results were obtained in other Bcr/Abl(+) cells (e.g., LAMA 84, BV-173) and with other MEK1/2 inhibitors (e.g., PD184352). Exposure of K562 cells to UCN-01 resulted in activation of ERK, an effect that was abrogated by co-administration of MEK1/2 inhibitors. Coadminstration of UCN-01 with U0126 produced multiple perturbations in signal transduction/cell cycle regulatory pathways, including diminished expression of Bcr/Abl, Mcl-1, cylin D(1), and activation of JNK and p34(cdc2). Coadministration of the JNK inhibitor SP600125 attenuated UCN-01/MEK inhibitor- associated lethality, suggesting a functional role for JNK activation in enhanced lethality. Finally, UCN-01 and MEK1/2 inhibitors effectively induced apoptosis in Bcr/Abl(+) cells (e.g., K562 and LAMA 84) overexpressing Bcr/Abl and resistant to STI571. These findings indicate that BcrAbl(+) leukemia cells are sensitive to a strategy combining UCN-01 with MEK/ERK inhibitors that simultaneously disrupts two signaling pathways.     

Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in mouse skin through suppression of extracellular signal-regulated kinase activity and NF-kappaB activation

Recently, there have been considerable efforts to search for naturally occurring substances for the intervention of carcinogenesis. Many components derived from dietary or medicinal plants have been found to possess substantial chemopreventive properties. Curcumin, a yellow coloring ingredient of turmeric (Curcuma longa L., Zingiberaceae), has been shown to inhibit experimental carcinogenesis and mutagenesis, but molecular mechanisms underlying its chemopreventive activities remain unclear. In the present work, we assessed the effects of curcumin on 12-O- tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2) in female ICR mouse skin. Topical application of the dorsal skin of female ICR mice with 10 nmol TPA led to maximal induction of cox-2 mRNA and protein expression at approximately 1 and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, curcumin inhibited the expression of COX-2 protein in a dose-related manner. Immunohistochemical analysis of TPA-treated mouse skin revealed enhanced expression of COX-2 localized primarily in epidermal layer, which was markedly suppressed by curcumin pre-treatment. Curcumin treatment attenuated TPA- stimulated NF-kappaB activation in mouse skin, which was associated with its blockade of degradation of the inhibitory protein IkappaBalpha and also of subsequent translocation of the p65 subunit to nucleus. TPA treatment resulted in rapid activation via phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein (MAP) kinases, which are upstream of NF-kappaB. The MEK1/2 inhibitor U0126 strongly inhibited NF-kappaB activation, while p38 inhibitor SB203580 failed to block TPA-induced NF-kappaB activation in mouse skin. Furthermore, U0126 blocked the IkappaBalpha phosphorylation by TPA, thereby blocking the nuclear translocation of NF-kappaB. Curcumin inhibited the catalytic activity of ERK1/2 in mouse skin. Taken together, suppression of COX-2 expression by inhibiting ERK activity and NF-kappaB activation may represent molecular mechanisms underlying previously reported antitumor promoting effects of this phytochemical in mouse skin tumorigenesis.     

Involvement of JNK-mediated pathway in EGF-mediated protection against paclitaxel-induced apoptosis in SiHa human cervical cancer cells.

We investigated the signalling pathways by which epidermal growth factor (EGF) modulates paclitaxel-induced apoptosis in SiHa human cervical cancer cells. SiHa cells exposed to paclitaxel underwent apoptosis, which was strongly inhibited by EGF. This inhibition of apoptosis by EGF was not altered by pharmacological blockade of phosphatidylinositol 3'-OH kinase (PI-3K) with the PI-3K specific inhibitor LY294002 or blockade of the mitogen-activated protein kinase (MAPK) kinase (MEK) with the MEK specific inhibitor PD98059, or by transfection of the cells with PI-3K or MEK dominant-negative expression vectors. EGF did not stimulate PI-3K/Akt, MEK/MAPK, or p38 MAPK activity in SiHa cells but did transiently activate the c-Jun NH2-terminal kinase (JNK). Co-exposure of SiHa cells to SB202190 at concentrations that inhibit JNK abolished the protective effect of EGF on SiHa cells against paclitaxel-induced apoptosis. Our findings indicate that the JNK signaling pathway plays an important role in EGF-mediated protection from paclitaxel-induced apoptosis in SiHa cells.     

JNK interacting protein 1 (JIP-1) protects LNCaP prostate cancer cells from growth arrest and apoptosis mediated by 12-0-tetradecanoylphorbol-13-acetate (TPA)

12-0-tetradecanoylphorbol-13-acetate (TPA) stimulates protein kinase C (PKC) which mediates apoptosis in androgen-sensitive LNCaP human prostate cancer cells. The downstream signals of PKC that mediate TPA-induced apoptosis in LNCaP cells are unclear. In this study, we found that TPA activates the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 pathway. To explore the possible role that the JNK/c-Jun/AP-1 signal pathway has on TPA-induced apoptosis in LNCaP cells, we stably transfected the scaffold protein, JNK interacting protein 1 (JIP-1), which binds to JNK inhibiting its ability to phosphorylate c-Jun. TPA (10(-9)-10(-7) mol l(-1)) caused phosphorylation of JNK in both wild-type and JIP-1-transfected (LNCaP-JIP-1) cells. It resulted in phosphorylation and upregulation of expression of c-Jun protein in the wild-type LNCaP cells, but not in the JIP-1-transfected LNCaP cells. In addition, upregulation of AP-1 reporter activity by TPA (10(-9) mol l(-1)) occurred in LNCaP cells but was abrogated in LNCaP-JIP-1 cells. Thus, TPA stimulated c-Jun through JNK, and JIP-1 effectively blocked JNK. TPA (10(-12)-10(-8) mol l(-1)) treatment of LNCaP cells caused their growth inhibition, cell cycle arrest, upregulation of p53 and p21waf1, and induction of apoptosis. All of these effects were significantly attenuated when LNCaP-JIP-1 cells were similarly treated with TPA. A previous study showed that c-Jun/AP-1 blocked androgen receptor (AR) signaling by inhibiting AR binding to AR response elements (AREs) of target genes including prostate-specific antigen (PSA). Therefore, we hypothesised that TPA would not be able to disrupt the AR signal pathway in LNCaP-JIP-1 cells. Contrary to expectation, TPA (10(-9)-10(-8) mol l(-1)) inhibited DHT-induced AREs reporter activity and decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, TPA, probably by stimulation of PKC, phosphorylates JNK, which phosphorylates and increases expression of c-Jun leading to AP-1 activity. Growth control of prostate cancer cells can be mediated through the JNK/c-Jun pathway, but androgen responsiveness of these cells can be independent of this pathway, suggesting that androgen independence in progressive prostate cancer may not occur through activation of this pathway.