Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells

Activated Ras utilises several downstream pathways, including the mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway and the phosphoinositide 3-kinase (PI-3k)/Akt pathway, to promote cell proliferation and to inhibit apoptosis. To investigate which pathway plays a major role in Ras-induced drug resistance to chemotherapeutic agents in breast cancer cells, we transfected MCF7 breast cancer cells with a constitutively active H-RasG12V and examined the toxicities of three commonly used breast cancer chemotherapeutic agents, paclitaxel, doxorubicin, and 5-fluorouracil in these cells under the conditions that PI-3K or MEK were selectively inhibited by their respective specific inhibitors or dominant negative expression vectors. We found that Ras-mediated drug resistance is well correlated with resistance to apoptosis induced by anticancer agents in MCF7 breast cancer cells. Although inhibition of MEK/MAPK or PI-3K/Akt can each enhance the cytotoxicity of paclitaxel, doxorubicin, or 5-fluorouracil, inhibition of the PI-3K/Akt pathway seems to have a greater effect than inhibition of the MEK/MAPK pathway in reversing Ras-mediated drug resistance. Our results indicate that the PI-3K pathway may play a more important role in receptor tyrosine kinase-mediated resistance to chemotherapy and suggest that PI-3K/Akt might be a critical target molecule for anticancer intervention in breast cancer.     

EGF调节MT1-MMP和MMP-2的表达及其信号传导机制的研究

目的:探讨表皮生长因子（EGF）在宫颈癌SiHa细胞中对膜型基质金属蛋白酶（MT1-MMP）和基质金属蛋白酶-2（MMP-2）表达的调节作用,明确其相关的信号传导机制。方法:利用EGF作为干预因素,信号通路阻断剂分别阻断表皮生长因子受体（EGFR）、AKT、ERK、p38和JNK的磷酸化,来观察EGF对MT1-MMP和MMP-2表达的影响及相关信号通路。结果:EGF在mRNA和蛋白水平上诱导MT1-MMP的表达增加和抑制MMP-2的表达;MAPK和ERK激酶抑制剂在mRNA和蛋白水平上可阻断这种诱导;PI3-K抑制剂不影响EGF对MT1-MMP的诱导,但可进一步抑制MMP-2的表达。结论:EGFR通过MAPK/ERK信号通路上调MP1-MMP的表达和下调MMP-2的表达,同时还通过PI3-K/AKT的信号通路轻度上调MMP-2的表达。此外,EGF可以提高细胞培养基中MMP-2的活性。     

Fibroblast growth factor and insulin-like growth factor differentially modulate the apoptosis and G1 arrest induced by anti-epidermal growth factor receptor monoclonal antibody

DiFi human colon carcinoma cells are stimulated by the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor (EGF) receptor autocrine loop. Exposure of DiFi cells to monoclonal antibody (mAb) 225, which blocks ligand-induced activation of the EGF receptor, induces G1 arrest and subsequent cell death via apoptosis. We investigated the signal pathways by which basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) modulate mAb 225-induced G1 arrest and apoptosis in DiFi cells. Both bFGF and IGF-1 activated the mitogen-activated protein kinase (MAPK) kinase (MEK) pathway in DiFi cells. Additionally, IGF-1 activated the phosphoinositide 3-kinase (PI-3K)/Akt pathway. Both bFGF and IGF-1 inhibited mAb 225-induced apoptosis; however, bFGF provided sustained protection against apoptosis, while the protection by IGF-1 was only temporary. Also, bFGF reversed the mAb 225-induced increase in the p27(Kip1) level, inhibition of cyclin-dependent kinase-2 (CDK-2) activity, dephosphorylation of the retinoblastoma (Rb) protein and the resultant G1 arrest of the cells. In contrast, IGF-1 did not reverse such effects by mAb 225. The prevention of mAb 225-induced G1 arrest and apoptosis in DiFi cells by bFGF was sensitive to the MEK/MAPK inhibitor PD98059 but not to the PI-3K inhibitor LY294002. In contrast, inhibition of apoptosis by IGF-1 in DiFi cells was sensitive only to LY294002 and not to PD98059. These results further our understanding of how mAb 225 induces apoptosis in DiFi cells.     

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.     

12-O-tetradecanoyl phorbol 13-acetate induces the expression of B7-DC, -H1, -H2, and -H3 in K562 cells

Induction of the B7 family molecules by 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been reported, however, the mechanism by which TPA up-regulates these molecules remains poorly understood. In this study, the expression of B7-DC, -H1, -H2, and -H3 in response to TPA was markedly induced in K562 cells. TPA also induced activation of ERK, p38 mitogen-activated protein kinase (MAPK), JNK, phosphatidylinositol-3-kinase (PI-3K), or nuclear factor (NF)-kappaB. Pre-treatments with protein kinase C (PKC) inhibitors significantly inhibited TPA-induced expression of B7-DC, -H1, -H2, and -H3 mRNA as well as TPA-induced phosphorylation of ERK, p38 MAPK, JNK, and PI-3K. TPA-induced expression of B7-DC, -H1, -H2, and -H3 mRNA was abrogated by pre-treatments with inhibitors of ERK and p38 MAPK. However, inhibition of PI-3K and JNK only caused decrease of TPA-induced B7-DC mRNA and B7-H3 mRNA, respectively. TPA-induced degradation of IkappaB-alpha was markedly abrogated by treatments with PKC inhibitors, but not by treatments with inhibitors of ERK, p38 MAPK, JNK, or PI-3K. NF-kappaB inhibitors significantly attenuated the expression of B7-DC, -H1, -H2, and -H3 mRNA in response to TPA. These results suggest that TPA induces the expression of B7-DC, -H1, -H2, and -H3 mRNA in K562 cells via activation of PKC, ERK, p38 MAPK, and NF-kappaB. Distinctly, the expression of B7-DC mRNA and -H3 mRNA in response to TPA is also PI-3K- and JNK-dependent, respectively.     

Expression and regulation of the novel vascular endothelial growth factor receptor neuropilin-1 by epidermal growth factor in human pancreatic carcinoma

BACKGROUND: It was recently shown that neuropilin-1 (NRP-1), which was described originally as a receptor for the semaphorins/collapsins (ligands involved in neuronal guidance), is a coreceptor for vascular endothelial growth factor (VEGF) and increases the affinity of specific isoforms of VEGF to its receptor, VEGF-R2. METHODS: The authors investigated the expression and regulation of NRP-1 in human pancreatic adenocarcinoma specimens and cell lines. RESULTS: Immunohistochemical analysis revealed that NRP-1 was expressed in 12 of 12 human pancreatic adenocarcinoma specimens but was absent in nonmalignant pancreatic tissue. Northern blot analysis revealed NRP-1 mRNA expression in 8 of 11 human pancreatic adenocarcinoma cell lines. NRP-1 mRNA expression was increased by epidermal growth factor (EGF) but not by tumor necrosis factor alpha in several of the human pancreatic adenocarcinoma cell lines studied. Treating human Panc-48 adenocarcinoma cells with EGF activated Akt and Erk but not P-38. Blockade of the phosphatidylinositol-3 kinase (PI-3K)/Akt, mitogen-activated protein kinase (MAPK)/Erk, or P-38 pathways abrogated EGF-induced NRP-1 expression. Finally, EGF receptor blockade in vivo led to a decrease in NRP-1 expression in an orthotopic model of human pancreatic carcinoma. CONCLUSIONS: NRP-1 is expressed in most human pancreatic adenocarcinomas and cell lines but not in nonmalignant pancreatic tissue. EGF regulates NRP-1 expression through the PI-3K/Akt and MAPK/Erk signaling pathways, and blockade of the EGF receptor is associated with decreased expression of NRP-1 in vivo. NRP-1 may act as a coreceptor for VEGF in pancreatic carcinoma, as it does in other tumor systems, thereby enhancing angiogenesis and the effect of VEGF on the growth of pancreatic adenocarcinoma.     

The mechanism of fucoidan‐induced apoptosis in leukemic cells: Involvement of ERK1/2, JNK,glutathione, and nitric oxide

Fucoidan, a sulfated polysaccharide in brown seaweed, has various biological activities including anti‐tumor activity. We investigated the effects of fucoidan on the apoptosis of human promyeloid leukemic cells and fucoidan‐mediated signaling pathways. Fucoidan induced apoptosis of HL‐60, NB4, and THP‐1 cells, but not K562 cells. Fucoidan treatment of HL‐60 cells induced activation of caspases‐8, ‐9, and ‐3, the cleavage of Bid, and changed mitochondrial membrane permeability. Fucoidan‐induced apoptosis, cleavage of procaspases, and changes in the mitochondrial membrane permeability were efficiently blocked by depletion of mitogen‐activated protein kinase (MAPK) kinase kinase 1 (MEKK1), and inhibitors of MAPK kinase 1 (MEK1) and c Jun NH₂‐terminal kinase (JNK). The phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and JNK was increased in fucoidan‐treated HL‐60, NB4, and THP‐1 cells, but not K562 cells. ERK1/2 activation occurred at earlier times than JNK activation and JNK activation was blocked by MEK1 inhibitor. In addition, fucoidan‐induced apoptosis was inhibited by addition of glutathione and/or L ‐NAME, and fucoidan decreased intracellular glutathione concentrations and stimulated nitric oxide (NO) production. Buthionine‐[R,S]‐sulfoximine rendered HL‐60 cells more sensitive to fucoidan. Depletion of MEKK1 and inhibition of MEK1 restored the intracellular glutathione content and abrogated NO production, whereas inhibition of JNK activation by SP600125 restored intracellular glutathione content but failed to inhibit NO production in fucoidan‐treated HL‐60 cells. These results suggest that activation of MEKK1, MEK1, ERK1/2, and JNK, depletion of glutathione, and production of NO are important mediators in fucoidan‐induced apoptosis of human leukemic cells. © 2010 Wiley‐Liss, Inc.     

Anti-proliferative and pro-apoptotic effects of 3,3'-diindolylmethane in human cervical cancer cells

The antitumor effects of Indo-3-carbinol (I3C) have been proven in many human carcinoma cells. However, the roles of 3,3-diindolylmethane (DIM), an important polymer converted from I3C under pH 5.0-7.0, on the growth and proliferation of cervical cancer HeLa and SiHa cells still remain unrevealed. In the present study, we investigated the potential anti-proliferative and pro-apoptotic effects of DIM on HeLa and SiHa cells. Cell proliferation was detected by Cell Counting kit-8 and apoptosis was analyzed by flow cytometry. In addition, morphological changes accompanying cell apoptosis were observed using an inverted microscope after Hoechst 33258 staining. In addition, expression changes of proteins involved in the MAPK and PI3K pathways were determined by western blotting. DIM treatment inhibited the proliferation and induced apoptosis of HeLa and SiHa cells significantly in a time- and dose-dependent manner. Moreover, SiHa cells were more sensitive to DIM treatment than HeLa cells (P<0.05). In addition, the expression of ERK, p38 and p-p38, which are involved in the MAPK pathway, was downregulated by DIM treatment. Another protein involved in the MAPK pathway, JNK, was upregulated. Furthermore, DIM treatment significantly suppressed the expression of Akt, p-Akt, PI3K p110α, PI3K p110β, PI3K class III, GSK3-β, p-PDK1 and p-c-Raf which are involved in the PI3K pathway. These results demonstrate that DIM exerts antitumor effects on HeLa and SiHa cells through its anti-proliferative and pro-apoptotic roles, especially for SiHa cells. The molecular mechanism for these effects may be related to its regulatory effects on MAPK and PI3K pathway and apoptosis proteins. DIM may be a preventive and therapeutic agent against cervical cancer.     

Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitor adaphostin proceeds through a RAF-1/MEK/ERK- and AKT-dependent process

Effects of the tyrphostin tyrosine kinase inhibitor adaphostin (NSC 680410) have been examined in human leukemia cells (Jurkat, U937) in relation to mitochondrial events, apoptosis, and perturbations in signaling and cell cycle regulatory events. Exposure of cells to adaphostin concentrations > or =0.75 microM for intervals > or =6 h resulted in a pronounced release of cytochrome c and AIF, activation of caspase-9, -8, and -3, and apoptosis. These events were accompanied by the caspase-independent downregulation of Raf-1, inactivation of MEK1/2, ERK, Akt, p70S6K, dephosphorylation of GSK-3, and activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK. Adaphostin also induced cleavage and dephosphorylation of pRb on CDK2- and CDK4-specific sites, as well as the caspase-dependent downregulation of cyclin D1. Inducible expression of a constitutively active MEK1 construct markedly diminished adaphostin-induced cytochrome c and AIF release, JNK activation, and apoptosis in Jurkat cells. Ectopic expression of Raf-1 or constitutively activated (myristolated) Akt also significantly attenuated adaphostin-induced apoptosis, but protection was less than that conferred by enforced activation of MEK. Lastly, antioxidants (e.g., L-N-acetylcysteine; L-NAC) opposed adaphostin-mediated mitochondrial dysfunction, Raf-1/MEK/ERK downregulation, JNK activation, and apoptosis. However, in contrast to L-NAC, enforced activation of MEK failed to block adaphostin-mediated ROS generation. Together, these findings demonstrate that the tyrphostin adaphostin induces multiple perturbations in signal transduction pathways in human leukemia cells, particularly inactivation of the cytoprotective Raf-1/MEK/ERK and Akt cascades, that culminate in mitochondrial injury, caspase activation, and apoptosis. They also suggest that adaphostin-related oxidative stress acts upstream of perturbations in these signaling pathways to trigger the cell death process.     

Aquaporin 8 Involvement in Human Cervical Cancer SiHa Migration via the EGFR-Erk1/2 Pathway

Overexpression of aquaporins (AQPs) has been reported in several human cancers. Epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinases 1/2 (Erk1/2) are associated with tumorigenesis and cancer progression and may upregulate AQP expression. In this study, we demonstrated that EGF (epidermal growth factor) induces SiHa cells migration and AQP8 expression. Wound healing results showed that cell migration was increased by 2.79-1.50-fold at 24h and 48h after EGF treatment. AQP8 expression was significantly increased(3.33-fold) at 48h after EGF treatment in SiHa cells. An EGFR kinase inhibitor, PD153035, blocked EGFinduced AQP8 expression and cell migration and AQP8 expression was decreased from 1.59-fold (EGF-treated) to 0.43-fold (PD153035-treated) in SiHa. Furthermore, the MEK (MAPK (mitogen-activated protein kinase)/ Erk (extracellular signal regulated kinase)/Erk inhibitor U0126 also inhibited EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 1.21-fold (EGF-treated) to 0.43-fold (U0126-treated).Immunofluorescence microscopy further confirmed the results. Collectively, our findings show that EGF induces AQP8 expression and cell migration in human cervical cancer SiHa cells via the EGFR/Erk1/2 signal transduction pathway.     

Epidermal growth factor (EGF) suppresses staurosporine-induced apoptosis by inducing mcl-1 via the mitogen-activated protein kinase pathway

Overexpression of epidermal growth factor receptor (EGFR) and establishment of transforming growth factor alpha (TGF alpha)/EGF autocrine system are frequently detected in tumor cells. In addition to mitogenic ability, we demonstrate in this report that EGF protects a human esophageal carcinoma (CE) cell line, CE81T/VGH, from staurosporine-induced apoptosis. The anti-apoptotic signal of EGF is alleviated by a MEK inhibitor PD98059 or an ERK2 dominant negative mutant but not by a phosphatidylinositol-3'-kinase (PI-3K) inhibitor wortmannin. Furthermore, v-raf blocks apoptosis induced by staurosporine. This evidence implies that the survival signal of EGF is mediated via the Raf-MEK-ERK pathway but not the PI3-K pathway. The survival effect of EGF is coincident with the induction of mcl-1, an antiapoptotic gene in the bcl-2 family. PD98059 also suppresses the induction of Mcl-1 by EGF, implying that EGF may up-regulate Mcl-1 via the MAP kinase pathway. Overexpression of mcl-1 is sufficient to protect against apoptosis, while transfection of a mcl-1 antisense plasmid causes cell death. The expression of mcl-1 antisense plasmid also suppresses the anti-apoptotic effect of EGF. Taken together, these results indicate that EGF may up-regulate Mcl-1 through the MAP kinase pathway to suppress apoptosis.     

Inhibition of extracellular signal-regulated kinase (ERK) mediates cell cycle phase independent apoptosis in vinblastine-treated ML-1 cells

Chemotherapeutic agents induce alterations in intracellular signal transduction cascades that culminate in the initiation of the apoptotic program. Here, the relationship between the mitogen-activated protein kinase (MAPK) response and apoptosis in ML-1 cells treated with vinblastine and paclitaxel was investigated. We show that these compounds elicit different effects on MAPKs with vinblastine, but not paclitaxel, increasing both c-Jun-NH2-terminal kinase (JNK) and p38 activity. However, vinblastine and paclitaxel both induced apoptosis with similar kinetics, suggesting that increased JNK and p38 activity is not required for apoptosis that is induced by microtubule interfering agents. Strikingly, the abrogation of extracellular signal-regulated kinase (ERK)-signaling by the MAPK/ERK kinase (MEK)1/2 inhibitor PD098059 in combination with vinblastine robustly induced apoptosis in ML-1 cells at a rate much faster than treatment with vinblastine alone and occurred at all phases of the cell cycle. This apoptotic induction was attributed to JNK activation because: (a) non-JNK-activating concentrations of vinblastine failed to increase apoptosis in the presence of PD098059; (b) apoptosis induced by paclitaxel, which did not activate JNK, was not potentiated by PD098059; and (c) transduction of an inhibitor of JNK activity partially suppressed both JNK activity and apoptosis induced by vinblastine plus PD098059. Additionally, we found that the activation of JNK by vinblastine occurred upstream of effector caspase activation because treatment with a pan-specific caspase inhibitor (valine-alanine-aspartate-fluoromethylketone) resulted in complete abrogation of apoptosis with no effect on MAPK signaling. Taken together, these data suggest that inhibition of the MEK-->ERK signal transduction cascade alleviates cell cycle dependence for vinblastine-induced apoptosis by a mechanism that requires JNK activation.     

Antitumor activity of pimasertib,a selective MEK 1/2 inhibitor,in combination with PI3K/mTOR inhibitors or with multi‐targeted kinase inhibitors in pimasertib‐resistant human lung and colorectal cancer cells

The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these pathways have been developed as molecular targeted anti‐cancer therapies. The in vitro and in vivo anti‐tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi‐targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib‐sensitive (IC₅₀ for cell growth inhibition of 0.001 µM) or pimasertib‐resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up‐regulated in pimasertib‐resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK‐ and AKT‐dependent signaling pathways in pimasertib‐resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.     

Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells

The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in apoptosis induction by phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived cancer chemopreventive agent, with DU145 and LNCaP human prostate cancer cells as a model. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. Exposure of DU145 and LNCaP cells to growth suppressive concentrations of PEITC resulted in activation of ERK1/2 and JNKs, but not p38 MAPK, in both cell lines. In DU145 cells, the apoptosis induction by PEITC was statistically significantly attenuated by pharmacological inhibition of JNKs with SP600125. Adenovirus-mediated overexpression of Flag-tagged JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), an inhibitor of JNK, also inhibited PEITC-induced apoptosis in DU145 cells. On the other hand, inhibition of ERK1/2 activation with MEK1 inhibitor PD98059 failed to offer protection against PEITC-induced apoptosis in DU145 cells. In LNCaP cells, the PEITC-induced cell death was not affected by either pretreatment with PD98059 or SP600125 or overexpression of JBD of JIP-1. These results indicate that involvement of MAPKs in apoptosis induction by PEITC in human prostate cancer cells is cell line-specific.     

Pharmacologic mitogen-activated protein/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase inhibitors interact synergistically with STI571 to induce apoptosis in Bcr/Abl-expressing human leukemia cells.

Interactions between the kinase inhibitor STI571 and pharmacological antagonists of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentrations of STI571 that minimally induced apoptosis (e.g., approximately 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e., > or =24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 microM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of DeltaPsim and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x(L) and Mcl-1; activation of JNK, p38 MAPK, and p34(cdc2); and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1-2 microM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy.     

The phosphatidylinositol 3'-kinase pathway is a dominant growth factor-activated cell survival pathway in LNCaP human prostate carcinoma cells.

Intracellular signaling pathways that mediate survival of prostate carcinoma (PCa) cells are poorly understood. We examined the potential role of the phosphatidylinositol 3' kinase (PI3K) pathway as a mediator of cell survival in LNCaP human PCa cells, which express a variety of properties characteristic of human prostate cancer. LNCaP cell cultures rapidly became apoptotic when treated with the specific PI3K inhibitors, wortmannin and LY294002. In contrast, apoptosis was not induced when the cells were treated with: (a) rapamycin, an inhibitor of the ribosomal S6 kinase pp70S6K, which acts downstream of PI3K; (b) PD98059, a specific inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) kinase (MEK); or (c) the antiandrogen, Casodex; or when the cells were cultured under androgen-depleted conditions. Apoptosis induced by PI3K inhibition was attenuated by: (a) dihydrotestosterone; or (b) the ErbB1 activating ligands [epidermal growth factor (EGF), transforming growth factor alpha, or heparin-binding EGF-like growth factor]. In response to ErbB1 activation by ligand, the p85 regulatory subunit of PI3K associated specifically with ErbB3 but not detectably with ErbB1. The anti-apoptotic effect of ErbB1 activation was significantly reduced when cells were treated simultaneously with wortmannin and PD98059. These data indicate that survival signals can be evoked in LNCaP cells by several distinct pathways and can be triggered by nuclear and cell-surface receptors. Constitutive signaling through the PI3K pathway is required to prevent cell death in LNCaP, whereas activation of the Erk/MAPK and androgen response pathways is not obligatory for cell survival. These results also show that survival signals, as distinguished from mitogenic signals, can be evoked in PCa cells by ErbB1 ligands known to be synthesized within the human prostate.     

Hypoxia increases resistance of human pancreatic cancer cells to apoptosis induced by gemcitabine.

PURPOSE: Hypoxia, frequently found in the center of solid tumor, is associated with resistance to chemotherapy by activation of signaling pathways that regulate cell pro-liferation, angiogenesis, and apoptosis. We determined whether hypoxia can increase the resistance of human pancreatic carcinoma cells to gemcitabine-induced apoptosis by activation of phosphatidylinositol 3'-kinase (PI3K)/Akt, MEK/mitogen-activated protein kinase (extracellular signal-regulated kinase) [MAPK(Erk) kinase (MEK)], and nuclear factor kappa B (NF-kappa B) signaling pathways. EXPERIMENTAL DESIGN: We evaluated the phosphorylation of Akt and MAPK(Erk), DNA binding activity of NF-kappa B, and apoptosis induced by gemcitabine in L3.6pl human pancreatic cancer cells under normoxic and hypoxic conditions. We then examined the effects of the PI3K inhibitor LY294002, MEK inhibitor U0126, and the epidermal growth factor receptor tyrosine kinase inhibitor PKI 166 on these signaling pathways and induction of apoptosis. RESULTS: Hypoxic conditions increased phosphorylation of Akt and MAPK(Erk) and NF-kappa B DNA binding activity in L3.6pl cells. The activation of Akt and NF-kappa B was prevented by LY294002, whereas the activity of MAPK(Erk), but not NF-kappa B, was inhibited by U0126. The increased activation of Akt, NF-kappa B, and MAPK(Erk) was inhibited by PKI 166. Under hypoxic conditions, L3.6pl cells were resistant to apoptosis induced by gemcitabine. The addition of LY294002 or PKI 166 abrogated cell resistance to gemcitabine, whereas U0126 only partially decreased this resistance. CONCLUSIONS: These data demonstrate that hypoxia can induce resistance of pancreatic cancer cells to gemcitabine mainly through the PI3K/Akt/NF-kappa B pathways and partially through the MAPK(Erk) signaling pathway. Because PKI 166 prevented the activation of PI3K/Akt/NF-kappa B and MAPK(Erk) pathways, the combination of this tyrosine kinase inhibitor with gemcitabine should be an effective therapy for pancreatic cancer.     

Phosphorylation of FADD is critical for sensitivity to anticancer drug-induced apoptosis

FADD has been shown to be phosphorylated at Ser194 at the G2/M transition of the cell cycle. Here we have investigated the contribution of this phosphorylation to apoptosis induced by anticancer drugs in two human prostate cancer cell lines, LNCaP and DU145. Both were arrested at G2/M and FADD was found to be phosphorylated at Ser194 on treatment with paclitaxel. Inhibition of paclitaxel-induced c-jun NH2-terminal kinase (JNK) activation by treatment with a specific inhibitor, SP600125, or overexpression of a dominant-negative mutant form of upstream kinases, MEK kinase 1 (MEKK1) and mitogen-activated protein kinase kinase (MKK) 7, significantly reduced the increase in phosphorylated FADD. It is noteworthy that pretreatment with paclitaxel significantly up-regulated MEKK1 expression, resulting in enhancement of etoposide- or cisplatin-induced MEKK1/MKK7-dependent JNK activation and apoptosis in LNCaP and DU145 cells. Interestingly, MEKK1 up-regulation and the synergistic effects of paclitaxel on anticancer drug-induced apoptosis were abolished by overexpression of mutant FADD (Ser194-->Ala). The results clearly show that FADD phosphorylation at Ser194 affects functions both upstream and downstream of the MEKK1/MKK7/JNK1 pathway and is closely associated with chemosensitivity in prostate cancer cells. This is the first report indicating that phosphorylated FADD plays an essential role in the mechanisms of amplifications of chemotherapy-induced apoptosis.     

Inhibition of ErbB-2 and ErbB-3 expression by quercetin prevents transforming growth factor alpha (TGF-alpha)- and epidermal growth factor (EGF)-induced human PC-3 prostate cancer cell proliferation

Because ErbB-2 receptor is involved in hormone-independency for growth and metastasis of prostate cancer cells, the aim was to investigate the effects of quercetin on ErbB-2 and ErbB-3 expression and its critical components such as MAP kinase and PI-3 kinase. Hemocytometric counts and [3H]-thymidine incorporation were used to determine the effects of quercetin, EGF and TGF-alpha on cell proliferation and DNA synthesis in PC-3 and LnCap cells. Changes in ErbB-2, ErbB-3 and components of MAPK and PI-3K pathways were analyzed by Western blot analysis. Treatment of PC-3 and LnCap cells with quercetin resulted in a dose-dependent growth inhibition. The rate of DNA synthesis was decreased by 40, 55 and 65% on treatment with 14.5, 29.0 and 58.0 microM of quercetin, respectively. Concomitantly, these treatments led to a dose-dependent decrease in ErbB-2, ErbB-3 and their basal autophosphorylation levels as compared to controls. Cyclin D1 expression and basal phosphorylation of c-Raf, MAPK, Elk-1 and Akt-1 in PC-3 cells was also inhibited by quercetin treatment. Co-treating PC-3 cells with quercetin significantly attenuated EGF- and TGF-alpha-induced growth and phosphorylation of ErbB-2, ErbB-3, c-Raf, MAPK kinase 1/2 (MEK1/2), MAPK, Elk-1 and Akt-1. Since ErbB receptor is important for growth, metastasis and drug resistance, inhibition of ErbB-2 and ErbB-3 by pharmacological doses of quercetin may provide a new approach for treatment of prostate cancers.     

Multiple signal pathways are involved in the mitogenic effect of 5(S)-HETE in human pancreatic cancer

Pancreatic carcinoma is characterized by poor prognosis and lack of response to conventional therapy. The reasons for this are not fully understood. We have reported that inhibition of 5-lipoxygenase abolished proliferation and induced apoptosis in pancreatic cancer cells while the 5-lipoxygenase metabolite, 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE] stimulated pancreatic cancer cell proliferation. The current study was designed to investigate the underlying mechanisms for 5(S)-HETE-stimulated proliferation of pancreatic cells. Two human pancreatic cancer cell lines, PANC-1 and HPAF, were used. Cell proliferation was monitored by thymidine incorporation and cell counting. Phosphorylation of P42/44(MAPK) (mitogen activated protein kinase, ERK), MEK (MAPK/ERK kinase), P38 kinase, JNK/SAPK (c-Jun N-terminal kinase/ stress-activated protein kinase), AKT and tyrosine residues of intracellular proteins was measured by Western blot using their corresponding phospho-specific antibodies. The results showed that (1) 5(S)-HETE markedly stimulated pancreatic cancer cell proliferation in a time- and concentration-dependent manner; (2) 5(S)-HETE induced tyrosine phosphorylation of multiple intracellular proteins while the tyrosine kinase inhibitor, genestein, blocked 5(S)-HETE-stimulated cell proliferation; (3) 5(S)-HETE significantly stimulated both MEK and P42/44(MAPK) phosphorylation and the MEK inhibitors, PD098059 and U0126, inhibited 5(S)-HETE-stimulated proliferation in these two cell lines; (4) 5(S)-HETE also stimulated P38 kinase phosphorylation but the P38 inhibitor, SB203580, did not effect 5(S)-HETE-stimulated cell proliferation; (5) 5(S)-HETE markedly stimulated AKT phosphorylation while the phosphatidylinositide-3 (PI3)-kinase inhibitor, wortmannin, blocked 5(S)-HETE-stimulated cell proliferation; (6) phosphorylation of JNK/SAPK was not induced by 5(S)-HETE, and (7) the general protein kinase C (PKC) inhibitor, GF109203X, did not affect 5(S)-HETE-stimulated cancer cell proliferation. These findings suggest that intracellular tyrosine kinases, MEK/ERK and PI3 kinase/AKT pathways are involved in 5(S)-HETE-stimulated pancreatic cancer cell proliferation but P38 kinase, JNK/SAPK and PKC are not involved in this mitogenic effect.