Activation of the RAF/mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway mediates apoptosis induced by chelerythrine in osteosarcoma

PURPOSE: Chelerythrine, a widely used broad-range protein kinase C inhibitor, induces apoptosis in many cell types. In this study, the mechanism of chelerythrine-induced apoptosis in osteosarcoma was investigated. EXPERIMENTAL DESIGN: Signaling pathways activated by chelerythrine in osteosarcoma were detected by Western blots. Impacts of RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK on apoptosis and cell survival were studied using genetic approaches and pharmacologic pathway-specific inhibitors. RESULTS: Osteosarcoma cells underwent apoptosis rapidly after treatment with chelerythrine. Three parallel MAPKs pathways, including the ERKs, c-Jun NH(2) kinases, and p38, were activated by chelerythrine in a dose-dependent and time-dependent fashion. For the ERKs, the activation was evident at the earliest time point tested (2 minutes) and sustained for >4 hours. Introduction of a dominant-negative H-RAS mutant (17N) partially attenuated ERK activation and delayed the onset of apoptosis induced by chelerythrine. The ERK activation and apoptotic effects of chelerythrine were greatly abrogated by the pharmaceutical inhibitors of MEK, but not by those of c-Jun NH(2) kinase or p38. Moreover, osteosarcoma cells were sensitized to chelerythrine by transient transfection with wild-type MEK1 or constitutively active MEK1 and became resistant with dominant-negative MEK1. Other protein kinase C inhibitors, including GF109203X or G?6976, did not cause ERK activation or apoptosis in the same timeframe tested. CONCLUSION: In osteosarcoma, chelerythrine-induced apoptosis is mediated through activation of the RAF/MEK/ERK pathway. These findings suggest that activating the ERK MAPK, as opposed to inhibiting it, may be a therapeutic strategy in osteosarcoma.     

Fas-induced expression of chemokines in human glioma cells: involvement of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase

Fas transduces not only apoptotic signals through various pathways but also angiogenic and proinflammatory responses in vivo. Human glioma cells express Fas although sensitivity to Fas-mediated cell death is variable, suggesting that Fas may have functions other than apoptosis in these cells. In this study, we addressed alternative functions of Fas expressed on human gliomas by Fas ligation in three human glioma cell lines, CRT-MG, U373-MG, and U87-MG, and the in vivo expression of Fas and chemokines in human glioblastoma multiforme (GBM). Herein, we demonstrate that: (a) stimulation with agonistic anti-Fas monoclonal antibody CH-11 and human recombinant soluble Fas ligand induces expression of the CC chemokine MCP-1 and the CXC chemokine interleukin-8 by human glioma cell lines at the mRNA and protein levels in a dose- and time-dependent manner; (b) selective pharmacological inhibitors of MEK1 (U0126 and PD98059) and p38 mitogen-activated protein kinase (MAPK) (SB202190) suppress Fas-mediated chemokine expression in a dose-dependent manner; (c) Fas ligation on human glioma cells leads to activation of both extracellular signal-regulated kinases ERK1/ERK2 and p38 MAPK; and (d) GBM samples express higher levels of Fas compared with normal control brain, which correlates with increased interleukin 8 expression. These findings indicate that Fas ligation on human glioma cells leads to the selective induction of chemokine expression, which involves the ERK1/ERK2 and p38 MAPK signaling pathways. Therefore, the Fas-Fas ligand system in human brain tumors may be involved not only in apoptotic processes but also in the provocation of angiogenic and proinflammatory responses.     

Big mitogen-activated protein kinase 1/extracellular signal-regulated kinase 5 signaling pathway is essential for tumor-associated angiogenesis

Although big mitogen-activated protein kinase 1 (BMK1) has been shown to be critical for embryonic angiogenesis, the role of BMK1 in tumor-associated neovascularization is poorly understood. Exogenous tumors were established in BMK1+/+, BMK1flox/+, or BMK1flox/flox mice carrying the Mx1-Cre transgene. Induced deletion of host BMK1 gene significantly reduced the volumes of B16F10 and LL/2 tumor xenografts in BMK1flox/flox mice by 63% and 72%, respectively. Examining the tumors in these induced BMK1-knockout animals showed a significant decrease in vascular density. Localized reexpression of BMK1 in BMK1-knockout mice by administration of adenovirus encoding BMK1 restored tumor growth and angiogenesis to the levels observed in wild-type mice. These observations were further supported by in vivo Matrigel plug assays in which vascular endothelial growth factor- and basic fibroblast growth factor-induced neovacularization was impaired by removing BMK1. Through screening with the Pepchip microarray, we discovered that in BMK1-knockout endothelial cells, phosphorylation of ribosomal protein S6 (rpS6) at Ser235/236 was mostly abrogated, and this BMK1-dependent phosphorylation required the activity of p90 ribosomal S6 kinase (RSK). Immunofluorescent analysis of tumor vasculature from BMK1-knockout and control animals revealed a strong correlation between the presence of BMK1 and the phosphorylation of rpS6 in tumor-associated endothelial cells of blood vessels. As both RSK and rpS6 are known to be important for cell proliferation and survival, which are critical endothelial cell functions during neovascularization, these findings suggest that the BMK1 pathway is crucial for tumor-associated angiogenesis through its role in the regulation of the RSK-rpS6 signaling module.     

p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1)-dependent signaling contributes to epithelial skin carcinogenesis

Extracellular signal-regulated kinases (ERK) regulate cellular functions in response to a variety of external signals. However, the specific functions of individual ERK isoforms are largely unknown. Hence, we have investigated the specific function of ERK1 in skin homeostasis and tumorigenesis in ERK1 knockout mice. They spontaneously develop cutaneous lesions and hyperkeratosis with epidermis thickness. Skin hyperproliferation and inflammation induced by application of 12-O-tetradecanoylphorbol-13-acetate (TPA) is strongly reduced in mutant mice. ERK1(-/-) mice are resistant to development of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by TPA. Tumor appearance was delayed, their formation was less frequent, and their number and size were reduced. Keratinocytes obtained from knockout mice showed reduced growth and resistance to apoptotic signals, accompanied by an impaired expression of genes implicated in growth control and invasiveness. These results highlight the importance of ERK1 in skin homeostasis and in the process of skin tumor development.     

Different effects of point mutations within the B-Raf glycine-rich loop in colorectal tumors on mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase and nuclear factor kappaB pathway and cellular transformation

Recently, mutations in the B-Raf gene have been identified in a variety of human cancers, such as melanoma and colorectal carcinoma, and more than 80% of the B-Raf mutations have been V599E. Although other mutations have been reported, their functional consequences are poorly understood. In our earlier study, we demonstrated that colon tumor-associated B-Raf mutations within the kinase activation segment are not necessarily associated with an increase in mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/Erk) or nuclear factor kappaB (NFkappaB) signaling activity or in NIH3T3-transforming ability. In this study, we examined the effect of colon tumor-associated mutations within the B-Raf glycine-rich loop (G loop) on MEK/Erk and NFkappaB signaling and on the transformation of NIH3T3 fibroblasts or IEC-6 intestinal epithelial cells. Of the six G loop mutations examined, only the B-Raf G468A significantly increased MEK/Erk and NFkappaB signaling and NIH3T3 transformation. Only this mutation induced transformed phenotypes of IEC-6 cells. In contrast, the B-Raf G468E mutation significantly decreased MEK/Erk signaling and NIH3T3 transformation and had no effect on NFkappaB signaling. The B-Raf F467C mutation moderately elevated MEK/Erk signaling and NIH3T3 transformation. The other three B-Raf mutations, R461I, I462S, and G463E, did not increase MEK/Erk or NFkappaB signaling or NIH3T3 transformation. Except for F467C, none of the tumors with B-Raf mutations examined in this study had K-Ras mutations. These results suggest that some of the B-Raf G loop mutations reported in colorectal tumors do not increase kinase or transforming activities but might contribute to carcinogenesis via other mechanisms or be irrelevant to carcinogenesis.     

p38 mitogen-activated protein kinase pathway is involved in protein kinase Calpha-regulated invasion in human hepatocellular carcinoma cells

Protein kinase Calpha (PKCalpha) has been suggested to play an important role in tumorigenesis, invasion, and metastasis. In this study, we investigated the signal pathways selectively activated by PKCalpha in human hepatocellular carcinoma (HCC) cells to determine the role of mitogen-activated protein kinases (MAPK) in PKCalpha-mediated HCC migration and invasion. A stable SK-Hep-1 cell clone (siPKCalpha-SK) expressing DNA-based small interfering RNA (siRNA) PKCalpha was established and was then characterized by cell growth, migration, and invasion. The expression of PKCalpha was decreased in siPKCalpha-SK, and cell growth, migration, and invasion were reduced. These changes were associated with the decrease in p38 MAPK phosphorylation level, but not in c-jun-NH(2)-kinase-1/2 (JNK-1/2) and extracellular signal-regulated kinase-1/2 (ERK-1/2). This phenomenon was confirmed in the SK-Hep-1 cells treated with antisense PKCalpha olignucleotide. The p38 MAPK inhibitor SB203580 or dominant negative p38 mutant plasmid (DN-p38) was used to evaluate the dependency of p38 MAPK in PKCalpha-regulated migration and invasion. Attenuation of cell migration and invasion was revealed in the SK-Hep-1 cells treated with the SB203580 or DN-p38, but not with ERK-1/2 inhibitor PD98059 or JNK-1/2 inhibitor SP600125. Overexpression of constitutively active MKK6 or PKCalpha may restore the inactivation of p38 and the attenuation of cell migration and invasion in siPKCalpha-SK. Similar findings were observed in the stable HA22T/VGH cell clone expressing siRNA PKCalpha. This study provides new insight into the role of p38 MAPK in PKCalpha-mediated malignant phenotypes, especially in PKCalpha-mediated cancer cell invasion, which may have valuable implications for developing new therapies for some PKCalpha-overexpressing cancers.     

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.     

Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway is involved in myostatin-regulated differentiation repression

The cytokines of transforming growth factor beta (TGF-beta) and its superfamily members are potent regulators of tumorigenesis and multiple cellular events. Myostatin is a member of TGF-beta superfamily and plays a negative role in the control of cell proliferation and differentiation. We now show that myostatin rapidly activated the extracellular signal-regulated kinase 1/2 (Erk1/2) cascade in C2C12 myoblasts. A more remarkable Erk1/2 activation stimulated by myostatin was observed in differentiating cells than proliferating cells. The results also showed that Ras was the upstream regulator and participated in myostatin-induced Erk1/2 activation because the expression of a dominant-negative Ras prevented myostatin-mediated inhibition of Erk1/2 activation and proliferation. Importantly, the myostatin-suppressed myotube fusion and differentiation marker gene expression were attenuated by blockade of Erk1/2 mitogen-activated protein kinase (MAPK) pathway through pretreatment with MAPK/Erk kinase 1 (MEK1) inhibitor PD98059, indicating that myostatin-stimulated activation of Erk1/2 negatively regulates myogenic differentiation. Activin receptor type IIb (ActRIIb) was previously suggested as the only type II membrane receptor triggering myostatin signaling. In this study, by using synthesized small interfering RNAs and dominant-negative ActRIIb, we show that myostatin failed to stimulate Erk1/2 phosphorylation and could not inhibit myoblast differentiation in ActRIIb-knockdown C2C12 cells, indicating that ActRIIb was required for myostatin-stimulated differentiation suppression. Altogether, our findings in this report provide the first evidence to reveal functional role of the Erk1/2 MAPK pathway in myostatin action as a negative regulator of muscle cell growth.     

Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway

Introduction  

The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion.     

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.     

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.     

Docetaxel-induced apoptosis of human melanoma is mediated by activation of c-Jun NH2-terminal kinase and inhibited by the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway.

PURPOSE: Our studies have shown variable sensitivity of cultured melanoma cells to docetaxel. To better understand this response, we studied the role of signal transduction pathways in modulating docetaxel-induced melanoma killing. EXPERIMENTAL DESIGN: Involvement of c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, and Akt signaling was studied by evaluating their extent of activation in melanoma cells after treatment with docetaxel. The effect of their activation on docetaxel-induced apoptosis was assessed using biochemical inhibitors of the pathways and Western blot analysis of proteins involved. RESULTS: Docetaxel induced activation of both JNK and ERK1/2 but not p38 mitogen-activated protein kinase or Akt kinases. Apoptosis was dependent on activation of JNK and mediated through activation of caspase-2 and caspase-dependent changes in Bax and Bak. The levels of activated JNK in individual lines showed a close correlation with the levels of apoptosis. In contrast, activation of ERK1/2 by docetaxel inhibited apoptosis and the levels of activation in individual lines were inversely correlated to the degree of apoptosis. Studies on the Bcl-2 family proteins seemed to reflect changes induced by activation of JNK and ERK1/2 pathways. Docetaxel-induced JNK activation was required for Bcl-2 phosphorylation as well as caspase-2-dependent activation of Bax and Bak and subsequent mitochondrial release of apoptosis-inducing factor and cytochrome c. In contrast, activation of ERK1/2 resulted in degradation of BH3-only protein Bim and phosphorylation of Bad. CONCLUSIONS: These studies provide further insights into sensitivity of melanoma cells to taxanes and provide a basis for the current rationale of combining taxanes with inhibitors of the Raf-ERK1/2 pathway.     

Green fluorescent protein tagging of extracellular signal-regulated kinase and p38 pathways reveals novel dynamics of pathway activation during primary and metastatic growth

We describe a novel approach that allows detection of primary and metastatic cells in vivo in which either the extracellular signal-regulated kinase (ERK) or the p38 pathway is activated. Our recent findings showed that ERK and p38 kinases regulate, respectively, programs dictating cell proliferation (high ERK-to-p38 ratio) or growth arrest and dormancy (low ERK-to-p38 ratio) in vivo. Thus, we were able to use green fluorescent protein (GFP) to reflect ERK and p38 activities and, consequently, the proliferative state of cancer cells. This was accomplished by transfecting tumorigenic T-HEp3 and HT1080 cells, and dormant D-HEp3 cells, with plasmids coding for Elk-GAL4 or CHOP-GAL4 fusion proteins that, when phosphorylated by either ERK or p38, respectively, transactivated a GFP-reporter gene. The fate of these cells was examined in culture, in primary sites, and in spontaneous metastasis in chick embryos and nude mice. In culture GFP level was directly proportional to the previously established levels of ERK or p38 activation. In contrast, during the first 24 hours of in vivo inoculation, both the tumorigenic and the dormant cells strongly activated the p38 pathway. However, in the tumorigenic cells, p38 activity was rapidly silenced, correcting the ERK/p38 imbalance and contributing to high ERK activity throughout the entire period of tumor growth. In contrast, in the small nodules formed by dormant cells, the level of ERK activity was dramatically reduced, whereas p38 activity remained high. Strong activation of ERK was evident in metastatic sites, whereas p38 activation was silenced in this anatomic location as well. These results show that it is possible to directly measure cancer cell response to microenvironment with this reporter system and that only proliferation-competent cells have the ability to rapidly adapt ERK and p38 signaling for proliferative success. This approach allows isolation and further characterization of metastatic cells with specific signaling signatures indicative of their phenotypes.