Semilicoisoflavone B Induces Apoptosis of Oral Cancer Cells by Inducing ROS Production and Downregulating MAPK and Ras/Raf/MEK Signaling.

Oral squamous cell carcinoma (OSCC) is the sixth most common type of cancer worldwide. Despite advancement in treatment, advanced-stage OSCC is associated with poor prognosis and high mortality. The present study aimed to investigate the anticancer activities of semilicoisoflavone B (SFB), which is a natural phenolic compound isolated from Glycyrrhiza species. The results revealed that SFB reduces OSCC cell viability by targeting cell cycle and apoptosis. The compound caused cell cycle arrest at the G2/M phase and downregulated the expressions of cell cycle regulators including cyclin A and cyclin-dependent kinase (CDK) 2, 6, and 4. Moreover, SFB induced apoptosis by activating poly-ADP-ribose polymerase (PARP) and caspases 3, 8, and 9. It increased the expressions of pro-apoptotic proteins Bax and Bak, reduced the expressions of anti-apoptotic proteins Bcl-2 and Bcl-xL, and increased the expressions of the death receptor pathway protein Fas cell surface death receptor (FAS), Fas-associated death domain protein (FADD), and TNFR1-associated death domain protein (TRADD). SFB was found to mediate oral cancer cell apoptosis by increasing reactive oxygen species (ROS) production. The treatment of the cells with N-acetyl cysteine (NAC) caused a reduction in pro-apoptotic potential of SFB. Regarding upstream signaling, SFB reduced the phosphorylation of AKT, ERK1/2, p38, and JNK1/2 and suppressed the activation of Ras, Raf, and MEK. The human apoptosis array conducted in the study identified that SFB downregulated survivin expression to induce oral cancer cell apoptosis. Taken together, the study identifies SFB as a potent anticancer agent that might be used clinically to manage human OSCC.

Dissecting the novel partners of nuclear c-Raf and its role in all-trans retinoic acid (ATRA)-induced myeloblastic leukemia cells differentiation.

All-trans retinoic acid (ATRA) is an anti-cancer differentiation therapy agent effective for acute promyelocytic leukemia (APL) but not acute myeloid leukemia (AML) in general. Using the HL-60 human non-APL AML model where ATRA causes nuclear enrichment of c-Raf that drives differentiation and G1/G0 cell cycle arrest, we now observe that c-Raf in the nucleus showed novel interactions with several prominent regulators of the cell cycle and cell differentiation. One is cyclin-dependent kinase 2 (Cdk2). ATRA treatment caused c-Raf to dissociate from Cdk2. This was associated with enhanced binding of Cdk2 with retinoic acid receptor α (RARα). Consistent with this novel Raf/CDK2/RARα axis contributing to differentiation, CD38 expression per cell, which is transcriptionally regulated by a retinoic acid response element (RARE), is enhanced. The RB tumor suppressor, a fundamental regulator of G1 cell cycle progression or arrest, was also targeted by c-Raf in the nucleus. RB and specifically the S608 phosphorylated form (pS608RB) complexed with c-Raf. ATRA treatment induced S608RB-hypophosphorylation associated with G1/G0 cell cycle arrest and release of c-Raf from RB. We also found that nuclear c-Raf interacted with SMARCD1, a pioneering component of the SWI/SNF chromatin remodeling complex. ATRA treatment diminished the amount of this protein bound to c-Raf. The data suggest that ATRA treatment to HL-60 human cells re-directed c-Raf from its historically pro-proliferation functions in the cytoplasm to pro-differentiation functions in the nucleus.

Chemoproteomic Discovery of a Ritanserin-Targeted Kinase Network Mediating Apoptotic Cell Death of Lung Tumor Cells.

Ritanserin was tested in the clinic as a serotonin receptor inverse agonist but recently emerged as a novel kinase inhibitor with potential applications in cancer. Here, we discovered that ritanserin induced apoptotic cell death of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells via a serotonin-independent mechanism. We used quantitative chemical proteomics to reveal a ritanserin-dependent kinase network that includes key mediators of lipid [diacylglycerol kinase α, phosphatidylinositol 4-kinase ß] and protein [feline encephalitis virus-related kinase, rapidly accelerated fibrosarcoma (RAF)] signaling, metabolism [eukaryotic elongation factor 2 kinase, eukaryotic translation initiation factor 2-α kinase 4], and DNA damage response [tousled-like kinase 2] to broadly kill lung tumor cell types. Whereas ritanserin exhibited polypharmacology in NSCLC proteomes, this compound showed unexpected specificity for c-RAF in the SCLC subtype, with negligible activity against other kinases mediating mitogen-activated protein kinase signaling. Here we show that ritanserin blocks c-RAF but not B-RAF activation of established oncogenic signaling pathways in live cells, providing evidence in support of c-RAF as a key target mediating its anticancer activity. Given the role of c-RAF activation in RAS-mutated cancers resistant to clinical B-RAF inhibitors, our findings may have implications in overcoming resistance mechanisms associated with c-RAF biology. The unique target landscape combined with acceptable safety profiles in humans provides new opportunities for repositioning ritanserin in cancer.

6-C-(E-phenylethenyl)naringenin induces cell growth inhibition and cytoprotective autophagy in colon cancer cells.

6-C-(E-phenylethenyl)naringenin (6-CEPN) is a small molecule found in naringenin fortified fried beef. It has been shown to suppress colon cancer cell proliferation, but the underlying mechanisms are not fully understood. Here we demonstrate that 6-CEPN suppresses tumour cell proliferation through cell cycle arrest in G1 phase, induces necrotic cell death and autophagy in colon cancer cells. Blockade of autophagy by knockdown of the essential autophagy proteins, Atg7 or beclin-1, resulted in aggravated cell death in response to 6-CEPN treatment. In addition, genome-wide transcriptome expression profiling by RNA-sequencing revealed that 6-CEPN-mediated gene expression pattern was extremely similar to the transcriptome response induced by a RAS inhibitor salirasib (farnesylthiosalicylic acid [FTS; salirasib]). Subsequent molecular biological and biochemical experiments demonstrated that 6-CEPN indeed strongly inhibited RAS activation, leading to the inhibition of the downstream effector pathways c-Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase kinase and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin. More importantly, our computational molecular docking data showed that 6-CEPN could bind to the active site of isoprenylcysteine carboxyl methyltransferase (Icmt), a critical enzyme for the activation of RAS. Icmt activity assay showed that 6-CEPN inhibited its activity significantly. Knockdown of Icmt by siRNA attenuated 6-CEPN-mediated autophagy and cell death. The present study demonstrates that 6-CEPN induces cell growth inhibition and cytoprotective autophagy in colon cancer cells, at least in part, though inhibition of the Icmt/RAS signalling pathways.

Crosstalk between 6-OHDA-induced autophagy and apoptosis in PC12 cells is mediated by phosphorylation of Raf-1/ERK1/2.

Parkinson's disease (PD) is a degenerative brain disorder characterized by motor symptoms and loss of dopaminergic (DA) neurons in the substantia nigra. The mechanisms for DA cell death in PD have been extensively investigated using PC12 cells treated with a dopamine neurotoxin 6-hydroxydopamine (6-OHDA). 6-OHDA may induce both autophagy and apoptosis in PC12 cells. However, it remains unclear whether crosstalk occurs between autophagy and apoptosis in PC12 cells treated with 6-OHDA and whether Raf-1/ERK1/2 and their phosphorylation status play a role in autophagy. In this study, we used MDC staining assay and flow cytometry and found that 6-OHDA induced autophagy in PC12 cells. This induction was inhibited by the autophagy inhibitor 3-MA. Our electron microscopy observations also supported 6-OHDA induced autophagy in PC12 cells. Apoptosis of PC12 cells was increased with inhibition of autophagy by 3-MA. In addition, Inhibition of Raf-1 resulted in a decreased 6-OHDA-induced autophagy rate among PC12 cells. Phosphorylation levels of Raf-1 and ERK1/2 were increased in PC12 cells treated with 6-OHDA and inhibited by co-treatment with 6-OHDA and 3-MA. These data suggest that crosstalk between 6-OHDA-induced apoptosis and autophagy in PC12 cells may be regulated via the Raf-1/ERK1/2 signaling pathway. Our data suggest a mechanism for 6-OHDA toxicity in PC12 cells, contributing to our understanding of the pathogenesis of PD.

Effects of insulin on human pancreatic cancer progression modeled in vitro.

BACKGROUND: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined. METHODS: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model. RESULTS: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling. CONCLUSIONS: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

The MEK1/2 inhibitor U0126 reverses imatinib resistance through down-regulating activation of Lyn/ERK signaling pathway in imatinib-resistant K562R leukemia cells.

Chronic myelogenous leukemia (CML) is triggered by the constitutively activated BCR-ABL oncoprotein and multiple downstream signaling pathways, including the Raf/MEK/ERK, Akt/mTOR, SRC, and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for CML. However, the development of imatinib resistance has become a new challenge for CML treatment. Here, we investigated the expression levels of the signaling pathways to explore the cause of imatinib resistance and seek new reversing drugs. Our results showed that abnormal activation of the BCR-ABL-independent Lyn/ERK signaling pathway was involved in imatinib-resistance of K562R cells. Furthermore, p-Lyn and p-ERK were up-regulated after treatment with imatinib alone. However, U0126, a MEK1/2 inhibitor, could counteract the up-regulation induced by imatinib, and the combination of imatinib and U0126 could overcome the resistance to imatinib in K562R cells. In conclusion, our studies suggest that the combination of imatinib and an inhibitor of the ERK signaling pathway may be effective in imatinib-resistant CML patients.

Novel C-terminal Hsp90 inhibitor for head and neck squamous cell cancer (HNSCC) with in vivo efficacy and improved toxicity profiles compared with standard agents.

BACKGROUND: Current therapies for HNSCC, especially platinum agents, are limited by their toxicities and drug resistance. This study evaluates a novel C-terminal Hsp90 inhibitor (CT-Hsp90-I) for efficacy and toxicity in vitro and in vivo in an orthotopic HNSCC model. Our hypothesis is that C-terminal inhibitors exhibit improved toxicity/efficacy profiles over standard therapies and may represent a novel group of anticancer agents. METHODS: MDA-1986 HNSCC cells were treated with doses of 17-AAG or KU363 (a CT-Hsp90-I) and compared for antiproliferation by GLO-Titer and trypan blue exclusion and for apoptosis by PARP cleavage and caspase-3 inactivation by Western analysis. In vivo studies in Nu/Nu mice examined an orthotopic model of MDA-1986 cells followed by drug dosing intraperitoneally for a 21-day period (mg/kg/dose: cisplatin = 3.5, low-dose KU363 = 5, high-dose KU363 = 25, 17-AAG = 175). Tumor size, weight, and toxicity (body score) were measured 3×/week. RESULTS: The IC(50) levels for KU363 = 1.2-2 µM in MDA-1986. KU363 induces apoptosis at 1 µM with cleavage of PARP and inactivation of caspase-3 levels after 24 h. Client proteins Akt and Raf-1 were also downregulated at 1-3 µM of drug. In vivo, 100% of controls had progressive disease, while 100% of cisplatin animals showed some response, all with significant systemic toxicity. High-dose KU363 showed 88% of animals responding and low-dose KU363 showed 75% responding. KU363 animals showed significantly less toxicity (P < 0.01) than cisplatin or 17-AAG. CONCLUSION: This novel CT-Hsp90-I KU363 manifests potent anticancer activity against HNSCC, showing excellent in vivo efficacy and reduced toxicity compared with standard agents justifying future translational evaluation.

Cyanidin suppresses ultraviolet B-induced COX-2 expression in epidermal cells by targeting MKK4, MEK1, and Raf-1.

Skin cancer is the most frequently diagnosed cancer in the United States. Ultraviolet B (UVB) rays (wavelength: 280-320nm) play a pivotal role in the development of skin cancer by inducing the expression of inflammatory proteins such as cyclooxygenase-2 (COX-2). Cyanidin, the most plentiful of the plant pigments known as anthocyanidins, is a potent chemopreventive agent. In the present study, we examined the molecular mechanisms underlying the chemopreventive activity of cyanidin and identified its molecular targets. Cyanidin inhibited UVB-induced COX-2 expression and prostaglandin E(2) secretion in the epidermal skin cell line JB6 P+ by suppressing the transactivation of nuclear factor-kappaB and activator protein-1 which are well-known transcription factors regulated by mitogen-activated protein kinase. Cyanidin markedly inhibited the phosphorylation of JNK1/2, ERK1/2, and MEK1/2 than the of MKK4 and Raf-1, two upstream kinases of JNK1/2, ERK1/2, and MEK1/2. Cyanidin significantly suppressed the activities of MKK4, MEK1, and Raf-1 through direct binding. Transient transfection of a small interfering RNA specific for MKK4 inhibited the UVB-induced expression of COX-2 in JB6 P+ cells, as did the expression of a dominant-negative ERK2 mutant. We conclude that MKK4, MEK1, and Raf-1 are targets of cyanidin for the suppression of UVB-induced COX-2 expression.

Reversal effect of Raf-1/Mdr-1 siRNAs co-transfection on multidrug resistance in KBv200 cell line.

Multidrug resistance (MDR) is a major barrier for chemotherapy of many cancers. Mdr-1 plays a key role in the development of MDR as extensively verified. However, the role of Raf-1 overexpression in the development of multidrug resistance in human squamous carcinoma (KBv200) cells remains largely unknown. The aim of this study was to investigate the correlation of Raf-1 overexpression with the development of multidrug resistance in KBv200 cells. Furthermore, we explored the reversal effect of Raf-1 siRNA transfection and Raf-1/Mdr-1 siRNAs co-transfection on the multidrug resistance of KBv200 cells and potential mechanism of reversing the multidrug resistance. MTT and flow cytometry assay were used to investigate the reversal effect of single transfection with either Raf-1 or Mdr-1 siRNA and double transfection with Raf-1/Mdr-1 siRNAs to vincristine of KBv200 cells. RT-PCR, immunofluorescence and Western Blot were used to detect mRNA and protein expression of Raf-1 and multidrug-resistant gene Mdr-1. The results of gene detection showed that the expression levels of both Raf-1 and Mdr-1 were greatly decreased upon Raf-1 silencing alone or in combination with Mdr-1 silencing. Raf-1 or Mdr-1 siRNA single transfection could reverse the multidrug resistance of KBv200 cells effectively. Compared with single transfection, Raf-1/Mdr-1 siRNAs co-transfection can significantly reduce IC(50) values and increase the apoptotic rates of KBv200 cells. The above results suggested that Raf-1 gene may be a novel target for reversing the multidrug resistance of human squamous carcinoma cells. Raf-1/Mdr-1 siRNAs co-transfection might be a promising approach to abrogate the multidrug resistance of cancer cells. The potential mechanism may be via inhibiting the multidrug-resistant gene Mdr-1 expression efficiently.

Epidermal growth factor receptor is involved in clusterin-induced astrocyte proliferation.

We previously reported that clusterin enhances astrocyte proliferation and extracellular signal-regulated kinase (ERK) activity. It, however, remains largely unknown how clusterin promotes cell growth. Here, we investigate the signaling pathway and related molecules underlying astrocyte proliferation by clusterin. Exogenous clusterin stimulates Ras-dependent Raf-1/mitogen-activated protein kinase kinase (MEK)/ERK activation. Clusterin-induced astrocyte proliferation and ERK1/2 phosphorylation were abrogated by either AG1478 (an inhibitor of epidermal growth factor receptor, EGFR) or EGFR small interfering RNA. Furthermore, clusterin treatment provoked tyrosine phosphorylation of EGFR (pY(1173)), which was also blocked by AG1478. These results suggest that clusterin requires EGFR activation to deliver its mitogenic signal through the Ras/Raf-1/MEK/ERK signaling cascade in astrocytes.

Sodium phenylacetate inhibits the Ras/MAPK signaling pathway to induce reduction of the c-Raf-1 protein in human and canine breast cancer cells.

An aromatic fatty acid, phenylacetate (PA), has been shown to have cytostatic, antitumor and cell differentiation-inducing effects on various kinds of tumors. Previously, we have demonstrated cell growth inhibition, malignant phenotype reduction and cell differentiation effects of sodium phenylacetate (NaPA) treatment in a canine mammary tumor cell line. To clarify the molecular mechanism of these effects, we examined the expression of Ras/MAPK signaling pathway-related molecules in human and canine breast cancer cell lines, and found that the level of c-Raf-1 protein was reduced by 5, 10 and 20 mM of NaPA treatments, though Ras activation was maintained. Dephosphorylation of c-Raf-1 at Serine (Ser) 259, Ser 338, and Ser 621 were also seen in NaPA-treated cells. Downstream factors in the pathway, such as mitogen-activated protein kinase/ERK kinase (MEK)1/2 and ERK1/2, showed decreased activity, and accordingly, expressions of cyclinD1, c-myc, and inactivation of p90 ribosomal S6 kinase (RSK), which are MAPK targets, were reduced. We also observed the reduction of cell-cycle-promoted molecules, such as cdc1/cdk2, cdk4, PCNA cyclin A, and cyclin B, and the increased expression of p27kip1. Furthermore, expression of an epithelial marker, E-cadherin, was increased by NaPA treatment. These results suggest that one of the molecular targets of NaPA treatment was the reduction of c-Raf-1 protein, and that its reduction results in the decrease of malignant characteristics of tumor cells through blockage of the Ras/MAPK signaling pathway.

Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

OBJECTIVE: To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)-induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied. METHODS: CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically. RESULTS: EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1)-CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts. CONCLUSION: By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis.

Activation of protein kinase Calpha signaling prevents cytotoxicity and mutagenicity following lead acetate in CL3 human lung cancer cells.

Protein kinase C (PKC) family of serine/threonine protein kinases is sensitive signaling transducers in response to lead acetate (Pb) that could transmit phosphorylation cascade for proliferation and de-differentiation of neural cells. However, little is known as to the impact of PKC on Pb genotoxicity. Here we investigate whether Pb activates the conventional/classical subfamily of PKC (cPKC) signaling to affect cytotoxicity and mutagenicity in CL3 human non-small-cell lung adenocarcinoma cells. Pb specifically promoted membrane localization of the alpha isoform of PKC in CL3 cells. Pb also elicited Raf-1 activation as measured by the induction of phospho-Raf-1S338 and the dissociation from the Raf-1 kinase inhibitor protein. Inhibition of cPKC activity using Gö6976 or depletion of PKCalpha by introducing specific small interfering RNA blocked the induction of phospho-Raf-1S338, phospho-MKK1/2 and phospho-ERK1/2 in cells exposed to Pb. Intriguingly, declining PKCalpha enhanced the Pb cytotoxicity and revealed the Pb mutagenicity at the hprt gene. The results suggest that PKCalpha is obligatory for activation of the Raf-1-MKK1/2-ERK1/2 signaling module and plays a defensive role against cytotoxicity and mutagenicity following Pb exposure. Results obtained in this study also support our previous report showing that ERK1/2 activity is involved in preventing Pb genotoxicity.

Involvement of Gq/11 in both integrin signal-dependent and -independent pathways regulating endothelin-induced neural progenitor proliferation.

We have previously shown that endothelin-B receptor stimulation increases neural progenitor proliferation, partly in G(i) and extracellular matrix molecule-dependent manner. In the present study, we investigated whether G(q/11) is also involved in this response and how G(i) and G(q/11) might regulate the extracellular signal-regulated kinase (ERK) pathway and integrin signaling. Endothelin-induced ERK phosphorylation was independent of integrin ligands, and an inhibitor of G(q/11), YM-254890, as well as pertussis toxin, partially inhibited endothelin-stimulated phosphorylation of Raf-1 and ERK. Endothelin-stimulated protein kinase C (PKC) was partially inhibited by both YM-254890 and pertussis toxin, while only pertussis toxin attenuated endothelin-induced Ras activation. In contrast, endothelin increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in an integrin ligand-dependent manner. Both YM-254890 and pertussis toxin partially inhibited endothelin-stimulated phosphorylation of these proteins. A PKC inhibitor and down-regulation of PKC prevented endothelin-induced phosphorylation of paxillin and ERK. In addition, endothelin-induced proliferation and DNA synthesis were partially inhibited by YM-254890 and pertussis toxin. Taken together, the results indicate that endothelin activates PKC via G(q/11) and G(i), and consequently stimulates the ERK cascade in cooperation with Ras signaling stimulated by G(i). PKC appears to increase tyrosine phosphorylation of paxillin to enhance integrin signaling, which further increases DNA synthesis and proliferation.

Geniposide, a novel agonist for GLP-1 receptor, prevents PC12 cells from oxidative damage via MAP kinase pathway.

Alzheimer's disease (AD) is the most common form of dementia. Glucagon-like peptide-1 (GLP-1) gives a new genre in therapeutic targets for intervention in AD with its neurotrophic and neuroprotective functions. In previous work, we identified that geniposide is a novel agonist for GLP-1 receptor, which shows neurotrophic characteristics to induce the neuronal differentiation of PC12 cells. The aim of this study is to determine whether geniposide prevents neurons from oxidative damage, and to explore its signaling pathways. The results demonstrated that geniposide increased the expression of anti-apoptotic proteins, including Bcl-2 and heme oxygenase-1 (HO-1), to antagonize the oxidative damage in PC12 cells induced by hydrogen peroxide. LY294002 (a PI3K inhibitor) inhibited the effect of geniposide increasing of Bcl-2 level by activation of MAPK, MEK and c-Raf phosphorylation in hydrogen peroxide treated PC12 cells. U0126 (a selective inhibitor of MEK) also attenuated the enhancement of geniposide on Bcl-2 level by inhibiting the phosphorylation of p90RSK in the hydrogen peroxide treated PC12 cells. All these data demonstrate that geniposide, an agonist for GLP-1 receptor, regulates expression of anti-oxidative proteins including HO-1 and Bcl-2 by activating the transcriptor of p90RSK via MAPK signaling pathway in PC12 cells.

A chemical biology approach identifies a beta-2 adrenergic receptor agonist that causes human tumor regression by blocking the Raf-1/Mek-1/Erk1/2 pathway.

A chemical biology approach identifies a beta 2 adrenergic receptor (beta2AR) agonist ARA-211 (Pirbuterol), which causes apoptosis and human tumor regression in animal models. beta2AR stimulation of cAMP formation and protein kinase A (PKA) activation leads to Raf-1 (but not B-Raf) kinase inactivation, inhibition of Mek-1 kinase and decreased phospho-extracellular signal-regulated kinase (Erk)1/2 levels. ARA-211 inhibition of the Raf/Mek/Erk1/2 pathway is mediated by PKA and not exchange protein activated by cAMP (EPAC). ARA-211 is selective and suppresses P-Erk1/2 but not P-JNK, P-p38, P-Akt or P-STAT3 levels. beta2AR stimulation results in inhibition of anchorage-dependent and -independent growth, induction of apoptosis in vitro and tumor regression in vivo. beta2AR antagonists and constitutively active Mek-1 rescue from the effects of ARA-211, demonstrating that beta2AR stimulation and Mek kinase inhibition are required for ARA-211 antitumor activity. Furthermore, suppression of growth occurs only in human tumors where ARA-211 induces cAMP formation and decreases P-Erk1/2 levels. Thus, beta2AR stimulation results in significant suppression of malignant transformation in cancers where it blocks the Raf-1/Mek-1/Erk1/2 pathway by a cAMP-dependent activation of PKA but not EPAC.

Conjugated linoleic acid inhibits Caco-2 cell growth via ERK-MAPK signaling pathway.

Conjugated linoleic acid (CLA) is a naturally occurring compound found in dairy and beef products. In recent years, it has received considerable attention because several studies showed a lower incidence of certain cancers in animals fed CLA-supplemented diets. In vitro studies further showed growth inhibitory activity on tumor cell proliferation, the CLA being effective above all against colon cancer cells. The aim of the present work was to investigate the growth inhibitory effect of CLA on Caco-2 cell line. Under our experimental conditions, CLA repressed Caco-2 cell proliferation, and the growth-inhibitory action increased by repeating treatments. However, in Caco-2 cells, CLA was unable to induce apoptosis, as revealed by cell-cycle analysis and Western blot studies. To determine the mechanism by which CLA inhibits cell growth, we studied its effect on extracellular-regulated kinase signaling. Conjugated linoleic acid reduced expression levels of Raf-1 and phosphorylation of ERK1/2, which was accompanied by a decrease in the expression of the downstream transcription factor c-myc. Our data suggest that CLA is dependent, at least in part, on the ERK kinase pathway for its ability to inhibit the growth of Caco-2 cancer cells.

Cyclophilin A is required for M-CSF-dependent macrophage proliferation.

The immunosuppressor sanglifehrin A (SfA) is a member of a family of immunophilin cyclophilin A-binding molecules and does not inhibit calcineurin activity. Sanglifehrin A inhibits M-CSF-dependent macrophage proliferation by arresting the G1 phase of the cell cycle but does not affect cell viability. This immunosuppressor exerts its action on proliferation by inactivating cyclin-dependent kinase 2 (Cdk2) activity. Moreover, c-myc expression is also repressed. In the early steps of M-CSF signaling, SfA inhibits the phosphorylation of Raf-1 and the external regulated kinases (ERK)1/2 and mitogen-activated protein kinase phosphatase-1, which are required for proliferation. The effects of SfA are not related to a block of the proteosome activity. These data show that immunophilin contributes to M-CSF-dependent proliferation through activation of the Raf-1/MEK/ERK pathway and the regulation of Cdk activities, which is required for cell cycle progression.

Cyclosporin-A inhibits ERK phosphorylation in B cells by modulating the binding of Raf protein to Bcl2.

Extracellular signal-related kinase (ERK) signaling is regulated by sequential phosphorylation of upstream kinases including Raf. We report herein that ERK phosphorylation is inhibited by a short incubation with Cyclosporin-A (CsA) in anti-IgM activated Daudi B cells. As Bcl2, through its BH4 domain, was previously shown to bind both Calcineurin (Can) and Raf proteins, we hypothesized that CsA inhibited Can binding to Bcl2 allowing the latter to bind more Raf at the mitochondria thereby diverting it from activating the ERK cascade. In support of this less Bcl2 coprecipitated with Can heterodimer in total lysates of cells treated with CsA as compared to controls. In parallel, Raf1 was augmented in both the mitochondrial fractions of cells treated with CsA and in Bcl2 immunoprecipitates under CsA. Finally, introduction of a Bcl2 BH4 domain into Daudi cells augmented ERK phosphorylation in unstimulated cells and this augmentation was unsensitive to CsA. We therefore suggest that CsA indirectly inhibited ERK activation through sequestration of Raf1, at the mitochondria.