B-RAF and PI-3 kinase signaling protect melanoma cells from anoikis

A hallmark feature of cancer is resistance to anoikis, apoptosis induced when cells either lose contact with or encounter an inappropriate extracellular matrix. Melanoma is inherently associated with a high degree of resistance to apoptosis. Mutations in B-RAF are prevalent in melanoma and promote constitutive MEK-ERK1/2 signaling and cell transformation. Acquisition of B-RAF mutations correlates with vertical phase growth when melanoma cells invade into the dermis, a collagen-rich environment that also contains fibronectin matrix. In addition, alterations in phosphoinositide-3 kinase (PI-3 kinase) signaling that lead to activation of AKT are detected in advanced melanomas. Here we show that knockdown of B-RAF expression by siRNA or pharmacological inhibition of MEK rendered melanoma cells susceptible to anoikis. Furthermore, adhesion to fibronectin but not collagen protected melanoma cells from anoikis through a PI-3 kinase-dependent pathway. Therefore, melanoma cells require either B-RAF or PI-3 kinase activation for protection from anoikis. Notably, AKT signaling in melanoma cells is substrate specific. These findings demonstrate that melanoma cells utilize multiple signaling pathways to provide resistance to apoptosis.     

Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients

BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients.BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi.A total of 23 melanoma patients (six females, 17 males, aged 47–80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2–23.2) and subsequent for BRAFi 4.5 (1.2–15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed.Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies.     

Protein kinase D2 promotes the proliferation of glioma cells by regulating Golgi phosphoprotein 3

Protein kinase D2 (PKD2) has been demonstrated to promote tumorigenesis in many types of cancers. However, how PKD2 regulates cancer cell growth is largely unknown. In this study, we found that over-expression of PKD2 promoted glioma cell growth but down-regulation of PKD2 inhibited it. Further investigation indicated that PKD2 down-regulation decreased the protein level of Golgi phosphoprotein 3(GOLPH3) as well as p-AKT level. On the contrary, over-expression of PKD2 increased the protein level of GOLPH3 and p-AKT. In addition, GOLPH3 exhibited similar effect on glioma cell growth to that of PKD2. Importantly, GOLPH3 down-regulation partially abolished glioma cell proliferation induced by PKD2 over-expression, while over-expression of GOLPH3 also partially rescued the inhibition effect of PKD2 down-regulation on glioma cell growth. Interestingly, the level of PKD2 and GOLPH3 significantly increased and was positively correlated in a cohort of glioma patients, as well as in patients from TCGA database. Taken together, these results reveal that PKD2 promotes glioma cell proliferation by regulating GOLPH3 and then AKT activation. Our findings indicate that both PKD2 and GOLPH3 play important roles in the progression of human gliomas and PKD2-GOLPH3-AKT signaling pathway might be a potential glioma therapeutic target.     

Breast cancer cells with acquired resistance to the EGFR tyrosine kinase inhibitor gefitinib show persistent activation of MAPK signaling

Although the epidermal growth factor receptor (EGFR) is frequently expressed in human primary breast carcinoma, the majority of breast cancer patients do not respond to treatment with EGFR tyrosine-kinase inhibitors such as gefitinib. We isolated through a stepwise dose escalation of the drug two gefitinib-resistant SK-Br-3 clones, ZD6 and ZD10 (ZD) cells, which showed, respectively, a three- to five-fold increase in the IC₅₀ for gefitinib as compared with parental cells. The levels of expression of EGFR were increased in ZD cells as compared with wild-type SK-Br-3 cells. The phosphorylation of EGFR, ErbB-2, ErbB-3 and Akt was significantly reduced in gefitinib-resistant cells. In contrast, ZD cells showed levels of MAPK phosphorylation similar to untreated wild-type cells when cultured in presence of gefitinib. Persistent activation of MAPK was also observed in gefitinib-resistant clones isolated from MDA-MB-175 and MDA-MB-361 breast cancer cell lines. ZD cells showed an increased sensitivity to the MEK inhibitor PD98059 as compared with SK-Br-3 cells, and a synergistic anti-tumor effect was observed when ZD cells were treated with a combination of gefitinib and PD98059. Overexpression of a constitutively activated form of p42-MAPK in SK-Br-3 cells resulted in an approximately 50% increase in the IC₅₀ to gefitinib. Finally, culture of ZD10 resistant cells in absence of gefitinib led to reversion of the resistant phenotype. These observations suggest that MAPK signaling might play a role in the resistance that develops in breast cancer cells after long-term exposure to gefitinib. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal-regulated kinase signaling in melanoma cell lines

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.     

Suppression of autophagy sensitizes multidrug resistant cells towards Src tyrosine kinase specific inhibitor PP2

Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we employed 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a selective SFK inhibitor, to determine the possible involvement of tyrosine phosphorylation in the modulation of autophagy, for overcoming multidrug resistance. We found that multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) were more susceptible to PP2 treatment than were their parental cells (Ras-NIH 3T3). The antiproliferative activity of PP2 appeared to be due to cell-cycle arrest at G1/S without induction of apoptosis. Interestingly, PP2 preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells, which implies that a high level of autophagy may protect PP2-treated cells from undergoing cell death. PP2-induced autophagy in Ras-NIH 3T3 cells is accompanied by an inhibition of the mTOR signaling pathway. However, we found that in Ras-NIH 3T3/Mdr cells, PP2-induced mTOR inhibition was uncoupled from the induction of autophagy-likely due to the hyperactivation of AMPK by delayed Raf activation. We also found that PP2-induced dissociation of Beclin 1 from Bcl-2 leads to autophagy in Ras-NIH 3T3 cells. Taken together, these results suggest that functional autophagy in response to PP2 may lead to cell survival in Ras-NIH 3T3 cells, while defective autophagy may contribute to inhibition of growth in Ras-NIH 3T3/Mdr cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of cancers with SFK inhibitors.     

The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells

Tumor cells with mutated PTEN proliferate in an EGFR-independent manner. Induction of PTEN sensitizes cells to EGFR inhibition, and the combination causes synergistic apoptosis. Synergy is due to inhibition of two parallel pathways that phosphorylate the proapoptotic protein BAD at distinct sites. Serine 112 phosphorylation is EGFR/MEK/MAPK dependent, whereas serine 136 phosphorylation is PI3K/Akt dependent. Either phosphorylation is sufficient to sequester BAD to 14-3-3. BAD is released and apoptosis is induced only if both serines are dephosphorylated in response to inhibition of both pathways. Reduction of BAD expression by RNA interference prevents apoptosis in response to pathway inhibition. Thus, BAD integrates the antiapoptotic effects of both pathways. Combined inhibition of EGFR and PI3K signaling may be a useful therapeutic strategy.     

Protein kinase C activation by anthracyclines in Swiss 3T3 cells.

The effects of the anti-cancer anthracyclines doxorubicin and daunorubicin on the activity of protein kinase C (PKC) were examined in intact Swiss 3T3 cells. The 2 drugs stimulated the phosphorylation of an 80K phosphoprotein found to be identical to that generated in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate as indicated by gel electrophoresis and peptide mapping. The effect of doxorubicin was dose-dependent in the range 10(-5) to 10(-3) M and was not associated with a detectable translocation of PKC activity from cytosol to the cell membrane. Doxorubicin and daunorubicin were found to increase the incorporation of phosphate into phosphatidic acid, phosphatidylinositol 4-monophosphate and phosphatidyl inositol 4,5-bisphosphate. In addition, the anthracyclines induced a rise in inositol phosphates, thus indicating a stimulation of the breakdown of phosphoinositides. These data are consistent with an indirect mechanism of PKC activation by anthracyclines. We propose that diacylglycerol, which is derived from the hydrolysis of phospholipids, (including the phosphoinositides), by activation of phospholipases, could mediate PKC activation. The described effects, involving cell-signal-transducing pathways, emphasize a new aspect of the cellular actions of these anti-tumor agents.     

Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells by stabilization of death receptors

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin (ADAM) activity. We have previously shown that adenovirally expressed tissue inhibitor of metalloproteinases-3 (TIMP-3) induces apoptosis in melanoma cells and inhibits growth of human melanoma xenografts. Here, we have studied the role of death receptors in apoptosis of melanoma cells induced by TIMP-3. Our results show, that the exposure of three metastatic melanoma cell lines (A2058, SK-Mel-5, and WM-266-4) to recombinant TIMP-3, N-terminal MMP inhibitory domain of TIMP-3, as well as to adenovirally expressed TIMP-3 results in stabilization of tumor necrosis factor receptor-1 (TNF-RI), FAS, and TNF-related apoptosis inducing ligand receptor-1 (TRAIL-RI) on melanoma cell surface and sensitizes these cells to apoptosis induced by TNF-alpha, anti-Fas-antibody and TRAIL. Stabilization of death receptors by TIMP-3 results in activation of caspase-8 and caspase-3, and subsequent apoptosis is blocked by specific caspase-8 inhibitor (Z-IETD-FMK) and by pan-caspase inhibitor (Z-DEVD-FMK). Adenovirus-mediated expression of TIMP-3 in human melanoma xenografts in vivo resulted in increased immunostaining for TNF-RI, FAS, and cleaved caspase-3, and in apoptosis of melanoma cells. Taken together, these results show that TIMP-3 promotes apoptosis in melanoma cells through stabilization of three distinct death receptors and activation of their apoptotic signaling cascade through caspase-8.     

IGFBP‐3 inhibits Wnt signaling in metastatic melanoma cells

In previous works, we have shown that insulin‐like growth factor‐binding protein‐3 (IGFBP‐3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP‐3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti‐tumoral effects of IGFBP‐3. We show that the anti‐tumoral effect of IGFBP‐3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP‐3 binds the Wnt signalosome interacting specifically with its component GSK‐3β. As a consequence, the β‐catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK‐3β is activated through dephosphorylation, becoming free to target cytoplasmic β‐catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP‐3 is a novel and effective inhibitor of Wnt signaling. As IGFBP‐3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.     

Downregulation of Noxa by RAF/MEK inhibition counteracts cell death response in mutant B-RAF melanoma cells

FDA approval of new therapies in 2011 has greatly expanded the treatment options for metastatic melanoma. Patients with V600 mutant v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) positive metastatic melanoma are now treated with the RAF inhibitor, vemurafenib (Zelboraf) as a first line therapy. Vemurafenib decreases tumor size by at least 30% in approximately 50% of patients and increases progression-free survival and overall patient survival compared to the previous standard-of-care, dacarbazine. However, some patients treated with vemurafenib fail to show significant tumor shrinkage, and most patients who initially respond to the drug eventually show disease progression. Therefore, there is a clinical need to improve efficacy and prevent resistance to vemurafenib. It has been previously shown that cell death resulting from RAF/mitogen-activated protein kinase kinase (MEK) inhibition is largely dependent on increased expression of pro-apoptotic, Bcl-2 homology domain (BH3)-only proteins, such as Bcl-2-like 11 (Bim-EL) and Bcl-2 modifying factor (Bmf). Here, we show that contrary to expression of Bim-EL and Bmf, the pro-apoptotic, BH3-only protein, phorbol-12-myristate-13-acetate-induced protein 1 (Noxa), is strongly downregulated after RAF/MEK inhibition. This downregulation occurs at both the protein and mRNA level of expression and is associated with the inhibition of cell cycle progression. Restoring expression of Noxa in combination with RAF/MEK inhibition enhances cell death. Co-expression of the pro-survival, B-cell CLL/lymphoma 2 (Bcl-2) family member, myeloid cell leukemia sequence 1 (Mcl-1), with Noxa fully mitigates the enhanced cell death associated with increased Noxa expression. These data indicate that manipulating the Noxa/Mcl-1 axis may enhance the efficacy of RAF/MEK inhibitors.     

Histone deacetylase inhibitor FK228 suppresses the Ras-MAP kinase signaling pathway by upregulating Rap1 and induces apoptosis in malignant melanoma

Histone deacetylase (HDAC) inhibitors are expected to be effective for refractory cancer because their mechanism of action differs from that of conventional antineoplastic agents. In this study, we examined the effect of the HDAC inhibitor FK228 on malignant melanoma, as well as its molecular mechanisms. FK228 was highly effective against melanoma compared with other commonly used drugs. By comparing the gene expression profiles of melanoma cells and normal melanocytes, we defined a subset of genes specifically upregulated in melanoma cells by FK228, which included Rap1, a small GTP-binding protein of the Ras family. The expression of Rap1 mRNA and protein increased in FK228-treated melanoma cells in both a dose- and a time-dependent manner. A decrease in the phosphorylation of c-Raf, MEK1/2, and ERK1/2 was accompanied by an increase in Rap1 expression in both FK228-treated and Rap1-overexpressing cells. Inhibition of Rap1 upregulation by small interfering RNA (siRNA) abrogated the induction of apoptosis and suppression of ERK1/2 phosphorylation in FK228-treated melanoma cells. These results indicate that the cytotoxic effects of FK228 are mediated via the upregulation of Rap1. Furthermore, we found that Rap1 was overexpressed and formed a complex with B-Raf in melanoma cell lines with a V599E mutation of B-Raf. The siRNA-mediated abrogation of Rap1 overexpression increased the viability of these cells, suggesting that Rap1 is also an endogenous regulator of Ras-MAP kinase signaling in melanomas.     

Protein kinase C-eta regulates resistance to UV- and gamma-irradiation-induced apoptosis in glioblastoma cells by preventing caspase-9 activation.

Both increased cell proliferation and apoptosis play important roles in the malignant growth of glioblastomas. We have demonstrated recently that the differential expression of protein kinase C (PKC)-eta increases the proliferative capacity of glioblastoma cells in culture; however, specific functions for this novel PKC isozyme in the regulation of apoptosis in these tumors has not been defined. In the present study of several glioblastoma cell lines, we investigated the role of PKC-eta in preventing UV- and gamma-irradiation-induced apoptosis and in caspase-dependent signaling pathways that mediate cell death. Exposure to UV or gamma irradiation killed 80% to 100% of PKC-eta-deficient nonneoplastic human astrocytes and U-1242 MG cells, but had little effect on the PKC-eta-expressing U-251 MG and U-373 MG cells. PKC-eta appears to mediate resistance to irradiation specifically such that when PKC-eta was stably expressed in U-1242 MG cells, more than 80% of these cells developed resistance to irradiation-induced apoptosis. Reducing PKC-eta expression by transient and stable expression of antisense PKC-eta in wild-type U-251 MG cells results in increased sensitivity to UV irradiation in a fashion similar to U-1242 MG cells and nonneoplastic astrocytes. Irradiation of PKC-eta-deficient glioblastoma cells resulted in the activation of caspase-9 and caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and a substantial increase in subdiploid DNA content that did not occur in PKC-eta-expressing tumor cells. A specific inhibitor (Ac-DEVD-CHO) of caspase-3 blocked apoptosis in PKC-eta-deficient U-1242 MG cells. The data demonstrate that resistance to UV and gamma irradiation in glioblastoma cell lines is modified significantly by PKC-eta expression and that PKC-eta appears to block the apoptotic cascade at caspase-9 activation.     

Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K

BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAF(V)???(E) melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.     

The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

Background  

By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition.     

The receptor tyrosine kinase inhibitor amuvatinib (MP470) sensitizes tumor cells to radio- and chemo-therapies in part by inhibiting homologous recombination

Background and purpose

RAD51 is a key protein involved in homologous recombination (HR) and a potential target for radiation- and chemotherapies. Amuvatinib (formerly known as MP470) is a novel receptor tyrosine kinase inhibitor that targets c-KIT and PDGFRα and can sensitize tumor cells to ionizing radiation (IR). Here, we studied amuvatinib mechanism on RAD51 and functional HR.

Materials and methods

Protein and RNA analyses, direct repeat green fluorescent protein (DR-GFP) assay and polysomal fractioning were used to measure HR efficiency and global translation in amuvatinib-treated H1299 lung carcinoma cells. Synergy of amuvatinib with IR or mitomycin c (MMC) was assessed by clonogenic survival assay.

Results

Amuvaninib inhibited RAD51 protein expression and HR. This was associated with reduced ribosomal protein S6 phosphorylation and inhibition of global translation. Amuvatinib sensitized cells to IR and MMC, agents that are selectively toxic to HR-deficient cells.

Conclusions

Amuvatinib is a promising agent that may be used to decrease tumor cell resistance. Our work suggests that this is associated with decreased RAD51 expression and function and supports the further study of amuvatinib in combination with chemotherapy and radiotherapy.     

An organometallic protein kinase inhibitor pharmacologically activates p53 and induces apoptosis in human melanoma cells

Unlike other tumors, melanomas harbor wild-type (WT) p53 but exhibit impaired p53-dependent apoptosis. The mechanisms for the impaired p53 activation are poorly understood but may be linked to the high expression of the p53 suppressor Mdm2, which is found in >50% of melanoma lesions. Here, we describe an organometallic glycogen synthase kinase 3beta (GSK3beta) inhibitor (DW1/2) as a potent activator of p53 and inducer of cell death in otherwise highly chemoresistant melanoma cells. Using RNA interference and pharmacologic approaches, we show that p53 is required for the cytotoxic effects of this organometallic inhibitor. The DW1/2 compound was barely able to induce cell death in melanoma cells with p53 mutations, further confirming the requirement for p53-WT in the cytotoxic effects of the GSK3beta inhibition. Mechanistic analysis of the p53-dependent cell death indicated an apoptotic mechanism involving depolarization of mitochondrial membrane potential, caspase cleavage, and elevated NOXA expression. The effect of p53 was not simply due to passive up-regulation of protein expression as adenoviral-mediated overexpression of p53 was not able to induce cell death. Treatment of melanoma cells with DW1/2 was instead found to decrease levels of Mdm2 and Mdm4. The importance of Mdm2 down-regulation in DW1/2-induced apoptosis was confirmed by treating the p53-WT cells with the p53/Mdm2 antagonist Nutlin-3. Taken together, our data provide a new strategy for the pharmacologic activation of p53 in melanoma, which may be a viable approach for overcoming apoptotic resistance in melanoma and offer new hope for rational melanoma therapy.     

U0126, a mitogen-activated protein kinase kinase inhibitor, inhibits the invasion of human A375 melanoma cells

The anti-invasive ability of the mitogen-activated protein kinase (MAPK) kinase inhibitor, U0126, was examined in human A375 melanoma cells in vitro. The effect was compared to that of PD98059, another commonly used MEK (MAPK kinase) inhibitor. U0126 or PD98059 showed a dose-dependent inhibition of A375 cell invasion through growth factor-reduced Matrigel. U0126 was more potent than PD98059 in suppressing tumor cell invasion. Both compounds significantly decreased urokinase plasminogen activator (uPA) and matrix metalloproteinases-9 (MMP-9) concentrations in conditioned media. At 5 microM, U0126 inhibited phosphorylation of the MEK 1/2 to a non-detectable level within 24 h. The phosphorylation of extracellular signal-related kinase 1/2 was also dramatically suppressed by the treatment with 10 microM U0126 or 40 microM PD98059. Both compounds suppressed the protein expression of c-Jun, but not c-Fos. The expression of uPA and MMP-9 was also inhibited. Our data suggest that U0126 is an effective agent in inhibiting human A375 melanoma cell invasion and that the effect is partially due to the decreased production of uPA and MMP-9.     

Inhibition of survivin and aurora B kinase sensitizes mesothelioma cells by enhancing mitotic arrests

PURPOSE: Survivin, a member of the inhibitor of apoptosis gene family, has also been shown to regulate mitosis. It binds Aurora B kinase and the inner centromere protein to form the chromosome passenger complex. Both Aurora B and survivin are overexpressed in many tumors. In this study, we examined whether irradiation affected survivin and Aurora B expression in mesothelioma cells, and how inhibition of these molecules affected radiosensitivity. METHODS AND MATERIALS: ZM447439 and survivin antisense oligonucleotides were used to inhibit survivin and Aurora B kinase respectively. Western blot was performed to determine the expression of survivin, Aurora B, phosphorylated-histone H3 (Ser 10), and caspase cleavage. Multinucleated cells were counted using flow cytometry, and cell survival after treatment was determined using clonogenic assay. RESULTS: At 3-Gy irradiation an increase was observed in levels of survivin and Aurora B as well as the kinase activity of Aurora B, with an increase in G2/M phase. The radiation-induced upregulation of these molecules was effectively attenuated by antisense oligonucleotides against survivin and a small-molecule inhibitor of Aurora B, ZM447439. Dual inhibition of survivin and Aurora B synergistically radiosensitized mesothelioma cells with a dose enhancement ratio of 2.55. This treatment resulted in increased formation of multinucleated cells after irradiation but did not increase levels of cleaved caspase 3. CONCLUSION: Inhibition of survivin and Aurora B induces mitotic cell arrest in mesothelioma cells after irradiation. These two proteins may be potential therapeutic targets for the enhancement of radiotherapy in malignant pleural mesothelioma.