RESUMO
There is an increasing evidence that the oncogenic kinase PAK1 is responsible not only for malignant transformation, but also for several other diseases such as inflammatory diseases (asthma and arthritis), infectious diseases including malaria, AIDS, and flu, as well as a series of neuronal diseases/disorders (neurofibromatosis, tuberous sclerosis, Alzheimer's diseases, Huntington's disease, epilepsy, depression, learning deficit, etc.) which often cause premature death. Interestingly, a few natural PAK1-blockers such as curcumin, caffeic acid (CA) and rosmarinic acid (RA) extend the lifespan of the nematode Caenorhabditis elegans or fruit flies. Here, to explore the possibility that C. elegans could provide us with a quick and inexpensive in vivo screening system for a series of more potent but safe (non-toxic) PAK1-blocking therapeutics, we examined the effects of PAK1-deficiency or down-regulation on a few selected functions of this worm, including reproduction, expression of HSP16.2 gene, and lifespan. In short, we found that PAK1 promotes reproduction, whereas it inactivates HSP16.2 gene and shortens lifespan, as do PI-3 kinase (AGE-1), TOR, and insulin-like signalling /ILS (Daf-2) in this worm. These findings not only support the "trade-off" theory on reproduction versus lifespan, but also suggest the possibility that the reduced reproduction (or HSP16.2 gene activation) of this worm could be used as the first indicator of extended lifespan for a quick in vivo screening for PAK1-blockers.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Choque Térmico/genética , Longevidade/genética , Quinases Ativadas por p21/antagonistas & inibidores , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Ácidos Cafeicos/farmacologia , Regulação para Baixo , Proteínas de Fluorescência Verde/genética , Longevidade/efeitos dos fármacos , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/farmacologia , Fenilpropionatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Reprodução/efeitos dos fármacos , Reprodução/genética , Transdução de Sinais/efeitos dos fármacos , Quinases Ativadas por p21/deficiência , Quinases Ativadas por p21/genéticaRESUMO
Neurofibromatosis (NF) is a family of genetic diseases which are caused by dysfunction of either NF1 gene or NF2 gene. One in 3,000 people suffer from this tumor-carrying NF. NF1 gene product is a RAS GTPase activating protein (GAP) of 2,818 amino acids, which normally attenuates the GTP-dependent signal transducing activity of the G protein RAS. Dysfunction of this GAP leads to the abnormal activation of RAS, and eventually an oncogenic kinase called PAK1 as well. NF2 gene product is ''Merlin'' which directly inactivates PAK1. Thus, dysfunction of Merlin causes the abnormal activation of PAK1. In other words, dysfunction of NF1 gene (causing type 1 NF) is basically the same as dysfunction of NF2 gene (causing type 2 NF). In fact the growth of both NF1 and NF2 tumors requires PAK1, and all PAK1 blockers, synthetic chemicals or natural products, suppress the growth of these NF tumor cells both in vitro (cell culture) and in vivo (mice). However, until recently, no FDA-approved effective NF therapeutics is available on the market. Here a series of anti-PAK1 products shall be introduced, which would be potentially useful for the life-long treatment of NF patients in the future. These include the most potent HDAC (histone deacetylase) inhibitor FK228 (IC50: around 1 nM), that eventually blocks PAK1, the direct PAK1 inhibitor PF3758309 (IC50: around 10 nM), a CAPE (caffeic acid phenethyl ester)-based propolis extract called ''Bio 30'' from NZ (New Zealand), and an ARC (artepillin C)-based green propolis extract (GPE) from Brazil. Although the first two drugs are potent, none of them is available on the market as yet. The last two natural (bee-made) products are available on the market, and have been used for the therapy of NF and tuberous sclerosis (TSC) as well as many PAK1-dependent solid cancers such as breast and pancreatic cancers as well as glioma, which altogether represent more than 70% of all human cancers. Since PAK1 is not essential for the normal cell growth, propolis extracts cause no side effects.
RESUMO
So far no effective therapeutic has been developed for the FDA-approved treatment of ovarian cancer patients. Recently we provided the first evidence indicating that an old antibiotic (antiparasitic drug) called Ivermectin suppresses the growth of a variety of human ovarian cancer cell lines in vitro by inactivating the oncogenic kinase PAK1 somehow (Hashimoto H, et al. Drug Discov Ther. 2009;3:243-246). This kinase is now known to be essential for the growth of more than 70% of all human cancers including breast, prostate, pancreatic, colon, gastric, lung, cervical, thyroid cancers as well as hepatoma, glioma, melanoma, MM (multiple myeloma) and NF (neurofibromatosis) tumors. In this study, using the cell-permeable PAK1-inactivating peptide TAT-PAK18 which blocks the essential PAK1-PIX interaction, we examined the relationship between the sensitivity of ovarian cancer cell lines to this anti-PAK1 peptide and the protein expression/autophosphorylation levels of PAK1 in these cell lines, and found that the more PAK1 is abnormally activated (autophosporylated at Thr 423), the more their growth is sensitive to this peptide, regardless of their PAK1 expression levels. This observation provides the first direct evidence that ovarian cancers also belong to the PAK1-dependent cancers which represent more than 70% of all human cancers, suggesting that anti-PAK1 drugs would be effective therapeutics for ovarian cancers.
RESUMO
Paul Ehrlich (1854-1915), a German microbiologist who was awarded a 1908 Nobel Prize in Physiology/Medicine for his pioneer work on the antibody production, pioneered the modern chemotherapy by discovering his magic bullet for syphilis, called "606" or "Salvarsan" in 1909 with a Japanese young scientist, Sahachiro Hata (1873-1938) from "Denken" (Institute for Infectious Diseases, now called IMS for Institute for Medical Sciences) in Tokyo. His magic bullet was used to eradicate syphilis for more than a half century until a more safe and effective antibiotic called "Penicillin" was introduced to this world towards the end of WWII by Howard Florey (1898-1968). Celebrating this year the 100th anniversary of his discovery, this brief review will discuss how Ehrlich, now known as the Father of Chemotherapy, managed to design the first effective therapeutic for this then formidable sexually transmitted disease, which is equivalent to AIDS, HIV-infection, in the present century, and how so many new chemotherapeutics have been successfully developed during the past 100 years for other formidable diseases such as cancers and AIDS by his followers (microbe hunters and oncogene hunters) such as Alexander Fleming (1881-1955), Hamao Umezawa (1914-1986) and Brian Druker, culminating in the first signal therapeutics of cancers such as "Gleevec" that block the oncogenic signaling, around the turn of this century.
RESUMO
Ivermectin is an old anti-parasitic antibiotic which selectively kills nematodes at a very low dose (0.2 mg/kg) by inhibiting their GABA (gamma-aminobutyric acid) receptor, but not mammalian counterpart. Interestingly, several years ago it was reported by a Russian group that Ivermectin can suppress almost completely the growth of human melanoma and a few other cancer xenografts in mice at the much higher doses (3-5 mg/kg) without any adverse effect on mice. However, its anti-cancer mechanism still remained to be clarified at the molecular levels, that would determine the specific type of cancers susceptible to this drug. The first hint towards its anti-PAK1 potential was a recent finding that Ivermectin at its sublethal doses dramatically reduces the litter size (number of eggs laid) of the tiny nematode C. elegans. Interestingly, either a PAK1-deficiency (gene knock-out) or treatment with natural anti-PAK1 products such as CAPE (caffeic acid phenethyl ester) and ARC (artepillin C), the major anti-cancer ingredients in propolis, also causes the exactly same effect on this nematode, suggesting the possibility that the kinase PAK1 might be a new target of Ivermectin. This kinase is required for the growth of more than 70% of human cancers such as pancreatic, colon, breast and prostate cancers and NF (neurofibromatosis) tumors. Here we demonstrate for the first time that Ivermectin blocks the oncogenic kinase PAK1 in human ovarian cancer and NF2-deficient Schwannoma cell lines to suppress their PAK1-dependent growth in cell culture, with the IC50 between 5-20 µM depending on cell lines.
RESUMO
Dysfunction of the NF1 gene coding a RAS GAP is the major cause of neurofibromatosis type 1 (NF1), whereas neurofibromatosis type 2 (NF2) is caused primarily by dysfunction of the NF2 gene product called merlin that inhibits directly PAK1, an oncogenic Rac/CDC42-dependent Ser/Thr kinase. It was demonstrated previously that PAK1 is essential for the growth of both NF1 and NF2 tumors. Thus, several anti-PAK1 drugs, including FK228 and CEP-1347, are being developed for the treatment of NF tumors. However, so far no effective NF therapeutic is available on the market. Since propolis, a very safe healthcare product from bee hives, contains anticancer ingredients called CAPE (caffeic acid phenethyl ester) or ARC (artepillin C), depending on the source, both of which block the oncogenic PAK1 signaling pathways, its potential therapeutic effect on NF tumors was explored in vivo. Here it is demonstrated that Bio 30, a CAPE-rich water-miscible extract of New Zealand (NZ) propolis suppressed completely the growth of a human NF1 cancer called MPNST (malignant peripheral nerve sheath tumor) and caused an almost complete regression of human NF2 tumor (Schwannoma), both grafted in nude mice. Although CAPE alone has never been used clinically, due to its poor bioavailability/water-solubility, Bio 30 contains plenty of lipids which solubilize CAPE, and also includes several other anticancer ingredients that seem to act synergistically with CAPE. Thus, it would be worth testing clinically to see if Bio 30 and other CAPE-rich propolis are useful for the treatment of NF patients.
Assuntos
Ácidos Cafeicos/farmacologia , Neurofibromatose 1/tratamento farmacológico , Neurofibromatose 2/tratamento farmacológico , Álcool Feniletílico/análogos & derivados , Própole/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Álcool Feniletílico/farmacologia , Quinases Ativadas por p21/antagonistas & inibidoresRESUMO
BACKGROUND: Both EGF family ligands and ErbB family receptor kinases act upstream of RAS to induce mitogenesis of normal cells, such as NIH 3T3 fibroblasts. However, oncogenically mutated RAS, such as v-Ha-RAS is constitutively activated and therefore no longer requires these ligands or receptors for its activation. Nevertheless, it up-regulates the expression of these EGF family ligands. To understand the biologic significance of RAS-induced up-regulation of these ligands in both RAS-induced PAK activation and malignant transformation, we have conducted the following studies, based on the previous observations that (1) the N-terminal SH3 domain of PIX selectively binds a Pro-rich domain of 18 amino acids of PAKs, CDC42/Rac-dependent Ser/Thr kinase family, and (2) this specific interaction is essential for both PAK activation and membrane ruffling RESULTS: Using four distinct, cell-permeable, and highly specific inhibitors, namely WR-PAK18, which blocks the PAK-PIX interaction; AG 1478, which inhibits ErbB1 kinase activity; and AG 825 or AG 879, which inhibits ErbB2 kinase activity, we demonstrate that (1) the PAK-PIX interaction is essential for v-Ha-RAS-induced malignant transformation; (2) v-Ha-RAS requires not only ErbB1 but also ErbB2, which are activated through two independent autocrine pathways to induce both the PIX/Rac/CDC42-dependent PAK activation and malignant transformation in vitro; and (3) a combination of AG 879 and the Src family kinase-specific inhibitor PP1 suppresses almost completely the growth of RAS-induced sarcomas in nude mice. CONCLUSION: These findings not only change our conventional view on the role of these RAS-inducible ligands and ErbB family receptors (serving as RAS activators) but also suggest a new avenue for the treatment of RAS-associated cancers by a combination of inhibitors specific for ERbB, Src, or PAK family kinases.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA , Inibidores Enzimáticos/uso terapêutico , Glicoproteínas/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Plantas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Receptor ErbB-2/efeitos dos fármacos , Tirfostinas/uso terapêutico , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Animais , Bovinos , Meios de Cultura Livres de Soro , Ativação Enzimática/efeitos dos fármacos , Feminino , Camundongos , Camundongos Nus , Fragmentos de Peptídeos/farmacologia , Quinazolinas , Receptor ErbB-2/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho , Sarcoma Experimental/tratamento farmacológico , Tirfostinas/farmacologia , Regulação para Cima , Quinases Ativadas por p21 , Quinases da Família srcRESUMO
Recent efforts in our laboratories have resulted in a synthetic approach toward C2'-alkylated K252a analogues via extension of a K252a cyclofuranosylation strategy. The bis-indole-N-glycosidic coupling of 6-N-(3,4-dimethoxybenzyl)-staurosporinone (21) with a number of highly functionalized carbohydrates has given access to previously unattainable, biologically relevant analogues.
Assuntos
Carbazóis/síntese química , Inibidores Enzimáticos/síntese química , Proteína Quinase C/antagonistas & inibidores , Alquilação , Indicadores e Reagentes , Alcaloides Indólicos , Conformação Molecular , EstereoisomerismoRESUMO
Insulin-like growth factor I receptor (IGFR) plays an important role in cell growth and transformation. We dissected the downstream signaling pathways of an oncogenic variant of IGFR, Gag-IGFR, called NM1. Loss of function mutants of NM1, Phe-1136 and dS2, that retain kinase activity but are attenuated in their transforming ability were used to identify signaling pathways that are important for transformation of NIH 3T3 cells. MAPK, phospholipase C gamma, and Stat3 were activated to the same extent by NM1 and its two mutants, suggesting that activation of these pathways, individually or in combination, was not sufficient for NM1-induced cell transformation. The mutant dS2 has decreased IRS-1 phosphorylation levels and IRS-1-associated phosphatidylinositol 3'-kinase activity, suggesting that this impairment may be in part responsible for the defectiveness of dS2. We show that Rho family members, RhoA, Rac1, and Cdc42 are activated by NM1, and this activation, particularly RhoA and Cdc42, is attenuated in both mutants of NM1. Dominant negative mutants of Rho, Rac, and Cdc42 inhibited NM1-induced cell transformation, as measured by focus and colony forming ability. Dominant negative Rho most potently inhibited the focus forming activity, whereas Cdc42 was most effective in inhibiting the colony forming ability of NM1-expressing cells. Conversely, constitutively activated (ca) Rho is more effective than ca Rac or ca Cdc42 in rescuing the focus forming ability of the mutants. By contrast, ca Cdc42 is most effective in rescuing the colony forming ability of both mutants.
Assuntos
Receptor IGF Tipo 1/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Células 3T3 , Animais , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Camundongos , Mutação , Transdução de SinaisRESUMO
The platelet-derived growth factor (PDGF) receptor (PDGFR) transactivates the epidermal growth factor (EGF) receptor (ErbB1) to stimulate the cell migration of fibroblasts through an unknown mechanism (Li, J., Kim, Y. N. & Bertics, P. (2000) J. Biol. Chem. 275, 2951-2958). In this paper we provide evidence that the transactivation of the EGF receptor (EGFR) by PDGFR is essential for PDGF to activate p21-activated kinase (PAK) family kinases. Fetal calf serum (10%) transiently stimulates the PAK activity in NIH 3T3 fibroblasts. The activation of PAK was completely inhibited by either PDGFR-specific inhibitor (AG1295) or EGFR-specific inhibitor (AG1478), suggesting that serum requires either the PDGF- or EGF-dependent pathway or the combination of both to activate PAK. PDGF-induced activation of PAK is completely inhibited by either AG1295 or AG1478, indicating that PDGF requires both PDGFR and EGFR for PAK activation. In support of this notion, a mouse embryo fibroblast cell line derived from the EGFR -/- mouse (from Dr. Erwin Wagner) doesn't activate PAK in response to PDGF. Expression of human EGFR in this cell line restores the ability of the PDGF to induce PAK activation. Our results indicate that PDGF activates PAK through transactivation of ErbB1.
Assuntos
Receptores ErbB/fisiologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3 , Animais , Sangue , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Ativação Enzimática , Fibroblastos/enzimologia , Humanos , Camundongos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de SinaisRESUMO
Drugs that inhibit important protein-protein interactions are hard to find either by screening or rational design, at least so far. Most drugs on the market that target proteins today are therefore aimed at well-defined binding pockets in proteins. While computer-aided design is widely used to facilitate the drug discovery process for binding pockets, its application to the design of inhibitors that target the protein surface initially seems to be limited because of the increased complexity of the task. Previously, we had started to develop a computational combinatorial design approach based on the well-known 'multiple copy simultaneous search' (MCSS) procedure to tackle this problem. In order to identify sequence patterns of potential inhibitor peptides, a three-step procedure is employed: first, using MCSS, the locations of specific functional groups on the protein surface are identified; second, after constructing the peptide main chain based on the location of favorite locations of N-methylacetamide groups, functional groups corresponding to amino acid side chains are selected and connected to the main chain C(alpha) atoms; finally, the peptides generated in the second step are aligned and probabilities of amino acids at each position are calculated from the alignment scheme. Sequence patterns of potential inhibitors are determined based on the propensities of amino acids at each C(alpha) position. Here we report the optimization of inhibitor peptides using the sequence patterns determined by our method. Several short peptides derived from our prediction inhibit the Ras--Raf association in vitro in ELISA competition assays, radioassays and biosensor-based assays, demonstrating the feasibility of our approach. Consequently, our method provides an important step towards the development of novel anti-Ras agents and the structure-based design of inhibitors of protein--protein interactions.
Assuntos
Técnicas de Química Combinatória , Peptídeos/química , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas ras/antagonistas & inibidores , Algoritmos , Sequência de Aminoácidos , Técnicas Biossensoriais , Desenho Assistido por Computador , Ensaio de Imunoadsorção Enzimática , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/farmacologia , Inibidores de Proteases/síntese química , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ensaio Radioligante , Alinhamento de Sequência , Proteínas ras/metabolismoRESUMO
BACKGROUND: We previously found that both PAK, a Rac/CDC42-activated Ser/Thr kinase, and its binding partner PIX are required for malignant transformation caused by oncogenic Ras mutants, such as v-Ha-Ras. Furthermore, oncogenic Ras requires an autocrine pathway to activate PAK. This pathway involves at least two distinct receptor kinases: EGF receptor (ErbB1) and ErbB2. Interestingly, both of these kinases are known to activate Src family kinases that phosphorylate CAT, another binding partner of PIX. PURPOSE: The major aim of this study was to determine whether Src family kinases are required for both Ras-induced PAK activation and malignant transformation. For this purpose, we used PP1, an inhibitor specific for Src family kinases, which does not inhibit either EGF receptor or ErbB2. METHODS AND RESULTS: We studied the effect of PP1 on the anchorage-dependent growth of normal and v-Ha-Ras transformed NIH 3T3 fibroblasts, PAK activation and anchorage-independent growth of Ras transformants, and development of Ras-induced sarcomas in nude mice. We found that PP1 (10 nM) strongly inhibits PAK activity in Ras transformants. PP1 at this concentration is known to inhibit c-Fyn kinase, but not c-Src kinase, and none of the three known Src family kinases (c-Src, c-Fyn, and c-Yes) expressed in fibroblasts is activated by v-Ha-Ras. Thus, it is most likely that the primary target of this drug is an as yet unidentified Ras-activated Tyr (Y) kinase or kinases, which we call "Ray." Although PP1 has no effect on their anchorage-dependent growth, it significantly inhibits their anchorage-independent growth in soft agar, as well as a rapid growth of Ras-induced sarcomas in mice. CONCLUSION: Like EGF receptor and ErbB2, a member of Src family kinases (most likely a new Src-related kinase called "Ray") is essential for the Ras-induced activation of PAK and the malignant transformation both in vitro and in vivo. These findings suggest that PP1 and other inhibitors specific for Src family kinases are potentially useful for the treatment of Ras-associated cancers.
Assuntos
Antineoplásicos/uso terapêutico , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Sarcoma/tratamento farmacológico , Quinases da Família src/antagonistas & inibidores , Células 3T3 , Animais , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Ativação Enzimática , Feminino , Camundongos , Camundongos Nus , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Quinases Ativadas por p21 , Quinases da Família src/fisiologiaRESUMO
To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains. Here we report the cloning of the full-length human p190-A cDNA and its first exon covering more than 80% of this protein, as well as its chromosomal mapping. Human p190-A encodes a protein of 1514 amino acids, and shares overall 97% sequence identity with rat p190-A. Like the p190-B exon, the first exon of p190-A is extremely large (3.7 kb in length), encoding both the GTPase and middle domains (residues 1-1228), but not the remaining GAP domain, suggesting a high conservation of genomic structure between two p190 genes. Using a well characterized monochromosome somatic cell hybrid panel, fluorescent in situ hybridization (FISH) and other complementary approaches, we have mapped the p190-A gene between the markers D19S241E and STD (500 kb region) of human chromosome 19q13.3. Interestingly, this chromosomal region is known to be rearranged in a variety of human solid tumors including pancreatic carcinomas and gliomas. Moreover, at least 40% glioblastoma/astrocytoma cases with breakpoints in this region were previously reported to show loss of the chromosomal region encompassing p190-A, suggesting the possibility that loss or mutations of this gene might be in part responsible for the development of these tumors.
Assuntos
Cromossomos Humanos Par 19/genética , Proteínas de Ligação a DNA , Proteínas de Ligação ao GTP , Genes Supressores de Tumor/genética , Glioma/genética , Proteínas Supressoras de Tumor , Proteínas Ativadoras de ras GTPase/genética , Sequência de Aminoácidos , Sequência de Bases , Bandeamento Cromossômico , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Éxons , Proteínas Ativadoras de GTPase , Deleção de Genes , Genes/genética , Fatores de Troca do Nucleotídeo Guanina , Humanos , Hibridização in Situ Fluorescente , Íntrons , Dados de Sequência Molecular , Mapeamento de Híbridos Radioativos , Proteínas Repressoras , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , ras-GRF1RESUMO
A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.
Assuntos
Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Análise Mutacional de DNA , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas c-raf/genética , Homologia de Sequência de AminoácidosRESUMO
A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.
Assuntos
Actinas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteína Oncogênica p21(ras)/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Células 3T3 , Actinas/química , Actinas/farmacologia , Actinas/ultraestrutura , Animais , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Membrana Celular/metabolismo , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas/farmacologia , Citoesqueleto/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Nus , Microscopia Eletrônica , Miosinas/metabolismo , Ligação Proteica , Sarcoma Experimental/tratamento farmacológico , Fatores de TempoRESUMO
MKT-077, a cationic rhodacyanine dye analogue has been under preclinical cancer therapeutical trials because of its selective toxicity to cancer cells. Its cellular targets and mechanism of action remain poorly understood. Here we report that MKT-077 binds to an hsp70 family member, mortalin (mot-2), and abrogates its interactions with the tumor suppressor protein, p53. In cancer cells, but not in normal cells, MKT-077 induced release of wild-type p53 from cytoplasmically sequestered p53-mot-2 complexes and rescued its transcriptional activation function. Thus, MKT-077 may be particularly useful for therapy of cancers with wild-type p53.
Assuntos
Antineoplásicos/toxicidade , Corantes/toxicidade , Proteínas de Choque Térmico HSP70/metabolismo , Piridinas/toxicidade , Tiazóis/toxicidade , Proteína Supressora de Tumor p53/metabolismo , Células 3T3 , Animais , Antineoplásicos/química , Cromatografia de Afinidade , Citoplasma/metabolismo , Genes Reporter , Humanos , Camundongos , Microscopia de Fluorescência , Proteínas Mitocondriais , Testes de Precipitina , Ligação Proteica , Piridinas/química , Tiazóis/química , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
Almost three decades have passed since actin-cytoskeleton (acto-myosin complex) was first discovered in non-muscle cells. A combination of cell biology, biochemistry, and molecular biology has revealed the structure and function of many actin-binding proteins and their physiological role in the regulation of cell motility, shape, growth, and malignant transformation. As molecular oncologists, we would like to review how the function of actin-cytoskeleton is regulated through Ras/Rho family GTPases- or phosphoinosites-mediated signaling pathways, and how malignant transformation is controlled by actin/phosphoinositides-binding proteins or drugs that block Rho/Rac/CDC42 GTPases-mediated signaling pathways.
Assuntos
Actinas/fisiologia , Transformação Celular Neoplásica/metabolismo , Citoesqueleto/química , GTP Fosfo-Hidrolases/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Inibidores Enzimáticos/farmacologia , Fosfatidilinositóis/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/efeitos dos fármacosRESUMO
UNLABELLED: For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. PURPOSE: This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. METHODS AND RESULTS: DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation. CONCLUSIONS: Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.
Assuntos
Inibidores de Caspase , Citocalasinas/farmacologia , Genes ras/genética , Indóis/farmacologia , Proteínas dos Microfilamentos/farmacologia , Proteínas Serina-Treonina Quinases , Proteínas ras/metabolismo , Células 3T3 , Animais , Apoptose , Transformação Celular Neoplásica/induzido quimicamente , DNA Complementar/metabolismo , Glucose/farmacocinética , Alcaloides Indólicos , Indóis/química , Camundongos , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tensinas , Fatores de Tempo , Transfecção , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf/Arg89 in the center of the binding interface plays an important role determining Ras-Raf binding affinity. We have investigated experimentally and computationally the Raf-R89K mutation, which abolishes signaling in vivo. The binding to [gamma-35S]GTP-Ras of a fusion protein between the Raf-binding domain (RBD) of Raf and GST was reduced at least 175-fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal/mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9+/-1.9 kcal/mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal/mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein-protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 A away, namely residues 37-41 in the conserved effector domain of Ras (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2-3 specific water molecules. This analysis will provide insights into the large experimental database of Ras-Raf mutations.