RESUMO
The PI3K/AKT signaling pathway is aberrant in a wide variety of cancers. Downstream effectors of AKT are involved in survival, growth and metabolic-related pathways. In contrast, contradictory data relating to AKT effects on cell motility and invasion, crucial prometastatic processes, have been reported pointing to a potential cell type and isoform type-specific AKT-driven function. By implication, study of AKT signaling should optimally be conducted in an appropriate intracellular environment. Prognosis in soft-tissue sarcoma (STS), the aggressive malignancies of mesenchymal origin, is poor, reflecting our modest ability to control metastasis, an effort hampered by lack of insight into molecular mechanisms driving STS progression and dissemination. We examined the impact of the cancer progression-relevant AKT pathway on the mesenchymal tumor cell internal milieu. We demonstrate that AKT1 activation induces STS cell motility and invasiveness at least partially through a novel interaction with the intermediate filament vimentin (Vim). The binding of AKT (tail region) to Vim (head region) results in Vim Ser39 phosphorylation enhancing the ability of Vim to induce motility and invasion while protecting Vim from caspase-induced proteolysis. Moreover, vimentin phosphorylation was shown to enhance tumor and metastasis growth in vivo. Insights into this mesenchymal-related molecular mechanism may facilitate the development of critically lacking therapeutic options for these devastating malignancies.
Assuntos
Movimento Celular , Mesoderma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sarcoma/metabolismo , Neoplasias de Tecidos Moles/metabolismo , Vimentina/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Imunoprecipitação , Mesoderma/patologia , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Sarcoma/genética , Sarcoma/patologia , Transdução de Sinais/fisiologia , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , TransfecçãoRESUMO
The antifolate combination pyrimethamine/sulphadoxine (PYR/SDX; Fansidar) is frequently used to combat chloroquine-resistant malaria. Its success depends upon pronounced synergy between the two components, which target dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folate pathway. This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained. Plasmodium falciparum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these conditions. However, we have measured parasite inhibition by SDX/PYR combinations in assays in which folate levels are strictly controlled. In parasites that use exogenous folate efficiently, SDX inhibition can be restored by levels of PYR significantly lower than those required to inhibit DHFR. Isobolograms show that the degree of synergy between PYR and SDX is highly dependent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was observed at physiological drug levels when PYR/SDX acted on purified DHFR, whether wild type or mutant. We conclude that the primary basis for antifolate synergy in these organisms arises from PYR targeting a site (or sites) in addition to DHFR, which restores DHPS as a relevant target for SDX.
Assuntos
Antimaláricos/farmacologia , Ácido Fólico/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Pirimetamina/farmacologia , Sulfadoxina/farmacologia , Ácido 4-Aminobenzoico/farmacologia , Animais , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Resistência a Medicamentos , Sinergismo Farmacológico , Humanos , Malária Falciparum , Plasmodium falciparum/metabolismo , Sulfadoxina/antagonistas & inibidores , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
A three-dimensional structure model of the dihydrofolate reductase (DHFR) domain of the bifunctional DHFR-thymidylate synthase of Plasmodium falciparum was used as a basis for computational screening of commercially available compounds for candidate inhibitors. Compounds which can stably dock to the model with strong ionic hydrogen bonds via protonation by an aspartic acid residue at the bottom of the active site were identified through docking simulation. Among compounds thus identified, 21 were assayed for inhibitory activity towards the recombinant DHFR domain. Two compounds, 2-amino-1,4-dihydro-4,4,7,8-tetramethyl-s-triazino(1,2-a)benzimida zole and Trp-P-2, inhibited the recombinant P. falciparum DHFR domain with Ki values of 0.54 and 8.7 microM, respectively. Kinetic analysis showed that these compounds competitively inhibited the enzyme with respect to the substrate dihydrofolate. These findings support the validity of both the modeled structure and the docking results. Furthermore, these compounds serve as leads for developing new DHFR inhibitors, since their skeletal structures are different from any of known DHFR inhibitors. This paper also reveals a new biological activity of Trp-P-2, a potent mutagen.
Assuntos
Benzimidazóis/química , Carbolinas/química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Plasmodium falciparum/enzimologia , Conformação Proteica , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo , Animais , Benzimidazóis/farmacologia , Sítios de Ligação , Carbolinas/farmacologia , Galinhas , Humanos , Ligação de Hidrogênio , Cinética , Lacticaseibacillus casei/enzimologia , Fígado/enzimologia , Modelos Moleculares , Complexos Multienzimáticos/química , Mutagênicos/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/químicaRESUMO
We describe the system for screening the effective antifolate antimalarials that uses the recombinant Plasmodium falciparum DHFR domain of the bifunctional DHFR-TS expressed in Escherichia coli, and were designed with amino acid alterations found in the DHFR genes of the antifolate resistant strains. The validity of the screen was verified by the subsequent examination of several substituted pyrrolo[2,3-d]pyrimidines for their antimalarial activity. Among the 120 chemical derivatives, 5 compounds were identified by their preferential inhibition of the drug sensitive pfDHFR to that of the mammalian isoenzyme. As compared to the sensitive enzyme, the decrease in response of the cycolguanil-resistant and pyrimethamine-resistant enzymes to the selected compounds were relatively moderate. This gave folds decrease in sensitivity of 0.8-7.5 and 3.6-29, respectively, while those for cycloguanil and pyrimethamine were 400 and 308. The compounds inhibited the growth of drug-sensitive cultured P. falciparum with 50% effective concentrations of the ranged 0.17-30 nM. As contrasted with the sensitive strain, the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds, while those for cycloguanil and pyrimethamine were 690 and 20,500. Moreover, the most selective pyrrolo-pyrimidine (P-1) showed in vivo activity against P. berghei in mice.