RESUMO
Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.
Assuntos
Aciltransferases/metabolismo , Toxoplasma/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/efeitos dos fármacos , Aciltransferases/genética , Sequência de Aminoácidos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Fibroblastos/parasitologia , Imunofluorescência , Prepúcio do Pênis/citologia , Prepúcio do Pênis/parasitologia , Humanos , Imunoprecipitação , Masculino , Filogenia , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Toxoplasma/classificação , Toxoplasma/enzimologia , Toxoplasma/genéticaRESUMO
MicroRNAs (miRNAs) play an important role in drug resistance and modulate the efficiency of chemotherapy. A recent study indicated that miR-340 functions as a tumor suppressor in various types of cancer. However, the role of miR-340 in chemotherapy has not been reported yet. In this study, we found that miR-340 enhanced cisplatin (CDDP)-induced cell death. Induction of miR-340-5p expression decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells. Moreover, miR-340-5p decreased the accumulation of MRP1 and MDR1. We further explored the mechanism underlying the promoting effects of miR-340-5p on CDDP-induced cell death. We identified a potential target of miR-340 in the 3' untranslated region of lysophosphatidic acid acyltransferase (LPAATß) using the online program Targetscan (http://www.microrna.org). Luciferase reporter assays showed that miR-340 binds to the 3'UTR of LPAATß. Enforced expression of miR-340-5p decreased the accumulation of LPAATß in both MG-63 and Saos-2 cells. Silencing LPAATß decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells, which is consistent with the effect of miR-340-5p on CDDP-induced cell death. Moreover, induced expression of LPAATß compromised the effects of miR-340-5p on CDDP-induced cell death and accumulation of MRP1 and MDR1. Taken together, our data indicated that miR-340-5p enhanced the sensitivity to CDDP by targeting LPAATß.
Assuntos
Aciltransferases/fisiologia , Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , MicroRNAs/fisiologia , Osteossarcoma/tratamento farmacológico , Aciltransferases/análise , Aciltransferases/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias Ósseas/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Luciferases , MicroRNAs/análise , MicroRNAs/efeitos dos fármacos , Osteossarcoma/fisiopatologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
MicroRNAs (miRNAs) play an important role in drug resistance and modulate the efficiency of chemotherapy. A recent study indicated that miR-340 functions as a tumor suppressor in various types of cancer. However, the role of miR-340 in chemotherapy has not been reported yet. In this study, we found that miR-340 enhanced cisplatin (CDDP)-induced cell death. Induction of miR-340-5p expression decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells. Moreover, miR-340-5p decreased the accumulation of MRP1 and MDR1. We further explored the mechanism underlying the promoting effects of miR-340-5p on CDDP-induced cell death. We identified a potential target of miR-340 in the 3′ untranslated region of lysophosphatidic acid acyltransferase (LPAATβ) using the online program Targetscan (http://www.microrna.org). Luciferase reporter assays showed that miR-340 binds to the 3′UTR of LPAATβ. Enforced expression of miR-340-5p decreased the accumulation of LPAATβ in both MG-63 and Saos-2 cells. Silencing LPAATβ decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells, which is consistent with the effect of miR-340-5p on CDDP-induced cell death. Moreover, induced expression of LPAATβ compromised the effects of miR-340-5p on CDDP-induced cell death and accumulation of MRP1 and MDR1. Taken together, our data indicated that miR-340-5p enhanced the sensitivity to CDDP by targeting LPAATβ.
Assuntos
Humanos , Aciltransferases/fisiologia , Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , MicroRNAs/fisiologia , Osteossarcoma/tratamento farmacológico , Aciltransferases/análise , Aciltransferases/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias Ósseas/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Luciferases , MicroRNAs/análise , MicroRNAs/efeitos dos fármacos , Osteossarcoma/fisiopatologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Hexachlorobenzene (HCB) induces porphyria both in humans and rodents, and hepatocarcinoma in rodents. In a previous work we observed that HCB produces a continuous decrease in hepatic sphingomyelin (SM) content in Wistar rats. A distinguishing characteristic of sphingolipids breakdown products is their participation in anti-proliferative and apoptotic processes and in the suppression of oncogenesis. As a first step to elucidate the role of SM decrease in the hepatotoxicity induced by HCB, the present study evaluates the metabolic causes of the continuous decrease in hepatic SM content observed in Wistar rats with HCB intoxication, and its relation with porphyria development. For this purpose, the time-course (3, 7, 15, 21 and 28 days) of the effects of HCB on hepatic SM levels and on some of the enzymes of SM synthesis (serine palmitoyltransferase, SPT) and catabolism (sphingomyelinases, SMases) was followed, using two strains of rats differing in their susceptibility to acquire porphyria: Chbb THOM (low) and Wistar (high). HCB (1 g kg(-1) b.w. per day) was administered by gastric intubation as an aqueous suspension. After 5 days of HCB treatment, animals were allowed a 2-day recovery period without HCB administration. Two phases in the HCB-induced damages to sphingolipid metabolism were observed. The first stage (7 days of treatment), common to both strains of rats, was characterized by a decrease in hepatic SM levels (17-25%) and in SPT activity (50-43%), while strain differences were found for the later stage. In Chbb THOM rats, hepatic SM content was restored to normal values concomitantly with an increase in SPT activity (44%, at day 28), and without any increase in SM catabolism. In addition, the level of the other phospholipids was not altered. In Wistar rats, hepatic SM levels decreased continuously throughout the experiment, accompanied by increases in SPT, acidic sphingomyelinase (A-SMase) and neutral sphingomyelinase (N-SMase) activities (86, 28.5 and 78% increase, respectively). A role for glutathione (GSH) in the interstrain differences or a direct effect of HCB on SM metabolism was not found. The present study: (a) demonstrates that N-SMase, A-SMase, and SPT are some of the enzymes that play a role in the HCB-induced decrease of hepatic SM content; (b) finds that HCB-induced alterations of SM metabolism do not correlate with HCB-induced accumulation of hepatic porphyrins; and (c) proposes a link between HCB-induced alterations in phospholipid pattern and in SM metabolism. The increased SM hydrolysis produced as a consequence of SMases induction could be regarded as a cellular response to liver injury elicited by HCB, perhaps acting through the activation of SM signal transduction pathway delaying the proliferative processes observed after long-term treatment with HCB in some rodent species. However, such protective mechanism appears to be strain-dependent.