RESUMO
Betulinic acid (BA) is a widely available plant-derived triterpene with reported activity against cancer cells of neuroectodermal origin and leukaemias. Treatment with BA was shown to protect mice against transplanted human melanoma and led to tumor regression. In contrast, cells from healthy tissues were resistant to BA and toxic side-effects in animals were absent. These findings have raised interest in the chemotherapeutical anti-cancer potential of BA. A comprehensive assessment of the efficacy of BA against the clinically most important cancer types is currently lacking. Therefore, we tested the in vitro sensitivity of broad cell line panels derived from lung, colorectal, breast, prostate and cervical cancer, which are the prevalent cancer types characterized with highest mortalities in woman and men. Multiple assays were used in order to allow a reliable assessment of anti-cancer efficacy of BA. After 48 h of treatment with BA, cell viability as assessed with MTT and cell death as measured with propidium iodide exclusion showed clear differences in sensitivity between cell lines. However, in all cell lines tested colony formation was completely halted at remarkably equal BA concentrations that are likely attainable in vivo. Our results substantiate the possible application of BA as a chemotherapeutic agent for the most prevalent human cancer types.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Triterpenos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Células Jurkat , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Triterpenos Pentacíclicos , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Ácido BetulínicoRESUMO
The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.
Assuntos
Antimaláricos/farmacologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resistência a Medicamentos/genética , Edição de Genes , Genoma de Protozoário , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Isoquinolinas/farmacologia , Plasmodium falciparum/genética , Resistência a Medicamentos/efeitos dos fármacos , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/genética , MutaçãoRESUMO
Tumors have several mechanisms to escape from the immune system. One of these involves expression of intracellular anticytotoxic proteins that modulate the execution of cell death. Previously, we have shown that the serine protease inhibitor (serpin) SPI-6, which inactivates the cytotoxic protease granzyme B (GrB), is capable of preventing cytotoxic T lymphocyte (CTL)-mediated apoptosis. Despite its potent antiapoptotic activity, SPI-6 does not prevent membranolysis induced by cytotoxic lymphocytes. We now provide evidence that several colon carcinoma cell lines do resist membranolysis and that this protection is dependent on SPI-6 but also requires expression of a closely related serpin called SPI-CI (serine protease inhibitor involved in cytotoxicity inhibition). Expression of SPI-CI is absent from normal colon but observed in placenta, testis, early during embryogenesis, and in cytotoxic lymphocytes. SPI-CI encodes a chymotrypsin-specific inhibitor and irreversibly interacts with purified granzyme M. Moreover, SPI-CI can protect cells from purified perforin/GrM-induced lysis. Our data therefore indicate that SPI-CI is a novel immune escape molecule that acts in concert with SPI-6 to prevent cytotoxic lymphocyte-mediated killing of tumor cells.