Browsing by Author "Nunes, Ana Raquel Santos"
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- Comparative study of the therapeutic effect of Doxorubicin and Resveratrol combination on 2D and 3D (spheroids) cell culture modelsPublication . Barros, Andreia; Costa, Elisabete C.; Nunes, Ana Raquel Santos; Diogo, Duarte Miguel de Melo; Correia, Ilídio Joaquim SobreiraThe assessment of drug-combinations for pancreatic cancer treatment is usually performed in 2D cell cultures. In this study, the therapeutic effect and the synergistic potential of a particular drug-combination towards 2D and 3D cell cultures of pancreatic cancer were compared for the first time. Thus, the effect of Doxorubicin:Resveratrol (DOX:RES) combinations (at molar ratios ranging from 5:1 to 1:5) in the viability of PANC-1 cells cultured as 2D monolayers and as 3D spheroids was analyzed. The results showed that the cells’ viability was more affected when DOX:RES combinations containing higher contents of RES (1:2–1:5 molar ratios) were used. This can be explained by the ability of RES to reduce the P-glycoprotein (P-gp)-mediated efflux of DOX. Further, it was also revealed that the synergic effect of this drug combination was different in 2D and in 3D cell cultures. In fact, despite of the 1:4 and 1:5 DOX:RES ratios being both synergistic for both types of PANC-1 cell cultures, their Combination Indexes (CI) in the monolayers were lower than those attained in spheroids. Overall, the obtained results revealed that the DOX:RES combination is promising for pancreatic cancer treatment and corroborate the emergent need to evaluate drug combinations in 3D cell cultures.
- Establishment of 2D Cell Cultures Derived From 3D MCF‐7 Spheroids Displaying a Doxorubicin Resistant ProfilePublication . Nunes, Ana Raquel Santos; Costa, Elisabete C.; Barros, Andreia; Diogo, Duarte Miguel de Melo; Correia, Ilídio Joaquim SobreiraIn vitro 3D cancer spheroids generally exhibit a drug resistance profile similar to that found in solid tumors. Due to this property, these models are an appealing for anticancer compounds screening. Nevertheless, the techniques and methods aimed for drug discovery are mostly standardized for cells cultured in 2D. The development of 2D cell culture models displaying a drug resistant profile is required to mimic the in vivo tumors, while the equipment, techniques, and methodologies established for conventional 2D cell cultures can continue to be employed in compound screening. In this work, the response of 3D‐derived MCF‐7 cells subsequently cultured in 2D in medium supplemented with glutathione (GSH) (antioxidant agent found in high levels in breast cancer tissues and a promoter of cancer cells resistance) to Doxorubicin (DOX) is evaluated. These cells demonstrated a resistance toward DOX closer to that displayed by 3D spheroids, which is higher than that exhibited by standard 2D cell cultures. In fact, the 50% inhibitory concentration (IC50) of DOX in 3D‐derived MCF‐7 cell cultures supplemented with GSH is about eight‐times higher than that obtained for conventional 2D cell cultures (cultured without GSH), and is only about two‐times lower than that attained for 3D MCF‐7 spheroids (cultured without GSH). Further investigation revealed that this improved resistance of 3D‐derived MCF‐7 cells may result from their increased P‐glycoprotein (P‐gp) activity and reduced production of intracellular reactive oxygen species (ROS).
- Modelo tumoral in vitro para a avaliação terapêutica de fármacosPublication . Nunes, Ana Raquel Santos; Correia, Ilídio Joaquim Sobreira; Costa, Elisabete Cristina da RochaThe anticancer therapies used nowadays in the clinic display a low efficacy. The ability of cancer cells to develop resistance to drugs used in chemotherapy, contributes to the high mortality associated with this disease. The development of new drug formulations is crucial for improving patient survival rates. In vitro 2D cancer cell cultures have been the main model used, in a first phase, for the development of these new therapies. However, these cultures are unable to mimic the main characteristics of in vivo tumors, such as their drug resistance mechanisms. On the other hand, in vitro 3D cancer models, in particular the cancer cell spheroids, have a drug resistance profile similar to that found in human solid tumors, such as breast cancer. Due to that, these models have been used by researchers as a tool to study anticancer drugs. Nevertheless, the techniques and methods used to analyze the therapeutic efficacy of drugs in in vitro models are developed and standardized only for 2D cells in culture. In this way, the develop of 2D cell culture models that display a drug resistant profile similar to the spheroids can be advantageous since these models can be studied through established equipment, techniques and methodologies. Further investigations revealed that 2D cell cultures obtained by spheroid disintegration and maintained in culture medium supplemented with glutathione (GSH) had the same phenotype as the cells present in 3D spheroids. In the present work, the resistance to doxorubicin (DOX) of 2D cultures of MCF-7 obtained by disaggregation of spheroids and then cultured in culture medium supplemented with GSH was evaluated. The results obtained demonstrated that this model of cell culture presents a DOX resistance profile closer to that presented by spheroids. In fact, the 50 % inhibitory concentration (IC50) of DOX in 3D-derived MCF-7 cell cultures supplemented with GSH was about 8-times higher than that obtained for conventional 2D cell cultures. In this work it was also possible to demonstrate that the increase in resistance of spheroid-derived MCF-7 cells results from increased P-glycoprotein (P-gp) activity and from the reduction of intracellular reactive oxygen species (ROS) levels in these cells. In summary, 2D cell culture obtained from spheroid disaggregation represents an improvement for the future development of anticancer therapies, owing to its ability to present in the 2D model a drug resistance similar to that exhibited by 3D models.