Evaluation of the antitumor effect of combined treatment with C6-ceramide and 7-ketocholesterol in a breast cancer cell line

A busca por novas terapias antitumorais é incessante. A ceramida, molécula central no metabolismo dos esfingolipídeos, vem recebendo grande atenção na literatura devido a seu papel na regulação de processos celulares como proliferação celular, apoptose, autofagia, entre outros. A C6-ceramida, um análogo de cadeia curta, vem sendo vastamente explorada por conta de sua capacidade de indução de morte celular tumoral, principalmente quando em associação com outro composto antitumoral. Nesses estudos, os achados apontam para uma inibição sinergística do tumor, com redução da toxicidade do tratamento e até reversão do fenótipo de múltipla resistência a drogas (MDR). O 7-cetocolesterol (7-KC), por sua vez, é um oxisterol com promissora atividade antitumoral, capaz de induzir um modo complexo de morte celular que envolve estresse oxidativo, apoptose e autofagia (oxiapoptofagia). Neste projeto, buscamos avaliar o potencial terapêutico antitumoral do tratamento combinada com as moléculas de C6-cer e 7-KC em uma linhagem de câncer de mama (MDA-MB-231), visando elucidar os mecanismos de morte celular envolvidos no processo. A eficácia da combinação dessas moléculas mostrou-se tempo-dependente, pois células tratadas por 24 horas apresentaram efeitos irrisórios de morte celular em comparação com tratamento por 48 horas. Em ambos os casos, as moléculas agiram de maneira sinérgica, evidenciado pelo cálculo do índice combinatório (CI). A apoptose com ativação das caspases 3/7 apresentou-se como protagonista na indução da morte dessas células, ocorrendo de forma dose-dependente. Além disso, pudemos verificar efeitos substanciais desse tratamento no potencial da membrana mitocondrial e da estrutura do citoesqueleto por meio da análise da organização estrutural da F-actina. Quanto à geração de ROS, a C6-cer, mas não o 7-KC, foi capaz de produzir ROS após o tratamento. Finalmente, a autofagia não foi desencadeada por nenhuma dessas moléculas, seja em administração individual ou em combinação. Acreditamos que nossos resultados sobre a eficácia deste tratamento combinado nesta linhagem celular específica podem abrir caminho para estudos em diferentes linhas de células cancerígenas ou modelos mais complexos daqui em diante, visando verificar se este efeito também é conservado em outros modelos tumorais bem como compreender mais a fundo os processos de sinalização celular e moléculas envolvidas no desencadeamento de morte celular tumoralNew alternatives for cancer treatment are continuously sought. Ceramide, the central molecule in sphingolipids metabolism, has received great attention in the literature due to its role in regulating cellular processes such as cell proliferation, apoptosis, autophagy, among others. C6-ceramide, a short-chain analog, has been widely explored because of its ability to induce tumor cell death, especially when in combination with another antitumor agent. In these studies, the findings point to synergistic inhibition of the tumor, with reduced treatment toxicity and even reversion of the multiple drug resistance (MDR) phenotype. 7-Ketocholesterol (7-KC), in turn, is an oxysterol with promising antitumor activity, capable of inducing a complex mode of cell death that involves oxidative stress, apoptosis and autophagy (oxypoptophagy). In this project, we sought to evaluate the antitumor therapeutic potential of the combined treatment with C6-cer and 7-KC in a breast cancer cell line (MDA-MB-231), and elucidate the mechanisms of cell death involved in this process. The effectiveness of the combination of these molecules proved to be time-dependent since cells treated for 24 hours showed little effect of cell death when compared to 48 hours treatment period. In both cases, molecules acted synergistically, evidenced by the calculation of the combination index (CI) Apoptosis with caspase 3/7 activation presented themselves as major players on these cells death induction, both occurring in a dose-dependent manner. Also, we were able to verify substantial effect of this treatment on the mitochondrial membrane potential and cytoskeleton structure by analyzing F-actin structural organization. As for ROS Generation, C6-cer but not 7-KC was able to produce ROS after treatment. Finally, autophagy was not triggered by any of these molecules, nor as single agents neither in combination. We believe that our findings on this combined regimen efficacy in this specific cell line may pave the way for studies on different cancer cell lines or more complex models henceforth, in order to verify whether this outcome is also conserved in other tumor models as well as to perform a more in-depth analysis on signaling pathways and molecules involved in the process of triggering cell deat

Ferroptosis Mechanisms Involved in Neurodegenerative Diseases

Ferroptosis is a type of cell death that was described less than a decade ago. It is caused by the excess of free intracellular iron that leads to lipid (hydro) peroxidation. Iron is essential as a redox metal in several physiological functions. The brain is one of the organs known to be affected by iron homeostatic balance disruption. Since the 1960s, increased concentration of iron in the central nervous system has been associated with oxidative stress, oxidation of proteins and lipids, and cell death. Here, we review the main mechanisms involved in the process of ferroptosis such as lipid peroxidation, glutathione peroxidase 4 enzyme activity, and iron metabolism. Moreover, the association of ferroptosis with the pathophysiology of some neurodegenerative diseases, namely Alzheimer’s, Parkinson’s, and Huntington’s diseases, has also been addressed

Constraining the magnitude of the Chiral Magnetic Effect with Event Shape Engineering in Pb-Pb collisions at sNN\sqrt{s_{\rm NN}} = 2.76$ TeV

In ultrarelativistic heavy-ion collisions, the event-by-event variation of the elliptic flow $v_2$ reflects fluctuations in the shape of the initial state of the system. This allows to select events with the same centrality but different initial geometry. This selection technique, Event Shape Engineering, has been used in the analysis of charge-dependent two- and three-particle correlations in Pb-Pb collisions at $\sqrt{s_{_{\rm NN}}} =2.76$ TeV. The two-particle correlator $\langle \cos(\varphi_\alpha - \varphi_\beta) \rangle$, calculated for different combinations of charges $\alpha$ and $\beta$, is almost independent of $v_2$ (for a given centrality), while the three-particle correlator $\langle \cos(\varphi_\alpha + \varphi_\beta - 2\Psi_2) \rangle$ scales almost linearly both with the event $v_2$ and charged-particle pseudorapidity density. The charge dependence of the three-particle correlator is often interpreted as evidence for the Chiral Magnetic Effect (CME), a parity violating effect of the strong interaction. However, its measured dependence on $v_2$ points to a large non-CME contribution to the correlator. Comparing the results with Monte Carlo calculations including a magnetic field due to the spectators, the upper limit of the CME signal contribution to the three-particle correlator in the 10-50% centrality interval is found to be 26-33% at 95% confidence level

Constraining the magnitude of the chiral magnetic effect with event shape engineering in Pb–Pb collisions at √sNN=2.76 TeV

In ultrarelativistic heavy-ion collisions, the event-by-event variation of the elliptic flow v2 reflects fluctuations in the shape of the initial state of the system. This allows to select events with the same centrality but different initial geometry. This selection technique, Event Shape Engineering, has been used in the analysis of charge-dependent two- and three-particle correlations in Pb–Pb collisions at √sNN=2.76 TeV. The two-particle correlator 〈cos⁡(φα−φβ)〉, calculated for different combinations of charges α and β, is almost independent of v2 (for a given centrality), while the three-particle correlator 〈cos⁡(φα+φβ−2Ψ2)〉 scales almost linearly both with the event v2 and charged-particle pseudorapidity density. The charge dependence of the three-particle correlator is often interpreted as evidence for the Chiral Magnetic Effect (CME), a parity violating effect of the strong interaction. However, its measured dependence on v2 points to a large non-CME contribution to the correlator. Comparing the results with Monte Carlo calculations including a magnetic field due to the spectators, the upper limit of the CME signal contribution to the three-particle correlator in the 10–50% centrality interval is found to be 26–33% at 95% confidence level