Rosemary (Rosmarinus officinalis L.) Glycolic Extract Protects Liver Mitochondria from Oxidative Damage and Prevents Acetaminophen-Induced Hepatotoxicity.

Rosmarinus officinalis L. (rosemary) is an aromatic culinary herb. Native to the Mediterranean region, it is currently cultivated worldwide. In addition to its use as a condiment in food preparation and in teas, rosemary has been widely employed in folk medicine and cosmetics. Several beneficial effects have been described for rosemary, including antimicrobial and antioxidant activities. Here, we investigated the mechanisms accounting for the antioxidant activity of the glycolic extract of R. officinalis (Ro) in isolated rat liver mitochondria (RLM) under oxidative stress conditions. We also investigated its protective effect against acetaminophen-induced hepatotoxicity in vivo. A crude extract was obtained by fractionated percolation, using propylene glycol as a solvent due to its polarity and cosmeceutical compatibility. The quantification of substances with recognized antioxidant action revealed the presence of phenols and flavonoids. Dereplication studies carried out through LC-MS/MS and GC-MS, supported by The Global Natural Product Social Molecular Networking (GNPS) platform, annotated several phenolic compounds, confirming the previous observation. In accordance, Ro decreased the production of reactive oxygen species (ROS) elicited by Fe2+ or t-BOOH and inhibited the lipid peroxidation of mitochondrial membranes in a concentration-dependent manner in RLM. Such an effect was also observed in liposomes as membrane models. Ro also prevented the oxidation of mitochondrial protein thiol groups and reduced glutathione (GSH). In model systems, Ro exhibited a potent scavenger activity toward 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radicals and superoxide anions. It also demonstrated an Fe2+ chelating activity. Moreover, Ro did not exhibit cytotoxicity or dissipate the mitochondrial membrane potential (∆Ψ) in rat liver fibroblasts (BRL3A cells). To evaluate whether such antioxidant protective activity observed in vitro could also be achieved in vivo, a well-established model of hepatotoxicity induced by acute exposure to acetaminophen (AAP) was used. This model depletes GSH and promotes oxidative-stress-mediated tissue damage. The treatment of rats with 0.05% Ro, administered intraperitoneally for four days, resulted in inhibition of AAP-induced lipid peroxidation of the liver and the prevention of hepatotoxicity, maintaining alanine and aspartate aminotransferase (ALT/AST) levels equal to those of the normal, non-treated rats. Together, these findings highlight the potent antioxidant activity of rosemary, which is able to protect mitochondria from oxidative damage in vitro, and effects such as the antioxidant and hepatoprotective effects observed in vivo.

Necroptosis activation is associated with greater methylene blue-photodynamic therapy-induced cytotoxicity in human pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinomas (PDAC) are the fourth leading cause of death due to neoplasms. In view of the urgent need of effective treatments for PDAC, photodynamic therapy (PDT) appears as a promising alternative. However, its efficacy against PDAC and the mechanisms involved in cell death induction remain unclear. In this study, we set out to evaluate PDT's cytotoxicity using methylene blue (MB) as a photosensitizer (PS) (MB-PDT) and to evaluate the contribution of necroptosis in its effect in human PDAC cells. Our results demonstrated that MB-PDT induced significant death of different human PDAC models presenting two different susceptibility profiles. This effect was independent of MB uptake or its subcellular localization. We found that the ability of triggering necroptosis was determinant to increase the treatment efficiency. Analysis of single cell RNA-seq data from normal and neoplastic human pancreatic tissues showed that specific necroptosis proteins RIPK1, RIPK3 and MLKL presented significant higher expression levels in cells displaying a transformed phenotype providing further support to the use of approaches that activate necroptosis, like MB-PDT, as useful adjunct to surgery of PDAC to tackle the problem of microscopic residual disease as well as to minimize the chance of local and metastatic recurrence.

HSPB1 Is Essential for Inducing Resistance to Proteotoxic Stress in Beta-Cells.

During type 1 diabetes mellitus (T1DM) development, beta-cells undergo intense endoplasmic reticulum (ER) stress that could result in apoptosis through the failure of adaptation to the unfolded protein response (UPR). Islet transplantation is considered an attractive alternative among beta-cell replacement therapies for T1DM. To avoid the loss of beta-cells that will jeopardize the transplant's outcome, several strategies are being studied. We have previously shown that prolactin induces protection against proinflammatory cytokines and redox imbalance-induced beta-cell death by increasing heat-shock protein B1 (HSPB1) levels. Since the role of HSPB1 in beta cells has not been deeply studied, we investigated the mechanisms involved in unbalanced protein homeostasis caused by intense ER stress and overload of the proteasomal protein degradation pathway. We tested whether HSPB1-mediated cytoprotective effects involved UPR modulation and improvement of protein degradation via the ubiquitin-proteasome system. We demonstrated that increased levels of HSPB1 attenuated levels of pro-apoptotic proteins such as CHOP and BIM, as well as increased protein ubiquitination and the speed of proteasomal protein degradation. Our data showed that HSPB1 induced resistance to proteotoxic stress and, thus, enhanced cell survival via an increase in beta-cell proteolytic capacity. These results could contribute to generate strategies aimed at the optimization of beta-cell replacement therapies.

Distinct photo-oxidation-induced cell death pathways lead to selective killing of human breast cancer cells.

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

Fluence Rate Determines PDT Efficiency in Breast Cancer Cells Displaying Different GSH Levels.

Photodynamic therapy (PDT) appears as a promising alternative in the treatment of breast cancer since it can be highly effective in curing cancer while preserving normal tissue. However, predicting outcomes in PDT still constitutes a great challenge. One of the parameters that are usually empirically determined is the rate of photon flux delivered to the tissue (light fluence rate). In the present study, we intended to understand why monolayers of human cells derived from mammary adenocarcinomas (MDA-MB-231 and MCF-7) respond quite differently to fluence rates (cells were irradiated either for 6 or for 16 min) at a fixed light dose (4.5 J cm-2 ) delivered with an array of LEDs in a typical methylene blue PDT protocol. While death rates of MDA-MB-231 cells were insensitive to the fluence rate, MCF-7 cells showed a quite impressive (three times) decrease in cell death levels in the shorter irradiation protocol. Independent on cell type cell death was invariably correlated with the depletion of reduced glutathione intracellular levels and consequently with widespread redox misbalance. Our data show the potential to optimize fluence rates to provide exhaustion of the cell antioxidant responses in order to circumvent therapy resistance of breast tumors.