Regulated cell death (RCD) has always been considered a tolerogenic event. Immunogenic cell death (ICD) occurs as a consequence of tumour cell death accompanied by the release of damage-associated molecular patterns (DAMPs), triggering an immune response. ICD plays a major role in stimulating the function of the immune system in cancer during chemotherapy and radiotherapy. ICD can therefore represent one of the routes to boost anticancer immune responses. According to the recommendations of the Nomenclature Committee on Cell Death (2018), apoptosis (type I cell death) and necrosis (type II cell death) represent are not the only types of RCD, which also includes necroptosis, pyroptosis, ferroptosis and others. Specific downstream signalling molecules and death-inducing stimuli can regulate distinct forms of ICD, which develop and promote the immune cell response. Dying cells deliver different potential immunogenic signals, such as DAMPs, which are able to stimulate the immune system. The acute exposure of DAMPs can prime antitumour immunity by inducing activation of antigen-presenting cells (APC), such as dendritic cells (DC), leading to the downstream response by cytotoxic T cells and natural killer cells (NK). As ICD represents an important target to direct and develop new pharmacological interventions, the identification of bioactive natural products, which are endowed with low side effects, higher tolerability and preferentially inducing immunogenic programmed cell death, represents a priority in biomedical research. The ability of ICD to drive the immune response depends on two major factors, neither of which is intrinsic to cell death: ‘Antigenicity and adjuvanticity’. Indeed, the use of natural ICD-triggering molecules, alone or in combination with different (immuno)therapies, can result in higher efficacy and tolerability. Here, we focused on natural (marine) compounds, particularly on marine microalgae derived molecules such as exopolysaccharides, sulphated polysaccharides, glycopeptides, glycolipids, phospholipids, that are endowed with ICD-inducing properties and sulfavants. Here, we discuss novel and repurposed small-molecule ICD triggers, as well as their ability to target important molecular pathways including the IL-6, TNF-α and interferons (IFNs), leading to immune stimulation, which could be used alone or in combinatorial immunotherapeutic strategies in cancer prevention and therapies.

Microalgal biotechnology is gaining importance. However, key issues in the pipeline from species selection towards large biomass production still require improvements to maximize the yield and lower the microalgal production costs. This study explores a co-cultivation strategy to improve the bioactive compounds richness of the harvested microalgal biomass. Based on their biotechnological potential, two diatoms (Skeletonema marinoi, Cyclotella cryptica) and one eustigmatophyte (Nannochloropsis oceanica) were grown alone or in combination. Concentrations of ten vitamins (A, B1, B2, B6, B12, C, D2, D3, E and H), carotenoids and polyphenols, together with total flavonoids, sterols, lipids, proteins and carbohydrates, were compared. Moreover, antioxidant capacity and chemopreventive potential in terms inhibiting four human tumor-derived and normal cell lines proliferation were evaluated. Co-cultivation can engender biomass with emergent properties regarding bioactivity or bioactive chemical profile, depending on the combined species. The high vitamin content of C. cryptica or N. oceanica further enhanced (until 10% more) when co-cultivated, explaining the two-fold increase of the antioxidant capacity of the combined C. cryptica and N. oceanica biomass. Differently, the chemopreventive activity was valuably enhanced when coupling the two diatoms C. cryptica and S. marinoi. The results obtained in this pilot study promote microalgal co-cultivation as a valuable strategy aiming to boost their application in eco-sustainable biotechnology.

The antioxidant activity of natural compounds consists in their ability to modulate gene and protein expression, thus inducing an integrated cell protective response and repair processes against oxidative stress. New screening tools and methodologies are crucial for the actual requirement of new products with antioxidant activity to boost endogenous oxidative stress responsive pathways, Reactive Oxygen Species (ROS) metabolism and immune system activity, preserving human health and wellness. In this study, we performed and tested an integrated oxidative stress analysis, using DPPH assay and PNT2 cells injured with DPPH. We firstly investigated the mechanism of action of the oxidising agent (DPPH) on PNT2 cells, studying the variation in cell viability, oxidative stress genes, inflammatory mediator and ROS levels. The results reveal that DPPH activated ROS production and release of Prostaglandin E2 in PNT2 at low and intermediate doses, while cells switched from survival to cell death signals at high doses of the oxidising agent. This new in vitro oxidative stress model was validated by using Trolox, β-carotene and total extract of the green microalga Testraselmis suecica. Only the T. suecica extract can completely counteract DPPH-induced injury, since its chemical complexity demonstrated a multilevel protecting and neutralising effect against oxidative stress in PNT2.

The exploration of natural antioxidants for nutraceuticals and pharmaceuticals industries has recently increased. In this scenario, marine microorganisms, represent underestimated sources of bioactive products endowed with beneficial effects on health, that include anti-oxidant, anti-tumor and anti-angiogenic activities. Here, we tested the potential cancer preventive activities of extracts from the marine coastal diatom Skeletonema marinoi Sarno and Zingone (CCMP 2092) on prostate cancer cells, in vitro. Skeletonema marinoi was grown under a sinusoidal light distribution with a midday peak of 150 μmol photons m−2 s−1, and then, once cells reached the exponential phase, shifted to a sinusoidal light distribution with a higher midday peak light intensity of 600 μmol photons m−2 s−1. This high light condition induces in cells the synthesis of antioxidant and protective molecules. Total wet biomass was collected and chemically extracted by methanol solvent. The dried extract was tested as a food supplement on prostate (PC-3), as compared to normal prostate epithelium. Gene expression analysis was performed by qPCR, measuring the variation of 365 different genes involved in the apoptosis and immunogenic cell death pathways. Treatments with Skeletonema marinoi extracts (1-10-100 µg ml-1) resulted in a statistically significant, dose dependent, antiproliferative (inhibition at 70% of cell viability at 100 µg ml-1) and pro-apoptotic effects in PC-3 cells, while sparing normal epithelial prostatic cells. Among the gene panel analysed as key indicators of the PC3 cell fate in terms of signalling pathway prediction, we observed up/down regulation of APAF1, BAD, BAG1, BAG3, BAK1, BAX, BCL2, BCL2L1, BCL2L13, BCL2L2, BCL3, BDNF, BECN1, BFAR, BID, BIK, BIRC2, BIRC3, BIRC6, BNIP1, BOK, BRAF, BRCA1, BRE, CARD17, CARD9, CASP1-2-3-4-5-6-7-10-14, CBX4, CRADD, DEDD, DHCR24, FADD, FAS, FASLG, FASTK. The integrated gene expression analysis revealed necroptosis activation in cells with the up-regulation of the inflammatory mediators such as F2R, F2, IL18-19-A-31RA, SOCS2, SOCS3 genes and the downregulation of the angiogenic factors such as ALOX12, ALOX15B, PIK3CA, PIK3R2, and VEGF genes. One of the main candidates as compounds responsible for such cancer preventive activities is the diatom-xanthophyll photoprotective diatoxanthin, probably in synergy with other molecules. Further metabolomics analysis on the extracts will help in understanding and interpretation of the results. These results demonstrate promising cancer preventive activities of Skeletonema marinoi extracts in PC3 cell lines. This confirms microalgal-derived drugs as potential relevant source of nutraceutical novel food and candidate as potential dietary intervention in cancer prevention approaches. Citation Format: Clementina Sansone, Piscitelli Concetta, Galasso Christian, Smerilli Arianna, Bruno Antonino, Noonan Douglas, Albini Adriana, Brunet Christophe. Skeletonema marinoi (diatom) extracts are endowed with promising cancer preventive properties in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 25.