Hydroxytyrosol Interference with Inflammaging via Modulation of Inflammation and Autophagy.

Inflammaging refers to a chronic, systemic, low-grade inflammation, driven by immune (mainly macrophages) and non-immune cells stimulated by endogenous/self, misplaced or altered molecules, belonging to physiological aging. This age-related inflammatory status is characterized by increased inflammation and decreased macroautophagy/autophagy (a degradation process that removes unnecessary or dysfunctional cell components). Inflammaging predisposes to age-related diseases, including obesity, type-2 diabetes, cancer, cardiovascular and neurodegenerative disorders, as well as vulnerability to infectious diseases and vaccine failure, representing thus a major target for anti-aging strategies. Phenolic compounds-found in extra-virgin olive oil (EVOO)-are well known for their beneficial effect on longevity. Among them, hydroxytyrosol (HTyr) appears to greatly contribute to healthy aging by its documented potent antioxidant activity. In addition, HTyr can modulate inflammation and autophagy, thus possibly counteracting and reducing inflammaging. In this review, we reference the literature on pure HTyr as a modulatory agent of inflammation and autophagy, in order to highlight its possible interference with inflammaging. This HTyr-mediated activity might contribute to healthy aging and delay the development or progression of diseases related to aging.

Perspectives on Populus spp. (Salicaceae) bud extracts as antioxidant and anti-inflammatory agents.

Hydroalcoholic extracts obtained from buds of P. nigra, P. deltoides and P. trichocarpa were characterized by HPLC-DAD-MS analysis and subsequently evaluated in vitro for their antioxidant and anti-inflammatory activities. ABTS and DPPH assays evidenced that P. nigra showed the best antioxidant activity in line with its highest total phenolic content. The analysis of the anti-inflammatory activity clearly demonstrated that all extracts suppressed the production of key pro-inflammatory cytokines (IL-6, Il-1ß and TNF-α) and HMGB1 inflammatory danger signal. These results show antioxidant and critical anti-inflammatory activities mediated by the extracts, emphasising their potentiality as therapeutic agents.

Omega-3 Polyunsaturated Fatty Acids (n-3 PUFAs) for Immunomodulation in COVID-19 Related Acute Respiratory Distress Syndrome (ARDS).

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), might be complicated by Acute Respiratory Distress Syndrome (ARDS) caused by severe lung damage. It is relevant to find treatments for COVID-19-related ARDS. Currently, DHA and EPA n-3 PUFAs, known for their immunomodulatory activities, have been proposed for COVID-19 management, and clinical trials are ongoing. Here, examining COVID-19-related ARDS immunopathology, we reference in vitro and in vivo studies, indicating n-3 PUFA immunomodulation on lung microenvironment (bronchial and alveolar epithelial cells, macrophages, infiltrating immune cells) and ARDS, potentially affecting immune responses in COVID-19-related ARDS. Concerning in vitro studies, evidence exists of the potential anti-inflammatory activity of DHA on airway epithelial cells and monocytes/macrophages; however, it is necessary to analyze n-3 PUFA immunomodulation using viral experimental models relevant to SARS-CoV-2 infection. Then, although pre-clinical investigations in experimental acute lung injury/ARDS revealed beneficial immunomodulation by n-3 PUFAs when extracellular pathogen infections were used as lung inflammatory models, contradictory results were reported using intracellular viral infections. Finally, clinical trials investigating n-3 PUFA immunomodulation in ARDS are limited, with small samples and contradictory results. In conclusion, further in vitro and in vivo investigations are needed to establish whether n-3 PUFAs may have some therapeutic potential in COVID-19-related ARDS.

Natural Polyphenols as Immunomodulators to Rescue Immune Response Homeostasis: Quercetin as a Research Model against Severe COVID-19.

The COVID-19 pandemic is caused by SARS-CoV-2 and is leading to the worst health crisis of this century. It emerged in China during late 2019 and rapidly spread all over the world, producing a broad spectrum of clinical disease severity, ranging from asymptomatic infection to death (4.3 million victims so far). Consequently, the scientific research is devoted to investigating the mechanisms of COVID-19 pathogenesis to both identify specific therapeutic drugs and develop vaccines. Although immunological mechanisms driving COVID-19 pathogenesis are still largely unknown, new understanding has emerged about the innate and adaptive immune responses elicited in SARS-CoV-2 infection, which are mainly focused on the dysregulated inflammatory response in severe COVID-19. Polyphenols are naturally occurring products with immunomodulatory activity, playing a relevant role in reducing inflammation and preventing the onset of serious chronic diseases. Mainly based on data collected before the appearance of SARS-CoV-2, polyphenols have been recently suggested as promising agents to fight COVID-19, and some clinical trials have already been approved with polyphenols to treat COVID-19. The aim of this review is to analyze and discuss the in vitro and in vivo research on the immunomodulatory activity of quercetin as a research model of polyphenols, focusing on research that addresses issues related to the dysregulated immune response in severe COVID-19. From this analysis, it emerges that although encouraging data are present, they are still insufficient to recommend polyphenols as potential immunomodulatory agents against COVID-19.

Granzyme B Expression in Visceral Adipose Tissue Associates With Local Inflammation and Glyco-Metabolic Alterations in Obesity.

Granzyme B (GrB) is a serine protease produced by immune and non-immune cells, able to promote multiple processes, like apoptosis, inflammation, extracellular matrix remodeling and fibrosis. GrB expression in visceral adipose tissue (VAT) was associated with tissue damage, local inflammation and insulin resistance in obesity murine model, but there is no data in humans. Aim of this study was to explore the expression of GrB in VAT from obese subjects in relation to adipose tissue injury, inflammation, metabolic alterations and GrB circulating levels. For this purpose, 85 obese individuals undergoing bariatric surgery and 35 healthy subjects (as control) were recruited at Sapienza University, Rome, Italy. Study participants underwent clinical work-up and routine biochemistry. mRNA expression of GrB in VAT and of a panel of VAT inflammatory markers was analyzed by real-time PCR. Serum GrB levels were measured by Elisa Affymetrix EBIO. We observed that 80% of obese patients expressed GrB mRNA in VAT, and GrB VAT expression was associated with the presence of local inflammation and glucose homeostasis alterations. Moreover, GrB serum levels, which were higher in obese subjects compared to non-obese healthy individuals, were associated with GrB expression in VAT and glyco-metabolic impairment. Our data show, for the first time in humans, that obese subjects with "sick" fat and altered glucose tolerance exhibit GrB expression in VAT, and suggest that GrB might contribute to obesity-related VAT inflammatory remodeling and glucose homeostasis dysregulation. Moreover, increased circulating GrB levels might represent a possible peripheral marker of VAT dysfunction in metabolic diseases.

Granzyme B in Inflammatory Diseases: Apoptosis, Inflammation, Extracellular Matrix Remodeling, Epithelial-to-Mesenchymal Transition and Fibrosis.

Inflammation is strictly interconnected to anti-inflammatory mechanisms to maintain tissue homeostasis. The disruption of immune homeostasis can lead to acute and chronic inflammatory diseases, as cardiovascular, pulmonary, metabolic diseases and cancer. The knowledge of the mechanisms involved in the development and progression of these pathological conditions is important to find effective therapies. Granzyme B (GrB) is a serine protease produced by a variety of immune, non-immune and tumor cells. Apoptotic intracellular and multiple extracellular functions of GrB have been recently identified. Its capability of cleaving extracellular matrix (ECM) components, cytokines, cell receptors and clotting proteins, revealed GrB as a potential multifunctional pro-inflammatory molecule with the capability of contributing to the pathogenesis of different inflammatory conditions, including inflammaging, acute and chronic inflammatory diseases and cancer. Here we give an overview of recent data concerning GrB activity on multiple targets, potentially allowing this enzyme to regulate a wide range of crucial biological processes that play a role in the development, progression and/or severity of inflammatory diseases. We focus our attention on the promotion by GrB of perforin-dependent and perforin-independent (anoikis) apoptosis, inflammation derived by the activation of some cytokines belonging to the IL-1 cytokine family, ECM remodeling, epithelial-to-mesenchymal transition (EMT) and fibrosis. A greater comprehension of the pathophysiological consequences of GrB-mediated multiple activities may favor the design of new therapies aim to inhibit different inflammatory pathological conditions such as inflammaging and age-related diseases, EMT and organ fibrosis.

In vitro studies on anti-inflammatory activities of kiwifruit peel extract in human THP-1 monocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Kiwifruit is native to eastern China and many are the references about the consumption of fruits and fruits extracts of the Actinidia plants in Chinese traditional medicine as therapeutic food supplements to prevent and/or counteract numerous disorders including inflammation-related diseases like cancer. AIM OF THE STUDY: Aim of the present work was to obtain a kiwifruit peel extract, rich in polyphenols, and to explore the anti-inflammatory potential by analyzing its capability to target multiple pathways involved in monocyte-mediated inflammatory response. MATERIALS AND METHODS: The extract was obtained from the fruit peel of Actinidia deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson, cv Hayward and characterized by HPLC-DAD-ESI-MS. Lipopolysaccharide-stimulated THP-1 monocytes were used as a model of human inflammation in vitro. RESULTS: Analytical data evidenced that procyanidins resulted the main polyphenols present in the extract, representing the 92% w/w of the total. The extract inhibited the production of inflammatory molecules such as IL-6, IL-1ß, TNF-α pro-inflammatory cytokines, HMGB1 danger signal and granzyme B serine protease by activated monocytes. In particular, an inhibitory activity of 81%, 68%, 63%, 76% and 60% on the extracellular release of IL-6, IL-1ß, TNF-α, HMGB1 and granzyme B, respectively, was observed by western blot analysis. Moreover, the extract prevented STAT3 activation and promoted autophagy. CONCLUSIONS: The reported findings demonstrated a strong and broad anti-inflammatory profile of the kiwifruit peel extract, which makes it a promising preventive and therapeutic natural ingredient for nutraceutical, cosmetic and pharmaceutical formulations to counteract multiple inflammatory disorders.

Increased circulating granzyme B in type 2 diabetes patients with low-grade systemic inflammation.

In metabolic diseases, like type 2 diabetes (T2D), adipose tissue (AT) is infiltrated by macrophages and other leukocytes - which secrete many bioactive peptides leading to local and systemic low-grade chronic inflammation - and undergoes remodeling and aberrant fibrosis. Granzyme B (GrB) is a serine protease produced by some leukocytes, including cytotoxic lymphocytes and macrophages. It exerts both intracellular apoptotic function and extracellular functions, leading to tissue injury, inflammation and repair. Elevated circulating GrB levels have been found in aging- and inflammation-associated diseases and a role for GrB in the pathogenesis of several chronic inflammatory diseases has been reported. Aims of this study were to investigate circulating GrB levels in T2D patients in relation to their systemic inflammatory profile and to unravel its correlates. For this cross-sectional study, we recruited 51 consecutive T2D patients referring to our diabetes outpatient clinics (Sapienza University, Rome, Italy) for metabolic evaluations, and 29 sex, age and body mass index comparable non-diabetic subjects as control group. Study participants underwent clinical work-up; fasting blood sampling was performed for routine biochemistry and for inflammatory profile (CRP, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, GM-CSF, adiponectin, WISP1); serum GrB was measured by Human Granzyme-B Platinum Elisa kit (Affymetrix EBIO). We found that T2D patients had serum levels of GrB significantly higher than the control group (10.17 ±â€¯12.6 vs 7.2 ±â€¯14.1 pg/ml, p = 0.03). Moreover, in T2D patients increased GrB correlated with unfavorable inflammatory profile, as described by elevated levels of validated adipokines such as IL-6 (p = 0.04), TNF-α (p = 0.019) and WISP1 (p = 0.005). Furthermore, multivariate linear regression analysis showed that increased GrB was associated with T2D diagnosis independently from possible confounders. In conclusion, our results show that increased levels of circulating GrB are associated with T2D diagnosis and correlates with markers of AT-linked systemic inflammation, suggesting a potential role for GrB in the inflammatory and reactive processes occurring in metabolic diseases.

Tyrosinase-Treated Hydroxytyrosol-Enriched Olive Vegetation Waste with Increased Antioxidant Activity Promotes Autophagy and Inhibits the Inflammatory Response in Human THP-1 Monocytes.

Treatment of olive vegetation waste with tyrosinase immobilized on multiwalled carbon nanotubes increased the antioxidant activity as a consequence of the conversion of phenols to corresponding catechol derivatives, as evaluated by DPPH, Comet assay, and micronucleus analyses. During this transformation, 4-hydroxyphenethyl alcohol (tyrosol) was quantitatively converted to bioactive 3,4-dihydroxyphenethyl alcohol (hydroxytyrosol). The hydroxytyrosol-enriched olive vegetation waste also promoted autophagy and inhibited the inflammatory response in human THP-1 monocytes.

Docosahexaenoic acid (DHA) promotes immunogenic apoptosis in human multiple myeloma cells, induces autophagy and inhibits STAT3 in both tumor and dendritic cells.

Docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid found in fish oil, is a multi-target agent and exerts anti-inflammatory and anticancer activities alone or in combination with chemotherapies. Combinatorial anticancer therapies, which induce immunogenic apoptosis, autophagy and STAT3 inhibition have been proposed for long-term therapeutic success. Here, we found that DHA promoted immunogenic apoptosis in multiple myeloma (MM) cells, with no toxicity on PBMCs and DCs. Immunogenic apoptosis was shown by the emission of specific DAMPs (CRT, HSP90, HMGB1) by apoptotic MM cells and the activation of their pro-apoptotic autophagy. Moreover, immunogenic apoptosis was directly shown by the activation of DCs by DHA-induced apoptotic MM cells. Furthermore, we provided the first evidence that DHA activated autophagy in PBMCs and DCs, thus potentially acting as immune stimulator and enhancing processing and presentation of tumor antigens by DCs. Finally, we found that DHA inhibited STAT3 in MM cells. STAT3 pathway, essential for MM survival, contributed to cancer cell apoptosis by DHA. We also found that DHA inhibited STAT3 in blood immune cells and counteracted STAT3 activation by tumor cell-released factors in PBMCs and DCs, suggesting the potential enhancement of the anti-tumor function of multiple immune cells and, in particular, that of DCs.

Epitelial-to-mesenchimal transition and invasion are upmodulated by tumor-expressed granzyme B and inhibited by docosahexaenoic acid in human colorectal cancer cells.

BACKGROUND: Granzyme B (GrB) is a serine protease, traditionally known as expressed by cytotoxic lymphocytes to induce target cell apoptosis. However, it is emerging that GrB, being also produced by a variety of normal and neoplastic cells and potentially acting on multiple targets, might represent a powerful regulator of a wide range of fundamental biological processes. We have previously shown that GrB is expressed in urothelial carcinoma tissues and its expression is associated to both pathological tumor spreading and EMT. We have also shown that docosahexaenoic acid (DHA), a dietary ω-3 polyunsaturated fatty acid with anti-tumor activity, while inhibiting urothelial and pancreatic carcinoma cell invasion also inhibited their GrB expression in vitro. In this study, we characterized a panel of colorectal carcinoma (CRC) cells, with different invasive capabilities, for GrB expression and for the contribution of GrB to their EMT and invasive phenotype. In addition, we investigated the effect of DHA on CRC cell-associated GrB expression, EMT and invasion. METHODS: The expression levels of GrB and EMT-related markers were evaluated by Western blotting. GrB knockdown was performed by Stealth RNAi small interfering RNA silencing and ectopic GrB expression by transfection of human GrB vector. Cell invasion was determined by the BioCoat Matrigel invasion chamber test. RESULTS: GrB was produced in 57.1% CRC cell lines and 100% CRC-derived Cancer Stem Cells. Although GrB was constitutive expressed in both invasive and noninvasive CRC cells, GrB depletion in invasive CRC cells downmodulated their invasion in vitro, suggesting a contribution of GrB to CRC invasiveness. GrB loss or gain of function downmodulated or upmodulated EMT, respectively, according to the analysis of cancer cell expression of three EMT biomarkers (Snail1, E-cadherin, N-cadherin). Moreover, TGF-ß1-driven EMT was associated to the enhancement of GrB expression in CRC cell lines, and GrB depletion led to downmodulation of TGF-ß1-driven EMT. In addition, DHA inhibited GrB expression, EMT and invasion in CRC cells in vitro. CONCLUSIONS: These findings present a novel role for GrB as upmodulator of EMT in CRC cells. Moreover, these results support the use of DHA, a dietary compound without toxic effects, as adjuvant in CRC therapy.

Omega-3 Fatty Acids and Cancer Cell Cytotoxicity: Implications for Multi-Targeted Cancer Therapy.

Cancer is a major disease worldwide. Despite progress in cancer therapy, conventional cytotoxic therapies lead to unsatisfactory long-term survival, mainly related to development of drug resistance by tumor cells and toxicity towards normal cells. n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can exert anti-neoplastic activity by inducing apoptotic cell death in human cancer cells either alone or in combination with conventional therapies. Indeed, n-3 PUFAs potentially increase the sensitivity of tumor cells to conventional therapies, possibly improving their efficacy especially against cancers resistant to treatment. Moreover, in contrast to traditional therapies, n-3 PUFAs appear to cause selective cytotoxicity towards cancer cells with little or no toxicity on normal cells. This review focuses on studies investigating the cytotoxic activity of n-3 PUFAs against cancer cells via apoptosis, analyzing the molecular mechanisms underlying this effective and selective activity. Here, we highlight the multiple molecules potentially targeted by n-3 PUFAs to trigger cancer cell apoptosis. This analysis can allow a better comprehension of the potential cytotoxic therapeutic role of n-3 PUFAs against cancer, providing specific information and support to design future pre-clinical and clinical studies for a better use of n-3 PUFAs in cancer therapy, mainly combinational therapy.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death.

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called "damage-associated molecular patterns" (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.

Hydroxytyrosol-Derived Compounds: A Basis for the Creation of New Pharmacological Agents for Cancer Prevention and Therapy.

Hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol, HTyr] is a phenolic compound found in olive leaves and fruits and extra-virgin olive oil, which has well-known strong antioxidant and radical-scavenging properties. Recently, it has received particular attention for its antiproliferative and apoptotic activities and its anti-inflammatory properties. During the past few years, more efforts have been focused on synthesizing HTyr-derived compounds with enhanced biological activities for their potential use in different chronic degenerative diseases. In this paper, we report a dissertation on the current knowledge of selected synthetic HTyr derivatives and analogues and their potential use in cancer prevention and therapy, which are related to their antioxidant, antiproliferative/apoptotic, and anti-inflammatory properties. On the basis of the perspective of using HTyr-derived compounds as anticancer agents, we have taken into account only studies that were performed in experimental cell-based models.

Capsaicin-mediated apoptosis of human bladder cancer cells activates dendritic cells via CD91.

OBJECTIVES: Immunostimulation by anticancer cytotoxic drugs is needed for long-term therapeutic success. Activation of dendritic cells (DCs) is crucial to obtain effective and long-lasting anticancer T-cell mediated immunity. The aim of this study was to explore the effect of capsaicin-mediated cell death of bladder cancer cells on the activation of human monocyte-derived CD1a+ immature DCs. METHODS: Immature DCs (generated from human peripheral blood-derived CD14+ monocytes cultured with granulocyte-macrophage colony stimulating factor and interleukin-4) were cocultured with capsaicin (CPS)-induced apoptotic bladder cancer cells. DC activation was investigated using immunofluorescence and flow cytometric analysis for key surface molecules. In some experiments, CD91 was silenced in immature DCs. RESULTS: We found that capsaicin-mediated cancer cell apoptosis upregulates CD86 and CD83 expression on DCs, indicating the induction of DC activation. Moreover, silencing of CD91 (a common receptor for damage-associated molecular patterns, such as calreticulin and heat-shock protein-90/70) in immature DCs led to the inhibition of DC activation. CONCLUSIONS: Our data show that CPS-mediated cancer cell apoptosis activates DCs via CD91, suggesting CPS as an attractive candidate for cancer therapy.

Dietary ω -3 polyunsaturated fatty acid DHA: a potential adjuvant in the treatment of cancer.

ω -3 Polyunsaturated fatty acids (PUFAs), mainly present in fish oil, are part of the human diet. Among PUFAs, docosahexaenoic acid (DHA) has received particular attention for its anti-inflammatory, antiproliferative, proapoptotic, antiangiogenetic, anti-invasion, and antimetastatic properties. These data suggest that DHA can exert antitumor activity potentially representing an effective adjuvant in cancer chemotherapy. This review is focused on current knowledge supporting the potential use of DHA for the enhancement of the efficacy of anticancer treatments in relation to its ability to enhance the uptake of anticancer drugs, regulate the oxidative status of tumor cells, and inhibit tumor cell invasion and metastasis.

Capsaicin as an inducer of damage-associated molecular patterns (DAMPs) of immunogenic cell death (ICD) in human bladder cancer cells.

Few conventional cytotoxic anticancer therapeutics induce immunogenic cell death (ICD). This means that they induce tumor cells to undergo apoptosis while eliciting the emission of a spatiotemporal-defined combination of damage-associated molecular patterns (DAMPs) decoded by the immune system to activate antitumor immunity effective for long-term therapeutic success. The neurotoxin capsaicin (CPS) can induce both cancer cell apoptosis and immune-mediated tumor regression. In the present study, we investigated whether CPS is capable of eliciting the emission of ICD hallmarks in human bladder cancer cell lines undergoing apoptosis. We demonstrated that CPS induces pre- and early apoptotic cell surface exposure of calreticulin (CRT), HSP90, and HSP70 as well as ATP release. Moreover, CRT exposure was prevented by inhibition of endoplasmic reticulum-Golgi traffic by brefeldin A. Furthermore, high-mobility group box 1, HSP90, and HSP70 were passively released at late apoptotic stages. We provide the first evidence that CPS is an inducer of ICD hallmarks, suggesting CPS as a novel potential immunogenic cytotoxic agent.

Docosahexaenoic acid inhibits invasion of human RT112 urinary bladder and PT45 pancreatic carcinoma cells via down-modulation of granzyme B expression.

Fish oil-derived n-3 polyunsaturated fatty acids (n-3 PUFAs) inhibit invasion of some tumor cell types in vitro and in vivo. The mechanisms underlying this activity are unclear. Here, we examined the capability of n-3 PUFA-docosahexaenoic acid (22:6n-3; DHA) to affect the invasiveness of human RT112 urinary bladder and PT45 pancreatic carcinoma cell lines in vitro and the mechanism underlying this activity; n-6 PUFA-arachidonic acid (20:4n-6; AA) served as control. We showed that, in contrast to AA, 25, 50 and 100 µM DHA significantly inhibited in a dose-dependent manner the invasion through Matrigel of both RT112 and PT45 cells. Then, we analyzed whether the serine proteinase granzyme B (GrB), originally known as cytotoxic molecule of lymphocytes and recently also characterized for its extracellular functions such as invasion promotion of bladder cancer cells, might be involved in the invasion inhibitory activity exerted by DHA. We demonstrated that, accordingly to RT112 bladder cancer cells, PT45 cells expressed GrB and GrB promoted their invasion, since stealth RNA interference-mediated down-regulation of GrB dramatically suppressed PT45 cell invasion. Notably, we also showed that, in contrast to AA, 25, 50 and 100 µM DHA induced a dose-dependent down-modulation of GrB expression in both RT112 and PT45 cells. In conclusion, DHA can reduce the invasive phenotype of bladder and pancreatic carcinoma cells, and we provide the first evidence for a possible causative role of GrB in DHA-induced inhibition of cancer cell invasion. The potential use of fish oil as adjuvant antibladder and antipancreatic cancer agent may be suggested.

The n3-polyunsaturated fatty acid docosahexaenoic acid induces immunogenic cell death in human cancer cell lines via pre-apoptotic calreticulin exposure.

Some anticancer chemotherapeutics, such as anthracyclines and oxaliplatin, elicit immunogenic apoptosis, meaning that dying cancer cells are engulfed by dendritic cells and tumor antigens are efficiently presented to CD8+ T cells, which control residual tumor cells. Immunogenic apoptosis is characterized by pre-apoptotic cell surface exposure of calreticulin (CRT), which usually resides into the endoplasmic reticulum. We investigated the ability of the n3-polyunsaturated fatty acid docosahexaenoic acid (22:6n3, DHA) to induce pre-apoptotic CRT exposure on the surface of the human PaCa-44 pancreatic and EJ bladder cancer cell lines. Cells were treated with 150 µM DHA for different time periods, and, by immunoblot and immunofluorescence, we showed that DHA induced CRT exposure, before the apoptosis-associated phosphatidylserine exposure. As for the known immunogenic compounds, CRT exposure was inhibited by the antioxidant GSH, the pan-caspase zVAD-FMK, and caspase-8 IETD-FMK inhibitor. We provide the first evidence that DHA induces CRT exposure, representing thus a novel potential anticancer immunogenic chemotherapeutic agent.