Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

Recombinant Toxoplasma gondii Calreticulin protein provides partial protection against acute and chronic toxoplasmosis.

Toxoplasma gondii, a highly prevalent apicomplexan pathogen, can cause serious or even fatal toxoplasmosis in both animals and humans. Immunoprophylaxis is considered a promising strategy for controlling this disease. Calreticulin (CRT) is known as a pleiotropic protein, which is critical for calcium storage and phagocytosis of apoptotic cells. Our study examined the protective effects of recombinant T. gondii Calreticulin (rTgCRT) as a recombinant subunit vaccine against the T. gondii challenge in mice. Here, rTgCRT was successfully expressed in vitro using prokaryptic expression system. Polyclonal antibody (pAb) has been prepared by immunizing Sprague Dawley rats with rTgCRT. Western blotting showed that rTgCRT and natural TgCRT protein were recognized by serum of T. gondii infected mice and rTgCRT pAb, respectively. T lymphocyte subsets and antibody response were monitored using flow cytometry and enzyme-linked immunosorbent assay (ELISA). The results showed that ISA 201 rTgCRT could stimulate lymphocyte proliferation and induce high levels of total and subclasses of IgG. After the RH strain challenge, a longer survival period was given by the ISA 201 rTgCRT vaccine compared to the control groups; after infection with the PRU strain, we observed a 100% survival rate and a significant reduction in cysts load and size. In the neutralization test, high concentrations of rat-rTgCRT pAb provided 100% protection, while in the passive immunization trial, only weak protection was observed after RH challenge, indicating that rTgCRT pAb needs further modification to improve its activity in vivo. Taken together, these data confirmed that rTgCRT can trigger strong cellular and humoral immune responses against acute and chronic toxoplasmosis.

Silver nanoparticles induce a non-immunogenic tumor cell death.

Immunogenic cell death (ICD) is a form of cell death characterized by the release of danger signals required to trigger an adaptive immune response against tumor-associated antigens. Silver nanoparticles (AgNP) display anti-proliferative and cytotoxic effects in tumor cells, but it has not been previously studied whether AgNP act as an ICD inductor. The present study evaluated the in vitro release of calreticulin as a damage-associated molecular pattern (DAMP) associated with the cytotoxicity of AgNP and their in vivo anti-cancer effects. In vitro, mouse CT26 colon carcinoma and MCA205 fibrosarcoma cells were exposed to AgNP and then cell proliferation, adhesion, and release of calreticulin were determined. The results indicated there were time- and concentration-related anti-proliferative effects of AgNP in both the CT26 and MCA205 lines. Concurrently, changes in cell adhesion were detected mainly in the CT26 cells. Regarding DAMP detection, a significant increase in calreticulin was observed only in CT26 cells treated with doxorubicin and AgNP; however, no differences were found in the MCA205 cells. In vivo, the survival and growth of subcutaneous tumors were monitored after vaccination of mice with cell debris from tumor cells treated with AgNP or after intra-tumoral administration of AgNP to established tumors. Consequently, anti-tumoral prophylactic immunization with AgNP-dead cells failed to protect mice from tumor re-challenge; intra-tumor injection of AgNP did not induce a significant effect. In conclusion, there was a noticeable anti-tumoral effect of AgNP in vitro in both CT26 and MCA205 cell lines, accompanied by the release of calreticulin in CT26 cells. In vivo, immunization with cell debris derived from AgNP-treated tumor cells failed to induce a protective immune response in the cancer model mice. Clearly, further research is needed to determine if one could combine AgNP with other ICD inducers to improve the anti-tumor effect of these nanoparticles in vivo.

Alantolactone induces concurrent apoptosis and GSDME-dependent pyroptosis of anaplastic thyroid cancer through ROS mitochondria-dependent caspase pathway.

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the fatal cancers and has not effective treatments. Alantolactone (ATL), a terpenoid extracted from traditional Chinese medicinal herb Inula helenium L., confers significant anti-inflammatory, antibacterial and antitumor activity. However, the activity and mechanisms of ATL in ATC remain unclear. PURPOSE: To investigate the potential anti-ATC effects in vitro and in vivo and the mechanisms involved. METHODS: The anti-proliferative activity of Alantolactone (ATL) against ATC cells was analyzed through CCK-8 and colony formation assays. Flow cytometry assay was performed to assess the cell cycle, cell apoptosis, ROS, and mitochondrial membrane potential (ΔΨm), whereas the cellular localization of cytochrome c and calreticulin were determined using cellular immunofluorescence assays. The lactate dehydrogenase (LDH) enzyme activity in the cell culture medium was measured using a commercial LDH kit, whereas ELISA was conducted to assess the secretory function of IL-1ß. Western blot assays were conducted to determine the expression or regulation of proteins associated with apoptosis and pyroptosis. Subcutaneous tumor model of nude mice was established to evaluate the anticancer activity of ATL in vivo. The expression of Ki67, cyclin B1, cleaved-PARP, cleaved-caspase 3, and IL-1ß in the animal tumor tissues was profiled using immunohistochemistry analyses. RESULTS: Our data showed that ATL significantly inhibited the proliferation and colony formation activity of ATC cells. ATL induced ATC cell cycle arrest at G2/M phase, and downregulated the expression of cyclin B1 and CDC2. Furthermore, ATL induced concurrent apoptosis and pyroptosis in the ATC cells, and the cleavage of PARP and GSDME. It also significantly increased the release of LDH and IL-1ß. Mechanically, ATL-mediated increase in ROS suppressed the Bcl-2/Bax ratio, downregulated the mitochondrial membrane potential and increased the release of cytochrome c, leading to caspase 9 and caspase 3 cleavage. We also found that ATL induced the translocation of an immunogenic cell death marker (calreticulin) to the cell membrane. In addition, it inhibited the growth of the ATC subcutaneous xenograft model, and activated proteins associated with apoptosis and pyroptosis, with a high safety profile. CONCLUSION: Taken together, these results firstly demonstrated that ATL exerted an anti-ATC activity by inducing concurrent apoptosis and GSDME-dependent pyroptosis through ROS-mediated mitochondria-dependent caspase activation. Meanwhile, these cell deaths exhibited obvious characteristics of immunogenic cell death, which may synergistically increase the potential of cancer immunotherapy in ATC. Further studies are needed to explore deeper mechanisms for the anti- ATC activity of ATL.

Clioquinol induces autophagy by down-regulation of calreticulin in human neurotypic SH-SY5Y cells.

Clioquinol (CQ) is considered as a promising drug of neurodegenerative diseases. However, the underlying mechanism is unclear. Our previous study has proved that CQ induces S-phase cell cycle arrest through the elevation of intracellular calcium concentration ([Ca2+]i) with high levels of SERCA2. Furthermore, it could induce autophagy in an intracellular calcium independent manner in human neurotypic SH-SY5Y cells. In this study, the involvement of calreticulin (CRT) in autophagy induced by CQ was investigated. Our results illustrated the endoplasmic reticulum (ER) stress induced by CQ and DTT led to the cell death in different manners. DTT, an ER stress positive control, induced UPR accompanied with up-regulation of CRT and apoptosis, while CQ inhibited UPR accompanied with down-regulation of CRT，resulting in autophagy. Then, overexpression of CRT was shown to cause UPR and decrease [Ca2+]i, leading to cell apoptosis and inhibition of S-phase arrest induced by CQ. While the UPR was alleviated and autophagy was further enhanced in CRT deficient cells by using targeted siRNA. Meanwhile, down-regulation of CRT resulted in [Ca2+]i overload and induction of S-phase arrest. Finally, we found that the effect of CQ on the HT22 cells was similar to that on the SH-SY5Y cells. Our data showed for the first time that CQ decreased expression of CRT, leading to autophagy, an increase of [Ca2+]i, and cell S-phase arrest in the neurotypic cells. The present study describes the cellular signal pathways regulating autophagy by CQ and highlights the potential therapeutic application of CQ in neurodegenerative disorders.

Tumor-targeting oxidative stress nanoamplifiers as anticancer nanomedicine with immunostimulating activity.

Compared to normal cells, cancer cells are more susceptible to insults of prooxidants that generate ROS (reactive oxygen species) or scavenge antioxidants such as glutathione (GSH). Cancer cells undergo immunogenic cell death (ICD) by elevated oxidative stress. Herein, we report rationally designed F-ssPBCA nanoparticles as a tumor-targeting prooxidant, which generates ROS and scavenges GSH simultaneously to cooperatively amplify oxidative stress, leading to ICD. Prooxidant F-ssPBCA nanoparticles are composed of a disulfide-bridged GSH scavenging dimeric prodrug (ssPB) that self-assembles to form nanoconstructs and encapsulates ROS-generating BCA (benzoyloxy cinnamaldehyde). F-ssPBCA nanoparticles significantly elevate oxidative stress to kill cancer cells and also evoke ICD featured by the release of CRT (calreticulin), HMGB-1 (high mobility group box-1), and adenosine triphosphate (ATP). Animal studies revealed that F-ssPBCA nanoparticles accumulate in tumors preferentially and suppress tumor growth effectively. The results of this study demonstrate that prooxidant-mediated oxidative stress elevation is a highly effective strategy to kill cancer cells selectively and even evoke abundant ICD. We anticipate that oxidative stress amplifying F-ssPBCA nanoparticles hold tremendous translational potential as a tumor targeted ICD-inducing anticancer nanomedicine.

Immunogenic Cell Death Role in Urothelial Cancer Therapy.

PURPOSE: Bladder cancer is the 13th most common cause of cancer death with the highest lifetime cost for treatment of all cancers. This scoping review clarifies the available evidence on the role of a novel therapeutic approach called immunogenic cell death (ICD) in urothelial cancer of the bladder. METHODS: In accordance with the recommendations of the Joanna Briggs Institute, we searched MEDLINE (Ovid), EMBASE, CENTRAL databases, and supplemented with manual searches through the conferences, Google scholar, and clinicaltrials.gov for published studies up to April 2022. We included literature that studied molecular mechanisms of ICD and the role of certain danger-associated molecular patterns (DAMPs) in generating ICD, safety and efficacy of different ICD inducers, and their contributions in combination with other urothelial cancer treatments. RESULTS: Oncolytic viruses, radiotherapy, certain chemo/chemo radiation therapy combinations, photodynamic therapy, and novel agents were studied as ICD-inducing treatment modalities in the included studies. ICD was observed in vitro (murine or human urothelial carcinoma) in ten studies, eight studies were performed on mouse models (orthotopic or subcutaneous), and five clinical trials assessed patient response to ICD inducing agents. The most common studied DAMPs were Calreticulin, HMGB1, ATP, and Heat Shock Proteins (HSP) 70 and 90, which were either expressed on the cancer cells or released. CONCLUSION: ICD inducers were able to generate lasting antitumor immune responses with memory formation in animal studies (vaccination effect). In clinical trials these agents generally had low side effects, except for one trial, and could be used alone or in combination with other cancer treatment strategies in urothelial cancer patients.

Light activation of iridium(III) complexes driving ROS production and DNA damage enhances anticancer activity in A549 cells.

The work aimed to synthesize and characterize two iridium(III) complexes [Ir(ppy)2(IPPH)](PF6) (Ir1, IPPH = (2S,3R,5S,6R)-2-(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol, ppy = 2-phenylpyridine), [Ir(piq)2(IPPH)](PF6) (Ir2, piq = 1-phenylisoquinoline). The cytotoxicity of the complexes against BEL-7402, A549, HCT-116, B16 cancer cells and normal LO2 was evaluated through 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) method. The complexes show no cytotoxic activity (IC50 > 100 µM) against these cancer cells, while their cytotoxicity can significantly be elevated upon illumination. The IC50 values range from 0.2 ± 0.05 to 35.5 ± 3.5 µM. The cellular uptake, endoplasmic reticulum and mitochondria localization, reactive oxygen species, the change of mitochondrial membrane potential, γ-H2AX levels, cycle arrest, apoptosis and the expression of B-cell lymphoma-2 were investigated. The calreticulin (CRT), heat shock protein 70 (HSP70), high mobility group box 1 (HMGB1) were explored. This study demonstrates that photoactivatable complexes induce cell death in A549 through ROS-mediated endoplasmic reticulum stress-mitochondrial pathway, DNA damage pathways, immunogenic cell death (ICD), activation of PI3K/AKT signaling pathway and inhibit the cell growth at S phase.

Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression.

BACKGROUND & AIMS: Liver fibrosis represents a hallmark of most chronic liver diseases (CLD) triggered by recurrent liver injury and subsequent myofibroblast transdifferentiations of resident hepatic stellate cells (HSCs). Mannan-binding lectin (MBL) is potentially involved in hepatic fibrosis in CLD through unclear mechanisms. Therefore, we investigated the crosstalk between MBL and HSCs, and the consequent effects on fibrosis progression. METHODS: Samples from patients with liver cirrhosis were collected. MBL deficiency (MBL-/-) and wild-type (WT) C57BL/6J mice were used to construct a CCl4-induced liver fibrosis model. Administration of MBL-expressing, liver-specific, adeno-associated virus was performed to restore hepatic MBL expression in MBL-/- mice. The human HSC line LX-2 was used for in vitro experiments. RESULTS: MBL levels in patients with liver cirrhosis were correlated with disease severity. In the CCl4-induced liver fibrosis model, MBL-/- mice showed severer liver fibrosis accompanied by reduced senescent activated HSCs in liver tissue compared with WT mice, which could be inhibited by administering MBL-expressing, liver-specific, adeno-associated virus. Moreover, depleting senescent cells with senolytic treatment could abrogate these differences owing to MBL absence. Furthermore, MBL could interact directly with calreticulin associated with low-density lipoprotein receptor-related protein 1 on the cell surface of HSCs, which further promotes senescence in HSCs by up-regulating the mammalian target of rapamycin/p53/p21 signaling pathway. CONCLUSIONS: MBL as a newfound senescence-promoting modulator and its crosstalk with HSCs in the liver microenvironment is essential for the control of hepatic fibrosis progression, suggesting its potential therapeutic use in treating CLD associated with liver fibrosis.

Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway.

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: ß-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

CALR-ETdb, the database of calreticulin variants diversity in essential thrombocythemia.

Essential thrombocythemia (ET) is a blood cancer defined by a strong increase of platelet numbers. A quarter of patients suffering from ET show mutations in the last exon of calreticulin (CALR) gene. Two variants named type 1 and type 2 represent 85% of these patients. However, a large number of other variants have been determined. In this study, we have compiled variants taken from COSMIC database and literature leading to 155 different variants. This large number of variants allowed redefining 5 new classes extending the classification of type 1-like and type 2-like to a finer description. These analyses showed that last class, named E, corresponding to more than 10% of CALR variants seemed not attached to ET. Structural properties analyzed showed that CALR variants associated to ET have common features. All the compiled and refined information had been included into a freely dedicated database CALR-ETdb (https://www.dsimb.inserm.fr/CALR-ET).

Immunomodulatory action of excretory-secretory products of Angiostrongylus cantonensis in a mouse tumour model.

Excretory-secretory products (ESPs) of parasitic helminths are well known to exert immunostimulation and immunomodulation in hosts. Immune regulation plays a key role in anti-tumour therapy. The present study explored the anti-tumour effect of ESPs released by Angiostrongylus cantonensis. In Hepa1-6 mouse tumour models, ESPs significantly reduced tumour growth. Tumour-bearing mice treated with ESPs had significantly higher CD3+, CD4+, and CD8+ T cell counts than those treated with Freund's adjuvant. In vitro, human hepatocarcinoma HepG2 cells, human lung cancer A549 cells, and normal human liver HL-7702 cells were co-incubated with ESPs for 24 h and 48 h. ESPs significantly accelerated HepG2 apoptosis but had no inhibitory effect on the proliferation of A549 and HL-7702 cells. Apoptotic HepG2 cells displayed condensed nuclei, apoptotic bodies, and swollen endoplasmic reticulum (ER). Expression of the endoplasmic reticulum stress (ERS)-related factors activating transcription factor 6 (ATF6) and C/EBP-homologous protein (CHOP) in HepG2 cells increased with increasing ESP concentration and treatment time. Calreticulin (CRT) is a key effector protein of ESPs, and recombinant calreticulin (rCRT) was produced in BL21 Escherichia coli (E. coli). In contrast to ESPs, rCRT markedly reduced the proliferation of HepG2 cells. The expression levels of ATF6 and CHOP in HepG2 cells treated with 30 µg/mL rCRT significantly increased at 48 h. Notably, these findings synergistically suggest that ESPs and rCRT are promising candidates for anti-tumour immunotherapy.

Antitumor effect of adenoviruses expressing mutant non-oncogenic E7 versions from HPV-16 fused to calreticulin.

PURPOSE: To compare the antitumor effect of adenoviruses that express mutant variants of the protein E7 from HPV-16 fused to calreticulin. METHODS: Recombinant adenoviruses were generated to express calreticulin fused to mutant versions of E7 (CRT/E7m and CRT/E7dm). Western blot and immunofluorescence assays were made to demonstrate protein expression. Antitumor assays were performed in C57BL6 mice injected with TC-1 cell line. RESULTS: When HEK293 cells were infected with these adenoviruses, we detected that all the recombinant proteins were expressed at endoplasmic reticulum, as expected. Next, the antitumor effect was tested on a murine tumor model established by inoculation of TC-1 cell line. We detected that both Ad CRT/E7m and Ad CRT/E7dm were capable of reducing the antitumor volume when compared to Ad LacZ, which was used as negative control. No significant difference was observed when compared to Ad CRT/E7, a positive control. CONCLUSIONS: Here we demonstrated that the mutant versions of E7 HPV-16 fused to calreticulin generate similar antitumor effect than the wild type version.

In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel Strategy Against Breast and Ovarian Cancer Stem-like Cells.

BACKGROUND AND AIMS: Calreticulin is a chaperone and master regulator of intracellular calcium homeostasis. Several additional functions have been discovered. Human and parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here, we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modulate cancer cell growth in vitro. METHODS: We used different concentrations of rTsCRT to treat cancer cell lines and analyzed viability and colony formation capacity. We also tested the combination of the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A was employed. The effect of the drug combinations was also assessed in cancer stem-like cells. Additionally, scavenger receptor ligands were employed to identify the role of this receptor in the rTsCRT anti-tumoral effect. RESULTS: rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used, MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher reduction in cell viability, while those from SKOV3 were more sensitive to colony disaggregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the reduction in viability induced by rTsCRT in both the parental and stem-like cells. CONCLUSION: Our data suggest that rTsCRT alone or in combination with 5-fluorouracil inhibits the growth of breast and ovarian cancer cell lines through its interaction with scavenger receptors.

Assessment of IFN-γ and granzyme-B production by in "sitro" technology.

Tumor neantigens (TNAs) and tumor-associated antigens (TAAs) are crucial triggers of anticancer immune responses. Through major histocompatibility complex, such antigens activate T cells, which, by releasing interferon gamma (IFN-γ) and granzyme B (GRZB), act as crucial effectors against tumor onset and progression. However, in response to immune pressure, cancer cells use different strategies to favor the establishment of an immunosuppressive tumor microenvironment (TME). Elucidating the dynamics of tumor-host co-evolution provides novel opportunities for personalized cancer immunotherapies. The in sitro (in vitro+in situ) technology is an experimental approach involving the preparation of heterocellular cell suspensions from fresh tumors and their use in vitro. The in sitro experimental setup offers the possibility to (1) analyze immune-related parameters (e.g., quantification of cytokines released in the TME), (2) reveal the mechanism of action of drugs, and (3) unveil crucial cell-intrinsic and cell-extrinsic processes boosting anticancer immune responses. Nonetheless, the in sitro technology does not fully recapitulate the complexity of the tissue "in situ" nor models the patterns of infiltrating immune cell localization, and hence parallel experimentation should be scheduled. In this chapter we discuss in sitro technology to analyze and quantify IFN-γ and GRZB production by T cells either co-cultured with cancer cells in the presence of exogenous adjuvant stimuli (i.e., an antibody targeting the immune checkpoint programmed cell death protein 1, and recombinant calreticulin) and boosting with TAAs (i.e., the model SIINFEKL ovalbumin antigen). Specifically, we describe IFN-γ and GRZB quantification by flow cytometry, ELISA and ELISpot technologies.

Calreticulin stabilizes F-actin by acetylating actin and protects microvascular endothelial cells against microwave radiation.

AIMS: Calreticulin (CRT) is a multifunctional protein that protects endothelial cells by alleviating actin cytoskeleton injury, but the underlying mechanism remains unclear. CRT was recently identified as a novel acyltransferase; acetylation at the N-terminus of actin monomers strengthens actin polymerization. This study was undertaken to determine whether CRT protects human microvascular endothelial cells (HMECs) against microwave radiation through actin acetylation. MATERIALS AND METHODS: We prepared a eukaryotic-derived recombinant CRT and incubated the HMECs with it prior to microwave exposure. We then assessed cell injury and endothelial function, detected actin polymerization and acetylation after HMECs exposure to S-band high-power microwaves. Coimmunoprecipitation, pull-down, and ex vitro acetylation reaction were performed to determine whether actin is a novel substrate of CRT acyltransferase. Finally, we employed the mutant experiments to demonstrate the acetylation sites contributing to CRT acetyltransferase activity. KEY FINDINGS: Microwave radiation induced severe cell injury and endothelial contact dysfunction, reduced the polymerization of actin filaments, and destroyed the actin arrangement, ultimately reducing acetylated actin expression. CRT treatment upregulated actin acetylation levels, promoted polymerization, and facilitated thicker and longer F-actin stress fibre formation. Pre-incubation with CRT rescued microwave-induced cell injury, decreased actin acetylation, and rendered the actin cytoskeleton radiation-retardant. The level of acetyl-actin was positively correlated with actin polymerization. Actin was identified as a novel substrate of CRT, being acetylated mainly through the CRT P-domain at lys-206 and -207. SIGNIFICANCE: This work provides a better understanding of the underlying mechanism of CRT-induced cytoprotection, and suggests a novel therapeutic target for microwave radiation-related diseases with endothelial dysfunction.

A peptide-modified solid lipid nanoparticle formulation of paclitaxel modulates immunity and outperforms dacarbazine in a murine melanoma model.

Melanoma is a highly aggressive skin cancer. A paclitaxel formulation of solid lipid nanoparticles modified with Tyr-3-octreotide (PSM) is employed to treat melanoma that highly expresses somatostatin receptors (SSTRs). PSM exerts more apoptotic and anti-invasive effects in B16F10 mice melanoma cells as compared to dacarbazine (DTIC), an approved chemotherapeutic drug for treating aggressive melanoma. Besides, PSM induces one of the biomarkers of immunogenic cell death in vitro and in vivo as confirmed by calreticulin exposure on the B16F10 cell surface. We observed a significant number of CD8 positive T cells in the tumor bed of the PSM treated group. As a result, PSM effectively reduces tumor volume in vivo as compared to DTIC. PSM also induces a favorable systemic immune response as determined in the spleen and sera of the treated animals. Importantly, PSM can reduce the number of nodule formations in the experimental lung metastasis model. Our experimentations indicate that the metronomic PSM exhibits remarkable anti-melanoma activities without any observable toxicity. This immune modulation behavior of PSM can be exploited for the therapy of melanoma and probably for other malignancies.

Evaluation of Opisthorchis viverrini calreticulin for potential host modulation.

The multifunctional calreticulin (CALR) was identified as a major calcium-binding protein of the endoplasmic reticulum before being recognized as a chaperone in the same place. Only later were activities of calreticulin outside the endoplasmic reticulum described that for example affect cell proliferation and the innate immune system. In the present work we have investigated those extracellular activities of CALR from the cancerogenic human liver fluke Opisthorchis viverrini (OvCALR), as they might be important in host/parasite interaction. We first demonstrate that OvCALR is released from the parasite and stimulates a specific humoral immune response. Recombinant OvCALR is then shown to suppress proliferation of primary endothelial cells, their motility and sprouting activities. The potential of OvCALR to interfere with the complement system is established, firstly by demonstrating its direct binding to C1q and, secondly by suppression of hemolysis of sensitized red blood cells. These findings suggest that OvCALR is an important parasite antigen that could modulate diverse host functions and support parasite survival.

Different Trypanosoma cruzi calreticulin domains mediate migration and proliferation of fibroblasts in vitro and skin wound healing in vivo.

Calreticulin is an endoplasmic reticulum-resident, calcium-binding, stress-produced, chaperone protein that serves multiple functions and is widely distributed in eukaryotic cells. Exogenously applied recombinant calreticulin solution, markedly enhanced the rate and quality of skin wound healing. These modulatory effects are more efficient than commercially available topic platelet-derived growth factor ointments (Regranex®). Trypanosoma cruzi calreticulin is more effective in equimolar terms to human counterpart in accelerating skin wound healing. While the effect of externally added recombinant parasite calreticulin on wound healing has been reported, the domains responsible for these modulatory effects have not yet been established. Here, recombinant parasite calreticulin and some of its domains were tested to assess their influence in increasing proliferation and migration of fibroblasts in vitro and rat skin wound healing in vivo. Herein, we propose that Trypanosoma cruzi whole calreticulin or some of its domains are differentially involved in the modulation of wound-healing cell migration and proliferation, and cosmetic outcome. Therefore, precise combination of the parasite protein and its domains could allow us to tailor-specific desired effects during the skin wound-healing process.

Antitumor Response by Endoplasmic Reticulum-Targeting DNA Vaccine Is Improved by Adding a KDEL Retention Signal.

Directing an antigen to the endoplasmic reticulum (ER) improves the antigen-specific immune response, revealing a potentially useful strategy in cancer immunotherapy using tumor-associated antigens (TAAs). This can be achieved by fusing the antigen to an ER chaperone protein, such as calreticulin (CRT). We previously reported the antitumor response by fusing the CRT signal peptide (SP) and its ER retention sequence (KDEL) to full-length human papillomavirus type 16 (HPV-16) E6 and E7 antigens, obtaining a potent antitumoral effect. In this article, we compare the antitumor response due to the use of each signal (SP and/or KDEL) fused to HPV16 E6 and E7 antigens in a DNA vaccination model. Using both SP and KDEL signals promotes higher interferon (IFN)-γ production and a faster antitumor response than using only the SP, resulting in better tumor growth restraint and higher survival, indicating that the KDEL addition to an ER-directed antigen helps by shortening the time to response. Meanwhile, antigens without signals or only the KDEL signal showed no induction of antigen-specific IFN-γ or antitumor response. Our results indicate that directing the E6E7m antigen to the ER by the SP signal is sufficient to promote an efficient antitumor response. Importantly, this effect is stronger and faster when the antigen also has an ER retention sequence, such as the KDEL signal.