The oncolytic peptide LTX-315 triggers necrotic cell death

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.     

Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.     

Opposing effects of the angiopoietins on the thrombin-induced permeability of human pulmonary microvascular endothelial cells

Background

Angiopoietin-2 (Ang-2) is associated with lung injury in ALI/ARDS. As endothelial activation by thrombin plays a role in the permeability of acute lung injury and Ang-2 may modulate the kinetics of thrombin-induced permeability by impairing the organization of vascular endothelial (VE-)cadherin, and affecting small Rho GTPases in human pulmonary microvascular endothelial cells (HPMVECs), we hypothesized that Ang-2 acts as a sensitizer of thrombin-induced hyperpermeability of HPMVECs, opposed by Ang-1.

Methodology/Principal Findings

Permeability was assessed by measuring macromolecule passage and transendothelial electrical resistance (TEER). Angiopoietins did not affect basal permeability. Nevertheless, they had opposing effects on the thrombin-induced permeability, in particular in the initial phase. Ang-2 enhanced the initial permeability increase (passage, P = 0.010; TEER, P = 0.021) in parallel with impairment of VE-cadherin organization without affecting VE-cadherin Tyr685 phosphorylation or increasing RhoA activity. Ang-2 also increased intercellular gap formation. Ang-1 preincubation increased Rac1 activity, enforced the VE-cadherin organization, reduced the initial thrombin-induced permeability (TEER, P = 0.027), while Rac1 activity simultaneously normalized, and reduced RhoA activity at 15 min thrombin exposure (P = 0.039), but not at earlier time points. The simultaneous presence of Ang-2 largely prevented the effect of Ang-1 on TEER and macromolecule passage.

Conclusions/Significance

Ang-1 attenuated thrombin-induced permeability, which involved initial Rac1 activation-enforced cell-cell junctions, and later RhoA inhibition. In addition to antagonizing Ang-1, Ang-2 had also a direct effect itself. Ang-2 sensitized the initial thrombin-induced permeability accompanied by destabilization of VE-cadherin junctions and increased gap formation, in the absence of increased RhoA activity.     

ATP and cancer immunosurveillance

While intracellular adenosine triphosphate (ATP) occupies a key position in the bioenergetic metabolism of all the cellular compartments that form the tumor microenvironment (TME), extracellular ATP operates as a potent signal transducer. The net effects of purinergic signaling on the biology of the TME depend not only on the specific receptors and cell types involved, but also on the activation status of cis‐ and trans‐regulatory circuitries. As an additional layer of complexity, extracellular ATP is rapidly catabolized by ectonucleotidases, culminating in the accumulation of metabolites that mediate distinct biological effects. Here, we discuss the molecular and cellular mechanisms through which ATP and its degradation products influence cancer immunosurveillance, with a focus on therapeutically targetable circuitries.     

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically

Background

Endothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to ‘classical’ biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied.

Methods

In an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces.

Results

Short intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011).

Conclusion

In conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent growing cell type in an automated and continuous fashion.     

Palmitate and oleate have distinct effects on the inflammatory phenotype of human endothelial cells

Free fatty acids may create a state of continuous and progressive damaging to the vascular wall manifested by endothelial dysfunction. In this study we determine the mechanisms by which fatty acids palmitate (C16:0) and oleate (C18:1) affect intracellular long chain acyl-CoA (LCAC) content, energy metabolism, cell survival and proliferation and activation of NF-kappaB in cultured endothelial cells. A 48-h exposure of human umbilical vein endothelial cells (HUVEC) to 0.5 mM palmitate or 0.5 mM oleate increased total long chain acyl-CoA (LCAC) content 1.7 and 2 fold, respectively and decreased ATP(total)/ADP(total) ratio by 26+/-5% (mean+/-SEM) and 15+/-2%, respectively, which was prevented by the acyl-CoA synthetase inhibitor triacsin C. Furthermore, palmitate inhibited cell proliferation by 34+/-5%, while oleate stimulated it by 12+/-2%. alpha-Tocopherol fully and triacsin C partially abolished the effect of palmitate on cell proliferation. Palmitate and oleate increased caspase-3 activity 3.2 and 1.4 fold, respectively. Palmitate-induced caspase-3 activation was prevented by triacsin C and slightly reduced by alpha-tocopherol and by the de novo ceramide synthesis inhibitor fumonisin B(1). Both fatty acids induced antioxidant-sensitive nuclear translocation of NF-kappaB after 72 h, but not after 48 h. In conclusion, we showed that fatty acids influence different aspects of HUVEC function resulting in amongst other activation of apoptotic and inflammatory pathways. Our results indicate that the effects depend on the fatty acid type and may be related to accumulation of LCAC.     

The oncolytic peptide LTX-315 triggers immunogenic cell death

LTX-315 is a cationic amphilytic peptide that preferentially permeabilizes mitochondrial membranes, thereby causing partially BAX/BAK1-regulated, caspase-independent necrosis. Based on the observation that intratumorally injected LTX-315 stimulates a strong T lymphocyte-mediated anticancer immune response, we investigated whether LTX-315 may elicit the hallmarks of immunogenic cell death (ICD), namely (i) exposure of calreticulin on the plasma membrane surface, (ii) release of ATP into the extracellular space, (iii) exodus of HMGB1 from the nucleus, and (iv) induction of a type-1 interferon response. Using a panel of biosensor cell lines and robotized fluorescence microscopy coupled to automatic image analysis, we observed that LTX-315 induces all known ICD characteristics. This conclusion was validated by several independent methods including immunofluorescence stainings (for calreticulin), bioluminescence assays (for ATP), immunoassays (for HMGB1), and RT-PCRs (for type-1 interferon induction). When injected into established cancers, LTX-315 caused a transiently hemorrhagic focal necrosis that was accompanied by massive release of HMGB1 (from close-to-all cancer cells), as well as caspase-3 activation in a fraction of the cells. LTX-315 was at least as efficient as the positive control, the anthracycline mitoxantrone (MTX), in inducing local inflammation with infiltration by myeloid cells and T lymphocytes. Collectively, these results support the idea that LTX-315 can induce ICD, hence explaining its capacity to mediate immune-dependent therapeutic effects.Although cytotoxic chemotherapeutics used for the treatment of cancer often fail to achieve their ultimate goal – namely curing the patient in a permanent manner, without later relapse of the disease – there are a few examples in which conventional chemotherapy achieves long-term effects.^{1, 2} Beyond hematopoietic cancers, this applies for example to anthracycline-based adjuvant chemotherapy of breast cancer, which achieves a marked reduction in the relapse rate.³ The extraordinary success of this treatment might be explained by the fact that anthracyclines mobilize the immune system against malignant cells. Thus, cancer cells treated with anthracyclines in vitro elicits a T lymphocyte-mediated immune response against tumor-associated antigens when they are injected subcutaneously into immunocompetent mice, thereby protecting mice against rechallenge with live tumor cells of the same kind.^{4, 5} In other words, anthracyclines trigger immunogenic cell death (ICD).^{6, 7, 8}At the immunological level, it turned out that several pattern recognition receptors are involved in the recognition of dying cancer cells, meaning that their knockout or loss-of-function mutation abolishes the anticancer immune response. This applies for example to toll-like receptor 4 (TLR4) and formyl peptide receptor 1 (FPR1), meaning that anthracyclines have a reduced efficacy on tumors growing in Tlr4^−/− or Fpr1^−/− mice (as compared with WT mice) and that breast cancer patients bearing loss-of-function mutations in TLR4 or FPR1 have a comparatively poor prognosis after adjuvant chemotherapy with anthracyclines.^{9, 10} Neoadjuvant chemotherapy with anthracyclines causes a favorable change in the ratio between cytotoxic T lymphocytes and immunosuppressive regulatory T cells, in particular in those patients who manifest a complete pathological response.¹¹ This constitutes a further proof in favor of the concept that anthracyclines mediate their antineoplastic effects via the induction of an anticancer immune response.Anthracycline-induced ICD relies on one of the biochemical hallmarks of apoptosis, namely caspase activation. Thus, the pharmacological pan-caspase inhibitor Z-VAD-fmk, as well as transfection with the baculovirus inhibitor p35, do not interfere with anthracycline-induced cell death (which apparently can proceed in the absence of caspase activation), yet do abolish the immunogenicity of anthracycline-induced cell death.⁴ Mechanistic studies revealed that caspase inhibition interferes with several of the hallmarks of anthracycline-induced ICD, namely the exposure of calreticulin (CALR) on the cell surface,^{5, 12} as well as with the release of ATP that is usually associated with the blebbing phase of apoptosis.^{13, 14} CALR acts as a potent ‘eat-me'' signal when it is exposed on the surface of stressed and dying cancer cells, facilitating the transfer of tumor antigens to dendritic cells.^{15, 16, 17} The mechanism of anthracycline-triggered CALR translocation to the cell surface is complex and involves the obligatory activation of caspase-8,^{18, 19} as well as the co-translocation of the disulfidisomerase PDIA3 (better known as ERp57).²⁰ ATP acts as a potent chemoattractant, hence causing the influx of myeloid cells into the tumor bed.^{21, 22} ATP is released through a partially autophagy-dependent mechanism that also involves the caspase-3-mediated cleavage of pannexin-1 channels.^{13, 21} Removal of CALR (by knockdown) or extracellular ATP (by expression of the ATP-degrading ectoenzyme ENTPDI, better known as CD39) abolishes the immunogenicity of anthracycline-triggered cell death, similarly as does caspase inhibition.^{5, 14} On the basis of these results, we have been assuming that ICD was intimately linked to caspase activation and that necrosis (which does not involve caspase activation) would be intrinsically incompatible with ICD. Indeed, induction of necrosis by freeze-thawing of otherwise untreated cancer cells failed to yield an immunogenic preparation; and freeze-thawing actually destroyed the immunogenic properties of anthracycline-treated cells.⁴ Other hallmarks of ICD, namely the exodus of HMGB1 from the nuclei of dead cells and the induction of a type-1 interferon response occur in a largely caspase-independent manner.^{9, 23}In apparent contradiction with our findings, Patricia Agostinis found that photodynamic therapy stimulates ICD without capase-8 activation,²⁴ establishing the existence of another type of ICD (called ‘type-2 ICD'' as opposed to the anthracycline-induced ‘type-1 ICD'') that differs in its signaling mechanisms.^{6, 25} In accord with this possibility, artificial activation of necroptosis by chemical dimerization of a modified transgenic RIPK3 (better known as RIP3) protein can induce ICD.²⁶ Hence, cell death that does not involve caspase activation can be perceived as immunogenic. Nonetheless, this cell death triggered by photodynamic therapy or RIP3 activation does exhibit all major signs of ICD including CALR exposure, ATP release, and HMGB1 exodus.^{24, 26}There is yet additional, though indirect evidence that necrosis may be immunogenic. Thus, LTX-315, a cationic amphiphilic peptide derivative that permeabilizes inner mitochondrial membranes and induces necrosis,^{27, 28} is a strong inducer of anticancer immune responses if injected into tumors developing on immunocompetent mice.^{29, 30} Indeed, intratumoral injection of LTX-315 can lead to the eradication of B16 melanomas, accompanied by an immune response that protects cured mice against rechallenge with live B16 cells.^{29, 30} Challenged by this observation, we investigated whether LTX-315 can trigger ICD by characterizing the hallmarks of ICD (CALR exposure, ATP release, HMGB1 exodus and type-1 interferon response). Our results indicate that LTX-315 can induce all characteristics of ICD in a caspase-independent manner in vitro. When injected in vivo, into tumors, LTX-315 induces massive focal necrosis, followed by immune infiltration.     

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune‐dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species‐dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA‐dependent secretory pathway. This led to a general inhibition of protein secretion by PDT‐treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin‐based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro‐apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.