Elisa Panzarini

Cell shape and plasma membrane alterations after static magnetic fields exposure

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy

Rose Bengal acetate photodynamic therapy (RBAc–PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT.

Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) Induces Exposure and Release of Damage-Associated Molecular Patterns (DAMPs) in Human HeLa Cells

The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.

Immunogenic Cell Death: Can It Be Exploited in PhotoDynamic Therapy for Cancer?

Immunogenic Cell Death (ICD) could represent the keystone in cancer management since tumor cell death induction is crucial as well as the control of cancer cells revival after neoplastic treatment. In this context, the immune system plays a fundamental role. The concept of Damage-Associated Molecular Patterns (DAMPs) has been proposed to explain the immunogenic potential of stressed or dying/dead cells. ICD relies on DAMPs released by or exposed on dying cells. Once released, DAMPs are sensed by immune cells, in particular Dendritic Cells (DCs), acting as activators of Antigen-Presenting Cells (APCs), that in turn stimulate both innate and adaptive immunity. On the other hand, by exposing DAMPs, dying cancer cells change their surface composition, recently indicated as vital for the stimulation of the host immune system and the control of residual ill cells. It is well established that PhotoDynamic Therapy (PDT) for cancer treatment ignites the immune system to elicit a specific antitumor immunity, probably linked to its ability in inducing exposure/release of certain DAMPs, as recently suggested. In the present paper, we discuss the DAMPs associated with PDT and their role in the crossroad between cancer cell death and immunogenicity in PDT.

Autophagy Contributes to the Death/Survival Balance in Cancer PhotoDynamic Therapy.

Autophagy is an important cellular program with a “double face” role, since it promotes either cell survival or cell death, also in cancer therapies. Its survival role occurs by recycling cell components during starvation or removing stressed organelles; when damage becomes extensive, autophagy provides another programmed cell death pathway, known as Autophagic Cell Death (ACD). The induction of autophagy is a common outcome in PhotoDynamic Therapy (PDT), a two-step process involving the irradiation of photosensitizer (PS)-loaded cancer cells. Upon tissue oxygen interaction, PS provokes immediate and direct Reactive Oxygen Species (ROS)-induced damage to Endoplasmic Reticulum (ER), mitochondria, plasma membrane, and/or lysosomes. The main biological effects carried out in cancer PDT are direct cytotoxicity to tumor cells, vasculature damage and induction of inflammatory reactions stimulating immunological responses. The question about the role of autophagy in PDT and its putative immunological impact is hotly controversial and largely studied in recent times. This review deals with the induction of autophagy in PDT protocols and its dual role, also considering its interrelationship with apoptosis, the preferential cell death program triggered in the photodynamic process.

Nanomaterials and autophagy: new insights in cancer treatment.

Autophagy represents a cell’s response to stress. It is an evolutionarily conserved process with diversified roles. Indeed, it controls intracellular homeostasis by degradation and/or recycling intracellular metabolic material, supplies energy, provides nutrients, eliminates cytotoxic materials and damaged proteins and organelles. Moreover, autophagy is involved in several diseases. Recent evidences support a relationship between several classes of nanomaterials and autophagy perturbation, both induction and blockade, in many biological models. In fact, the autophagic mechanism represents a common cellular response to nanomaterials. On the other hand, the dynamic nature of autophagy in cancer biology is an intriguing approach for cancer therapeutics, since during tumour development and therapy, autophagy has been reported to trigger both an early cell survival and a late cell death. The use of nanomaterials in cancer treatment to deliver chemotherapeutic drugs and target tumours is well known. Recently, autophagy modulation mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as adjuvant in chemotherapy or in the development of cancer vaccines or as a potential anti-cancer agent. Herein, we summarize the effects of nanomaterials on autophagic processes in cancer, also considering the therapeutic outcome of synergism between nanomaterials and autophagy to improve existing cancer therapies.

Nanomaterial-Induced Autophagy: A New Reversal MDR Tool in Cancer Therapy?

Most of the therapeutic strategies to counteract cancer imply killing of malignant cells. The most exploited cell death mechanism in cancer therapies is apoptosis, but recently, a lot of papers report that other mechanisms, mainly autophagy, could represent a new line of attack in the fight against cancer. One of the limitations for the effectiveness of the approved clinical treatments is the phenomenon of multidrug resistance (MDR) which enables the cancer cells to develop resistance to therapy, especially for chemotherapy. The MDR mechanisms include (a) decreased uptake of drug, (b) reduced intracellular drug concentration by efflux pumps, (c) altered cell cycle checkpoints, (d) altered drug targets, (e) increased metabolism of drugs, (f) induced emergency response genes to impair apoptotic pathway, and (g) altered drug detoxification. Great efforts have been made to reverse MDR. Currently, autophagy and nanosized drug delivery systems (DDSs) belonging to nanomaterials (NMs) provide alternative strategies to circumvent MDR. Nanosized DDSs are very promising tools to accumulate chemotherapeutics at targeting sites and control temporal and spatial drug release into tumor cells. On the other hand, autophagy could overrule drug resistance upon its activation by ensuring cell death via switching its prosurvival role to a prodeath one or by mediating the occurrence of cell death, i.e., apoptosis or necrosis. Likewise, the autophagy inhibition could counteract MDR by sensitizing the cells to anticancer molecules, i.e., Src family tyrosine kinase (SFK) inhibitors or 5-fluorouracil. Noteworthy, autophagy has been recently indicated to be a common cellular response to NMs, corroborating the fascinating idea of the exploitation of NM-induced autophagy in nanomedicine therapy. This review focuses on recently published literature about the relationship between MDR reversal and NMs or autophagy pointing to hypothesize a pivotal role of autophagy modulation induced by NMs in counteracting MDR.

In Vitro Analysis of the Anti-Inflammatory Effect of Inhomogeneous Static Magnetic Field-Exposure on Human Macrophages and Lymphocytes

The effect of inhomogeneous static magnetic field (SMF)-exposure on the production of different cytokines from human peripheral blood mononuclear cells (PMBC), i.e., lymphocytes and macrophages, was tested in vitro. Some cultures were activated with lipopolysaccharide (LPS) at time point −3 h and were either left alone (positive control) or exposed to SMF continuously from 0 until 6, 18, or 24 h. The secretion of interleukin IL-6, IL-8, tumor necrosis factor TNF-α, and IL-10 was tested by ELISA. SMF-exposure caused visible morphological changes on macrophages as well as on lymphocytes, and also seemed to be toxic to lymphocytes ([36.58; 41.52]%, 0.308≤p≤0.444), but not to macrophages (<1.43%, p≥0.987). Analysis of concentrations showed a significantly reduced production of pro-inflammatory cytokines IL-6, IL-8, and TNF-α from macrophages compared to negative control ([56.78; 87.52]%, p = 0.031) and IL-6 compared to positive control ([45.15; 56.03]%, p = 0.035). The production of anti-inflammatory cytokine IL-10 from macrophages and from lymphocytes was enhanced compared to negative control, significantly from lymphocytes ([−183.62; −28.75]%, p = 0.042). The secretion of IL-6 from lymphocytes was significantly decreased compared to positive control ([−115.15; −26.84]%, p = 0.039). This massive in vitro evidence supports the hypotheses that SMF-exposure (i) is harmful to lymphocytes in itself, (ii) suppresses the release of pro-inflammatory cytokines IL-6, IL-8, and TNF-α, and (iii) assists the production of anti-inflammatory cytokine IL-10; thus providing a background mechanism of the earlier in vivo demonstrated anti-inflammatory effects of SMF-exposure.

Overview of Cell Death Mechanisms Induced by Rose Bengal Acetate-Photodynamic Therapy

Photodynamic Therapy (PDT) is a non-invasive treatment for different pathologies, cancer included, using three key components: non-toxic light-activated drug (Photosensitizer, PS), visible light, and oxygen. Their interaction triggers photochemical reactions leading to Reactive Oxygen Species (ROS) generation, that mediate cytotoxicity and cell death. In the present paper, the most important findings about the synthetic dye Rose Bengal Acetate (RBAc), an emerging photosensitizer for its efficient induction of cell death, will be reported with the aim to integrate RBAc phototoxicity to novel therapeutic PDT strategies against tumour cells. After its perinuclear intracellular localization, RBAc causes multiple subcellular organelles damage, that is, mitochondria, Endoplasmic Reticulum (ER), lysosomes, and Golgi complex. Indeed, RBAc exerts long-term phototoxicity through activation of both caspase-independent and- dependent apoptotic pathways and autophagic cell death. In particular, this latter cell death type may promote cell demise when apoptotic machinery is defective. The deep knowledge of RBAc photocytotoxicity will allow to better understand its potential photomedicine application in cancer.

Photodynamic Therapy‐Induced Apoptosis of HeLa Cells

Photodynamic therapy (PDT), which is a treatment for cancer and certain noncancerous conditions, requires exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength. By using Rose Bengal Acetate (RBAc) as the photosensitizer and an innovative green light‐emitting diode, we investigated the efficiency with which apoptosis is induced in HeLa cells, focusing our study on mitochondria alteration and cytochrome c release. Indeed, RBAc is a very efficient fluorogenic substrate and easily enters the cells where the original photoactive molecule is restored by specific esterases. HeLa cells after PDT underwent a consistent rate of apoptosis (peaked at 12 h of recovery post‐PDT). Necrosis was observed at the longest times of recovery as a result of secondary necrosis. PDT gave rise to a series of shape modifications, mainly referable to apoptotic‐related changes (i.e., extensive blebs formation) involving both F‐actin and tubulin networks. Soon after PDT, mitochondria lose their potential membranes and release large quantities of cytochrome c.