Wound Repair and Ca2+ Signalling Interplay: The Role of Ca2+ Channels in Skin.

The process of wound healing is intricate and tightly controlled, involving a number of different cellular and molecular processes. Numerous cellular functions, especially those related to wound healing, depend critically on calcium ions (Ca2+). Ca2+ channels are proteins involved in signal transduction and communication inside cells that allow calcium ions to pass through cell membranes. Key Ca2+ channel types involved in wound repair are described in this review.

Chronic Inflammation, Oxidative Stress and Metabolic Plasticity: Three Players Driving the Pro-Tumorigenic Microenvironment in Malignant Mesothelioma.

Malignant pleural mesothelioma (MPM) is a lethal and rare cancer, even if its incidence has continuously increased all over the world. Asbestos exposure leads to the development of mesothelioma through multiple mechanisms, including chronic inflammation, oxidative stress with reactive oxygen species (ROS) generation, and persistent aberrant signaling. Together, these processes, over the years, force normal mesothelial cells' transformation. Chronic inflammation supported by "frustrated" macrophages exposed to asbestos fibers is also boosted by the release of pro-inflammatory cytokines, chemokines, growth factors, damage-associated molecular proteins (DAMPs), and the generation of ROS. In addition, the hypoxic microenvironment influences MPM and immune cells' features, leading to a significant rewiring of metabolism and phenotypic plasticity, thereby supporting tumor aggressiveness and modulating infiltrating immune cell responses. This review provides an overview of the complex tumor-host interactions within the MPM tumor microenvironment at different levels, i.e., soluble factors, metabolic crosstalk, and oxidative stress, and explains how these players supporting tumor transformation and progression may become potential and novel therapeutic targets in MPM.

Endoplasmic Reticulum Stress and Cancer: Could Unfolded Protein Response Be a Druggable Target for Cancer Therapy?

Unfolded protein response (UPR) is an adaptive response which is used for re-establishing protein homeostasis, and it is triggered by endoplasmic reticulum (ER) stress. Specific ER proteins mediate UPR activation, after dissociation from chaperone Glucose-Regulated Protein 78 (GRP78). UPR can decrease ER stress, producing an ER adaptive response, block UPR if ER homeostasis is restored, or regulate apoptosis. Some tumour types are linked to ER protein folding machinery disturbance, highlighting how UPR plays a pivotal role in cancer cells to keep malignancy and drug resistance. In this review, we focus on some molecules that have been revealed to target ER stress demonstrating as UPR could be a new target in cancer treatment.

ER Stress Response and Induction of Apoptosis in Malignant Pleural Mesothelioma: The Achilles Heel Targeted by the Anticancer Ruthenium Drug BOLD-100.

Malignant mesothelioma is a rare cancer arising from the serosal surfaces of the body, mainly from the pleural layer. This cancer is strongly related to asbestos exposure and shows a very inauspicious prognosis, because there are scarce therapeutic options for this rare disease. Thus, there is an urgent need to develop novel therapeutic approaches to treat this form of cancer. To explore the biology of malignant pleural mesothelioma (MPM), we previously observed that MPM cell lines show high expression of the GRP78 protein, which is a chaperone protein and the master regulator of the unfolded protein response (UPR) that resides in the endoplasmic reticulum (ER). Based on our previous studies showing the importance of GRP78 in MPM, we observed that BOLD-100, a specific modulator of GRP78 and the UPR, shows cytotoxicity against MPM cells. Our studies demonstrated that BOLD-100 increases ROS production and Ca2+ release from the ER, leading to ER stress activation and, ultimately, to cell death. Our in vitro data strongly suggest that BOLD-100 inhibits the growth of MPM cell lines, proposing the application as a single agent, or in combination with other standard-of-care drugs, to treat MPM.

Aquaporin-6 May Increase the Resistance to Oxidative Stress of Malignant Pleural Mesothelioma Cells.

Malignant pleural mesothelioma (MPM) is an aggressive cancer of the pleural surface and is associated with previous asbestos exposure. The chemotherapy drug is one of the main treatments, but the median survival ranges from 8 to 14 months from diagnosis. The redox homeostasis of tumor cells should be carefully considered since elevated levels of ROS favor cancer cell progression (proliferation and migration), while a further elevation leads to ferroptosis. This study aims to analyze the functioning/role of aquaporins (AQPs) as a hydrogen peroxide (H2O2) channel in epithelial and biphasic MPM cell lines, as well as their possible involvement in chemotherapy drug resistance. Results show that AQP-3, -5, -6, -9, and -11 were expressed at mRNA and protein levels. AQP-6 was localized in the plasma membrane and intracellular structures. Compared to normal mesothelial cells, the water permeability of mesothelioma cells is not reduced by exogenous oxidative stress, but it is considerably increased by heat stress, making these cells resistant to ferroptosis. Functional experiments performed in mesothelioma cells silenced for aquaporin-6 revealed that it is responsible, at least in part, for the increase in H2O2 efflux caused by heat stress. Moreover, mesothelioma cells knocked down for AQP-6 showed a reduced proliferation compared to mock cells. Current findings suggest the major role of AQP-6 in providing mesothelioma cells with the ability to resist oxidative stress that underlies their resistance to chemotherapy drugs.

"Green" Biomaterials: The Promising Role of Honey.

The development of nanotechnology has allowed us to better exploit the potential of many natural compounds. However, the classic nanotechnology approach often uses both dangerous and environmentally harmful chemical compounds and drastic conditions for synthesis. Nevertheless, "green chemistry" techniques are revolutionizing the possibility of making technology, also for tissue engineering, environmentally friendly and cost-effective. Among the many approaches proposed and among several natural compounds proposed, honey seems to be a very promising way to realize this new "green" approach.

Cancer Therapy Challenge: It Is Time to Look in the "St. Patrick's Well" of the Nature.

Cancer still remains a leading cause of death despite improvements in diagnosis, drug discovery and therapy approach. Therefore, there is a strong need to improve methodologies as well as to increase the number of approaches available. Natural compounds of different origins (i.e., from fungi, plants, microbes, etc.) represent an interesting approach for fighting cancer. In particular, synergistic strategies may represent an intriguing approach, combining natural compounds with classic chemotherapeutic drugs to increase therapeutic efficacy and lower the required drug concentrations. In this review, we focus primarily on those natural compounds utilized in synergistic approached to treating cancer, with particular attention to those compounds that have gained the most research interest.

Multidisciplinary analysis of Italian Alpine wildflower honey reveals criticalities, diversity and value.

Wildflower honeys produced in mountain grasslands are an expression of the biodiversity of these fragile habitats. Despite its importance, the botanical origin of honey is often defined without performing formal analysis. The aim of the study was to characterize six wildflower mountain honeys produced in the Italian Alps with different analytic techniques (SPME-GC-MS, HPLC-Orbitrap, cicatrizing and antioxidant activity) alongside melissopalynological analysis and botanical definition of the production area. Even though the apiaries were in mountain grasslands rich in Alpine herbaceous species, the honey could be defined as rhododendron/raspberry unifloral or raspberry and rhododendron bifloral while the honey produced at the lowest altitude differed due to the presence of linden, heather and chestnut. The non-compliance of the honey could be due to habitat (meadows and pastures) fragmentation, but also to specific compounds involved in the plant-insect relationship, such as kynurenic acid, present in a high quantity in the sample rich in chestnut pollen. 255 volatile compounds were detected as well as some well-known markers of specific botanic essences, in particular chestnut, linden and heather, also responsible for most of the differences in aroma profiling. A high correlation between nicotinaldehyde content and percentage of raspberry pollen (r = 0.853, p < 0.05) was found. Phenolic acid and hydroxy-fatty acid were predominant in the chestnut pollen dominant honey, which presented the highest antioxidant activity and the lowest cicatrizing activity, while the flavonoid fraction was accentuated in one sample (rhododendron pollen prevalent), that was also the one with the highest effect on wound closure, although all samples had similar cicatrizing effects apart from the chestnut pollen dominant honey (lowest cicatrizing activity). Our study highlighted the difficulty of producing mountain wildflower honey and the importance of a thorough characterization of this product, also to encourage its production and valorisation.

Endothelial and Vascular Health: A Tale of Honey, H2O2 and Calcium.

Intracellular Ca2+ regulation plays a pivotal role in endothelial biology as well as during endothelial restoration processes. Interest in honey utilization in wound approaches is rising in recent years. In order to evaluate the positive effects of buckwheat honey on endothelial responses, we utilized an immortalized endothelial cell line to evaluate cellular responses upon honey exposure, with particular interest in Ca2+ signaling involvement. The results highlight the positive effects of buckwheat honey on endothelial cells' responses and the central role played by Ca2+ signaling as an encouraging target for more efficacious clinical treatments.

Mediterranean Diet Polyphenols: Anthocyanins and Their Implications for Health.

The Mediterranean diet (MD) is becoming a milestone for the prevention of chronic diseases, such as cardiovascular diseases (CVDs), Alzheimer's and Parkinson's disease. Ancel Keys in the 1950's showed a low mortality rate, particularly for coronary heart disease, among people resident in the Mediterranean area. The MD is characterized by the intake of the high amount of vegetables, fruit, and cereals and regular but moderate consumption of wine, fish, and dairy products, while olive oil is the main source of culinary fat. Therefore, it is principally a plant-based diet rich in polyphenols, a heterogeneous category of compounds with different properties and bioavailabilities. Among polyphenols, anthocyanins have been combined into the human food regime for centuries. They have been utilized as traditional herbal remedies for their ability to treat several conditions, as potent anti-oxidants, anti-diabetic and anti-carcinogenic compounds. This review summarizes our knowledge on the health-enhancing component of the anthocyanins-rich diet.

Manuka Honey Induces Apoptosis of Epithelial Cancer Cells through Aquaporin-3 and Calcium Signaling.

Honey is a natural product with a long use in traditional medicine and is well recognized to regulate different biological events. It is an important source of various biological or pharmacological molecules and, therefore, there is a strong interest to explore their properties. Evidence is growing that honey may have the potential to be an anticancer agent acting through several mechanisms. Here we observed for the first time in a cancer cell line a possible mechanism through which honey could induce an alteration in the intracellular reactive oxygen species and homeostatic balance of intracellular calcium concentration leading to cell death by apoptosis. This mechanism seems to be enhanced by manuka honey's ability to maintain high H2O2 permeability through aquaporin-3.

FAM46C and FNDC3A Are Multiple Myeloma Tumor Suppressors That Act in Concert to Impair Clearing of Protein Aggregates and Autophagy.

Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is FAM46C that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.

Epidermal Stem Cells in Regenerative Medicine.

Stem cells present in the epidermis, and hair follicle, guarantee the conservation of adult skin maintenance and hair renewal, but they also play a pivotal role in wound repair and tissue regeneration. Adult stem cells present in the epidermis are also responsible for epidermis different layers' regeneration.We here summarize the epidermal stem cells information in term of their central features in stem cells niche, their signalling pathways and their maintenance, and activation.

Endothelial response boosted by platelet lysate: the involvement of calcium toolkit.

Wound repair is a dynamic process during which crucial signaling pathways are regulated by growth factors and cytokines released by several kinds of cells directly involved in the healing process. However, the limited applications and heterogeneous clinical results of single growth factors in wound healing encouraged the use of a mixture of bioactive molecules such as platelet derivatives for best results in wound repair. An interesting platelet derivative, obtained from blood samples, is platelet lysate (PL), which has shown potential clinical application. PL is obtained from freezing and thawing of platelet-enriched blood samples. Intracellular calcium (Ca2+) signals play a central role in the control of endothelial cell survival, proliferation, motility, and differentiation. We investigated the role of Ca2+ signaling in the PL-driven endothelial healing process. In our experiments, the functional significance of Ca2+ signaling machinery was highlighted performing the scratch wound assay in presence of different inhibitors or specific RNAi. We also pointed out that the PL-induced generation of intracellular ROS (reactive oxygen species) via NOX4 (NADPH oxidase 4) is necessary for the activation of TRPM2 and the resulting Ca2+ entry from the extracellular space. This is the first report of the mechanism of wound repair in an endothelial cell model boosted by the PL-induced regulation of [Ca2+]i.

Scratch Wound Healing Assay.

Cell migration is a crucial step for wound healing. Assays able to evaluate cell migration are very useful to evaluate in vitro wound healing. Scratch wound assay creates a gap in confluent monolayer of keratinocytes to mimic a wound. The protocol of scratch wound is based on few steps: cell culture preparation, scratch wound assay, data acquisition, and data analysis.

Targeting Calcium Signalling in Malignant Mesothelioma.

Calcium ions (Ca2+) are central in cancer development and growth, serving as a major signaling system determining the cell's fate. Therefore, the investigation of the functional roles of ion channels in cancer development may identify novel approaches for determining tumor prognosis. Malignant mesothelioma is an aggressive cancer that develops from the serosal surface of the body, strictly related to asbestos exposure. The treatment of malignant mesothelioma is complex and the survival outcomes, rather than the overall survival data are, to date, disappointedly daunting. Nevertheless, conventional chemotherapy is almost ineffective. The alteration in the expression and/or activity of Ca2+ permeable ion channels seems to be characteristic of mesothelioma cells. In this review, we explore the involvement of the Ca2+toolkit in this disease. Moreover, the established sensitivity of some Ca2+channels to selective pharmacological modulators makes them interesting targets for mesothelioma cancer therapy.

Propolis Induces AQP3 Expression: A Possible Way of Action in Wound Healing.

Propolis is the generic name of a complex of resinous compound collected by honeybees and it has been utilized for many years in folk medicine. As other products generated by honeybees (such as royal jelly, pollen, honey), propolis has great therapeutic properties, but very little scientific information is available. Therefore, this study was aimed at exploring the potential wound healing properties of propolis. To that end, we utilized an in vitro scratch wound healing model consisting of human immortalized keratinocytes. Our scratch wound data clearly demonstrated that propolis induced a pronounced increase in the wound repair abilities of keratinocytes. A cell migration assay showed that propolis stimulated keratinocytes to close the wound. We revealed the role of H2O2 as the main mediator of propolis regenerative properties. We showed that this extracellularly released H2O2 could pass across the plasma membrane through a specific aquaporin (i.e., AQP3) modulating intracellular responses. The data offer a biological characterization of propolis positive effects suggesting that propolis could also be utilized in wound treatment within clinical settings.

Honey-Mediated Wound Healing: H2O2 Entry through AQP3 Determines Extracellular Ca2+ Influx.

Since Biblical times, honey has been utilized in "folk medicine", and in recent decades the positive qualities of honey have been re-discovered and are gaining acceptance. Scientific literature states that honey has been successfully utilized on infections not responding to classic antiseptic and antibiotic therapy, because of its intrinsic H2O2 production. In our study, we demonstrated the involvement of H2O2 as a main mediator of honey regenerative effects on an immortalized human keratinocyte cell line. We observed that this extracellularly released H2O2 could pass across the plasma membrane through a specific aquaporin (i.e., AQP3). Once in the cytoplasm H2O2, in turn, induces the entry of extracellular Ca2+ through Melastatin Transient Receptor Potential 2 (TRPM2) and Orai1 channels. Honey-induced extracellular Ca2+ entry results in wound healing, which is consistent with the role played by Ca2+ signaling in tissue regeneration. This is the first report showing that honey exposure increases intracellular Ca2+ concentration ([Ca2+]i), due to H2O2 production and redox regulation of Ca2+-permeable ion channels, opening up a new horizon for the utilization of the honey as a beneficial tool.

Tailored functionalization of poly(L-lactic acid) substrates at the nanoscale to enhance cell response.

Poly(L-lactic) acid (PLLA) has been widely employed in tissue engineering due to its mechanical properties, biodegradability and biocompatibility. The layer-by-layer (LbL) technique was here proposed as a simple method to impart bioactivity to the surface of PLLA substrates. Aminolysis treatment was applied to introduce amino groups on the surface of PLLA solvent cast films. Then, PLLA films were coated with heparin (HE)/chitosan (CH) multilayer by the LbL technique. Each functionalization step was characterized through physico-chemical and morphological analyses. Aminolysis treatment increased film surface wettability (64.8° ± 2.4° against 74.6° ± 1.3° for untreated PLLA) due to the formation of surface amino groups, which were quantified by acid orange colorimetric assay (0.05 nmol/mm2). After the deposition of 9 layers, the static contact angle varied between values close to 40° C (HE-based layer) and 60 °C (CH-based layer), showing the typical alternate trend of LbL coating. The successful HE/CH deposition was confirmed by ATR-FTIR and X-ray photoelectron spectroscopy (XPS) analyses. Particularly, XPS spectra of coated samples showed the presence of nitrogen (indicative of HE and CH deposition), and sulfur (indicative of HE deposition). The amount of deposited HE was quantified by Taylor's Blue colorimetric method: after the deposition of 19 and 20 layers the HE concentration was around 33 µg/cm2. Finally, in vitro studies performed using HaCaT immortalized human skin keratinocytes, C2C12 immortalized mouse myoblasts and human fibroblasts demonstrated that HE/CH multilayer-coated PLLA is a promising substrate for soft tissue engineering, as cell response may be modulated by changing the surface chemical properties.

Honey: An Effective Regenerative Medicine Product in Wound Management.

Honey has successfully been used in the treatment of a broad spectrum of injuries including burns and non-healing wounds. It acts as an antibacterial and anti-biofilm agent with anti/pro-inflammatory properties. However, besides these traditional properties, recent evidence suggests that honey is also an immunomodulator in wound healing and contains several bee and plant-derived components that may speed up wound healing and tissue regeneration process. Identifying their exact mechanism of action allows better understanding of honey healing properties and promotes its wider translation into clinical practice. This review will discuss the physiological basis for the use of honey in wound management, its current clinical uses, as well as the potential role of honey bioactive compounds in dermal regenerative medicine and tissue re-modeling.