ß2-AR blockade potentiates MEK1/2 inhibitor effect on HNSCC by regulating the Nrf2-mediated defense mechanism.

The ß2-Adrenergic receptor (ß2-AR) is a G protein-coupled receptor (GPCR), involved in the development of many cancers, among which HNSCC. In this contest, ß2-AR signaling interacts with different pathways, such as PI3K and MAPK, commonly activated by TK receptors. For this reason, TK blockade is one of the most adopted therapeutic strategies in HNSCC patients. In our study we investigated the effects of the ß2-AR blocking in HNSCC cell lines, using the selective inhibitor ICI118,551 (ICI), in combination with the MAPK inhibitor U0126. We found that ICI leads to the blocking of p38 and NF-kB oncogenic pathways, strongly affecting also the ERK and PI3K pathways. Cotreatment with U0126 displays a synergic effect on cell viability and pathway alteration. Interestingly, we found that the ß2-AR blockade affects Nrf2-Keap1 stability and its nuclear translocation leading to a drastic ROS increase and oxidative stress. Our results are confirmed by a TCGA dataset analysis, showing that NFE2L2 gene is commonly overexpressed in HNSC, and correlated with a lower survival rate. In our system, the PI3K pathway inhibition culminated in the blocking of pro-survival autophagy, a mechanism normally adopted by cancer cells to became less responsive to the therapies. The mTOR expression, commonly upregulated in HNSC, was reduced in patients with disease-recurrence. It is well known that mTOR has a strong autophagy inhibition effect, therefore its downregulation promoted pro-survival autophagy, with a related increase recurrence rate. Our findings highlight for the first time the key role of ß2-AR and related pathway in HNSCC cell proliferation and drug resistance, proposing it as a valuable therapeutic molecular target.

The role of autophagy in resistance to targeted therapies.

Autophagy is a self-degradative cellular process, involved in stress response such as starvation, hypoxia, and oxidative stress. This mechanism balances macro-molecule recycling to regulate cell homeostasis. In cancer, autophagy play a role in the development and progression, while several studies describe it as one of the key processes in drug resistance. In the last years, in addition to standard anti-cancer treatments such as chemotherapies and irradiation, targeted therapy became one of the most adopted strategies in clinical practices, mainly due to high specificity and reduced side effects. However, similar to standard treatments, drug resistance is the main challenge in most patients. Here, we summarize recent studies that investigated the role of autophagy in drug resistance after targeted therapy in different types of cancers. We highlight positive results and limitations of pre-clinical and clinical studies in which autophagy inhibitors are used in combination with targeted therapies.

Glucose-6-phosphate dehydrogenase blockade potentiates tyrosine kinase inhibitor effect on breast cancer cells through autophagy perturbation.

BACKGROUND: Glucose-6-phospate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphate pathway (PPP) correlated to cancer progression and drug resistance. We previously showed that G6PD inhibition leads to Endoplasmic Reticulum (ER) stress often associated to autophagy deregulation. The latter can be induced by target-based agents such as Lapatinib, an anti-HER2 tyrosine kinase inhibitor (TKI) largely used in breast cancer treatment. METHODS: Here we investigate whether G6PD inhibition causes autophagy alteration, which can potentiate Lapatinib effect on cancer cells. Immunofluorescence and flow cytometry for LC3B and lysosomes tracker were used to study autophagy in cells treated with lapatinib and/or G6PD inhibitors (polydatin). Immunoblots for LC3B and p62 were performed to confirm autophagy flux analyses together with puncta and colocalization studies. We generated a cell line overexpressing G6PD and performed synergism studies on cell growth inhibition induced by Lapatinib and Polydatin using the median effect by Chou-Talay. Synergism studies were additionally validated with apoptosis analysis by annexin V/PI staining in the presence or absence of autophagy blockers. RESULTS: We found that the inhibition of G6PD induced endoplasmic reticulum stress, which was responsible for the deregulation of autophagy flux. Indeed, G6PD blockade caused a consistent increase of autophagosomes formation independently from mTOR status. Cells engineered to overexpress G6PD became resilient to autophagy and resistant to lapatinib. On the other hand, G6PD inhibition synergistically increased lapatinib-induced cytotoxic effect on cancer cells, while autophagy blockade abolished this effect. Finally, in silico studies showed a significant correlation between G6PD expression and tumour relapse/resistance in patients. CONCLUSIONS: These results point out that autophagy and PPP are crucial players in TKI resistance, and highlight a peculiar vulnerability of breast cancer cells, where impairment of metabolic pathways and autophagy could be used to reinforce TKI efficacy in cancer treatment.

A new inhibitor of glucose-6-phosphate dehydrogenase blocks pentose phosphate pathway and suppresses malignant proliferation and metastasis in vivo.

Pentose phosphate pathway (PPP) is a major glucose metabolism pathway, which has a fundamental role in cancer growth and metastasis. Even though PPP blockade has been pointed out as a very promising strategy against cancer, effective anti-PPP agents are not still available in the clinical setting. Here we demonstrate that the natural molecule polydatin inhibits glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP. Polydatin blocks G6PD causing accumulation of reactive oxygen species and strong increase of endoplasmic reticulum stress. These effects are followed by cell cycle block in S phase, an about 50% of apoptosis, and 60% inhibition of invasion in vitro. Accordingly, in an orthotopic metastatic model of tongue cancer, 100 mg/kg polydatin induced an about 30% tumor size reduction with an about 80% inhibition of lymph node metastases and 50% reduction of lymph node size (p < 0.005). Polydatin is not toxic in animals up to a dose of 200 mg/kg and a phase II clinical trial shows that it is also well tolerated in humans (40 mg twice a day for 90 days). Thus, polydatin may be used as a reliable tool to limit human cancer growth and metastatic spread.

Evaluation and Isolation of Cancer Stem Cells Using ALDH Activity Assay.

The aldehyde dehydrogenase (ALDH) is a polymorphic enzyme responsible for the oxidation of aldehydes to carboxylic acids. In this chapter, it is described the role of ALDH in the identification of cancer stem cells (CSCs), having been shown that stem cells express high levels of ALDH. Here, we present a method called ALDEFLUOR assay used for the identification, evaluation, and isolation of normal, cancer stem and progenitor cells.

Synchrotron Phase Tomography: An Emerging Imaging Method for Microvessel Detection in Engineered Bone of Craniofacial Districts.

The engineering of large 3D constructs, such as certain craniofacial bone districts, is nowadays a critical challenge. Indeed, the amount of oxygen needed for cell survival is able to reach a maximum diffusion distance of ~150-200 µm from the original vascularization vector, often hampering the long-term survival of the regenerated tissues. Thus, the rapid growth of new blood vessels, delivering oxygen and nutrients also to the inner cells of the bone grafts, is mandatory for their long-term function in clinical practice. Unfortunately, significant progress in this direction is currently hindered by a lack of methods with which to visualize these processes in 3D and reliably quantify them. In this regard, a challenging method for simultaneous 3D imaging and analysis of microvascularization and bone microstructure has emerged in recent years: it is based on the use of synchrotron phase tomography. This technique is able to simultaneously identify multiple tissue features in a craniofacial bone site (e.g., the microvascular and the calcified tissue structure). Moreover, it overcomes the intrinsic limitations of both histology, achieving only a 2D characterization, and conventional tomographic approaches, poorly resolving the vascularization net in the case of an incomplete filling of the newly formed microvessels by contrast agents. Indeed, phase tomography, being based on phase differences among the scattered X-ray waves, is capable of discriminating tissues with similar absorption coefficients (like vessels and woven bone) in defined experimental conditions. The approach reviewed here is based on the most recent experiences applied to bone regeneration in the craniofacial region.

NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression.

Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact.

Three-Dimensional Imaging of the Developing Vasculature within Stem Cell-Seeded Scaffolds Cultured in ovo.

Successful tissue engineering requires functional vascularization of the three-dimensional constructs with the aim to serve as implants for tissue replacement and regeneration. The survival of the implant is only possible if the supply of oxygen and nutrients by developing capillaries from the host is established. The chorioallantoic membrane (CAM) assay is a valuable tool to study the ingrowth and distribution of vessels into scaffolds composed by appropriate biomaterials and stem cell populations that are used in cell-based regenerative approaches. The developing vasculature of chicken embryos within cell-seeded scaffolds can be visualized with microcomputed tomography after intravenous injection of MicroFil®, which is a radiopaque contrast agent. Here, we provide a step-by-step protocol for the seeding of stem cells into silk fibroin scaffolds, the CAM culture conditions, the procedure of MicroFil® perfusion, and finally the microcomputed tomography scanning. Three-dimensional imaging of the vascularized tissue engineered constructs provides an important analytical tool for studying the potential of cell seeded scaffolds to attract vessels and form vascular networks, as well as for analyzing the number, density, length, branching, and diameter of vessels. This in ovo method can greatly help to screen implants that will be used for tissue regeneration purposes before their in vivo testing, thereby reducing the amount of animals needed for pre-clinical studies.

Strain Analysis in the Assessment of a Mouse Model of Cardiotoxicity due to Chemotherapy: Sample for Preclinical Research.

BACKGROUND: In recent years, the development of more effective anticancer drugs has provided great benefits in patients' quality of life by improving both prognosis and disease-free survival. Nevertheless, the frequency and severity of side-effects, with particular reference to cardiac toxicity, have gained particular attention. The purpose of this study was to create a precise and sensitive preclinical model, able to identify early contractile dysfunction in mice treated with chemotherapy, through use of speckle-tracking echocardiography. MATERIALS AND METHODS: We generated a mouse model of cardiotoxicity induced by doxorubicin. C57BL 6 mice were divided into two groups, treated for 7 days by intraperitoneal injections of placebo (vehicle) or doxorubicin (2.17 mg/kg), in order to characterize the cardiac phenotype in vivo. RESULTS: We demonstrated that doxorubicin caused ealy remodeling of the left ventricle: after two days of therapy, the radial, circumferential and strain rates were reduced respectively by 35%, 34%, and 39% (p-value ≤0.001). Moreover, histological analysis revealed that doxorubicin treatment increased fibrosis, cardiomyocyte diameter and apoptosis. CONCLUSION: In a murine model of doxorubicin-induced cardiac injury, we detected left ventricular dysfunction followed by alterations in conventional echocardiographic indices. Our study suggests that a change in strain could be an effective early marker of myocardial dysfunction for new anticancer treatments and, in preclinical studies, it might also be a valuable indicator for the assessment of activity of cardioprotective agents.

Changing Paradigms in Cranio-Facial Regeneration: Current and New Strategies for the Activation of Endogenous Stem Cells.

Craniofacial area represent a unique district of human body characterized by a very high complexity of tissues, innervation and vascularization, and being deputed to many fundamental function such as eating, speech, expression of emotions, delivery of sensations such as taste, sight, and earing. For this reasons, tissue loss in this area following trauma or for example oncologic resection, have a tremendous impact on patients' quality of life. In the last 20 years regenerative medicine has emerged as one of the most promising approach to solve problem related to trauma, tissue loss, organ failure etc. One of the most powerful tools to be used for tissue regeneration is represented by stem cells, which have been successfully implanted in different tissue/organs with exciting results. Nevertheless, both autologous and allogeneic stem cell transplantation raise many practical and ethical concerns that make this approach very difficult to apply in clinical practice. For this reason different cell free approaches have been developed aiming to the mobilization, recruitment, and activation of endogenous stem cells into the injury site avoiding exogenous cells implant but instead stimulating patients' own stem cells to repair the lesion. To this aim many strategies have been used including functionalized bioscaffold, controlled release of stem cell chemoattractants, growth factors, BMPs, Platelet-Rich-Plasma, and other new strategies such as ultrasound wave and laser are just being proposed. Here we review all the current and new strategies used for activation and mobilization of endogenous stem cells in the regeneration of craniofacial tissue.