Modulation of AMPK Significantly Alters Uveal Melanoma Tumor Cell Viability.

INTRODUCTION: Uveal melanoma (UM) responds poorly to targeted therapies or immune checkpoint inhibitors. Adenosine monophosphate-activated protein kinase (AMPK) is a pivotal serine/threonine protein kinase that coordinates vital processes such as cell growth. Targeting AMPK pathway, which represents a critical mechanism mediating the survival of UM cells, may prove to be a novel treatment strategy for UM. We aimed to demonstrate the effects of AMPK modulation on UM cells. METHODS: In silico analyses were performed to compare UM and normal melanocyte cells via Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA). The effects of AMPK modulation on cell viability and proliferation in UM cell lines with different molecular profiles (i.e., 92-1, MP46, OMM2.5, and Mel270) were investigated via XTT cell viability and proliferation assays after treating the cells with varying concentrations of A-769662 (AMPK activator) or dorsomorphin (AMPK inhibitor). RESULTS: KEGG/GSEA studies demonstrated that genes implicated in the AMPK signaling pathway were differentially regulated in UM. Gene sets comprising genes involved in AMPK signaling and genes involved in energy-dependent regulation of mammalian target of rapamycin by liver kinase B1-AMPK were downregulated in UM. We observed gradual decreases in the numbers of viable UM cells as the concentration of A-769662 treatment increased. All UM cells demonstrated statistically significant decreases in cell viability when treated with 200 µm A-769662. Moreover, the effects of AMPK inhibition on UM cells were potent, since low doses of dorsomorphin treatment resulted in significant decreases in viabilities of UM cells. The half maximal inhibitory concentration (IC50) values confirmed the potency of dorsomorphin treatment against UM in vitro. CONCLUSION: AMPK may act like a friend or a foe in cancer depending on the context. As such, the current study contributes to the literature in determining the effects of therapeutic strategies targeting AMPK in several UM cells. We propose a new perspective in the treatment of UM. Targeting AMPK pathway may open up new avenues in developing novel therapeutic approaches to improve overall survival in UM.

BODIPY-GO nanocomposites decorated with a biocompatible branched ethylene glycol moiety for targeted PDT.

The properties of graphene oxide (GO) have received much attention and been applied to the exploration of potential applications in disease-related diagnostics and non-invasive therapy. One application, photodynamic therapy (PDT), involves the killing of cancer cells where singlet oxygen is generated with light irradiation of the appropriate wavelength. In this work, three new BODIPY derivatives (13-15), decorated with carbohydrate moieties for active targeting and branched ethylene glycol for biocompatibility, and their GO based nanocarriers were designed to study the singlet oxygen production and PDT efficiency. First, BODIPYs were prepared, followed by the fabrication of GO layers with BODIPY dyes via a non-covalent method. Detailed characterizations of the materials were carried out with mass spectrometry, FT-IR spectroscopy, 1H NMR, 13C NMR, elemental analysis, Raman spectroscopies, EDX analysis and TEM and AFM microscopies. The efficiency of singlet oxygen generation in organic and water-based solutions was determined by photobleaching with 1,3-diphenylisobenzofuran (DPBF) and 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA), respectively. The results in in vitro PDT analysis against K562 human cancer cells indicate the prepared materials are highly promising in PDT anticancer therapy and the IC50 values of GO loaded BODIPY derivatives bearing heavy atoms, GO-14 and GO-15, were calculated as 40.59 nM and 39.21 nM, respectively.

NI-BODIPY-GO Nanocomposites for Targeted PDT.

Three multifunctional targeted NI-BODIPYs (10-12) and GO-(10-12) nanocarriers were fabricated. NI-BODIPYs are designed to facilitate non-covalent interaction with graphene oxide (GO) and target toward cancer cells for specific recognition with glucose moieties while efficiently producing singlet oxygen. We probed detailed characterization, fundamental photophysical/photochemical properties, and interactions with GO of such triplet photosensitizers and nanocarriers. The effect of the formation of nanohybrids with GO on singlet oxygen formation as well as on the efficacies of the molecules in terms of in vitro killing of cancer cells was evaluated with K562 human chronic myelogenous leukemia cells. Amazingly, it was observed that GO exhibited favorable interactions with the NI-BODIPY dyads and promoted the formation of singlet oxygen, while not showing any dark toxicity.

Candidate Angiogenesis-Related Biomarkers in Patients with Laryngeal Carcinoma (AngLaC): A Prospective Cohort Study.

OBJECTIVE: Angiogenesis is indeed a vital process in the progression of carcinomas, including that of larynx. Therefore, this study (AngLaC) aimed to identify candidate angiogenesis-related biomarkers in laryngeal carcinoma patients. STUDY DESIGN: Prospective controlled cohort study. SETTING: Tertiary referral center. METHODS: In silico analyses of angiogenesis-related genes in laryngeal carcinoma were performed to determine candidate biomarkers. Serum levels of candidate biomarkers were determined via enzyme-linked immunosorbent assay in laryngeal carcinoma patients as well as in an age and gender-matched control group. The associations of the biomarkers with clinical parameters were investigated. RESULTS: The study included 60 laryngeal carcinoma patients and 20 healthy controls. The serum levels of osteopontin, IGFBP-3, VEGF, sVEGFR-1, and VEGFR-2 were significantly higher in the patient group (p < .001, p ≤ .001, p < .001, p < .01, p < .01, respectively). High osteopontin and sVEGFR-1 levels were associated with locoregional-recurrence (p = .024, p = .016, respectively). IGFBP-3 had the highest diagnostic sensitivity (81.4%) and specificity (80%) among the molecules that were investigated (p < .001). High sVEGFR-1 and low VEGFR-2 levels were associated with poor overall-survival (p = .037, p = .027, respectively). High osteopontin and sVEGFR-1 levels were associated with poor disease-specific survival rates (p = .035, p = .018, respectively). CONCLUSION: High serum levels of sVEGFR-1 and osteopontin as well as low serum levels of VEGFR-2 proved to be poor prognostic in terms of survival in laryngeal carcinoma. VEGF, sVEGFR1, VEGFR2, IGFBP-3, and osteopontin levels were found to be significantly increased in larynx cancer patients compared to the normal population. Further studies on osteopontin and sVEGFR-1 are required in order to determine their associations with recurrence.

Amphiphilic Fullerene-BODIPY Photosensitizers for Targeted Photodynamic Therapy.

Nanotheranostic tailor-made carriers are potent platforms for the treatment of cancer that propound a number of advantages over conventional agents for photodynamic therapy (PDT). Herein, four new heavy atom free amphiphilic glucose-BODIPY-fullerene dyads (14-17) endowed with carbohydrate units in the styryl units, which can also form nanomicelles (14-17NM) with Tween 80 for PDT are reported. Glucose-BODIPY-fullerene systems (14-17) and related nanomicelles (14-17NM) have been prepared to emcee efficient singlet oxygen generation upon light irradiation. In vitro anti-tumor effects of the compounds 14-17 and 14-17NM in the presence of light and in darkness have been investigated with K562 human chronic myelogenous leukemia suspension cells. Anti-tumor toxicity upon light irradiation was due to the formation of singlet oxygen and reactive oxygen species (ROS). This study may provide an accomplished example of efficient PDT applications based on nanovehicles fabricated with universal spin converter, fullerene, light harvesting unit, BODIPY dyes conjugated with targeting units to fight against cancer.

Photodynamic Therapy for the Treatment and Diagnosis of Cancer-A Review of the Current Clinical Status.

Photodynamic therapy (PDT) has been used as an anti-tumor treatment method for a long time and photosensitizers (PS) can be used in various types of tumors. Originally, light is an effective tool that has been used in the treatment of diseases for ages. The effects of combination of specific dyes with light illumination was demonstrated at the beginning of 20th century and novel PDT approaches have been developed ever since. Main strategies of current studies are to reduce off-target effects and improve pharmacokinetic properties. Given the high interest and vast literature about the topic, approval of PDT as the first drug/device combination by the FDA should come as no surprise. PDT consists of two stages of treatment, combining light energy with a PS in order to destruct tumor cells after activation by light. In general, PDT has fewer side effects and toxicity than chemotherapy and/or radiotherapy. In addition to the purpose of treatment, several types of PSs can be used for diagnostic purposes for tumors. Such approaches are called photodynamic diagnosis (PDD). In this Review, we provide a general overview of the clinical applications of PDT in cancer, including the diagnostic and therapeutic approaches. Assessment of PDT therapeutic efficacy in the clinic will be discussed, since identifying predictors to determine the response to treatment is crucial. In addition, examples of PDT in various types of tumors will be discussed. Furthermore, combination of PDT with other therapy modalities such as chemotherapy, radiotherapy, surgery and immunotherapy will be emphasized, since such approaches seem to be promising in terms of enhancing effectiveness against tumor. The combination of PDT with other treatments may yield better results than by single treatments. Moreover, the utilization of lower doses in a combination therapy setting may cause less side effects and better results than single therapy. A better understanding of the effectiveness of PDT in a combination setting in the clinic as well as the optimization of such complex multimodal treatments may expand the clinical applications of PDT.

Photodynamic Therapy-Current Limitations and Novel Approaches.

Photodynamic therapy (PDT) mostly relies on the generation of singlet oxygen, via the excitation of a photosensitizer, so that target tumor cells can be destroyed. PDT can be applied in the settings of several malignant diseases. In fact, the earliest preclinical applications date back to 1900's. Dougherty reported the treatment of skin tumors by PDT in 1978. Several further studies around 1980 demonstrated the effectiveness of PDT. Thus, the technique has attracted the attention of numerous researchers since then. Hematoporphyrin derivative received the FDA approval as a clinical application of PDT in 1995. We have indeed witnessed a considerable progress in the field over the last century. Given the fact that PDT has a favorable adverse event profile and can enhance anti-tumor immune responses as well as demonstrating minimally invasive characteristics, it is disappointing that PDT is not broadly utilized in the clinical setting for the treatment of malignant and/or non-malignant diseases. Several issues still hinder the development of PDT, such as those related with light, tissue oxygenation and inherent properties of the photosensitizers. Various photosensitizers have been designed/synthesized in order to overcome the limitations. In this Review, we provide a general overview of the mechanisms of action in terms of PDT in cancer, including the effects on immune system and vasculature as well as mechanisms related with tumor cell destruction. We will also briefly mention the application of PDT for non-malignant diseases. The current limitations of PDT utilization in cancer will be reviewed, since identifying problems associated with design/synthesis of photosensitizers as well as application of light and tissue oxygenation might pave the way for more effective PDT approaches. Furthermore, novel promising approaches to improve outcome in PDT such as selectivity, bioengineering, subcellular/organelle targeting, etc. will also be discussed in detail, since the potential of pioneering and exceptional approaches that aim to overcome the limitations and reveal the full potential of PDT in terms of clinical translation are undoubtedly exciting. A better understanding of novel concepts in the field (e.g. enhanced, two-stage, fractional PDT) will most likely prove to be very useful for pursuing and improving effective PDT strategies.

Proof-of-principle for two-stage photodynamic therapy: hypoxia triggered release of singlet oxygen.

We propose to overcome oxygen deficiency and light attenuation problems in photodynamic therapy (PDT), by separating photoexcitation and singlet oxygen delivery of the PDT process into two distinct operations to be carried out sequentially, at different locations. We now demonstrate the viability of this approach, using 2-pyridone derivative which yields a relatively stable endoperoxide. The initial storage endoperoxide obtained is transformed enzymatically into a more labile compound when placed in hypoxic cell cultures, and releases singlet oxygen significantly faster. The potential of this approach in advancing PDT beyond its current limits is exciting.

Cancer associated fibroblasts sculpt tumour microenvironment by recruiting monocytes and inducing immunosuppressive PD-1+ TAMs.

Fibroblasts turn into cancer associated fibroblasts (CAFs) in the tumour microenvironment. CAFs have recently attracted attention for their function as a regulator of immune cell recruitment and function in addition to their tumour-promoting roles. In this study, we aimed to determine the role of CAFs on monocyte recruitment and macrophage polarization in breast cancer. CAFs, which were α-SMA expressing fibroblasts in contrast to normal fibroblasts (NFs), effectively recruited monocytes. Recruitment of monocytes by CAFs might be mediated by monocyte chemotactic protein-1 (MCP-1) as well as stromal cell-derived factor-1 (SDF-1) cytokines. CAFs differentiated the recruited monocytes into M2-like macrophages which are capable of exerting their immunosuppressive roles via the PD-1 axis. CAF-educated monocytes exhibited strong immune suppression unlike NF-educated monocytes and enhanced the motility/invasion of breast cancer cells in addition to increasing the expressions of epithelial-mesenchymal transition (EMT)-related genes and vimentin protein in cancer cells. CAF-educated M1 macrophages displayed increased expression of M2 markers and production of anti-inflammatory cytokine IL-10 in contrast to decreased production of pro-inflammatory cytokine IL-12 compared with control M1 macrophages; suggesting that CAFs were also able to induce the trans-differentiation of M1 macrophages to M2 macrophages. We then investigated the relationship between the infiltration of CAFs and tumour associated macrophages (TAMs) using tissue samples obtained from breast cancer patients. High grade of CAFs significantly correlated with the number of TAMs in human breast cancer tissue samples. It was also associated with higher Ki-67 proliferation index, and higher tumour volume. This result is in line with our finding of increased breast cancer cell proliferation due to the effects of CAF-educated monocytes in vitro. Our results concluded that CAFs play pivotal roles in sculpturing the tumour microenvironment in breast cancer, and therapeutic strategies to reverse the CAF-mediated immunosuppressive microenvironment should be taken into consideration.