Identification of novel biomarkers in the early diagnosis of malignant melanoma by untargeted liquid chromatography coupled to high-resolution mass spectrometry-based metabolomics: a pilot study.

BACKGROUND: Malignant melanoma (MM) is a highly aggressive form of skin cancer whose incidence continues to rise worldwide. If diagnosed at an early stage, it has an excellent prognosis, but mortality increases significantly at advanced stages after distant spread. Unfortunately, early detection of aggressive melanoma remains a challenge. OBJECTIVES: To identify novel blood-circulating biomarkers that may be useful in the diagnosis of MM to guide patient counselling and appropriate disease management. METHODS: In this study, 105 serum samples from 26 healthy patients and 79 with MM were analysed using an untargeted approach by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to compare the metabolomic profiles of both conditions. Resulting data were subjected to both univariate and multivariate statistical analysis to select robust biomarkers. The classification model obtained from this analysis was further validated with an independent cohort of 12 patients with stage I MM. RESULTS: We successfully identified several lipidic metabolites differentially expressed in patients with stage I MM vs. healthy controls. Three of these metabolites were used to develop a classification model, which exhibited exceptional precision (0.92) and accuracy (0.94) when validated on an independent sample. CONCLUSIONS: These results demonstrate that metabolomics using LC-HRMS is a powerful tool to identify and quantify metabolites in bodily fluids that could serve as potential early diagnostic markers for MM.

Melanoma is a type of skin cancer that can be deadly if it is not detected at an early stage. Unfortunately, the early detection of melanoma is challenging. Our team has developed a model that could be used to predict whether a person has stage I malignant melanoma based on blood serum analysis. The model was trained on data from a group of people with melanoma and it was found to be accurate in predicting melanoma at an early stage. This means that the model could be used to identify people who have skin cancer before it progresses and becomes more complicated to treat. Although the researchers recommend that further studies are conducted to validate the model in a larger population of people, this research could help with the early diagnosis of melanoma and work toward improving survival rates.

Interferon-Alpha Decreases Cancer Stem Cell Properties and Modulates Exosomes in Malignant Melanoma.

Malignant melanoma (MM) can spread to other organs and is resistant in part due to the presence of cancer stem cell subpopulations (CSCs). While a controversial high dose of interferon-alpha (IFN-α) has been used to treat non-metastatic high-risk melanoma, it comes with undesirable side effects. In this study, we evaluated the effect of low and high doses of IFN-α on CSCs by analyzing ALDH activity, side population and specific surface markers in established and patient-derived primary cell lines. We also assessed the clonogenicity, migration and tumor initiation capacities of IFN-α treated CSCs. Additionally, we investigated genomic modulations related to stemness properties using microRNA sequencing and microarrays. The effect of IFN-α on CSCs-derived exosomes was also analyzed using NanoSight and liquid chromatography (LC-HRMS)-based metabolomic analysis, among others. Our results showed that even low doses of IFN-α reduced CSC formation and stemness properties, and led to a significant decrease in the ability to form tumors in mice xenotransplants. IFN-α also modulated the expression of genes and microRNAs involved in several cancer processes and metabolomics of released exosomes. Our work suggests the utility of low doses of interferon, combined with the analysis of metabolic biomarkers, as a potential clinical approach against the aggressiveness of CSCs in melanoma.

Biogeneration of silver nanoparticles from Cuphea procumbens for biomedical and environmental applications.

Nanotechnology is one of the most important and relevant disciplines today due to the specific electrical, optical, magnetic, chemical, mechanical and biomedical properties of nanoparticles. In the present study we demonstrate the efficacy of Cuphea procumbens to biogenerate silver nanoparticles (AgNPs) with antibacterial and antitumor activity. These nanoparticles were synthesized using the aqueous extract of C. procumbens as reducing agent and silver nitrate as oxidizing agent. The Transmission Electron Microscopy demonstrated that the biogenic AgNPs were predominantly quasi-spherical with an average particle size of 23.45 nm. The surface plasmonic resonance was analyzed by ultraviolet visible spectroscopy (UV-Vis) observing a maximum absorption band at 441 nm and Infrared Spectroscopy (FT IR) was used in order to structurally identify the functional groups of some compounds involved in the formation of nanoparticles. The AgNPs demonstrated to have antibacterial activity against the pathogenic bacteria Escherichia coli and Staphylococcus aureus, identifying the maximum zone of inhibition at the concentration of 0.225 and 0.158 µg/mL respectively. Moreover, compared to the extract, AgNPs exhibited better antitumor activity and higher therapeutic index (TI) against several tumor cell lines such as human breast carcinoma MCF-7 (IC50 of 2.56 µg/mL, TI of 27.65 µg/mL), MDA-MB-468 (IC50 of 2.25 µg/mL, TI of 31.53 µg/mL), human colon carcinoma HCT-116 (IC50 of 1.38 µg/mL, TI of 51.07 µg/mL) and melanoma A-375 (IC50 of 6.51 µg/mL, TI of 10.89 µg/mL). This fact is of great since it will reduce the side effects derived from the treatment. In addition, AgNPs revealed to have a photocatalytic activity of the dyes congo red (10-3 M) in 5 min and malachite green (10-3 M) in 7 min. Additionally, the degradation percentages were obtained, which were 86.61% for congo red and 82.11% for malachite green. Overall, our results demonstrated for the first time that C. procumbens biogenerated nanoparticles are excellent candidates for several biomedical and environmental applications.

Anti-Cancerous Potential of Polysaccharides Derived from Wheat Cell Culture.

There is a global need to discover effective anti-cancerous compounds from natural sources. Cultivated wheat cells can be a valuable source of non-toxic or low toxic plant-derived polysaccharides. In this study, we evaluated the anti-cancer ability of seven fractions of wheat cell culture polysaccharides (WCCPSs) in the HCT-116 colon cancer cell line. Almost all (6/7) fractions had an inhibitory effect on the proliferation of colon cancer cells, and two fractions (A-b and A-f) had considerable therapeutic indexes. The WCCPS fractions induced cell cycle arrest in the G1 phase and induced different rates of apoptosis (≤48%). Transmission and scanning electron microscopy revealed that WCCPS fractions caused apoptotic changes in the nucleus and cytoplasm, including damage to mitochondria and external morphological signs of apoptosis. In addition, the WCCPSs induced an increase in the levels of Bax, cytochrome c, and caspases 8 and 3, indicating that cell death progressed through intrinsic and extrinsic pathways of apoptosis. Furthermore, some fractions caused a significant decrease of c-Myc, b-catenin, NFkB2, and HCAM (CD 44) levels, indicating enhanced cell differentiation. Thus, for the first time, our results provide a proof of concept of the anti-cancer capacity of WCCPS fractions in colorectal cancer.

Luminescent Citrate-Functionalized Terbium-Substituted Carbonated Apatite Nanomaterials: Structural Aspects, Sensitized Luminescence, Cytocompatibility, and Cell Uptake Imaging.

This work explores the preparation of luminescent and biomimetic Tb3+-doped citrate-functionalized carbonated apatite nanoparticles. These nanoparticles were synthesized employing a citrate-based thermal decomplexing precipitation method, testing a nominal Tb3+ doping concentration between 0.001 M to 0.020 M, and a maturation time from 4 h to 7 days. This approach allowed to prepare apatite nanoparticles as a single hydroxyapatite phase when the used Tb3+ concentrations were (i) ≤ 0.005 M at all maturation times or (ii) = 0.010 M with 4 h of maturation. At higher Tb3+ concentrations, amorphous TbPO4·nH2O formed at short maturation times, while materials consisting of a mixture of carbonated apatite prisms, TbPO4·H2O (rhabdophane) nanocrystals, and an amorphous phase formed at longer times. The Tb3+ content of the samples reached a maximum of 21.71 wt%. The relative luminescence intensity revealed an almost linear dependence with Tb3+ up to a maximum of 850 units. Neither pH, nor ionic strength, nor temperature significantly affected the luminescence properties. All precipitates were cytocompatible against A375, MCF7, and HeLa carcinogenic cells, and also against healthy fibroblast cells. Moreover, the luminescence properties of these nanoparticles allowed to visualize their intracellular cytoplasmic uptake at 12 h of treatment through flow cytometry and fluorescence confocal microscopy (green fluorescence) when incubated with A375 cells. This demonstrates for the first time the potential of these materials as nanophosphors for living cell imaging compatible with flow cytometry and fluorescence confocal microscopy without the need to introduce an additional fluorescence dye. Overall, our results demonstrated that Tb3+-doped citrate-functionalized apatite nanoparticles are excellent candidates for bioimaging applications.

Design, synthesis, HER2 inhibition and anticancer evaluation of new substituted 1,5-dihydro-4,1-benzoxazepines.

A series of 11 new substituted 1,5-dihydro-4,1-benzoxazepine derivatives was synthesised to study the influence of the methyl group in the 1-(benzenesulphonyl) moiety, the replacement of the purine by the benzotriazole bioisosteric analogue, and the introduction of a bulky substituent at position 6 of the purine, on the biological effects. Their inhibition against isolated HER2 was studied and the structure-activity relationships have been confirmed by molecular modelling studies. The most potent compound against isolated HER2 is 9a with an IC50 of 7.31 µM. We have investigated the effects of the target compounds on cell proliferation. The most active compound (7c) against all the tumour cell lines studied (IC50 0.42-0.86 µM) does not produce any modification in the expression of pro-caspase 3, but increases the caspase 1 expression, and promotes pyroptosis.

Silver Nanoparticles from Annona muricata Peel and Leaf Extracts as a Potential Potent, Biocompatible and Low Cost Antitumor Tool.

Cancer is one of the most prevalent diseases in the world and requires new therapies for its treatment. In this context, the biosynthesis of silver nanoparticles (AgNPs) has been developed to treat different types of tumors. The Annona muricata plant is known for having anticancer activity. Its main compounds present in the leaves, stems and skin, allowing for its use as reducing agents. In this manuscript, AgNPs with leaf extract (AgNPs-LE) and fruit peel extract (AgNPs-PE) of A. muricata were biosynthesized obtaining an average nanoparticle diameter sizes smaller than 50 nm, being 19.63 ± 3.7 nm and 16.56 ± 4.1 nm, and with a surface plasmonic resonance (SPR) at 447 and 448 nm, respectively. The lactone functional group present in the LE and PE extracts was identified by the FTIR technique. The behavior and antiproliferation activity of AgNPs-LE and AgNPs-PE were evaluated in breast, colon and melanoma cancer cell lines. Our results showed that Annona muricata fruit peel, which is a waste product, has an antitumor effect more potent than leaf extract. This difference is maintained with AgNPs where the destruction of cancer cells was, for the first time, achieved using concentrations that do not exceed 3 µg/mL with a better therapeutic index in the different tumor strains. In conclusion, we present a low-cost one-step experimental setup to generate AgNPs-PE whose in-vitro biocompatibility and powerful therapeutic effect make it a very attractive tool worth exploiting.

Metabolomic profile of cancer stem cell-derived exosomes from patients with malignant melanoma.

Malignant melanoma (MM) is the most aggressive and life-threatening form of skin cancer. It is characterized by an extraordinary metastasis capacity and chemotherapy resistance, mainly due to melanoma cancer stem cells (CSCs). To date, there are no suitable clinical diagnostic, prognostic or predictive biomarkers for this neoplasia. Therefore, there is an urgent need for new MM biomarkers that enable early diagnosis and effective disease monitoring. Exosomes represent a novel source of biomarkers since they can be easily isolated from different body fluids. In this work, a primary patient-derived MM cell line enriched in CSCs was characterized by assessing the expression of specific markers and their stem-like properties. Exosomes derived from CSCs and serums from patients with MM were characterized, and their metabolomic profile was analysed by high-resolution mass spectrometry (HRMS) following an untargeted approach and applying univariate and multivariate statistical analyses. The aim of this study was to search potential biomarkers for the diagnosis of this disease. Our results showed significant metabolomic differences in exosomes derived from MM CSCs compared with those from differentiated tumour cells and also in serum-derived exosomes from patients with MM compared to those from healthy controls. Interestingly, we identified similarities between structural lipids differentially expressed in CSC-derived exosomes and those derived from patients with MM such as the glycerophosphocholine PC 16:0/0:0. To our knowledge, this is the first metabolomic-based study aimed at characterizing exosomes derived from melanoma CSCs and patients' serum in order to identify potential biomarkers for MM diagnosis. We conclude that metabolomic characterization of CSC-derived exosomes sets an open door to the discovery of clinically useful biomarkers in this neoplasia.

Large-Scale Production of Lentiviral Vectors: Current Perspectives and Challenges.

Lentiviral vectors (LVs) have gained value over recent years as gene carriers in gene therapy. These viral vectors are safer than what was previously being used for gene transfer and are capable of infecting both dividing and nondividing cells with a long-term expression. This characteristic makes LVs ideal for clinical research, as has been demonstrated with the approval of lentivirus-based gene therapies from the Food and Drug Administration and the European Agency for Medicine. A large number of functional lentiviral particles are required for clinical trials, and large-scale production has been challenging. Therefore, efforts are focused on solving the drawbacks associated with the production and purification of LVsunder current good manufacturing practice. In recent years, we have witnessed the development and optimization of new protocols, packaging cell lines, and culture devices that are very close to reaching the target production level. Here, we review the most recent, efficient, and promising methods for the clinical-scale production ofLVs.

Targeted Gene Delivery Therapies for Cervical Cancer.

Despite being largely preventable through early vaccination and screening strategies, cervical cancer is the most common type of gynecological malignancy worldwide and constitutes one of the leading causes of cancer deaths in women. Patients with advanced or recurrent disease have a very poor prognosis; hence, novel therapeutic modalities to improve clinical outcomes in cervical malignancy are needed. In this regard, targeted gene delivery therapy is presented as a promising approach, which leads to the development of multiple strategies focused on different aspects. These range from altered gene restoration, immune system potentiation, and oncolytic virotherapy to the use of nanotechnology and the design of improved and enhanced gene delivery systems, among others. In the present manuscript, we review the current progress made in targeted gene delivery therapy for cervical cancer, the advantages and drawbacks and their clinical application. At present, multiple targeted gene delivery systems have been reported with encouraging preclinical results. However, the translation to humans has not yet shown a significant clinical benefit due principally to the lack of efficient vectors. Real efforts are being made to develop new gene delivery systems, to improve tumor targeting and to minimize toxicity in normal tissues.

miRNAs as radio-response biomarkers for breast cancer stem cells.

In breast cancer (BC), the presence of cancer stem cells (CSCs) has been related to relapse, metastasis, and radioresistance. Radiotherapy (RT) is an extended BC treatment, but is not always effective. CSCs have several mechanisms of radioresistance in place, and some miRNAs are involved in the cellular response to ionizing radiation (IR). Here, we studied how IR affects the expression of miRNAs related to stemness in different molecular BC subtypes. Exposition of BC cells to radiation doses of 2, 4, or 6 Gy affected their phenotype, functional characteristics, pluripotency gene expression, and in vivo tumorigenic capacity. This held true for various molecular subtypes of BC cells (classified by ER, PR and HER-2 status), and for BC cells either plated in monolayer, or being in suspension as mammospheres. However, the effect of IR on the expression of eight stemness- and radioresistance-related miRNAs (miR-210, miR-10b, miR-182, miR-142, miR-221, miR-21, miR-93, miR-15b) varied, depending on cell line subpopulation and clinicopathological features of BC patients. Therefore, clinicopathological features and, potentially also, chemotherapy regimen should be both taken into consideration, for determining a potential miRNA signature by liquid biopsy in BC patients treated with RT. Personalized and precision RT dosage regimes could improve the prognosis, treatment, and survival of BC patients.

Phenformin as an Anticancer Agent: Challenges and Prospects.

Currently, there is increasing evidence linking diabetes mellitus (especially type 2 diabetes mellitus) with carcinogenesis through various biological processes, such as fat-induced chronic inflammation, hyperglycemia, hyperinsulinemia, and angiogenesis. Chemotherapeutic agents are used in the treatment of cancer, but in most cases, patients develop resistance. Phenformin, an oral biguanide drug used to treat type 2 diabetes mellitus, was removed from the market due to a high risk of fatal lactic acidosis. However, it has been shown that phenformin is, with other biguanides, an authentic tumor disruptor, not only by the production of hypoglycemia due to caloric restriction through AMP-activated protein kinase with energy detection (AMPK) but also as a blocker of the mTOR regulatory complex. Moreover, the addition of phenformin eliminates resistance to antiangiogenic tyrosine kinase inhibitors (TKI), which prevent the uncontrolled metabolism of glucose in tumor cells. In this review, we evidence the great potential of phenformin as an anticancer agent. We thoroughly review its mechanism of action and clinical trial assays, specially focusing on current challenges and future perspectives of this promising drug.

LdrB Toxin with In Vitro and In Vivo Antitumor Activity as a Potential Tool for Cancer Gene Therapy.

Due to the high prevalence of cancer in recent years, it is necessary to develop new and more effective therapies that produce fewer side effects. Development of gene therapy for cancer based on the use of suicide genes that can damage the tumor cell, without requiring a prodrug for its lethal effect, is one of the recent foci of gene therapy strategies. We evaluated the cytotoxic impact of the LdrB toxin from Escherichia coli k12 as a possible tool for cancer gene therapy. For that, colorectal and breast cancer cells were transfected under the control of a TRE3G promoter inducible by doxycycline. Our results showed that ldrB gene expression induced a drastic inhibition of proliferation in vitro, in both 2D and 3D experimental models. Moreover, unlike conventional chemotherapy, the ldrB gene induced a severe loss of proliferation in vivo without any side effects in our animal model. This antitumor outcome was modulated by cell cycle arrest in the G0/G1 phase and apoptotic death. Scanning electronic microscopy demonstrates that the LdrB toxin conserves its pore-forming ability in HCT-116 cells as in E. coli k12. Taken together, our results provide, for the first time, a proof of concept of the antitumor capacity of the ldrB gene in colorectal and breast cancer.

Deciphering the Mechanism of Action Involved in Enhanced Suicide Gene Colon Cancer Cell Killer Effect Mediated by Gef and Apoptin.

Despite the great advances in cancer treatment, colorectal cancer has emerged as the second highest cause of death from cancer worldwide. For this type of tumor, the use of suicide gene therapy could represent a novel therapy. We recently demonstrated that co-expression of gef and apoptin dramatically inhibits proliferation of the DLD-1 colon cell line. In the present manuscript, we try to establish the mechanism underlying the enhanced induction of apoptosis by triggering both gef and apoptin expression in colon tumor cells. Scanning microscopy reveals that simultaneous expression of gef and apoptin induces the apparition of many "pores" in the cytoplasmic membrane not detected in control cell lines. The formation of pores induced by the gef gene and accentuated by apoptin results in cell death by necrosis. Moreover, we observed the presence of apoptotic cells. Performing protein expression analysis using western blot, we revealed an activation of mitochondrial apoptosis (increased expression of Pp53, cytochrome c, Bax, and caspase 9) and also the involvement of the extrinsic pathway through caspase 8activation. In conclusion, in this manuscript we demonstrate for the first time that the extrinsic pathway of apoptosis and pore formation is also involved in the cell death caused by the co-expression of the gef and apoptin genes. Our results suggest that co-expression of gef and apoptin genes induces an increase in post-apoptotic necrotic cell death and could be a valuable tool in the design of new antitumor strategies focused on the enhancement of the immune response against cancer cell death.

Clinical Trials of Thermosensitive Nanomaterials: An Overview.

Currently, we are facing increasing demand to develop efficient systems for the detection and treatment of diseases that can realistically improve distinct aspects of healthcare in our society. Sensitive nanomaterials that respond to environmental stimuli can play an important role in this task. In this manuscript, we review the clinical trials carried out to date on thermosensitive nanomaterials, including all those clinical trials in hybrid nanomaterials that respond to other stimuli (e.g., magnetic, infrared radiation, and ultrasound). Specifically, we discuss their use in diagnosis and treatment of different diseases. At present, none of the existing trials focused on diagnosis take advantage of the thermosensitive characteristics of these nanoparticles. Indeed, almost all clinical trials consulted explore the use of Ferumoxytol as a current imaging test enhancer. However, the thermal property is being further exploited in the field of disease treatment, especially for the delivery of antitumor drugs. In this regard, ThermoDox®, based on lysolipid thermally sensitive liposome technology to encapsulate doxorubicin (DOX), is the flagship drug. In this review, we have evidenced the discrepancy existing between the number of published papers in thermosensitive nanomaterials and their clinical use, which could be due to the relative novelty of this area of research; more time is needed to validate it through clinical trials. We have no doubt that in the coming years there will be an explosion of clinical trials related to thermosensitive nanomaterials that will surely help to improve current treatments and, above all, will impact on patients' quality of life and life expectancy.

Thermo-Sensitive Nanomaterials: Recent Advance in Synthesis and Biomedical Applications.

Progress in nanotechnology has enabled us to open many new fronts in biomedical research by exploiting the peculiar properties of materials at the nanoscale. The thermal sensitivity of certain materials is a highly valuable property because it can be exploited in many promising applications, such as thermo-sensitive drug or gene delivery systems, thermotherapy, thermal biosensors, imaging, and diagnosis. This review focuses on recent advances in thermo-sensitive nanomaterials of interest in biomedical applications. We provide an overview of the different kinds of thermoresponsive nanomaterials, discussing their potential and the physical mechanisms behind their thermal response. We thoroughly review their applications in biomedicine and finally discuss the current challenges and future perspectives of thermal therapies.

Enhancement of Tumor Cell Death by Combining gef Gene Mediated Therapy and New 1,4-Benzoxazepin-2,6-Dichloropurine Derivatives in Breast Cancer Cells.

New treatment modalities are urgently needed to better manage advanced breast cancer. Combination therapies are usually more effective than monotherapy. In this context, the use of cyclic and acyclic O,N-acetals derivative compounds in combination with the suicide gef gene shown a potent anti-tumor activity and represent a new generation of anticancer agents. Here, we evaluate the use of the gef gene to promote and increase the anti-tumor effect of cyclic and acyclic O,N-acetals purine derivatives and elucidate their mechanisms of action. Among all compounds tested, those with a nitro group and a cyclic pattern structures (FC-30b2, FC-29c, and bozepinib) are the most benefited from the gef gene effect. These compounds, in combination with gef gene, were able to abolish tumor cell proliferation with a minimal dose leading to more effective and less toxic chemotherapy. The effect of this combined therapy is triggered by apoptosis induction which can be found deregulated in the later stage of breast cancer. Moreover, the combined therapy leads to an increase of cell post-apoptotic secondary necrosis that is able to promote the immunogenicity of cancer cells leading to a successful treatment. This data suggests that this novel combination therapy represents a promising candidate for breast cancer treatment.

Recent Progress in Gene Therapy for Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy in developed countries. This is due to the lack of specific symptoms that hinder early diagnosis and to the high relapse rate after treatment with radical surgery and chemotherapy. Hence, novel therapeutic modalities to improve clinical outcomes in ovarian malignancy are needed. Progress in gene therapy has allowed the development of several strategies against ovarian cancer. Most are focused on the design of improved vectors to enhance gene delivery on the one hand, and, on the other hand, on the development of new therapeutic tools based on the restoration or destruction of a deregulated gene, the use of suicide genes, genetic immunopotentiation, the inhibition of tumour angiogenesis, the alteration of pharmacological resistance, and oncolytic virotherapy. In the present manuscript, we review the recent advances made in gene therapy for ovarian cancer, highlighting the latest clinical trials experience, the current challenges and future perspectives.

Mesenchymal stem cell's secretome promotes selective enrichment of cancer stem-like cells with specific cytogenetic profile.

Cancer stem cells (CSCs) are responsible for tumor initiation, metastasis and cancer recurrence, however the involvement of microenvironment is crucial. Here, we have analyzed how human mesenchymal stem cells (MSCs)-derived conditioned medium (CM) affect colon and melanoma CSCs enrichment and maintenance. Our results strongly suggest that the secretome of CM-MSCs selects and maintains subpopulations with high expression of CSCs markers and ALDH1 activity, low proliferation rates with G1 phase arrest, and notably retain in vivo these properties. Cytogenetic analyses indicated that CM-cultured cells contain alterations in chromosome 17 (17q25). Subsequent SKY-FISH analyses suggested that genes located in 17q25 might be involved in stem-cell maintenance. The characterization of secreted proteins present in CM-MSCs revealed that four cytokines and seven growth factors are directly linked to the CSCs enrichment reported in this study. Further analyses revealed that the combination of just IL6 and HGF is enough to provide cancer cells with better stemness properties. In conclusion, this study demonstrates how specific chromosomal alterations present in CSCs subpopulations might represent an advantage for their in vitro maintenance and in vivo stemness properties.