Tryptophan Derivatives by Saccharomyces cerevisiae EC1118: Evaluation,Optimization, and Production in a Soybean-Based Medium

Given the pharmacological properti es and the potential role of kynurenic acid (KYNA) in human physiology and the pleiotropic activity of the neurohormone melatonin (MEL) involved in physiological and immunological functions and as regulator of antioxidant enzymes, this study aimed at evaluating the capability of Saccharomyces cerevisiae EC1118 to release tryptophan derivatives (dTRPs) from the kynurenine (KYN) and melatonin pathways. The setting up of the spectroscopic and chromatographic conditions for the quantification of the dTRPs in LC-MS/MS system, the optimization of dTRPs’ production in fermentative and whole-cell biotransformation approaches and the production of dTRPs in a soybean-based cultural medium naturally enriched in tryptophan, as a case of study, were included in the experimental plan. Variable amounts of dTRPs, with a prevalence of metabolites of the KYN pathway, were detected. The LC-MS/MS analysis showed that the compound synthesized at highest concentration is KYNA that reached 9.146 ± 0.585 mg/L in fermentation trials in a chemically defined medium at 400 mg/L TRP. Further experiments in a soybean-based medium confirm KYNA as the main dTRPs, whereas the other dTRPs reached very lower concentrations. While detectable quantities of melatonin were never observed, two MEL isomers were successfully measured in laboratory media.     

Tryptophan Metabolites at the Crossroad of Immune-Cell Interaction via the Aryl Hydrocarbon Receptor: Implications for Tumor Immunotherapy

The Aryl hydrocarbon receptor (AhR) is a critical regulator of both innate and adaptive immune responses, with potent immunomodulatory effects that makes this receptor an attractive molecular target for novel therapeutics. Accumulating evidence indicates that diverse—both host’s and microbial—tryptophan metabolites profoundly regulate the immune system in the host via AhR, promoting either tolerance or immunity, largely as a function of the qualitative and quantitative nature of the metabolites being contributed by either source. Additional findings indicate that host and microbiota-derived tryptophan metabolic pathways can influence the outcome of immune responses to tumors. Here, we review recent studies on the role and modalities of AhR activation by various ligands, derived from either host-cell or microbial-cell tryptophan metabolic pathways, in the regulation of immune responses. Moreover, we highlight potential implications of those ligands and pathways in tumor immunotherapy, with particular relevance to checkpoint-blockade immune intervention strategies.     

The Role of Altered Nucleotide Excision Repair and UVB-Induced DNA Damage in Melanomagenesis

UVB radiation is the most mutagenic component of the UV spectrum that reaches the earth’s surface and causes the development of DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts. UV radiation usually results in cellular death, but if left unchecked, it can affect DNA integrity, cell and tissue homeostasis and cause mutations in oncogenes and tumour-suppressor genes. These mutations, if unrepaired, can lead to abnormal cell growth, increasing the risk of cancer development. Epidemiological data strongly associates UV exposure as a major factor in melanoma development, but the exact biological mechanisms involved in this process are yet to be fully elucidated. The nucleotide excision repair (NER) pathway is responsible for the repair of UV-induced lesions. Patients with the genetic disorder Xeroderma Pigmentosum have a mutation in one of eight NER genes associated with the XP complementation groups XP-A to XP-G and XP variant (XP-V). XP is characterized by diminished repair capacity, as well as a 1000-fold increase in the incidence of skin cancers, including melanoma. This has suggested a significant role for NER in melanoma development as a result of UVB exposure. This review discusses the current research surrounding UVB radiation and NER capacity and how further investigation of NER could elucidate the role of NER in avoiding UV-induced cellular death resulting in melanomagenesis.     

Dieckol Inhibits Autophagic Flux and Induces Apoptotic Cell Death in A375 Human Melanoma Cells via Lysosomal Dysfunction and Mitochondrial Membrane Impairment

Dieckol is a natural brown algal-derived polyphenol and its cytotoxic potential against various types of cancer cells has been studied. However, the effects of dieckol on autophagy in cancer cells remain unknown. Here, we show that dieckol inhibits the growth of A375 human melanoma cells by inducing apoptotic cell death, which is associated with lysosomal dysfunction and the inhibition of autophagic flux. Dieckol induces autophagosome accumulation by inhibiting autophagosome-lysosome fusion. Moreover, dieckol not only triggers lysosomal membrane permeabilization, followed by an increase in lysosomal pH and the inactivation of cathepsin B and D, but also causes the loss of mitochondrial membrane potential. Importantly, a cathepsin D inhibitor partially relieved dieckol-induced mitochondrial membrane impairment and caspase-mediated apoptosis. Collectively, our findings indicate that dieckol is a novel autophagy inhibitor that induces apoptosis-mediated cell death via lysosomal dysfunction and mitochondrial membrane impairment in A375 human melanoma cells. This suggests the novel potential value of dieckol as a chemotherapeutic drug candidate for melanoma treatment.     

Novel and Potent Small Molecules against Melanoma Harboring BRAF Class I/II/III Mutants for Overcoming Drug Resistance

Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling. The most frequently occurring class I BRAF V600 mutations are sensitive to vemurafenib whereas class II and class III mutants, non-V600 BRAF mutants are resistant to vemurafenib. Herein we report six pyrimido[4,5-d]pyrimidin-2-one derivatives possessing highly potent anti-proliferative activities on melanoma cells harboring BRAF class I/II/III mutants. Novel and most potent derivative, SIJ1777, possesses not only two-digit nanomolar potency but also 2 to 14-fold enhanced anti-proliferative activities compared with reference compound, GNF-7 against melanoma cells (SK-MEL-2, SK-MEL-28, A375, WM3670, WM3629). Moreover, SIJ1777 substantially inhibits the activation of MEK, ERK, and AKT and remarkably induces apoptosis and significantly blocks migration, invasion, and anchorage-independent growth of melanoma cells harboring BRAF class I/II/II mutations while both vemurafenib and PLX8394 have little to no effects on melanoma cells expressing BRAF class II/III mutations. Taken together, our six GNF-7 derivatives exhibit highly potent activities against melanoma cells harboring class I/II/III BRAF mutations compared with vemurafenib as well as PLX8394.     

Lignin Nanoparticles Deliver Novel Thymine Biomimetic Photo-Adducts with Antimelanoma Activity

We report here the synthesis of novel thymine biomimetic photo-adducts bearing an alkane spacer between nucleobases and characterized by antimelanoma activity against two mutated cancer cell lines overexpressing human Topoisomerase 1 (TOP1), namely SKMEL28 and RPMI7951. Among them, Dewar Valence photo-adducts showed a selectivity index higher than the corresponding pyrimidine-(6-4)-pyrimidone and cyclobutane counterpart and were characterized by the highest affinity towards TOP1/DNA complex as evaluated by molecular docking analysis. The antimelanoma activity of novel photo-adducts was retained after loading into UV photo-protective lignin nanoparticles as stabilizing agent and efficient drug delivery system. Overall, these results support a combined antimelanoma and UV sunscreen strategy involving the use of photo-protective lignin nanoparticles for the controlled release of thymine dimers on the skin followed by their sacrificial transformation into photo-adducts and successive inhibition of melanoma and alert of cellular UV machinery repair pathways.     

Influence of Complexation of Thiosemicarbazone Derivatives with Cu (II) Ions on Their Antitumor Activity against Melanoma Cells

A series of thiosemicarbazone derivatives was prepared and their anti-tumor activity in vitro was tested. The X-ray investigation performed for compounds T2, T3 and T5 confirmed the synthesis pathway and assumed molecular structures of analyzed thiosemicarbazones. The conformational preferences of the thiosemicarbazone system were characterized using theoretical calculations by AM1 method. Selected compounds were converted into complexes of Cu (II) ions. The effect of complexing on anti-tumor activity has been investigated. The copper(II) complexes, with Schiff bases T1, T10, T12, T13, and T16 have been synthesized and characterized by chemical and elemental analysis, FTIR spectroscopy and TGA method. Thermal properties of coordination compounds were studied using TG-DTG techniques under dry air atmosphere. G361, A375, and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells were treated with tested compounds and their cytotoxicity was evaluated with MTT test. The compounds with the most promising anti-tumour activity were then selected and their cytotoxicity was verified with cell cycle analysis and apoptosis/necrosis detection. Additionally, DNA damages in the form of a basic sites presence and the expression of oxidative stress and DNA damage response genes were evaluated. The obtained results indicate that complexation of thiosemicarbazone derivatives with Cu (II) ions improves their antitumor activity against melanoma cells. The observed cytotoxic effect is associated with DNA damage and G2/M phase of cell cycle arrest as well as disorders of the antioxidant enzymes expression.     

Developmental Programming and Reprogramming of Hypertension and Kidney Disease: Impact of Tryptophan Metabolism

The concept that hypertension and chronic kidney disease (CKD) originate in early life has emerged recently. During pregnancy, tryptophan is crucial for maternal protein synthesis and fetal development. On one hand, impaired tryptophan metabolic pathway in pregnancy impacts fetal programming, resulting in the developmental programming of hypertension and kidney disease in adult offspring. On the other hand, tryptophan-related interventions might serve as reprogramming strategies to prevent a disease from occurring. In the present review, we aim to summarize (1) the three major tryptophan metabolic pathways, (2) the impact of tryptophan metabolism in pregnancy, (3) the interplay occurring between tryptophan metabolites and gut microbiota on the production of uremic toxins, (4) the role of tryptophan-derived metabolites-induced hypertension and CKD of developmental origin, (5) the therapeutic options in pregnancy that could aid in reprogramming adverse effects to protect offspring against hypertension and CKD, and (6) possible mechanisms linking tryptophan metabolism to developmental programming of hypertension and kidney disease.     

Ablation of Acid Ceramidase Impairs Autophagy and Mitochondria Activity in Melanoma Cells

Cutaneous melanoma is often resistant to therapy due to its high plasticity, as well as its ability to metabolise chemotherapeutic drugs. Sphingolipid signalling plays a pivotal role in its progression and metastasis. One of the ways melanoma alters sphingolipid rheostat is via over-expression of lysosomal acid ceramidase (AC), which catalyses the hydrolysis of pro-apoptotic long-chain ceramides into sphingosine and fatty acid. In this report, we examine the role of acid ceramidase in maintaining cellular homeostasis through the regulation of autophagy and mitochondrial activity in melanoma cell lines. We show that under baseline conditions, wild-type melanoma cells had 3-fold higher levels of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3 II), compared to AC-null cells. This difference was further magnified after cell starvation. Moreover, we noticed autophagy impairment in A375 AC-null cells, possibly due to local accumulation of non-metabolized ceramides. Nonetheless, we observed that AC-null cells exhibited a significant increase in mitochondrial membrane potential compared to control cells. Consistent with this observation, we found that, after total starvation, ~30% of AC-null cells undergo apoptosis compared to ~6% of wild-type cells. As expected, AC transfection restored viability in A375 AC-null cells. Together, these findings suggest that AC-null melanoma cells change and adapt their metabolism to survive in the absence of AC, although in a way that does not allow them to cope with the stress of nutrient deprivation.     

An Emerging Cross-Species Marker for Organismal Health: Tryptophan-Kynurenine Pathway

Tryptophan (TRP) is an essential dietary amino acid that, unless otherwise committed to protein synthesis, undergoes metabolism via the Tryptophan-Kynurenine (TRP-KYN) pathway in vertebrate organisms. TRP and its metabolites have key roles in diverse physiological processes including cell growth and maintenance, immunity, disease states and the coordination of adaptive responses to environmental and dietary cues. Changes in TRP metabolism can alter the availability of TRP for protein and serotonin biosynthesis as well as alter levels of the immune-active KYN pathway metabolites. There is now considerable evidence which has shown that the TRP-KYN pathway can be influenced by various stressors including glucocorticoids (marker of chronic stress), infection, inflammation and oxidative stress, and environmental toxicants. While there is little known regarding the role of TRP metabolism following exposure to environmental contaminants, there is evidence of linkages between chemically induced metabolic perturbations and altered TRP enzymes and KYN metabolites. Moreover, the TRP-KYN pathway is conserved across vertebrate species and can be influenced by exposure to xenobiotics, therefore, understanding how this pathway is regulated may have broader implications for environmental and wildlife toxicology. The goal of this narrative review is to (1) identify key pathways affecting Trp-Kyn metabolism in vertebrates and (2) highlight consequences of altered tryptophan metabolism in mammals, birds, amphibians, and fish. We discuss current literature available across species, highlight gaps in the current state of knowledge, and further postulate that the kynurenine to tryptophan ratio can be used as a novel biomarker for assessing organismal and, more broadly, ecosystem health.     

人恶性黑色素瘤A375细胞黑素刺激素受体(MC1R)的检测

目的检测人恶性黑色素瘤A375细胞膜上MC1R蛋白及其cDNA,为研究黑色素瘤细胞表面受体及靶向配体奠定基础。方法利用放射配体分析法检测人恶性黑色素瘤A375细胞膜上的MC1R蛋白;用RTPCR方法扩增人恶性黑色素瘤A375细胞MC1R的cDNA,并克隆至pMD18T质粒,进行酶切分析鉴定及扩增片段DNA序列测定。结果放射配体分析结果表明,125IαMSH可与人恶性黑色素瘤A375细胞膜特异结合。RTPCR结果显示,扩增产物的大小与MC1R的cDNA相符。测序结果证实扩增片段为MC1R基因的特异片段。结论证实人恶性黑色素瘤A375细胞膜上表达MC1R蛋白。MC1R基因的成功克隆为其进一步研究提供了必要条件。     

The Role of AKT/mTOR Pathway in Stress Response to UV-Irradiation: Implication in Skin Carcinogenesis by Regulation of Apoptosis,Autophagy and Senescence

Induction of DNA damage by UVB and UVA radiation may generate mutations and genomic instability leading to carcinogenesis. Therefore, skin cells being repeatedly exposed to ultraviolet (UV) light have acquired multilayered protective mechanisms to avoid malignant transformation. Besides extensive DNA repair mechanisms, the damaged skin cells can be eliminated by induction of apoptosis, which is mediated through the action of tumor suppressor p53. In order to prevent the excessive loss of skin cells and to maintain the skin barrier function, apoptotic pathways are counteracted by anti-apoptotic signaling including the AKT/mTOR pathway. However, AKT/mTOR not only prevents cell death, but is also active in cell cycle transition and hyper-proliferation, thereby also counteracting p53. In turn, AKT/mTOR is tuned down by the negative regulators being controlled by the p53. This inhibition of AKT/mTOR, in combination with transactivation of damage-regulated autophagy modulators, guides the p53-mediated elimination of damaged cellular components by autophagic clearance. Alternatively, p53 irreversibly blocks cell cycle progression to prevent AKT/mTOR-driven proliferation, thereby inducing premature senescence. Conclusively, AKT/mTOR via an extensive cross talk with p53 influences the UV response in the skin with no black and white scenario deciding over death or survival.     

Combined Therapy with Dacarbazine and Hyperthermia Induces Cytotoxicity in A375 and MNT-1 Melanoma Cells

Melanoma is a drug-resistant cancer, representing a serious challenge in cancer treatment. Dacarbazine (DTIC) is the standard drug in metastatic melanoma treatment, despite the poor results. Hyperthermia has been proven to potentiate chemotherapy. Hence, this work analyzed the combined action of hyperthermia and DTIC on A375 and MNT-1 cell lines. First, temperatures between 40 °C and 45 °C were tested. The effect of DTIC on cell viability was also investigated after exposures of 24, 48, and 72 h. Then, cells were exposed to 43 °C and to the respective DTIC IC10 or IC20 of each time exposure. Overall, hyperthermia reduced cell viability, however, 45 °C caused an excessive cell death (>90%). Combinational treatment revealed that hyperthermia potentiates DTIC’s effect, but it is dependent on the concentration and temperature used. Also, it has different mechanisms from the treatments alone, delaying A375 cells at the G2/M phase and MNT-1 cells at the S and G2/M phases. Intracellular reactive oxygen species (ROS) levels increased after treatment with hyperthermia, but the combined treatment showed no additional differences. Also, hyperthermia highly increased the number of A375 early apoptotic cells. These results suggest that combining hyperthermia and DTIC should be more explored to improve melanoma treatment.     

Structure--activity relationships in human interleukin-1{alpha}: identification of key residues for expression of biological activities

To identify the sites important for the different biologicalactivities of human interleukin-l (hIL-1), 56 single-amino acid-substitutedmutants of hIL-l were produced in Escherichia coli using site-directedmutagenesis, and were examined for their biological activitiessuch as mouse lymphocyte activating factor activity (LAF activity),cytostatic activity against human melanoma cells A-375 (A375activity) and prostaglandin E2 (PGE2) inducing activity in humanosteosarcoma cells MG-63 (PEI activity). Two amino acid residues,Asp26 and Asp151, were found to be important for these activities.The replacement of Asp26 by Val caused a decrease in LAF andA375 activities by one or two orders of magnitude and a slightdecrease in A375 activity. The Tyr or Phe substitution for Asp151caused decreases in LAF and A375 activities by one or two ordersof magnitude and complete loss of PEI activity. The change fromAsp151 to Lys or Arg resulted in marked decrease in LAF activityand complete loss of A375 and PEI activities. Since Asp26 andAsp151 are close to each other in the three-dimensional structure,the region involving these amino acids seems to be importantfor the biological activities of hIL-1.     

Development of Sesamol Carbamate-L-Phenylalanine Prodrug Targeting L-Type Amino Acid Transporter1 (LAT1) as a Potential Antiproliferative Agent against Melanoma

Sesamol is a compound reported to have anti-melanogenesis and anti-melanoma actions. Sesamol, however, has low intracellular drug concentration and fast excretion, which can limit its benefits in the clinic. To overcome this drawback and increase intracellular delivery of sesamol into the target melanoma, research has focused on L-type amino acid transporter 1 (LAT1)-mediated prodrug delivery into melanoma cells. The sesamol prodrug was designed by conjugating sesamol with L-phenylalanine at the para position with a carbamate bond. LAT1 targeting was evaluated vis-à-vis a competitive [¹⁴C]-leucine uptake inhibition. The sesamol prodrug has a higher [¹⁴C]-leucine uptake inhibition than sesamol in human LAT1-transfected HEK293 cells. Moreover, the sesamol prodrug was taken up by LAT1-mediated transport into SK-MEL-2 cells more effectively than sesamol. The sesamol prodrug underwent complete hydrolysis, releasing the active sesamol at 72 h, which significantly exerted its cytotoxicity (IC₅₀ of 29.3 µM) against SK-MEL-cells more than sesamol alone. Taken together, the strategy for LAT1-mediated prodrug delivery has utility for the selective uptake of sesamol, thereby increasing its intracellular concentration and antiproliferation activity, targeting melanoma SK-MEL-2 cells that overexpress the LAT1 protein. The sesamol prodrug thus warrants further evaluation in an in vivo model.