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Melanoma, a highly aggressive skin cancer, is characterized by rapid progression and high mortality. Recent advances in molecular pathogenesis have shed light on genetic and epigenetic changes that drive melanoma development. This review provides an overview of these developments, focusing on molecular mechanisms in melanoma genesis. It highlights how mutations, particularly in the BRAF, NRAS, c-KIT, and GNAQ/GNA11 genes, affect critical signaling pathways. The evolution of diagnostic techniques, such as genomics, transcriptomics, liquid biopsies, and molecular biomarkers for early detection and prognosis, is also discussed. The therapeutic landscape has transformed with targeted therapies and immunotherapies, improving patient outcomes. This paper examines the efficacy, challenges, and prospects of these treatments, including recent clinical trials and emerging strategies. The potential of novel treatment strategies, including neoantigen vaccines, adoptive cell transfer, microbiome interactions, and nanoparticle-based combination therapy, is explored. These advances emphasize the challenges of therapy resistance and the importance of personalized medicine. This review underlines the necessity for evidence-based therapy selection in managing the increasing global incidence of melanoma. [ABSTRACT FROM AUTHOR]

A series of novel echinatin derivatives with 1,3,4-oxadiazole moieties were designed and synthesized. Most of the newly synthesized compounds exhibited moderate antiproliferative activity against the four cancer cell lines. Notably, Compound T4 demonstrated the most potent activity, with IC50 values ranging from 1.71 µM to 8.60 µM against the four cancer cell lines. Cell colony formation and wound healing assays demonstrated that T4 significantly inhibited cell proliferation and inhibited migration. We discovered that T4 exhibited moderate binding affinity with the c-KIT protein through reverse docking. The results were effectively validated through subsequent molecular docking and c-KIT enzyme activity assays. In addition, Western blot analysis revealed that T4 inhibits the phosphorylation of downstream proteins of c-KIT. The results provide valuable inspiration for exploring novel insights into the design of echinatin-related hybrids as well as their potential application as c-KIT inhibitors to enhance the efficacy of candidates. [ABSTRACT FROM AUTHOR]

Studying primary melanoma and its corresponding metastasis has twofold benefits. Firstly, to better understand tumor biology, and secondly, to determine which sample should be examined in assessing drug targets. This study systematically analyzed all the literature on primary melanoma and its matched metastasis. Following PRISMA guidelines, we searched multiple medical databases for relevant publications from January 2000 to December 2022, assessed the quality of the primary-level studies using the QUIPS tool, and summarized the concordance rate of the most reported genes using the random-effects model. Finally, we evaluated the inter-study heterogeneity using the subgroup analysis. Thirty-one studies investigated the concordance of BRAF and NRAS in 1220 and 629 patients, respectively. The pooled concordance rate was 89.4% [95% CI: 84.5; 93.5] for BRAF and 97.8% [95% CI: 95.8; 99.4] for NRAS. When high-quality studies were considered, only BRAF mutation status consistency increased. Five studies reported the concordance status of c-KIT (93%, 44 patients) and TERT promoter (64%, 53 patients). Lastly, three studies analyzed the concordance of cancer genes involved in the signaling pathways, apoptosis, and proliferation, such as CDKN2A (25%, four patients), TP53 (44%, nine patients), and PIK3CA (20%, five patients). Our study found that the concordance of known drug targets (mainly BRAF) during melanoma progression is higher than in previous meta-analyses, likely due to advances in molecular techniques. Furthermore, significant heterogeneity exists in the genes involved in the melanoma genetic makeup; although our results are based on small patient samples, more research is necessary for validation. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are tissue-resident immune cells of a hematopoietic origin that play vital roles in innate and adaptive immunity. Human MCs can be isolated and differentiated from various tissue sources, including cord blood, when supplemented with cytokines such as stem cell factor, interleukin 3, and interleukin 6. Our current research study has shown significant differences in the marker expressions of human cord blood-derived mast cells (hCBMCs) based on donor dependency and the type of medium used for culturing and differentiation. These findings are particularly relevant given the challenges of obtaining specialty media influencing MC phenotypic marker expressions. We found that hCBMCs cultured in StemSpanTM-XF medium had a moderate expression of mast/stem cell growth factor receptor Kit (c-KIT) (mRNA and protein), low expressions of FcεRI (mRNA) and TLR2 (mRNA and protein) but had high levels of MRGPRX2 (mRNA and protein) expressions. In contrast, hCBMCs cultured in Stem Line II medium expressed FcεRI and TLR2 (mRNA and protein) with higher c-KIT but had lower MRGPRX2 expressions compared to the hCBMCs cultured in the StemSpanTM-XF medium. These results suggest that it is crucial to consider both donor dependency and the medium when investigating MC functions and that further research is needed to fully understand the impact of these factors on the hCBMC marker expressions. [ABSTRACT FROM AUTHOR]

Allergic sensitization to cannabis is an emerging public health concern and is difficult to clinically establish owing to lack of standardized diagnostic approaches. Attempts to develop diagnostic tools were largely hampered by the Schedule I restrictions on cannabis, which limited accessibility for research. Recently, however, hemp was removed from the classified list, and increased accessibility to hemp allows for the evaluation of its practical clinical value for allergy diagnosis. We hypothesized that the proteomic profile is preserved across different cannabis chemotypes and that hemp would be an ideal source of plant material for clinical testing. Using a proteomics-based approach, we examined whether distinct varieties of cannabis plant contain relevant allergens of cannabis. Cannabis extracts were generated from high tetrahydrocannabinol variety (Mx), high cannabidiol variety (V1-19) and mixed profile variety (B5) using a Plant Total Protein Extraction Kit. Hemp extracts were generated using other standardized methods. Protein samples were subjected to nanoscale tandem mass spectrometry. Acquired peptides sequences were examined against the Cannabis sativa database to establish protein identity. Non-specific lipid transfer protein (Can s 3) level was measured using a recently developed ELISA 2.0 assay. Proteomic analysis identified 49 distinct potential allergens in protein extracts from all chemotypes. Most importantly, clinically relevant and validated allergens, such as profilin (Can s 2), Can s 3 and Bet v 1-domain-containing protein 10 (Can s 5), were identified in all chemotypes at label-free quantification (LFP) intensities > 106. However, the oxygen evolving enhancer protein 2 (Can s 4) was not detected in any of the protein samples. Similarly, Can s 2, Can s 3 and Can s 5 peptides were also detected in hemp protein extracts. The validation of these findings using the ELISA 2.0 assay indicated that hemp extract contains 30–37 ng of Can s 3 allergen per µg of total protein. Our proteomic studies indicate that relevant cannabis allergens are consistently expressed across distinct cannabis chemotypes. Further, hemp may serve as an ideal practical substitute for clinical testing, since it expresses most allergens relevant to cannabis sensitization, including the validated major allergen Can s 3. [ABSTRACT FROM AUTHOR]

The visual appearance of humans is derived significantly from our skin and hair color. While melanin from epidermal melanocytes protects our skin from the damaging effects of ultraviolet radiation, the biological value of pigmentation in the hair follicle, particularly on the scalp, is less clear. In this study, we explore the heterogeneity of pigment cells in the human scalp anagen hair follicle bulb, a site conventionally viewed to be focused solely on pigment production for transfer to the hair shaft. Using c-KIT/CD117 microbeads, we isolated bulbar c-KIT-positive and c-KIT-negative melanocytes. While both subpopulations expressed MITF, only the c-KIT-positive fraction expressed SOX10. We further localized bulbar melanocyte subpopulations (expressing c-KIT, SOX10, MITF, and DCT) that exhibited distinct/variable expression of downstream differentiation-associated melanosome markers (e.g., gp100 and Melan-A). The localization of a second 'immature' SOX10 negative melanocyte population, which was c-KIT/MITF double-positive, was identified outside of the melanogenic zone in the most peripheral/proximal matrix. This study describes an approach to purifying human scalp anagen hair bulb melanocytes, allowing us to identify unexpected levels of melanocyte heterogeneity. The function of the more immature melanocytes in this part of the hair follicle remains to be elucidated. Could they be in-transit migratory cells ultimately destined to synthesize melanin, or could they contribute to the hair follicle in non-melanogenic ways? [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a complex disorder characterized by vascular remodeling and a consequent increase in pulmonary vascular resistance. The histologic hallmarks of PAH include plexiform and neointimal lesions of the pulmonary arterioles, which are composed of dysregulated, apoptosis-resistant endothelial cells and myofibroblasts. Platelet-derived growth factor receptors (PDGFR) α and β, colony stimulating factor 1 receptor (CSF1R), and mast/stem cell growth factor receptor kit (c-KIT) are closely related kinases that have been implicated in PAH progression. In addition, emerging data indicate significant crosstalk between PDGF signaling and the bone morphogenetic protein receptor type 2 (BMPR2)/transforming growth factor β (TGFβ) receptor axis. This review will discuss the importance of the PDGFR-CSF1R-c-KIT signaling network in PAH pathogenesis, present evidence that the inhibition of all three nodes in this kinase network is a potential therapeutic approach for PAH, and highlight the therapeutic potential of seralutinib, currently in development for PAH, which targets these pathways. [ABSTRACT FROM AUTHOR]

The proto-oncogenic protein, c-KIT, plays a crucial role in regulating cellular transformation and differentiation processes, such as proliferation, survival, adhesion, and chemotaxis. The overexpression of, and mutations, in c-KIT can lead to its dysregulation and promote various human cancers, particularly gastrointestinal stromal tumors (GISTs); approximately 80–85% of cases are associated with oncogenic mutations in the KIT gene. Inhibition of c-KIT has emerged as a promising therapeutic target for GISTs. However, the currently approved drugs are associated with resistance and significant side effects, highlighting the urgent need to develop highly selective c-KIT inhibitors that are not affected by these mutations for GISTs. Herein, the recent research efforts in medicinal chemistry aimed at developing potent small-molecule c-KIT inhibitors with high kinase selectivity for GISTs are discussed from a structure–activity relationship perspective. Moreover, the synthetic pathways, pharmacokinetic properties, and binding patterns of the inhibitors are also discussed to facilitate future development of more potent and pharmacokinetically stable small-molecule c-KIT inhibitors. [ABSTRACT FROM AUTHOR]

Intimal sarcomas (IS) are rare malignant mesenchymal tumors arising in large blood vessels of the systemic and pulmonary circulation and also in the heart. They are morphologically similar to other spindle cell, poorly differentiated sarcomas. The prognosis is poor and depends mainly on surgical options. Three cases of IS were collected from two institutions. Clinical data were retrieved and histological study was performed. A wide immunohistochemical panel was analyzed. FISH of MDM2 gene was performed, and a molecular study with NGS was implemented in all cases. The mean age of our cases was 54 years. Histologically, the tumors presented a diffuse growth pattern with heterogeneous atypical epithelioid or spindle cells and extensive thrombosed areas. All cases presented intense immunoexpression for MDM2, CDK4, CD117, c-myc, PDGFRA, and p16. PDGFRA, HTERT, and pan-TRK gained expression, while p16 lost intensity, being weaker in both the local recurrences and xenografts. The three cases showed amplification of MDM2 by FISH. NGS analysis revealed amplifications in the CDK4, PDGFRA, and KIT genes, together with BRAF mutation and KRAS amplification. P16 was expressed in all cases, losing intensity in local recurrence and xenografts. Two new alterations, a BRAF mutation and a KRAS amplification, were detected by NGS in different tumors, opening up new therapeutic options for these patients. [ABSTRACT FROM AUTHOR]

Anaplastic thyroid cancer (ATC) is a rare and rapidly fatal human cancer. Its usual treatment includes the combination of surgery, external hyperfractionated radiation therapy, and chemotherapy. These treatments permit achieving about 6–10 months of median survival. For this reason, it is challenging to predict the ATC patient clinical therapy responsiveness. Pazopanib is a multitarget tyrosine kinase inhibitor of VEGF receptors, PDGF, and c-Kit. Until now, the effect of pazopanib in primary human ATC cells (pATC) has not been reported in the literature. The aim of our study was to evaluate in vitro the antineoplastic effect of pazopanib in pATC. Surgical thyroidal tissues were collected from five patients with ATC, from thyroid biopsy at the moment of first surgical operation. An inhibition of proliferation, migration, and invasion, and an increase in apoptosis were demonstrated upon treating pATC cells with pazopanib (p < 0.05). Moreover, pazopanib was able to significantly decrease the VEGF expression in pATC cells (p < 0.05). To conclude, in this study, we demonstrate the antineoplastic activity of the antiangiogenic inhibitor, pazopanib, in human pATC in vitro. [ABSTRACT FROM AUTHOR]

Hyperpigmentation is a skin condition where patches of skin become darker in color due to excess melanin production upon UV exposure leading to melasma, which are lentigines or post inflammatory hyperpigmentation that psychologically affecting a great number of people. The present study investigates the anti-melanogenic effect of Butyroside D and the underling mechanism. After the confirmation of the non-cytotoxic effect of Butyroside D on B16F10 cells, we proceeded with analyzing the impact of the treatment at low and high concentration (i.e., 0.2 μM and 2 μM) using gene profiling analysis and examined the differentiation in gene expression. Our results identify cyclic adenosine monophosphate (cAMP), Wnt/β-catenin and Mitogen-Activated Protein Kinase (MAPK) signaling pathways to be downregulated upon treatment with Butyroside D. These pathways were targeted to further validate the effect of Butyroside D on membrane receptors melanocortin 1 receptor (MC1R) and receptor tyrosine kinase (c-Kit), related microphthalmia-associated transcription factor (MITF) and consequently tyrosinase (TYR), and tyrosine-related protein-1 (TYRP-1) that were all shown to be downregulated and, therefore, leading to the repression of melanin biosynthesis. Finally, the anti-melanogenic effect of Butyroside D was confirmed on human epidermal melanocytes (HEM) cells by inhibiting the activation of cAMP pathway generally mediated through α-melanocyte-stimulating hormone (α-MSH) and MC1R. Overall, this study suggests the potential applicability of this purified compound for the prevention of hyperpigmentation conditions. [ABSTRACT FROM AUTHOR]

G-quadruplexes are nucleotide sequences present in the promoter region of numerous oncogenes, having a key role in the suppression of gene transcription. Recently, the binding of anthraquinones from Aloe vera to G-quadruplex structures has been studied through various physico-chemical techniques. Intrigued by the reported results, we investigated the affinity of aloe emodin, aloe emodin-8-glucoside, and aloin to selected G-quadruplex nucleotide sequences by NMR spectroscopy. The structural determinants for the formation of the ligand/nucleotide complexes were elucidated and a model of the interactions between the tested compounds and C-Kit and c-Myc G-quadruplex DNA structures was built by integrated NMR and molecular modeling studies. Overall, the obtained results confirmed and implemented the previously reported findings, pointing out the complementarity of the different approaches and their contribution to a more detailed overview of the ligand/nucleotide complex formation. Furthermore, the proposed models of interaction could pave the way to the design of new nature-derived compounds endowed with increased G-quadruplex stabilizing activity. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a major source of morbidity and mortality, characterized by intratumoral heterogeneity and the presence of cancer stem cells (CSCs). Bufalin has potent activity against many tumors, but studies of its effect on CRC stemness are limited. We explored bufalin's function and mechanism using CRC patient-derived organoids (PDOs) and cell lines. In CRC cells, bufalin prevented nuclear translocation of β-catenin and down-regulated CSC markers (CD44, CD133, LGR5), pluripotency factors, and epithelial–mesenchymal transition (EMT) markers (N-Cadherin, Slug, ZEB1). Functionally, bufalin inhibited CRC spheroid formation, aldehyde dehydrogenase activity, migration, and invasion. Network analysis identified a C-Kit/Slug signaling axis accounting for bufalin's anti-stemness activity. Bufalin treatment significantly downregulated C-Kit, as predicted. Furthermore, overexpression of C-Kit induced Slug expression, spheroid formation, and bufalin resistance. Similarly, overexpression of Slug resulted in increased expression of C-Kit and identical functional effects, demonstrating a pro-stemness feedback loop. For further study, we established PDOs from diagnostic colonoscopy. Bufalin differentially inhibited PDO growth and proliferation, induced apoptosis, restored E-cadherin, and downregulated CSC markers CD133 and C-Myc, dependent on C-Kit/Slug. These findings suggest that the C-Kit/Slug axis plays a pivotal role in regulating CRC stemness, and reveal that targeting this axis can inhibit CRC growth and progression. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is one of the most common hematological malignancies with high heterogeneity, characterized by a differentiating block at the early progenitor stage. The selective BCL-2 inhibitor, Venetoclax (Ven), has shown exciting clinical results in a certain group of AML patients. However, Ven alone is insufficient to reach an enduringly complete response, which leads to the concern of Ven resistance. Alternative combined therapies with Ven are demanded in AML. Here, we reported the synergistic effect and molecular mechanism of the enhancer of zeste homolog 2 (EZH2) inhibitor DZNeP with Ven in AML cells. Results showed that the combination of DZNeP with Ven significantly induces cell proliferation arrest compared to single-drug control in AML cells and primary samples, and CalcuSyn analysis showed their significant synergy. The combination also significantly promotes apoptosis and increases the expression of pro-apoptotic proteins. The whole transcriptome analysis showed that phosphoinositide-3-kinase-interacting protein1 (PIK3IP1), the PI3K/AKT/mTOR signaling suppressor, is upregulated upon DZNeP treatment. Moreover, EZH2 is upregulated but PIK3IP1 is downregulated in 88 newly diagnosed AML cohorts compared to 70 healthy controls, and a higher expression of EZH2 is associated with poor outcomes in AML patients. Particularly, the combination of DZNeP with Ven dramatically eliminated CD117 (c-KIT) (+) AML blasts, suggesting the effect of the combination on tumor stem cells. In summary, our data indicated that DZNeP increases the sensitivity of Ven in AML by affecting PI3K and c-KIT signaling in AML. Our results also suggested that the therapeutic targeting of both EZH2 and BCL-2 provides a novel potential combined strategy against AML. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is associated with a poor prognosis and the absence of targeted therapy. c-Kit, a receptor tyrosine kinase (RTK), is considered a molecular target for anticancer drugs. Tyrosine kinase inhibitors (TKIs) recognizing c-Kit are used for the treatment of c-Kit-expressing tumors. However, the expression, function, and therapeutic potential of c-Kit have been little explored in TNBC. Here, we studied the expression and effects of c-Kit in TNBC through in vitro and in silico analysis, and evaluated the response to TKIs targeting c-Kit. Analysis of TNBC cells showed the expression of functional c-Kit at the cell membrane. The stimulation of c-Kit with its ligand induced the activation of STAT3, Akt, and ERK1/2, increasing cell migration, but had no effect on cell proliferation or response to Doxorubicin. Analysis of public datasets showed that the expression of c-Kit in tumors was not associated with patient survival. Finally, TNBC cells were susceptible to TKIs, in particular the effect of Nilotinib was stronger than Doxorubicin in all cell lines. In conclusion, TNBC cells express functional c-Kit, which is a targetable molecule, and show a strong response to Nilotinib that may be considered a candidate drug for the treatment of TNBC. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play an important role in gene regulation by degradation or translational inhibition of the targeted mRNAs. It has been experimentally shown that the way miRNAs interact with their targets can be used to explain the indirect interactions among their targets, i.e., competing endogenous RNA (ceRNA). However, whether the protein translated from the targeted mRNAs can play any role in this ceRNA network has not been explored. Here we propose a deterministic model to demonstrate that in a network of one miRNA interacting with multiple-targeted mRNAs, the competition between miRNA-targeted mRNAs is not sufficient for the significant change of those targeted mRNA levels, while dramatic changes of these miRNA-targeted mRNAs require transcriptional inhibition of miRNA by its target proteins. When applied to estrogen receptor signaling pathways, the miR-193a targets E2F6 (a target of estrogen receptor), c-KIT (a marker for cancer stemness), and PBX1 (a transcriptional activator for immunosuppressive cytokine, IL-10) in ovarian cancer, such that epigenetic silencing of miR-193a by E2F6 protein is required for the significant change of c-KIT and PBX1 mRNA level for cancer stemness and immunoevasion, respectively, in ovarian cancer carcinogenesis [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15–25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study, we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit and specifically binds to SCLC cells expressing c-Kit with a binding affinity of KD = 5.5 × 10−9 M. Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently induced apoptosis in SCLC with an IC50 ranging from 158 pM to 4 nM. An in vivo assay using a xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59% (5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment. [ABSTRACT FROM AUTHOR]

RTK KIT regulates a variety of crucial cellular processes via its cytoplasmic domain (CD), which is composed of the tyrosine kinase domain, crowned by the highly flexible domains—the juxtamembrane region, kinase insertion domain, and C-tail, which are key recruitment regions for downstream signalling proteins. To prepare a structural basis for the characterization of the interactions of KIT with its signalling proteins (KIT INTERACTOME), we generated the 3D model of the full-length CD attached to the transmembrane helix. This generic model of KIT in inactive state was studied by molecular dynamics simulation under conditions mimicking the natural environment of KIT. With the accurate atomistic description of the multidomain KIT dynamics, we explained its intrinsic (intra-domain) and extrinsic (inter-domain) disorder and represented the conformational assemble of KIT through free energy landscapes. Strongly coupled movements within each domain and between distant domains of KIT prove the functional interdependence of these regions, described as allosteric regulation, a phenomenon widely observed in many proteins. We suggested that KIT, in its inactive state, encodes all properties of the active protein and its post-transduction events. [ABSTRACT FROM AUTHOR]

Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity. [ABSTRACT FROM AUTHOR]