Search Results (83)

Background and Objectives: Neuronal nitric oxide synthase (nNOS) overexpressed in melanoma plays a critical role in disease progression. Our previous studies demonstrated that nNOS inhibitors exhibited potent anti-melanoma activity and regulated PD-L1 expressions in the presence of interferon-gamma (IFN-γ). However, the role of nNOS in the melanoma immune response has not been well defined. Methods: Changes in gene expression profiles after nNOS inhibitor treatment were determined by transcriptomic analysis. A melanoma mouse model was used to determine the effects of nNOS inhibition on peripheral T cells and the in vivo anti-tumor activity of combining nNOS inhibitors with immune checkpoint blockade. Changes in human T cell activation through interleukin-2 (IL-2) production were investigated using an ex vivo co-culture system with human melanoma cells. Results: Cellular RNA analysis revealed significant changes in the genes involved in key signaling pathways after nNOS inhibitor HH044 treatment. Immunophenotyping of mouse peripheral blood mononuclear cells (PBMCs) after prolonged HH044 treatment showed marked increases in CD4⁺ and CD8⁺PD-1⁺ T cells. Ex vivo studies demonstrated that co-culturing human PBMCs with melanoma cells inhibited T cell activation, decreasing IL-2-secreting T cells both in the presence and absence of IFN-γ. PBMCs from a significant portion of donors (7/11, 64%), however, were reactivated by nNOS inhibitor pretreatment, displaying a significant increase in IL-2⁺ T cells. Distinctive T cell characteristics were noted at baseline among the responders with increased CD4⁺RORγt⁺ and reduced CD4 naïve T cells. In vivo mouse studies demonstrated that nNOS inhibitors, when combined with PD-1 blockade, significantly reduced tumor growth more effectively than monotherapy. Additionally, the median survival was extended from 43 days in the control mice to 176.5 days in mice co-treated with HH044 and anti-PD-1. Conclusions: Targeting nNOS is a promising approach to enhancing the anti-melanoma activity of immune checkpoint inhibitors, not only interfering with melanoma biological activities but also regulating the tumor microenvironment, which subsequently affects T cell activation and tumor immune response. Full article

Background/Objectives: Triple-negative breast cancer (TNBC) is a major cause of cancer-related deaths among women. The Hedgehog (Hh) signaling pathway plays a critical role in tumor development, and targeting this pathway may provide new therapeutic opportunities for TNBC. TPB15 is a novel smoothened inhibitor of the Hh pathway, showing promising tumor reduction and low-toxicity properties in vivo/vitro. This study aims to evaluate TPB15’s protein binding rates, metabolic stability, and metabolism across different species, including mice, rats, dogs, monkeys, and humans. Methods: TPB15 was synthesized, and its pharmacokinetic profile was assessed. Plasma protein binding was determined using ultrafiltration across multiple species. Stability studies were conducted in plasma and liver microsomes from each species. Additionally, metabolic enzymes in human liver microsomes were characterized with selective CYP450 inhibitors, and high-resolution mass spectrometry was employed to identify metabolites. Results: Plasma protein binding of TPB15 was consistent across species, ranging from 81.5% to 82.4% in humans and rats. After 120 min, TPB15 remained stable in plasma, with retention rates of 97.2–98.3%. The elimination half-life (t_1/2) varied from 88 min in monkeys to 630 min in dogs. In human liver microsomes, metabolism was significantly inhibited by sulfaphenazole and ketoconazole, indicating the involvement of CYP3A4 and CYP2C9 enzymes. TPB15 underwent phase I metabolism, producing a major metabolite with a molecular weight of 468.9. Conclusions: TPB15 demonstrates stable pharmacokinetic properties across species, with consistent protein binding and significant variability in half-life. The observed differences in metabolism are primarily attributed to CYP2C9 and CYP3A4, offering valuable insights into its drug development potential. Full article

The Hedgehog (Hh) signaling pathway plays a crucial role in the initiation and progression of tumors, and Hh inhibitors have been used as potential chemotherapeutic agents for the treatment of basal cell carcinomas (BCCs). Vitamin D₃ (VD₃) and its derivatives have been identified as potent Hh inhibitors. However, the selectivity of VD₃ derivatives to vitamin D receptor (VDR) and the Hh signaling pathway still needs optimization. In this study, a series of aromatic A-ring mimics VD₃ analogues that contain a C-23 oxygen atom or incorporate C-25 hydroxyl on side chains were designed and synthesized. These compounds were tested in various cell lines for anti-Hh activity, with analogues 3j and 4i identified as potent inhibitors. Mechanism studies showed their anti-Hh effects are mainly due to targeting Smoothened (Smo) without binding to the cyclopamine site. Structure-activity relationship (SAR) studies revealed that VD₃-based inhibitors enhance anti-Hh activity by adding a hydroxyl group at C25 while reducing VDR activity by incorporating an oxygen atom into the side chain. Full article

The Hedgehog (Hh) and PI3K/Akt/mTOR signaling pathways play a pivotal role in driving the initiation and progression of various cancers, including hematologic malignancies such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL). These pathways are often dysregulated in leukemia cells, leading to increased cell growth, survival, and drug resistance while also impairing mechanisms of cell death. In leukemia, the Hh pathway can be abnormally activated by genetic mutations. Additionally, the PI3K/Akt/mTOR pathway is frequently overactive due to genetic changes. A key aspect of these pathways is their interaction: activation of the PI3K/Akt pathway can trigger a non-canonical activation of the Hh pathway, which further promotes leukemia cell growth and survival. Targeted inhibitors of these pathways, such as Gli inhibitors and PI3K/mTOR inhibitors, have shown promise in preclinical and clinical studies. Full article

Background/Objectives: Interferon gamma (IFN-γ) in the melanoma tumor microenvironment plays opposing roles, orchestrating both pro-tumorigenic activity and anticancer immune responses. Our previous studies demonstrated the role of neuronal nitric oxide synthase (nNOS) in IFN-γ-stimulated melanoma progression. However, the underlying mechanism has not been well defined. This study determined whether the nNOS/NO and COX-2/PGE₂ signaling pathways crosstalk and augment the pro-tumorigenic effects of IFN-γ in melanoma. Methods: Bioinformatic analysis of patient and cellular proteomic data was conducted to identify proteins of interest associated with IFN-γ treatment in melanoma. Changes in protein expression were determined using various analytical techniques including western blot, flow cytometry, and confocal microscopy. The levels of PGE₂ and nitric oxide (NO) were analyzed by HPLC chromatography and flow cytometry. In vivo antitumor efficacy was determined utilizing a human melanoma xenograft mouse model. Results: Our omics analyses revealed that the induction of COX-2 was significantly predictive of IFN-γ treatment in melanoma cells. In the presence of IFN-γ, PGE₂ further enhanced PD-L1 expression and amplified the induction of nNOS, which increased intracellular NO levels. Cotreatment with celecoxib effectively diminished these changes induced by PGE₂. In addition, nNOS blockade using a selective small molecule inhibitor (HH044), efficiently inhibited IFN-γ-induced PGE₂ and COX-2 expression levels in melanoma cells. STAT3 inhibitor napabucasin also inhibited COX-2 expression both in the presence and absence of IFN-γ. Furthermore, celecoxib was shown to enhance HH044 cytotoxicity in vitro and effectively inhibit human melanoma tumor growth in vivo. HH044 treatment also significantly reduced tumor PGE₂ levels in vivo. Conclusions: Our study demonstrates the positive feedback loop linking nNOS-mediated NO signaling to the COX-2/PGE₂ signaling axis in melanoma, which further potentiates the pro-tumorigenic activity of IFN-γ. Full article

Hedgehog (Hh) signaling is a well-established developmental pathway; it is crucial for early embryogenesis, cell differentiation, and damage-driven regeneration. It is being increasingly recognized that dysregulated Hh signaling is also involved in fibrotic diseases, which are characterized by excessive extracellular matrix deposition that compromises tissue architecture and function. As in-depth insights into the mechanisms of Hh signaling are obtained, its complex involvement in fibrosis is gradually being illuminated. Notably, some Hh-targeted inhibitors are currently under exploration in preclinical and clinical trials as a means to prevent fibrosis progression. In this review, we provide a concise overview of the biological mechanisms involved in Hh signaling. We summarize the latest advances in our understanding of the roles of Hh signaling in fibrogenesis across the liver, kidneys, airways, and lungs, as well as other tissues and organs, with an emphasis on both the shared features and, more critically, the distinct functional variations observed across these tissues and organs. We thus highlight the context dependence of Hh signaling, as well as discuss the current status and the challenges of Hh-targeted therapies for fibrosis. Full article

HER2 overexpression occurs in 20–30% of breast cancers and is associated with poor prognosis. Trastuzumab is a standard treatment for HER2-positive breast cancer; however, resistance develops in approximately 50% of patients within a year. The Hedgehog (Hh) signalling pathway, known for its role in maintaining stemness in various cancers, may contribute to trastuzumab resistance in HER2-positive breast cancer. This study aimed to investigate the role of Hedgehog signalling in maintaining stemness and contributing to trastuzumab resistance in HER2-positive breast cancer cell lines. Trastuzumab-resistant HER2-positive breast cancer cell lines, SKBR3 and HCC1954, were developed through continuous trastuzumab exposure. Cells were treated with GANT61 (Hh inhibitor, IC₅₀:10 µM) or SAG21K (Hh activator, IC₅₀:100 nM) for 24 h to evaluate the Hedgehog signalling response. Stemness marker expression (Nanog, Sox2, Bmi1, Oct4) was measured using qRT-PCR. The combination index (CI) of GANT61 with trastuzumab was calculated using CompuSyn software (version 1.0) to identify synergistic doses (CI < 1). The synergistic concentrations’ impact on stemness markers was assessed. Data were analysed using two-way ANOVA and Tukey’s post hoc test (p < 0.05). Trastuzumab-resistant cells exhibited increased Hedgehog signalling activity. Treatment with GANT61 significantly downregulated stemness marker expression, while SAG21K treatment led to their upregulation in both SKBR3-R and HCC1954-R cells. The combination of GANT61 and trastuzumab demonstrated a synergistic effect, markedly reducing the expression of stemness markers. These findings indicate that Hedgehog signalling plays a pivotal role in maintaining stemness in trastuzumab-resistant cells, and that the inhibition of this pathway may prevent tumour progression. Hedgehog signalling is crucial in regulating stemness in trastuzumab-resistant HER2-positive breast cancer. Targeting this pathway could overcome resistance and enhance trastuzumab efficacy. Further studies should explore the clinical potential of Hedgehog inhibitors in combination therapies. Full article

Activation of the Hedgehog (Hh) signaling pathway is often associated with the progression of various types of cancer. The purpose of study was to search for inhibitors of the Hh signaling pathway among eight compounds belonging to the group of isoxazolyl steroids. The evaluation of the effectiveness of the compounds was based on the analysis of their cytotoxicity, effect on the cell cycle, on the expression of key Hh-signaling-pathway genes (Ptch1, Smo, and Gli1) and putative target genes MMP-2 and MMP-9. Four compounds with the most pronounced cytotoxic effect were identified: compounds 1, 2 (HeLa cells) and 3, 4 (A549 cells). Compounds 1 and 2 significantly reduced the expression of the Ptch1, Smo, Gli1 genes, but had the opposite effect on MMP-2 gene expression: Compound 1 increased it, and compound 2 decreased it. Compounds 3 and 4 did not have a noticeable inhibitory effect on the expression of the Shh pathway receptors, but significantly inhibited MMP-2 and MMP-9 expression. Thus, it was shown that inhibition of the Shh signaling pathway by isoxazolyl steroids can have the opposite effect on MMPs gene expression, which is what should be taken into account in further studies of these compounds as therapeutic agents. Full article

Histiocytic sarcoma (HS) is a rare and highly aggressive cancer in humans and dogs. In dogs, it has a high prevalence in certain breeds, such as Bernese mountain dogs (BMDs) and flat-coated retrievers. Hemophagocytic histiocytic sarcoma (HHS) is a unique form of HS that presents with erythrophagocytosis. Due to its rareness, the study of HHS is very limited, and mutations in canine HHS patients have not been studied to date. In previous work, our research group identified two major PTPN11/SHP2 driver mutations, E76K and G503V, in HS in dogs. Here, we report additional mutations located in exon 3 of PTPN11/SHP2 in both HS and HHS cases, further supporting that this area is a mutational hotspot in dogs and that mutations in tumors and liquid biopsies should be evaluated utilizing comprehensive methods such as Sanger and NextGen sequencing. The overall prevalence of PTPN11/SHP2 mutations was 55.8% in HS and 46.2% in HHS. In addition, we identified mutations in KRAS, in about 3% of HS and 4% of HHS cases. These findings point to the shared molecular pathology of activation of the MAPK pathway in HS and HHS cases. We evaluated the efficacy of the highly specific MEK inhibitor, cobimetinib, in canine HS and HHS cell lines. We found that the IC₅₀ values ranged from 74 to 372 nM, which are within the achievable and tolerable ranges for cobimetinib. This finding positions cobimetinib as a promising potential candidate for future canine clinical trials and enhances our understanding of the molecular defects in these challenging cancers. Full article

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors. Full article

Melanoma is the principal cause of death in skin cancer due to its ability to invade and cause metastasis. Hypoxia, which characterises the tumour microenvironment (TME), plays an important role in melanoma development, as cancer cells can adapt and acquire a more aggressive phenotype. Carbonic anhydrases (CA) activity, involved in pH regulation, is related to melanoma cell migration and invasion. Furthermore, the Hedgehog (Hh) pathway, already known for its role in physiological processes, is a pivotal character in cancer cell growth and can represent a promising pharmacological target. In this study, we targeted Hh pathway components with cyclopamine, glabrescione B and C22 in order to observe their effect on carbonic anhydrase XII (CAXII) expression especially under hypoxia. We then performed a migration and invasion assay on two melanoma cell lines (SK-MEL-28 and A375) where Smoothened, the upstream protein involved in Hh regulation, and GLI1, the main transcription factor that determines Hh pathway activation, were chemically inhibited. Data suggest the existence of a relationship between CAXII, hypoxia and the Hedgehog pathway demonstrating that the chemical inhibition of the Hh pathway and CAXII reduction resulted in melanoma migration and invasion impairment especially under hypoxia. As in recent years drug resistance to small molecules has arisen, the development of new chemical compounds is crucial. The multitarget Hh inhibitor C22 proved to be effective without signs of cytotoxicity and, for this reason, it can represent a promising compound for future studies, with the aim to reach a better melanoma disease management. Full article

Veratrum californicum contains steroidal alkaloids that function as inhibitors of hedgehog (Hh) signaling, a pathway involved in the growth and differentiation of cells and normal tissue development. This same Hh pathway is abnormally active for cell proliferation in more than 20 types of cancer. In this current study, alkaloids have been extracted from the root and rhizome of V. californicum, followed by their separation into five fractions using high performance liquid chromatography. Mass spectrometry was used to identify the presence of twenty-five alkaloids, nine more than are commonly cited in literature reports, and the Bruker Compass Data Analysis software was used to predict the molecular formula for every detected alkaloid. The Gli activity of the raw extract and each fraction were compared to 0.1 µM cyclopamine, and fractions 1, 2, and 4 showed increased bioactivity through suppression of the Hh signaling pathway. Fractions 2 and 4 had enhanced bioactivity, but fraction 1 was most effective in inhibiting Hh signaling. The composition of fraction 1 consisted of veratrosine, cycloposine, and potential isomers of each. Full article

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading malignancy worldwide, with a poor prognosis and limited treatment options. Molecularly targeted therapies for HNSCC are still lacking. However, recent reports provide novel insights about many molecular alterations in HNSCC that may be useful in future therapies. Therefore, it is necessary to identify new biomarkers that may provide a better prediction of the disease and promising targets for personalized therapy. The poor response of HNSCC to therapy is attributed to a small population of tumor cells called cancer stem cells (CSCs). Growing evidence indicates that the Hedgehog (HH) signaling pathway plays a crucial role in the development and maintenance of head and neck tissues. The HH pathway is normally involved in embryogenesis, stem cell renewal, and tissue regeneration. However, abnormal activation of the HH pathway is also associated with carcinogenesis and CSC regulation. Overactivation of the HH pathway was observed in several tumors, including basal cell carcinoma, that are successfully treated with HH inhibitors. However, clinical studies about HH pathways in HNSCC are still rare. In this review, we summarize the current knowledge and recent advances regarding the HH pathway in HNSCC and discuss its possible implications for prognosis and future therapy. Full article

The Wnt/β-catenin, EGFR, and PI3K pathways frequently undergo upregulation in head and neck squamous carcinoma (HNSCC) cells. Moreover, the Wnt/β-catenin pathway together with Hedgehog (Hh) signaling regulate the activity of cancer stem cells (CSCs). The aim of this study was to investigate the effects of the combinatorial use of the Wnt/β-catenin and Hh pathway inhibitors on viability, cell cycle progression, apoptosis induction, cell migration, and expression of CSC markers in tongue (CAL 27) and hypopharynx (FaDu) cancer cells. Co-inhibition of Wnt signaling with EGFR or PI3K pathways was additionally tested. The cells were treated with selective inhibitors of signaling pathways: Wnt/β-catenin (PRI-724), Hh (vismodegib), EGFR (erlotinib), and PI3K (HS-173). Cell viability was evaluated by the resazurin assay. Cell cycle progression and apoptosis induction were tested by flow cytometric analysis after staining with propidium iodide and Annexin V, respectively. Cell migration was detected by the scratch assay and CSC marker expression by the R-T PCR method. Mixtures of PRI-724 and vismodegib affected cell cycle distribution, greatly reduced cell migration, and downregulated the transcript level of CSC markers, especially POU5F1 encoding OCT4. Combinations of PRI-724 with erlotinib or HS-173 were more potent in inducing apoptosis. Full article

A crucial regulator in melanoma progression and treatment resistance is tumor microenvironments, and Hedgehog (Hh) signals activated in a tumor bone microenvironment are a potential new therapeutic target. The mechanism of bone destruction by melanomas involving Hh/Gli signaling in such a tumor microenvironment is unknown. Here, we analyzed surgically resected oral malignant melanoma specimens and observed that Sonic Hedgehog, Gli1, and Gli2 were highly expressed in tumor cells, vasculatures, and osteoclasts. We established a tumor bone destruction mouse model by inoculating B16 cells into the bone marrow space of the right tibial metaphysis of 5-week-old female C57BL mice. An intraperitoneal administration of GANT61 (40 mg/kg), a small-molecule inhibitor of Gli1 and Gli2, resulted in significant inhibition of cortical bone destruction, TRAP-positive osteoclasts within the cortical bone, and endomucin-positive tumor vessels. The gene set enrichment analysis suggested that genes involved in apoptosis, angiogenesis, and the PD-L1 expression pathway in cancer were significantly altered by the GANT61 treatment. A flow cytometry analysis revealed that PD-L1 expression was significantly decreased in cells in which late apoptosis was induced by the GANT61 treatment. These results suggest that molecular targeting of Gli1 and Gli2 may release immunosuppression of the tumor bone microenvironment through normalization of abnormal angiogenesis and bone remodeling in advanced melanoma with jaw bone invasion. Full article