Search Results (69)

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a high rate of recurrence and a dismal prognosis. Studies have shown that pancreatic cancer stem cells (PCSCs) are a subpopulation that contributes to tumor progression, resistance to therapeutics, and metastasis, making them a key subpopulation to target for treatment. Gedunin (GD), a natural compound derived from Azadirachta indica (neem), has shown anticancer properties in pancreatic cancer cells, but its effects on PCSCs remains unclear. This study evaluated the effects of GD in pancreatic cancer stem cells, highlighting its impacts on tumor growth and progression and focusing on its impact on the sonic hedgehog (Shh) signaling pathway. Methods: Functional assays were performed to assess the effect of GD on the sphere-forming ability, colony formation, and self-renewal of PCSCs. Athymic mice xenograft models were utilized to evaluate the tumor suppression effect of GD in vivo. Furthermore, the anticancer effect of GD on PCSCs was assessed using both in vitro and in vivo limiting dilution assay. GD-induced changes in Shh signaling and key stem cell marker expressions in PCSCs were evaluated. Results: GD effectively inhibited tumor growth in xenograft models and reduced the percentage of PCSCs. GD was effective in decreasing PCSCs’ proliferative, self-renewal, and colony-forming capacity. GD decreased the protein expression levels of key Shh signaling markers Gli1 and Shh, stem cell markers SOX2, Nanog, and Oct4, metastasis-related proteins MMP-2, MMP-3, and MMP-9, and EMT markers Tgf1, Slug, Snail, and Twist in both PDAC cells and PCSCs. We demonstrated a significant decrease in the spheroid formation and self-renewal capacity of the (ALDH+) PCSC population following GD treatment in HPAC cells, indicating its potential antagonistic effects on PCSCs. GD was highly effective in reducing tumor volume, stemness, and metastasis in both early and late chemotherapy. In vivo limiting dilution assay using CD133+/LGR5+ PCSC xenografts demonstrated that GD reduces tumor growth, metastasis, and stemness associated with PCSCs by downregulating the expression of Shh and Gli1. GD treatment also reduced micrometastatic lesions in the lung, liver, and brain, as identified using H&E staining. Conclusions: The findings highlight GD’s potential as a promising therapeutic candidate for PDAC, with the ability to target both bulk tumor cells and PCSCs. By simultaneously suppressing tumor growth, stemness, and metastatic spread, GD may contribute to more effective treatment strategies and improved patient outcomes. Full article

Purpose: This study aims to investigate whether two stress keratins, KRT6A or KRT17, label self-renewing stem cells (SCs) in adult mouse Meibomian gland (MG), the palpebral conjunctiva (PC) homeostasis, and to explore the mechanisms regulating their expression. Methods: KRT6A and KRT17 expression in adult mouse MG and PC were examined by single-nucleus RNA sequencing and immunofluorescence (IF). Lineage-tracing experiments were performed using Krt6a-Cre^ERT2 and Krt17-Cre^ERT2 mice carrying the Rosa26R^nTnG or Rosa26R^mTmG reporter. As Hedgehog (Hh) signaling, the histone deacetylase HDAC3, and the transcription factor KLF4 regulate KRT6A and KRT17 in other contexts, IF was conducted to assess the in vivo effects of overexpression of the Hh pathway activator GLI2ΔN, and inducible epithelial deletion of Hdac3 or Klf4 on KRT6A and KRT17 expression in the MG and PC. Results: KRT6A and KRT17 are primarily expressed in the MG central duct and ductules. KRT6A also shows robust expression in PC. Lineage tracing indicated that Krt17 labels self-renewing SCs in the MG, whereas Krt6a labels SCs in the PC. GLI2ΔN overexpression induced ectopic KRT17 expression in MG acini and PC but did not affect KRT6A expression in either MG or PC. Hdac3 deficiency caused expanded expression of KRT6A and KRT17 in MG acini, ectopic KRT17 expression in PC, and increased KRT6A expression in PC basal layer. Klf4 deletion resulted in ectopic KRT17 expression in PC but did not influence KRT6A expression in MG or PC. Conclusions: Krt6a- and Krt17-expressing cells contribute to adult PC and MG homeostasis, respectively. KRT17 expression is enhanced by GLI2ΔN, and suppressed by HDAC3 and KLF4, whereas KRT6A expression is controlled only by HDAC3. These findings provide important biological insight into tissue-specific maintenance mechanisms and may inform future therapeutic strategies for regenerating MG and PC tissues affected by SC exhaustion or dysregulation. Full article

The Hedgehog (HH) signaling pathway is an evolutionarily conserved, multi-component signaling pathway. Its activation is initiated by the Hh protein, which signals upstream regulators PATCH and SMO to activate the transcription factor GLI. Upon activation, GLI translocates to the nucleus to induce the transcription of Hh/GLI target genes. Under normal conditions, the HH pathway plays a crucial role in embryogenesis, development, tissue patterning, and stem cell maintenance. Deregulation of the HH signaling pathway leads to various diseases, including cancer. However, in many human cancers, GLI1 is upregulated through a non-canonical pathway (independent of the HH pathway). This aberrant regulation of GLI1 via a non-canonical pathway is linked to the increased expression of various oncogenes. Aberrant expression of GLI not only affects the genes of several DNA repair pathways but also cancer stem cell pathways, which can contribute to genome instability and ultimately lead to cancer. The ineffectiveness of current HH pathway inhibitors in clinical trials necessitates the discovery of new HH pathway inhibitors. In this review, we will discuss our current understanding of the aberrant signaling of the HH-GLI pathway and focus on GLI1-mediated HH signaling in cancers, cancer stem cells, and carcinogenesis. We will also discuss the effectiveness of current HH inhibitors/drugs and combination therapies based on recent advances in this field. Furthermore, we will also review the role of HH-GLI in cancer stem cell markers, DNA damage response, gene regulation, tumor initiation, metastasis, cancer pathogenesis, and the role of drugs/inhibitors on this pathway. Full article

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. In particular, patients with chronic myeloid leukemia (CML) often require lifelong therapy with ABL1 tyrosine kinase inhibitors (TKIs), partly due to a persistent population of TKI-resistant leukemic stem cells (LSCs). Therefore, identifying specific pathways crucial for LSC maintenance is necessary. The Hedgehog (HH) pathway, especially the protein Smoothened (SMO), has been found to be essential for CML LSCs, but its role in TKI resistance is still largely unknown. We have now demonstrated that the expression of HH pathway genes SMO and GLI2 is increased in CD34⁺ CML stem/progenitor cells compared to healthy counterparts, and is higher in TKI-nonresponders than in responders by transcriptome profiling and qRT-PCR analysis. Interestingly, they are most highly expressed in LSCs compared to progenitors and mature cells in TKI-nonresponders. Inhibition of SMO through genetic knockdown or with a potent, selective SMO inhibitor, Glasdegib, reduces the survival of cells from nonresponder patients. Notably, SMO inhibition also sensitizes TKI-nonresponder stem/progenitor cells to Bostutinib, a second-generation TKI, both in vitro and in a patient-derived xenotransplantation (PDX) model. These findings present a promising therapeutic target and a model for curative combination therapies in stem-cell-driven cancers. Full article

This study investigated the potential of Thymus serpyllum L. and Mentha × piperita L. essential oils (EOs), known for their bioactive properties, as adjunctive treatments targeting Basal cell carcinoma cancer stem cells (BCC CSCs). Primary cultures were established from ten BCC tumor samples and their distant resection margins as controls. The chemical composition of the EOs was analyzed by gas chromatography–mass spectroscopy (GC-MS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The biological effects were evaluated via colony and spheroid formation, scratch assays, MTT and neutral red cytotoxicity assays, and qRT-PCR for Hh (SHH, PTCH1, SMO, and GLI1) and Notch (Notch1 and JAG1) gene expression. GC analysis identified thymol, p-cymene, and linalool as the main components of the EO of T. serpyllum L., and menthone and menthol in the EO of M. × piperita L. IC50 values were 262 µg/mL for T. serpyllum L. and 556 µg/mL for M. × piperita L. and were applied in all experiments. Both EOs significantly reduced CSC clonogenicity and migration (p < 0.05). The EO of T. serpyllum L. downregulated SMO and GLI1, while the EO of M. × piperita L. upregulated PTCH1, Notch1, and JAG1 (p < 0.05). These findings suggest that both EOs exhibit anticancer effects in BCC CSCs by modulating key oncogenic pathways, supporting their potential in BCC therapy. Full article

Glioblastoma (GBM) is the most aggressive and challenging brain cancer, in terms of diagnosis and therapy. The highly infiltrative glioblastoma stem cells (GSCs) are difficult to visualize and surgically remove with the current diagnostic tools, which often lead to misdiagnosis and false-positive results. In this study, we focused on a groundbreaking tool for specifically visualizing and removing GSCs. We exploited the specific binding of A40s aptamer to EphA2 for the selective delivery of Near-Infrared Dyes (NIR-Dyes), like IR700DX and ICG, both in vitro and in vivo. The A40s aptamer, engineered through the NIR-Dye conjugation, did not affect aptamer binding ability; indeed, A40s-NIR-Dye conjugates bound GLI261 stem-like cells and patient-derived GSCs in vitro; moreover, they induced cell death upon photodynamic therapy treatment (PDT). Additionally, when systemically administrated, the A40s-NIR-Dye conjugates allowed GSC visualization and accumulated in tumor mass. This allows GSCs detection and treatment. Our findings demonstrate the potential use of A40s aptamer as a targeted therapeutic approach and imaging tool in vivo for GSCs, paving the way for improved, more effective, and less invasive GBM management. Full article

Sutures such as fibrous joints in craniofacial bones provide a niche for Gli1+ mesenchymal stem cells (MSCs) in promoting calvarial bone development and growth. However, the underlying molecular mechanism behind the fate of the Wnt/β-catenin regulation of Gli1+ MSCs during calvarial bone formation remains unclear. Here, we showed that β-catenin was colocalized with Gli1+ lineage cells near the osteogenic front within a suture, and postnatal skull development was delayed via a conditional knockout of Ctnnb1 in Gli1+ MSCs. Calcein–Alizarin Red dual staining revealed that Wnt/β-catenin signal inhibition impaired the rate of bone formation. Furthermore, immunofluorescent staining indicated that Wnt/β-catenin signaling was crucial in facilitating the proliferative capacity of Gli1+ MSCs and their commitment to the osteogenic lineage. Notably, activating hedgehog (Hh) signaling partially restored the suture morphology in Ctnnb1 knockout mice. Collectively, our findings revealed the crosstalk between Wnt and Hh signaling modulates the fate of Gli1+ MSCs during calvarial bone formation. Full article

Adult mesenchymal stem cells (MSCs) play a crucial role in maintaining bone health and promoting regeneration. In our previous research, we identified Gli1⁺ MSCs as key contributors to the formation of most trabecular bone in adulthood and as essential for healing bicortical fractures. However, the mechanisms behind the maintenance and differentiation of Gli1⁺ MSCs are still not fully understood. O-linked N-acetylglucosamine modification (O-GlcNAcylation), mediated by O-GlcNAc glycosyltransferase (OGT), is involved in various biological processes and diseases. Our earlier work also demonstrated that O-GlcNAcylation is necessary for Wnt-stimulated bone formation. Nonetheless, the specific functions of O-GlcNAcylation in MSCs have not been completely elucidated. In this study, we found that the absence of OGT in Gli1⁺ MSCs led to a decrease in O-GlcNAcylation, which impaired both the bone formation and regeneration following fractures. Mechanistically, the Hedgehog signaling pathway induced O-GlcNAcylation through the insulin-like growth factor (Igf)-mTORC2 axis. This process stabilized the Gli2 protein at a specific site Ser355 and promoted osteogenesis in MSCs in vitro. Our findings reveal a significant mechanism by which O-GlcNAcylation regulates bone development and repair in mammals. Full article

Cadmium accumulation in the body can damage a variety of organs and impair their development and functions. In the present study, we investigated the effect of cadmium on the stemness and proliferation of normal bovine mammary epithelial cells (BMECs). Normal bovine mammary epithelial cells treated with cadmium chloride were assessed for the expression of stemness-related proteins and cell proliferation. Western blotting results found that exposure to different concentrations of cadmium (0, 1.25, 2.5, and 5 μm) for 48 h significantly increased Gli1 expression but unexpectedly decreased the expression of downstream stem cell-related proteins including BMI1, SOX2, and ALDH. However, we also observed that treatment with 5 μm cadmium for 48 h inhibited mammosphere formation using microscopy. In this study, cadmium exposure significantly reduced cell viability and mobility. Flow cytometry detection found that cadmium decreased the percentage of cells in the G0 phase but increased the percentage of cells in the S phase and the apoptosis rate. Furthermore, cadmium exposure significantly increased the levels of caspase-8, caspase-3, and PARP cleavage as assessed by western blotting. Our study uncovers a previously unrecognized role of cadmium in mammary cell stemness and suggests that cadmium may affect breast development by impairing normal stem cell self-renewal and inducing their apoptosis. Therefore, this study provides important scientific significance regarding whether heavy metal cadmium affects normal breast development. Full article

Breast cancer stem cells (CSCs) are resistant to most cancer therapeutics and contribute to tumor recurrence and metastasis. Two breast CSC-promoting transcription factors, truncated glioma-associated oncogene homolog 1 (tGLI1) and signal transducer and activator of transcription 3 (STAT3), have been reported to be frequently co-expressed in HER2-enriched breast cancer and triple-negative breast cancer (TNBC), undergo protein-protein interactions for gene regulation and activation, and functionally cooperate to promote breast CSCs. STAT3 can be activated by activated interleukin-6 receptor/glycoprotein-130 (IL-6R/GP130). Co-targeting of tGLI1 and IL-6R/GP130 has not been investigated in breast cancer or any tumor type. Here, we report that tGLI1 and GP130 are co-overexpressed in the majority of HER2-enriched breast cancers and TNBCs at 53.8% and 44.4%, respectively. tGLI1+IL-6/IL-6R/GP130 signaling is frequently co-enriched and co-activated in HER2-enriched breast cancer and TNBC when compared to luminal subtypes. tGLI1+GP130 co-overexpression strongly promotes CSCs of HER2-enriched breast cancer and TNBC. FDA-approved tGLI1 inhibitor Ketoconazole and GP130 inhibitor Bazedoxifene synergize against breast cancer proliferation and CSC phenotypes in vitro and reduce TNBC tumor growth and metastatic burden in vivo. Our study demonstrates, for the first time, that co-targeting tGLI1 and IL-6R/GP130/STAT3 signaling pathways is synergistic against HER2-enriched breast cancer and TNBC, warranting future clinical investigations. Full article

Some oxysterols were shown to promote osteogenic differentiation of mesenchymal stem cells (MSCs). Little is known about the effects of 7-ketocholesterol (7-KC) in this process. We describe its impact on human adipose tissue-derived MSC (ATMSC) osteogenic differentiation. ATMSCs were incubated with 7-KC in osteogenic or adipogenic media. Osteogenic and adipogenic differentiation was evaluated by Alizarin red and Oil Red O staining, respectively. Osteogenic (ALPL, RUNX2, BGLAP) and adipogenic markers (PPARƔ, C/EBPα) were determined by RT-PCR. Differentiation signaling pathways (SHh, Smo, Gli-3, β-catenin) were determined by indirect immunofluorescence. ATMSCs treated with 7-KC in osteogenic media stained positively for Alizarin Red. 7-KC in adipogenic media decreased the number of adipocytes. 7-KC increased ALPL and RUNX2 but not BGLAP expressions. 7-KC decreased expression of PPARƔ and C/EBPα, did not change SHh, Smo, and Gli-3 expression, and increased the expression of β-catenin. In conclusion, 7-KC favors osteogenic differentiation of ATMSCs through the expression of early osteogenic genes (matrix maturation phase) by activating the Wnt/β-catenin signaling pathway, while inhibiting adipogenic differentiation. This knowledge can be potentially useful in regenerative medicine, in treatments for bone diseases. Full article

HCC remains one of the leading causes of cancer-related death globally. The main challenges in treatments of hepatocellular carcinoma (HCC) primarily arise from high rates of postoperative recurrence and the limited efficacy in treating advanced-stage patients. Various signaling pathways involved in HCC have been reported. Among them, the Sonic hedgehog (SHH) signaling pathway is crucial. The presence of SHH ligands is identified in approximately 60% of HCC tumor tissues, including tumor nests. PTCH-1 and GLI-1 are detected in more than half of HCC tissues, while GLI-2 is found in over 84% of HCC tissues. The SHH signaling pathway (including canonical and non-canonical) is involved in different aspects of HCC, including hepatocarcinogenesis, tumor growth, tumor invasiveness, progression, and migration. The SHH signaling pathway also contributes to recurrence, metastasis, modulation of the cancer microenvironment, and sustaining cancer stem cells. It also affects the resistance of HCC cells to chemotherapy, target therapy, and radiotherapy. Reappraisal of the roles of the SHH signaling pathway in HCC may trigger some novel therapies for HCC. Full article

A characteristic feature of repair processes in mammals is the formation of scar tissue at the site of injury, which is designed to quickly prevent contact between the internal environment of the organism and the external environment. Despite this general pattern, different organs differ in the degree of severity of scar changes in response to injury. One of the areas in which regeneration after wounding leads to the formation of a structure close to the original one is the abdominal skin of laboratory rats. Finding out the reasons for such a phenomenon is essential for the development of ways to stimulate full regeneration. The model of skin wound healing in the abdominal region of laboratory animals was reproduced in this work. It was found that the wound surface is completely epithelialized on the abdomen by 20 days, while on the back—by 30 days. The qPCR method revealed higher expression of marker genes of skin stem cells (Sox9, Lgr6, Gli1, Lrig1) in the intact skin of the abdomen compared to the back, which corresponded to a greater number of hairs with which stem cells are associated on the abdomen compared to the back. Considering that some stem cell populations are associated with hair, it can be suggested that one of the factors in faster regeneration of abdominal skin in the rat is the greater number of stem cells in this area. Full article

Glioma-associated oncogene homolog 1 (Gli1) is a transcriptional activator of hedgehog (Hh) signaling that regulates target gene expression and several cellular biological processes. Cell lineage tracing techniques have highlighted Gli1 as an ideal marker for mesenchymal stem cells (MSCs) in vivo. Gli1⁺ MSCs are critical for the osteogenesis of the craniofacial bone; however, the regulatory mechanism by which Gli1⁺ MSCs mediate the bone development and tissue regeneration of craniofacial bone has not been systematically outlined. This review comprehensively elucidates the specific roles of Gli1⁺ MSCs in craniofacial bone osteogenesis. In addition to governing craniofacial bone development, Gli1⁺ MSCs are associated with the tissue repair of craniofacial bone under pathological conditions. Gli1⁺ MSCs promote intramembranous and endochondral ossification of the craniofacial bones, and assist the osteogenesis of the craniofacial bone by improving angiopoiesis. This review summarizes the novel role of Gli1⁺ MSCs in bone development and tissue repair in craniofacial bones, which offers new insights into bone regeneration therapy. Full article

Growing evidence indicates that cancer stem cells (CSCs) endow the tumor with stem-like properties. Recently, induced pluripotent stem cells (iPSCs) have gained increased attention because of their easy derivation and availability and their potential to differentiate into any cell type. A CSC model derived from iPSCs of human origin would help understand the driving force of tumor initiation and early progression. We report the efficient generation of feeder-free SSEA4, TRA-1-60 and TRA-1-81 positive iPSCs from amniotic membrane-derived mesenchymal stem cells (AMMSCs), which successfully differentiated into three germ layers. We then developed human iPSC-derived glioblastoma multiforme (GBM) model using conditioned media (CM) from U87MG cell line and CSCs derived from U87MG, which confer iPSCs with GBM and GSC-like phenotypes within five days. Both cell types overexpress MGMT and GLI2, but only GSCs overexpress CD133, CD44, ABCG2 and ABCC2. We also observed overexpression of LEF1 and β-catenin in both cell types. Down-regulation of Wnt antagonist secreted frizzled-related protein 4 (sFRP4) in GBM and GSCs, indicating activation of the Wnt/β-catenin pathway, which could be involved in the conversion of iPSCs to CSCs. From future perspectives, our study will help in the creation of a rapid cell-based platform for understanding the complexity of GBM. Full article