(1) Background: Activation of the PI3K-AKT pathway controls most hallmarks of cancer, and the hedgehog (HH) pathway has been associated with oral squamous cell carcinoma (OSCC) development and progression. We hypothesized that fibroblast-derived insulin-like growth factor-1 (IGF-1) acts in oral squamous cell carcinoma (OSCC) cells, leading to the non-canonical activation of the HH pathway, maintaining AKT activity and promoting tumor aggressiveness. (2) Methods: Primary fibroblasts (MF1) were genetically engineered for IGF-1 overexpression (MF1-IGF1) and CRISPR/Cas9-mediated IGF1R silencing was performed in SCC-4 cells. SCC-4 cells were co-cultured with fibroblasts or incubated with fibroblast conditioned medium (CM) or rIGF-1 for functional assays and the evaluation of AKT and HH pathways. (3) Results: Gene expression analysis confirmed IGF-1 overexpression in MF1-IGF1 and the absence of IGF-1 expression in SCC-4, while elevated IGF1R expression was detected. IGF1R silencing was associated with decreased survival of SCC-4 cells. Ihh was expressed in both MF1 and MF1-IGF1, and increased levels of GLI1 mRNA were observed in SCC-4 after stimulation with CM-MF1. Activation of both PI3K-AKT and the HH pathway (GLI1, Ihh and SMO) were identified in SCC-4 cells cultured in the presence of MF1-IGF1-CM. rIGF-1 promoted tumor cell proliferation, migration, invasion and tumorsphere formation, whereas CM-MF1 significantly stimulated angiogenesis. (4) Conclusions: IGF-1 exerts pro-tumorigenic effects by stimulating SCC-4 cell proliferation, migration, invasion and stemness. AKT and HH pathways were activated by IGF-1 in SCC-4, reinforcing its influence on the regulation of these signaling pathways.

Due to its importance in the pathogenesis of oral squamous cell carcinoma (OSCC), the Hedgehog (HH) pathway is considered a potential therapeutic target. We investigated the effects of GANT61, a GLI inhibitor, on HH gene expression, as well as on metastatic OSCC cell proliferation and death. Following culture in DMEM medium, cytotoxicity of GANT61 against different tumor and non-tumor cell types was assessed by alamarBlue assays. Cytotoxicity analysis revealed that the metastatic HSC3 cell line was the most sensitive (IC₅₀: 36 µM) to the tested compound. The compound’s effects on the expression of HH pathways components were analyzed by qPCR and Western blot; cell viability was analyzed by trypan blue assay and flow cytometry were used to investigate cell cycle phase, morphology, and death patterns in HSC3 cells. A significant reduction in mRNA levels of the GLI1 transcription factor was found after 12 h of treatment withGANT61. Protein expression levels of other HH pathway components (PTCH1, SHH, and Gli1) and HSC3 cell viability also decreased after 24 h of treatment. Cell cycle analysis and death pattern evaluations revealed significantly increased nuclear fragmentation in sub-G1 phase, as well as cell death due to apoptosis. In conclusion, the significantly reduced GLI1 gene expression seen in response to the GLI inhibitor indicates diminished downstream activation in HH pathway components. GANT61 significantly reduced cell viability in the metastatic cell line of OSCC and promoted a significant increase in nuclear fragmentation and cell death by apoptosis.

Background: Glioblastomas (GBMs) are the most aggressive and common neoplasms that affect glial cells, presenting rapid growth, invasion, and resistance to treatments. Studies have demonstrated the potentially inhibitory effect of flavonoids on glioblastoma cells’ stemness and viability. However, further research is needed to explore sensitivity and the mechanism of action in chemoresistant cells. Methods: In this study, we characterized the impact of apigenin treatment on the viability and differentiation of human GBM cells in vitro and its effects on tumorigenesis and regulation of the inflammatory response in vivo. Results: The flavonoid apigenin reduced the viability of U-251 cells, patient-derived cells TG-1 and OB-1 stem cells in a dose-dependent manner, associated with the induction of acidic vesicle organelles formation and apoptosis. Treatment with apigenin also inhibited migration and induced neural differentiation in the remaining viable cells, characterized by a decrease in the expression of the precursor marker nestin and an increase in the expression of astrocyte and neuron markers, GFAP and β-III tubulin, respectively. The xenotransplantation of apigenin-pretreated U251 cells into rat brains did not lead to tumor formation, unlike untreated cells. The surrounding area of transplanted untreated U251 cells exhibited reactive microglia and astrocytes, along with increased VEGF expression, which was absent in implant sites of apigenin-pretreated GBM cells. Moreover, in this implant area, we observed a significant decrease in the expression of mRNA for inflammatory factors IL-1β, TNF, and NOS2, and the downregulation of IL-10 and IL-4. Conclusions: These results demonstrate that apigenin inhibits the growth of tumoral cells, affecting the viability of tumor stem cells and impairing tumorigenicity, while altering the regulatory profile of immunomodulatory proteins. Therefore, this flavonoid can be considered for further studies to determine its use as an adjuvant to the treatment of human GBMs.