Abstract
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.
Highlights
Cancer remains the second leading cause of mortality worldwide, accounting for one in six, or 9.6 million, deaths reported in 2018 [1]
It has been reported that the downstream inhibition of the HH pathway through the targeting of GLI1 can alternatively attenuate the expression of genes involved in the biological behavior of cancer, as well as interfere with other signaling cascades involved in tumor progression, such as MAPK, PI3K, and TGFβ [11,16]
At the half-maximal inhibitory concentration of GANT61 (36 μM), a statistically significant reduction was observed in mRNA levels of the GLI1 transcription factor in comparison to negative (DMSO 0.2%) and positive (5-FU) controls
Summary
Cancer remains the second leading cause of mortality worldwide, accounting for one in six, or 9.6 million, deaths reported in 2018 [1]. It has been reported that the downstream inhibition of the HH pathway through the targeting of GLI1 can alternatively attenuate the expression of genes involved in the biological behavior of cancer, as well as interfere with other signaling cascades involved in tumor progression, such as MAPK, PI3K, and TGFβ [11,16]. The downstream inhibition of HH pathway regulators by GLI transcription factor antagonists, especially the efficacious activity demonstrated by the experimental agent GANT61, may present a promising strategic alternative to SMO inhibitors [17]. Studies have shown that GANT61 significantly decreases the transcriptional production and gene expression of GLI1, PTCH1, and other HH pathway target genes, as evidenced by inventoried GLI assays in a range of cancer cell types [17,18,19,20]. HH pathway, on the gene and protein expression of HH pathway compoTnheunst,sth(ePTpCurHp1o,seGoLfIt1h,isGwLoI2rk, awnads tGoLeIv3a)l,uaastewtheellanastittuummoorralcaecltlivpirtoyloiffeGraAtiNonT6a1n,da ddoewatnhstirneaam minehtaisbtiatotircoOf SthCeCHcHellplainthew. ay, on the gene and protein expression of HH pathway components (PTCH1, GLI1, GLI2, and GLI3), as well as tumor cell proliferation and death in a metastatic OSCC cell line
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