RESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Neoplasias de la Boca/patología , Piridinas/farmacología , Pirimidinas/farmacología , Proteína con Dedos de Zinc GLI1/genética , Adulto , Apoptosis/genética , Carcinoma de Células Escamosas/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias de la Boca/genética , Metástasis de la Neoplasia , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
This work evaluated the in vitro cytotoxic activity of laticifer proteins (LP) recovered from the latex of the medicinal plant Calotropis procera. The LP displayed considerable cytotoxicity with IC(50) values ranging from 0.42 to 1.36 microg/ml to SF295 and MDA-MB-435 cell lines, respectively. In healthy peripheral blood mononuclear cells exposed to LP (10 microg/ml) for 72 h, no noticeable effects on viability or cell morphology were seen. The fractionating of LP on an ion exchange chromatography gave rise to a new fraction (PI) that retained almost all cytotoxicity. The cytotoxic effects of both LP and PI were diminished when previously treated with pronase, or 2-mercaptoethanol, suggesting a protein nature of active molecules, however, pre-incubation with dithiothreitol (DTT) only reduced PI activity. PI did not exhibit cysteine proteinase activity, indicating that cysteine proteinases, abundantly found in LP, are not implicated in LP cytotoxicity. In this study, using HL-60 cell as a model, LP was shown to inhibit DNA synthesis. This is probably due to alterations in the topology of DNA, since it was observed that LP is able to interfere in topoisomerase I activity by somehow acting upon DNA. LP provoked reduction in cell number but it did not cause any significant increase in the number of non-viable cells. These findings corroborated with the morphologic analysis, where cells treated with LP showed morphology of apoptotic process with abundant vacuoles, chromatin condensation and fragmentation of the nuclei. The results of this study suggests that LP is a target for DNA topoisomerase I triggering apoptosis in cancer cell lines.
