Abstract
Glioblastoma (GBM) is the most common adult primary tumor of the CNS characterized by rapid growth and diffuse invasiveness into the brain parenchyma. The GBM resistance to chemotherapeutic drugs may be due to the presence of cancer stem cells (CSCs). The CSCs activate the same molecular pathways as healthy stem cells such as WNT, Sonic hedgehog (SHH), and Notch. Mutations or deregulations of those pathways play a key role in the proliferation and differentiation of their surrounding environment, leading to tumorigenesis. Here we investigated the effect of SHH signaling pathway inhibition in human GBM cells by using GANT-61, considering stem cell phenotype, cell proliferation, and cell death. Our results demonstrated that GANT-61 induces apoptosis and autophagy in GBM cells, by increasing the expression of LC3 II and cleaved caspase 3 and 9. Moreover, we observed that SHH signaling plays a crucial role in CSC phenotype maintenance, being also involved in the epithelial-mesenchymal transition (EMT) phenotype. We also noted that SHH pathway modulation can regulate cell proliferation as revealed through the analysis of Ki-67 and c-MYC expressions. We concluded that SHH signaling pathway inhibition may be a promising therapeutic approach to treat patients suffering from GBM refractory to traditional treatments.
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The authors declare that there are no data to share.
Abbreviations
- AO:
-
Acridine orange
- ATTC:
-
American Tissue Culture Collection
- AVOs:
-
Acidic vacuolar organelles
- BSA:
-
Bovine serum albumin
- CNS:
-
Central nervous system
- CSCs:
-
Cancer stem cells
- DAPI:
-
4–6-Diamino-2-phenylindole
- DMEM/F-12:
-
Dulbecco’s medium supplemented with F-12
- DMSO:
-
Dimethyl-sulfoxide
- EMT:
-
Epithelial-mesenchymal transition
- FBS:
-
Fetal bovine serum
- GBM:
-
Glioblastoma
- GANT-61:
-
GLI-ANTagonist 61
- IECPN:
-
Instituto Estadual do Cérebro Paulo Niemeyer
- IF:
-
Immunofluorescence
- MTT:
-
3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
- PBS:
-
Phosphate-buffered saline
- PFA:
-
Paraformaldehyde
- PI:
-
Propidium iodide
- PTCH:
-
Patched
- PVDF:
-
Polyvinylidene difluoride
- qRT-PCR:
-
Quantitative PCR
- rh-SHH:
-
Recombinant Sonic Hedgehog/SHH(C24II)
- SHH:
-
Sonic hedgehog
- SMO:
-
Smoothened
- SuFu:
-
Suppressor of Fused
- TBS-T:
-
Tris-buffered saline with 0.1% Tween-20
- TGF-β:
-
Transforming growth factor-beta
- TMZ:
-
Temozolomide
- WB:
-
Western Blot
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Acknowledgements
Thanks are to Professor Vivaldo Moura Neto for his careful manuscript reading and all the support given.
Funding
This work was supported by the National Institute for Translational Neuroscience (INNT) of Ministry of Science and Technology; Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES) (number 2/2017) of the Ministry of Education; National Council for Scientific and Technological Development (CNPq) (EDITAL UNIVERSAL 2018 INFRA 04/2018); Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ) (number APQ1 2016 E010.002216/2016); and Ary Frauzino Foundation for Cancer Research (ONCOBIOLOGIA 2017); Pro‐Saúde Associação Beneficente de Assistencia Social e Hospitalar and Mahatma Gandhi Foundation.
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The manuscript was conceptualized, written, edited, and critically evaluated by each of the authors. TS and GBC performed most of the experiments. JH, VF, GF, and RSD performed a part of the experiments (part of the western blots, qRT-PCRs, cytometry, and micro-RNA analysis). The work was supervised by TS. All authors read and approved the final submitted version of the manuscript.
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The written informed consent for the isolation of cells to establish a new cell line for future research was obtained from the patient himself before the surgery. The approval for the isolation of patient cells was approved by the Clinical Research Ethics Committee of Instituto Estadual do Cérebro Paulo Niemeyer (CAAE: 90670018.4.0000.8110), as we have established a cell line for future studies.
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Carballo, G.B., Ribeiro, J.H., Lopes, G.P.F. et al. GANT-61 Induces Autophagy and Apoptosis in Glioblastoma Cells despite their heterogeneity. Cell Mol Neurobiol 41, 1227–1244 (2021). https://doi.org/10.1007/s10571-020-00891-6
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DOI: https://doi.org/10.1007/s10571-020-00891-6