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. 2021 Aug 12;10(8):767.
doi: 10.3390/biology10080767.

Connexin 43 and Sonic Hedgehog Pathway Interplay in Glioblastoma Cell Proliferation and Migration

Filippo Torrisi  1 , Cristiana Alberghina  1 , Debora Lo Furno  1 , Agata Zappalà  1 , Samuel Valable  2 , Giovanni Li Volti  3 , Daniele Tibullo  3 , Nunzio Vicario  1 , Rosalba Parenti  1
Affiliations

Connexin 43 and Sonic Hedgehog Pathway Interplay in Glioblastoma Cell Proliferation and Migration

Filippo Torrisi et al. Biology (Basel). .

Abstract

Glioblastoma (GBM) represents the most common primary brain tumor within the adult population. Current therapeutic options are still limited by high rate of recurrences and signalling axes that promote GBM aggressiveness. The contribution of gap junctions (GJs) to tumor growth and progression has been proven by experimental evidence. Concomitantly, tumor microenvironment has received increasing interest as a critical process in dysregulation and homeostatic escape, finding a close link between molecular mechanisms involved in connexin 43 (CX43)-based intercellular communication and tumorigenesis. Moreover, evidence has come to suggest a crucial role of sonic hedgehog (SHH) signalling pathway in GBM proliferation, cell fate and differentiation. Herein, we used two human GBM cell lines, modulating SHH signalling and CX43-based intercellular communication in in vitro models using proliferation and migration assays. Our evidence suggests that modulation of the SHH effector smoothened (SMO), by using a known agonist (i.e., purmorphamine) and a known antagonist (i.e., cyclopamine), affects the CX43 expression levels and therefore the related functions. Moreover, SMO activation also increased cell proliferation and migration. Importantly, inhibition of CX43 channels was able to prevent SMO-induced effects. SHH pathway and CX43 interplay acts inducing tumorigenic program and supporting cell migration, likely representing druggable targets to develop new therapeutic strategies for GBM.

Keywords: GBM; GLI1; connexin; gap junction; smoothened.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SMO modulation in U-251 MG and T98-G cells impacts on surviving fraction. (a) Rate of cell growth of human U-251 MG and T98-G cell lines, data are shown as violin plot of R.C.G. of n = 3 replicates over n = 6 passages. (b) Representative microphotographs of human U-251 MG andT98-G cells expressing GFAP and SMO; nuclei were counterstained with DAPI; scale bar = 20 μm. (c,d) Surviving fraction and representative wells of U-251 MG (c) and T98-G cell lines (d) exposed to increasing concentration of purmorphamine and cyclopamine (0.1–10 µM). Data are expressed as mean ± SEM of n = 3 independent experiments; * p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001 versus control cell cultures treated with vehicle; one-way ANOVA with Holm–Šídák post-hoc test. R.C.G.: rate of cell growth. P: passage.
Figure 2
Figure 2
SHH pathway activation is related to CX43 increased expression levels in U-251 MG. (a–c) CX43 and GLI1 expression levels (a,b) and representative blots (c) on human U-251 MG cells exposed to 1 µM of purmorphamine and/or 1 µM cyclopamine. Data are mean FC over control ± SEM of n = 3 independent experiments; * p-value < 0.05, ** p-value < 0.01 versus control or between groups; one-way ANOVA with Holm–Šídák post-hoc test. FC: fold change.
Figure 3
Figure 3
SHH pathway activation increase immunofluorescence intensity of CX43 in human GBM cell lines. (a–c) Representative microphotographs of CX43 and GFAP (a), CX43 frequency distribution (b) and quantification of CX43 MFI (c) of control human U-251 MG cells and U-251 MG cells exposed to 1 µM of purmorphamine and/or cyclopamine. (d–f) Representative pictures of CX43 and GFAP (d), CX43 frequency distribution (e) and quantification of CX43 MFI (f) of control human T98-G cells and T98-G cells exposed to 1 µM of purmorphamine and/or cyclopamine. Data in (b,e) are shown as violin plot of n ≥ 20 cells and data in (c,f) are shown via standard box and whiskers plot of n = 4 independent experiments; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 versus control or between groups; one-way ANOVA with Holm–Šídák post-hoc test. In (a,d) nuclei were counterstained with DAPI; scale bar in (a,d) = 10 μm. MFI.
Figure 4
Figure 4
SHH pathway activation increase Ki-67 in U-251 MG cell line. (a,b) Representative microphotographs of Ki-67 and GFAP immunofluorescence analysis (a) and quantification of Ki-67 MFI (b) of control U-251 MG cells and U-251 MG cells exposed to 1 µM of purmorphamine and/or cyclopamine, treated with either vehicle and ioxynil octanoate; nuclei were counterstained with DAPI; scale bar = 20 μm. Data are mean ± SEM of n = 3 independent experiments; * p-value < 0.05 and ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001 versus control or between groups; one-way ANOVA with Holm–Šídák post-hoc test. MFI.
Figure 5
Figure 5
SHH-CX43 axis induces migration enhancement in human GBM cell line. (a,b) Representative images (a) and migration index (b) of control human U-251 MG cells and U-251 MG exposed to 1 µM of purmorphamine and/or cyclopamine, treated with either vehicle or ioxynil octanoate. (c,d) Representative images (c) and migration index (d) of control human T98-G cells and T98-G exposed to 1 µM of purmorphamine and/or cyclopamine, treated with either vehicle or ioxynil octanoate. Data are shown as mean fold change over 0 hrs ± SEM of n = 3 independent experiments; * p-value < 0.05 and ** p-value < 0.01 versus control; one-way ANOVA with Holm–Šídák post-hoc test.
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