The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-Patched1 (PTCH1) and Gli1 in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEP1), respectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines. Cyclopamine decreased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G(0)/G(1) phase, while Shh induced the reverse effects and could partially prevent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic approach to NB.

The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H(2)O(2), 100 μmol/L) was used to treat neurons for 24 h to induce oxidative stress. Exogenous SHH (3 μg/mL) was employed to activate the SHH pathway, and cyclopamine (20 μmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 μmol/L) were used to pre-treat the neurons 30 min before H(2)O(2) treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H(2)O(2) treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the pro-apoptotic gene Bax by 12% and down-regulate the expression of the anti-apoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H(2)O(2)-induced apoptosis.

The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H(2)O(2), 100 μmol/L) was used to treat neurons for 24 h to induce oxidative stress. Exogenous SHH (3 μg/mL) was employed to activate the SHH pathway, and cyclopamine (20 μmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 μmol/L) were used to pre-treat the neurons 30 min before H(2)O(2) treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H(2)O(2) treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the pro-apoptotic gene Bax by 12% and down-regulate the expression of the anti-apoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H(2)O(2)-induced apoptosis.
Animals ; Cerebral Cortex ; metabolism ; Hedgehog Proteins ; metabolism ; Neurons ; metabolism ; Neuroprotective Agents ; metabolism ; Oxidative Stress ; physiology ; Phosphatidylinositol 3-Kinase ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-- Patchedl (PTCH1) and Glil in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Glil in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEPI), re-spectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concen-trations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expres-sion of PTCH1 and Gill was detected in both NB samples and S-type NB cell lines. Cyclopamine de-creased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopaminc could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase, while Shh induced the reverse effects and could partially pre-vent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies re-vealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic ap-proach to NB.