Preferential involvement of mitochondria in Toll-like receptor 3 agonist-induced neuroblastoma cell apoptosis, but not in inhibition of cell growth

Abstract

Double-stranded RNA (dsRNA) can mediate its therapeutic effect through Toll-like receptor 3 (TLR3) expressed on tumor cells including neuroblastoma. We used synthetic dsRNA polyinosinic-polycytidylic acid [Poly(I:C)] as a TLR3 agonist to treat TLR3-expressing SK-N-AS neuroblatoma (NB) cells. We found up-regulation of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 and inositol-requiring enzyme 1. Bafilomycin A1, an inhibitor of ER function, effectively blocked poly(I:C)-induced activation of caspase-8, -9, and -3, MnSOD and glutathione peroxidase 1 and reduced poly(I:C)-induced SK-N-AS apoptosis. Pan caspase inhibitor and inhibitor of caspase-9, but not of caspase-8, inhibited poly(I:C)-induced activated caspase-3 expression. Rho zero (ρ(0))-SK-N-AS cells were resistant to poly(I:C)-induced mitochondrial reactive oxygen species production and apoptosis, but not to inhibition of cell growth, as compared to parent SK-N-AS cells. Taking together, these findings suggest that mitochondria are preferentially involved in poly(I:C)-induced NB cell apoptosis, but not in inhibition of cell growth. A crosstalk between mitochondria and ER is implicated.

Downregulation of c-Myc is involved in TLR3-mediated tumor death of neuroblastoma xenografts

“…Transcription of MnSOD is regulated by NF-κB, and involved in TLR3-mediated NB cell death. 26,35 A significantly increased MnSOD expression was found in poly (I:C)-treated AS xenografts (Po0.001, Figures 4c and d). Furthermore, ROS was visualized by 8-hydroxy-2'-deoxyguanosine (8-OHdG), revealing a stronger immunostaining of 8-OHdG in nuclei of tumor cells in poly(I:C)-treated xenografts than in controls (Po0.001, Figures 4c and e).…”

“…32 Our previous studies have revealed that the levels of c-Myc are associated with TLR3 expression in NB patients and TLR3-induced signaling in NB cells. 21,26,27 However, the role of c-Myc in the innate immune system is still unknown. To uncover whether c-Myc expression is influenced by TLR3-mediated innate immunity, we first compared c-Myc and TLR3 expressions by IHC between the control and poly(I:C) treatment groups of AS xenografts.…”

“…26 To explore whether poly(I:C) could also regulate the activation of NF-κB in AS xenografts, p-NF-κB p65 expressions between treated and untreated xenografts were analyzed by IHC. The results showed that p-NF-κB p65 expression was increased in the treatment group compared with that in the control group (Figures 4a and b).…”

“…25 In NB, we have found that poly(I:C) may induce tumor cells apoptosis, preferentially through the mitochondrial pathway. 21,26 Several TLR3 agonist-induced signals in suppression of NB cells have been shown, including activation of protein kinase R, interferon regulatory factor 3 (IRF3), mitochondrial antioxidant manganese superoxide dismutase (MnSOD), and caspase 3. 21,26 In addition, we found that another viral RNA sensor, melanoma differentiation-associated antigen-5 (MDA5), can complement TLR3 to improve poly(I:C) suppression of NB.…”

“…21,26 Several TLR3 agonist-induced signals in suppression of NB cells have been shown, including activation of protein kinase R, interferon regulatory factor 3 (IRF3), mitochondrial antioxidant manganese superoxide dismutase (MnSOD), and caspase 3. 21,26 In addition, we found that another viral RNA sensor, melanoma differentiation-associated antigen-5 (MDA5), can complement TLR3 to improve poly(I:C) suppression of NB. 27 These studies demonstrate that poly(I:C) is a potential therapeutic strategy for NB, and may be applied in clinical treatment in the future.…”