Abstract
BackgroundNeuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.Methodology/Principal FindingsTo develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.ConclusionsGiven that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.
Highlights
Neuroblastoma is the most frequent extra-cranial solid tumor in children and high-risk cases still face poor prognosis due to therapy-resistant relapse [1,2]
Consistent with previous reports on other neuroblastoma cell lines [10,15,16], we found that MG132 induces apoptosis in SK-N-BE(2) cells in a dosedependent manner (Figure 1A)
When retinoic acid (RA) was combined with MG132 for 3 days, the apoptosis rate was around 40%, only slightly lower than cells treated with MG132 alone (Figure 1B and 1D)
Summary
Neuroblastoma is the most frequent extra-cranial solid tumor in children and high-risk cases still face poor prognosis due to therapy-resistant relapse [1,2]. To control minimal residual disease, high risk neuroblastoma is currently treated with the differentiating agent 13-cis-retinoic acid (RA) at completion of cytotoxic therapy [3,4]. Recent reports have shown that cellular response to RA can be increased by inhibiting proteasome-mediated RARα degradation which thereby increases RARα transcriptional activity This further promotes retinoic acid-induced differentiation in both acute myeloid leukemia cells [5] and neuroblastoma cells [6]. Conclusions: Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma
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