Abstract
Neuroblastoma is the most common pediatric extracranial tumor, and advanced-stage cases are highly resistant to conventional chemotherapy. Heat-shock protein-70 (Hsp-70) is overexpressed in several human malignancies, and its inhibition has been shown to kill cancer cells. We have previously shown that triptolide, an inhibitor of Hsp-70, induces apoptotic cell death of pancreatic tumors. In this chapter, we explore the effectiveness of triptolide therapy in the treatment of neuroblastoma, both in vitro and in vivo. Additionally, we discuss the effect of triptolide treatment on levels of Hsp-70 and markers of apoptosis in neuroblastoma cell lines. We aim to clarify the mechanism by which triptolide induces cell death in neuroblastoma. After exposing neuroblastoma cells to triptolide, cellular viability was assessed, with results indicating that triptolide causes both dose- and time-dependent cell killing. In order to ascertain whether triptolide-mediated cell death occurs via an apoptotic pathway, caspase-3 and -9 activities as well as annexin staining were measured; these markers of apoptosis were found to be elevated, implicating an apoptotic pathway. As triptolide is a known inhibitor of Hsp-70, Western blot analysis and RT-PCR were performed following triptolide treatment, resulting in decreases of both Hsp-70 protein and mRNA levels. To clarify the cause-effect relationship, Hsp-70 was specifically silenced in neuroblastoma cells via siRNA, and viability, caspase activity, and phophatidylserine externalization were subsequently measured. The effects on cellular viability and markers of apoptosis were similar to those which are seen following triptolide therapy, supporting the hypothesis that Hsp-70 inhibition serves a key role in triptolide-mediated cell death. To examine the effects of triptolide on neuroblastoma in vivo, an orthotopic tumor model was developed, and, following randomization, mice in treatment and control groups received daily injections of triptolide and vehicle, respectively. At 21 days, mice were sacrificed, and tumors were measured. Mice receiving triptolide therapy had significantly smaller tumors than control, with average tumors in control mice 6 times the size of tumors in mice treated with triptolide. Immunohistochemisty and Western blotting were used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis. Hsp-70 immunohistochemistry showed greater staining in control tumors than those from treated mice. Tumors from triptolide-receiving mice demonstrated significant TUNEL staining, while those receiving vehicle showed no evidence of apoptosis. In summary, treatment of neuroblastoma with triptolide shows efficacy in decreasing cellular viability in vitro and in inhibiting tumor growth in vivo through apoptotic pathways. Correlated drops in levels of Hsp-70 suggest that triptolide’s effects are associated with inhibition of Hsp-70 expression. Our findings suggest that triptolide may provide a novel therapy for neuroblastoma and further studies are certainly warranted.
