Molecular mechanisms for regression of tumors in rapamycin-treated neuroblastoma SK-N-BE2 and IMR-32 xenografts in nude mice after therapeutic treatments.

<p>Both SK-N-BE2 and IMR32 cells were harvested, counted, and suspended in an equal volume of highly-concentrated Matrigel and then cell suspension (100 µl) was injected in nude mice for development of xenografts during 3 weeks. Xenograft bearing animals were pre-treated with rapamycin (2 mg/kg body weight/day) for 7 days and then randomly assigned to four therapeutic treatment groups: CTL shRNA plasmid, LC3 shRNA plasmid, GST, and LC3 shRNA plasmid + GST. Animals were daily injected with CTL shRNA plasmid (50 µg/mouse), LC3 shRNA plasmid (50 µg/mouse), or/and GST (2 mg/kg body weight) for 15 days. The data are representative of at least 3 independent experiments using 4 mice in each therapeutic treatment group. (a) Mice with SK-N-BE2 and IMR-32 xenografts. (b) Tumors after surgical removal from the animals. (c) Estimation of tumor volumes in both neuroblastoma xenografts. Significant difference between CTL shRNA plasmid and another treatment was indicated by *<i>P</i><0.05 while significant difference between single treatment and double treatment was indicated by #<i>P</i><0.05. (d) H&E staining to show hisopathological alterations in tumor sections after the therapeutic treatments. (e) Representative Western blots to show changes in expression of LC3 II, Beclin 1, Bax, Bcl-2, active caspase-3, PARP fragment, andβ-actin.