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Abstract
Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.
Highlights
Malignant neuroblastoma is the most frequently diagnosed and highly aggressive extracranial solid tumor that mainly occurs in children
Rapamycin for induction of autophagy in human malignant neuroblastoma cells We first investigated whether rapamycin could induce autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cell lines (Figure 1)
We employed combination of light chain 3 (LC3) short hairpin RNA (shRNA) plasmid transfection and GST treatment to inhibit rapamycin-induced autophagy and increase apoptosis in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells both in vitro and in vivo. This uniquely consummate combination therapy involving the genetic approach using shRNA technology to knockdown the expression of LC3 along with the pharmacological intervention using the anti-cancer isoflavone GST was highly effective in controlling the growth of human malignant neuroblastoma cells in culture and animal models
Summary
Malignant neuroblastoma is the most frequently diagnosed and highly aggressive extracranial solid tumor that mainly occurs in children. It most commonly arises from adrenal medulla or abdominal sympathetic ganglia and exhibits very complex biological and clinical heterogeneity [1,2]. The role of autophagy in various cancers is highly complex and not well understood yet It appears that autophagy is an important process in solid tumors to utilize nutrients and provide building blocks for growth of tumor cells during adverse circumstances such as oxygen depletion and starvation and autophagy contributes to overall survival of tumor cells [7,8]. Rapamycin is a widely known autophagy inducer and specific inhibitor of mTOR signaling, and rapamycin blocks the functions of mTOR by inhibiting phosphorylation of downstream signaling molecules to induce autophagy [11,12]
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