The use of novel chemical or bioengineered bacteria as treatment strategy for neuroblastoma

Abstract

Neuroblastoma is a relatively common childhood tumor with poor prognosis by current therapeutic approaches. A novel group of thiosemicarbazones, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) have been found to be a promising novel anti-cancer agents. Recently, a newly synthesized agent, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), showed greater activity than Dp44mT in vivo and a lower toxicity profile than Dp44mT. The anti-proliferative efficacy of DpC and Dp44mT were examined using a panel of neuroblastoma (SK-N-LP/SK-N-AS/BE(2)-C/SH-SY5Y) and cardiac (H9C2), hepatocyte (MIHA), kidney (HK2) and mesenchymal stem cells. DpC demonstrated more potent cytotoxicity than Dp44mT against neuroblastoma cells in a dose- and time- dependent manner. DpC also demonstrated some anti-proliferative activity against the other cell lines tested. DpC significantly increased expression of phosphorylated JNK, neuroglobin (Ngb), cytoglobin (Cygb), cleaved Caspase 3 and 9 and a decrease in IkBα levels could be identified in vitro. The contribution of the JNK signaling was verified by JNK, NFκB and caspase inhibitors. Orthotopic SK-N-LP/Luciferase nude mice were used as an in vivo model. After three weeks of treatment, no mice (n=4) died and tumor growth was significantly (P<0.05) reduced by DpC (4 mg/kg/day). Apoptosis in tumors was confirmed by Annexin V/PI Flow cytometry and H & E staining. Xenograft tissues showed significantly higher expression of Ngb, Cygb, Caspase 3 and secretory TNFα levels. The anti-cancer activity of this agent appeared to be related to JNK pathway activation with the activation of apoptosis. Another new strategy by targeting neuroblastoma with genetically engineered anaerobic Salmonella (Sal-YB1). Nude and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) orthotopic mouse models were used and Sal-YB1 was administered via tail vein. The therapeutic effectiveness, bio-safety and mechanisms were studied. No mice died of therapy related complications. Tumor regression rates were 70% and 30% in nude and NOD-SCID mice, respectively. No Salmonella was detected in the urine; 75% mice had positive stool culture if diaminopimelic acid was added but all turned negative subsequently. Tumor tissues had more Sal-YB1 infiltration, necrosis and shrinkage in Sal-YB1-treated mice. Significantly higher expression of TLR4, TSG6 and cleaved Caspase 1, 3, 8 and 9 was found in the tumor masses of the Sal-YB1-treated group with a decrease of IRAK and IkBα. There was a high release of TNFα both in human macrophages and mouse tumor tissues with Sal-YB1 treatment. The antitumor effect of the supernatant derived from macrophages treated with Sal-YB1 could be reversed with TNFα and pan-Caspase inhibitors. In summary, DpC has a better cytotoxic profile for neuroblastoma in vivo despite having high in vitro toxicity to normal cell lines. The possible anti-cancer mechanisms can be partly due to induced oxidative stress and JNK pathway activation with the resultant apoptosis. Also, bioengineered Sal-YB1 should provide a new paradigm in targeting the hypoxic neuroblastoma tumor core. The macrophages may play a critical role in Sal-YB1 directed biotherapy.