Combining low-dose
chemotherapies is a strategy for designing less toxic and more potent childhood
cancer treatments. We examined the effects of combining the novel
thiosemicarbazones,
di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), or its analog,
di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (
Dp44mT), with the standard
chemotherapies,
celecoxib (CX),
etoposide (ETO), or
temozolomide (TMZ). These combinations were analyzed for synergism to inhibit proliferation of three pediatric
tumor cell-types, namely
osteosarcoma (Saos-2),
medulloblastoma (Daoy) and
neuroblastoma (SH-SY5Y). In terms of mechanistic dissection, this study discovered novel
thiosemicarbazone targets not previously identified and which are important for considering possible
drug combinations. In this case, DpC and
Dp44mT caused: (1) up-regulation of a major
protein target of CX, namely
cyclooxygenase-2 (COX-2); (2) down-regulation of the DNA repair
protein,
O6-methylguanine DNA methyltransferase (MGMT), which is known to affect TMZ resistance; (3) down-regulation of mismatch repair (MMR)
proteins, MSH2 and MSH6, in Daoy and SH-SY5Y cells; and (4) down-regulation in all three cell-types of the MMR repair
protein, MLH1, and also topoisomerase 2α (Topo2α), the latter of which is an ETO target. While
thiosemicarbazones up-regulate the
metastasis suppressor, NDRG1, in adult
cancers, it is demonstrated herein for the first time that they induce NDRG1 in all three pediatric
tumor cell-types, validating its role as a potential target. In fact,
siRNA studies indicated that NDRG1 was responsible for MGMT down-regulation that may prevent TMZ resistance. Examining the effects of combining
thiosemicarbazones with CX, ETO, or TMZ, the most promising synergism was obtained using CX. Of interest, a positive relationship was observed between NDRG1 expression of the cell-type and the synergistic activity observed in the combination of
thiosemicarbazones and CX. These studies identify novel
thiosemicarbazone targets relevant to childhood
cancer combination chemotherapy.