Decitabine is a prototype for epigenetic therapy in cancer which targets DNA methyltransferase. While it was known that decitabine monotherapy has been associated with a relative low rate of complete remission rates in acute myeloid leukemia and myelodysplastic syndrome. Several groups have attempted to increase the response rate with decitabine-based therapy by developing combinations. In the preliminary experiments, we investigated the effect of anti-leukemia drugs (idarubicin, daunorubicin, cytarabine, thalidomide and homoharringtonine) in combination simultaneously or sequentially with decitabine of inhibiting proliferation of acute myeloid leukemia cell lines. The result showed that sequential combination of decitabine and idarubicin induced a significant synergistic effect in cell apoptosis, inhibition of cell proliferation. With the gene chip technology, we found that the gene expression of Wnt pathway changed obviously after the treatment of DAC combined with IDA. Further we confirm that sequential combination of DAC and IDA caused depression of wnt pathway in vitro. In this study, we evaluate the anti-leukemic effect and the combination mechanism of DAC in sequential combination with IDA in vivo.
The AML cell line U937 (14×107 cells per animal) were injected subcutaneously into the right flank of 6 to 8-week old NOD-SCID mice. After tumor growth, decitabine(0.5mg/kg/day) were injected intraperitoneal of a five consecutive days followed by a three days of idarubicin(0.5 mg/kg/day). The tumor growth inhibition effect was evaluated by microPET to reveal the synergistic effect of decitabine and idarubicin. Using TUNEL method and electron microscopy, we detected the apoptosis of leukemic cells from tumor bearing mice. The expression of β-catenin which is the key gene of Wnt/β-catenin pathway was also examined by immunochemistry. We then detected the expression of protein levels of the other genes(cyclinD1, c-myc, SFRP1,HDPR1 and DKK3) of Wnt/β-catenin pathway in tumor cells of tumor-bearing mice by western blot.
In vivo, the effect of sequential combination of decitabine and idarubicin was initially examined in a subcutaneous AML mouse model. The AML cell line U937(1×107 cell per animal) was transplanted subcutaneously into the right flank of 2-week-old female NOD-SCID mouse. The cell line developed into a rapidly growing tumor. Treatment was initiated in 7th days after injection of the leukemic cells, included with 5 days of decitabine alone, 3 days of idarubicin alone and 5 days of decitabine followed by 3 days of idarubicin. A significant inhibition of tumor growth was demonstrated with microPET in animals treated with sequential combination of decitabine and idarubicin compared with the other treatment and control group animals. The apoptosis of tumor cells was also significantly increased in the sequential combination treatment group detected by TUNEL and electron microscope. We also found that the sequential treatment group induced synergistic effect in re-expression or up-expression of the Wnt inhibitor genes (SFRP1, HDPR1 and DKK3) in tumor cells. Furthermore, the down-steam genes of wnt pathway including β-catenin, c-myc and cyclinD1 were down-regulated which suggest depression of wnt/β-catenin pathway. These results suggest that sequential combination of decitabine and idarubicin has a powerful anti-leukemic effect not only in vitro but also in vivo.
Our results demonstrate decitabine in sequential combination with idarubicin in mouse model of AML and suggest that this combination may be of clinical value in the treatment of patients with AML.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.