Itraconazole (ITCZ) has been widely used for the treatment of fungal infection in the treatment of hematological malignancies. Recently, an anti-proliferative effect of ITCZ has been reported in solid tumors (Kim J et al. Cancer Cell 2010;17:388–399, Blake T et al. Cancer Res 2011;71:6764–6772). However, there has been no report on the effect of ITCZ on hematological malignancies. Therefore, we aimed to investigate the anti-proliferative effect of ITCZ on hematological malignancies in the present study.
Daudi (Human Burkitt's lymphoma cell line), Jurkat (Human T cell lymphoblast cell line), K562 (erythroleukemia), Karpas299 (Human ALK-positive Anaplastic large cell lymphoma cell line) cell lines were used as models of hematological malignancies. Growth inhibition by ITCZ was assessed by MTT assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega). To determine if ITCZ possessed anti-proliferative effect on the cell growth, we treated these cell lines with ITCZ. First, the concentrations of ITCZ were settled as ranging from 0 to 80 μM. To analyze the gene expressions concerning the cellular signal transductions, we treated cells with different concentrations of ITCZ for 24 hours. After treatment, cells were collected and lysed, followed by Western blot analysis to investigate molecular signals that associated with cell proliferation. To assess in vivo effects of ITCZ, 2.0×107 viable Karpas299 cells were suspended in 200 μl of Matrigel (Becton, Dickinson and Company) and implanted subcutaneously in severe immunodeficient mice (Crli:SHO-PrkcdscidHrhr). When tumor volumes reached more than 500 mm3 (tumor diameters were measured with caliper and tumor volumes were calculated by following formula: Volume= d short2×d long), we divided these mouse into 3 groups. In the control group, mice were given water for 14 days. In the treatment of ITCZ group, mice were given 100 mg/kg/day or 200 mg/kg/day of ITCZ orally for 14 days. We measured each tumor size for every 14 days and compared tumor size between 3 groups. After treatment, we retrieved each tumors and performed Western blot analysis and immunostaining for markers of cell survival.
ITCZ significantly inhibited the cell proliferation in Karpas299 cells compared with other cell lines. We focused on NPM-ALK which mediates oncogenesis in ALK- positive Anaplastic large cell lymphoma. It has been reported that NPM-ALK induces activation of mTOR signal pathway through activation of PI3K/Akt signal pathway (Marzec M. et al Oncogene 2007;26:5606–5614, Singh R. et al. Cancer Res 2009;69:2550–2558). So we examined molecules of signal pathway associated with NPM-ALK and PI3K/Akt, mTOR signal pathway by Westernblot analysis. The phosphorylation of ALK, Akt, 4EBP-1 and p70S6 were reduced from 30% to 80% by treatment of ITCZ in Karpas299. This result suggests that ITCZ might inhibit mTOR signal pathway through inhibition of NPM-ALK. In xenograft models, ITCZ suppressed tumor progression about one third of control group. The immunostaining of xenograft tissues obtained from tumors which treated with ITCZ showed increase of apoptosis.
Aberrant activation of ALK is considered as a source of proliferative signal pathways in Karpas299 cells. In our present study, ITCZ inhibited the growth of Karpas299 cells and phosphorylation of ALK and signal moleculesof mTOR pathway. These results indicate that ITCZ might inhibit activation of ALK and have therapeutic effects on ALK-positive Anaplastic large cell lymphoma.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.