We provide evidence for the first time that DIM significantly impairs the growth of human T-ALL cells in vitro
and in vivo
. Moreover, we show that DIM blocks growth of T-ALL cell types that represent the spectrum of T-cell differentiation arrest occurring within this disease, ranging from least differentiated to nearly mature (HSB2>CEM>SUP-T1>Jurkat). All four T-ALL cell types studied responded to DIM treatment in a dose-dependent manner, as shown by inhibition of cell proliferation and viability and increased levels of apoptosis; in addition, a G1
cell cycle arrest was observed in HSB2 and CEM cells, lines that represent early (pre-T) differentiated cells. In this study, we also show that the I3C derivative, DIM, was far more potent than its precursor and exhibited therapeutic effects on a variety of highly aggressive juvenile T-ALL cell lines, including Jurkat and CEM, at physiological concentrations. Others have reported that I3C suppressed NFκB stimulation by TNF and downstream gene products, including CCND1, BCL-2 and TRAF1, in myeloid and leukemia (Jurkat) cells 
, but I3C was not capable of blocking the growth of T-cell lines that were not infected with human T-cell leukemia virus type-1 (MOLT-4, Jurkat and CCRF-CEM) 
The SCID mouse model supports the solid growth of subcutaneously injected human acute leukemia blast cells in a manner that is easily measurable and exhibits a dissemination pattern analogous to the human disease 
. We supplemented this pre-clinical model with dietary indoles to determine the extent of xenograft growth suppression following absorption, metabolism and disposition to the grafted cells. This study is the first to employ continuous exposure of I3C or DIM through the diet, as opposed to bolus administration via gavage or injection, with a human cell xenograft model in SCID mice. In the present study, growth of human CEM cell xenografts in mice consuming DIM (approximately 0.4 mg/day) was only about half that of the control animals, an observation that is comparable to other studies with breast cancer cell xenografts that employed even greater amounts of DIM or more direct routes of exposure. For example, oral gavage of about 1 mg DIM/day (3.5 mg BioResponse-DIM/day) decreased growth of MDA-MB-231 xenografted cells by approximately 30% after 3 weeks of exposure 
whereas daily 5 mg/kg s.c.
injections of DIM at the site of MCF-7 xenografts reduced tumor volume by about 45% 
We selected a dietary concentration of 2000 ppm I3C level based on the apparent anticancer effects at this level observed in our previous studies 
. The likely proportion of this I3C diet to be converted to DIM following in vivo
condensation corresponds to a diet concentration of 350 ppm DIM, based on a 2
1 molar ratio and assuming a 20% conversion rate 
. The BioResponse-DIM formulation is a commercially available dietary supplement, sold for human consumption, that is also used in animal studies and clinical trials; thus, 350 ppm BioResponse-DIM (100 ppm DIM) was selected in anticipation of comparable bioactivity to the 2000 ppm I3C treatment 
. Pharmacokinetic studies in mice comparing this formulated DIM to crystalline DIM demonstrate a 50% improvement in adsorption 
. This difference in bioavailability, along with the rapid elimination of I3C and formation of additional bioactive I3C derivatives, may account for the reduced efficacy of 2000 ppm I3C in vivo
compared to 100 ppm DIM.
DIM was also significantly more potent than I3C in vitro
based on the relative IC50
values for inhibition of cell proliferation and viability across all cell lines tested. Moreover, the anti-proliferative effect of I3C was delayed compared to DIM, suggesting that conversion of I3C to DIM and other ACPs in the culture media may contribute to the physiological effects of I3C. A recent report by Bradlow and Zeligs 
showed that addition of 100 µM I3C to culture media at a neutral pH resulted in concentrations of DIM of about 25 µM within 24 hours. However, because the degree of difference in potency of DIM and I3C varied across the four T-ALL cell lines tested and by the endpoint examined (viability, proliferation) in this study, the apparent lower potency of I3C compared to DIM cannot be fully explained by conversion of I3C to DIM in the culture media.
Other plausible explanations exist for the distinctive responses to DIM observed in the four T-ALL cell lines studied, which are characterized by different lineages of T-cell differentiation (pre-T, cortical-T and mature-T), as well as different ages and genders of the source patients (). Gene deletions and mutations as well as epigenetic mechanisms of gene dysregulation are commonly implicated in the oncogenesis of T-cells and in therapeutic outcome 
. Common leukemic signature genes include those involved in normal T-cell receptor signaling and T-cell differentiation such as NOTCH1
. A selection of these therapeutically relevant targets and their status in HSB2, CEM, SUP-T1 and Jurkat cells are listed in .
Although beyond the scope of this study, the different combinations of these aberrations across the cell lines tested are likely to play a role in the therapeutic effect of DIM. The variable responses to both targeted and conventional chemotherapeutic drugs that have been previously observed in T-ALL cells are likely a consequence of the respective mutations harbored by the cell lines (e.g., 
). Thus, the observation that DIM had variable potency for blocking growth of T-ALL cells (though effective in all four cell lines tested) is not necessarily unexpected given the apparent variability in response of T-ALL cells to drug therapies.
Treatment of CEM and HSB cells with DIM caused a blockade of cell-cycle progression at the G1
phase checkpoint, although this effect was not observed in more differentiated T-ALL cell lines (SUP-T1 or Jurkat); DIM (and I3C) also suppressed expression of CCND3, CDK4 and CDK6 cell cycle regulatory proteins in CEM cells. Early progression of the eukaryotic cell cycle is positively regulated by the coupling of D-type cyclins with the highly homologous CDK4 or CDK6 proteins and negatively regulated by cyclin dependent kinase inhibitors and phosphatases 
. I3C and DIM inhibit proliferation and cell cycle progression of various tumor cells, including breast 
, prostate 
and colon 
, via down-regulation of cyclins and cyclin dependent kinases and/or up-regulation of cyclin dependent kinase inhibitors, such as p21 or p27 
. The INK4A
gene locus, which encodes the cyclin dependent kinase inhibitors p16 and p19, is inactivated in up to 80% of T-ALL cases and in all T-ALL cell lines tested 
. CDK6 is the initial CDK induced during T-lymphocyte activation/proliferation and is highly expressed in T-cell lymphoblastic leukemias/lymphomas 
; similarly, over-expression of cyclin D3 is oncogenic in an array of mouse and human T-ALL cell lines 
. Aberrant expression of cyclin D3 and cyclin dependent kinases during leukemic transformation underscores their relevance as therapeutic targets for I3C/DIM.
DIM treatment effectively induced apoptosis in human T-ALL cells in vitro
and in vivo
, although the apoptotic response to in vitro
treatment with DIM varied greatly among the cell lines tested. HSB2 cells, which represent T-ALL originating from T-cells at a very early stage of differentiation, were highly sensitive to DIM-induced apoptosis compared to other T-ALL cell types that were only modestly affected. This observation reinforces a general conclusion of this report that HSB2 cells are more sensitive to the anticancer effects of DIM in vitro
. DIM treatment of CEM cells in vitro
also altered expression of mRNA transcripts belonging to the BCL-2 superfamily or involved in TNF signaling, suggesting the involvement of both intrinsic and extrinsic apoptotic pathways. Others have shown that in vitro
treatment with I3C or DIM inhibits NFκB activity in human breast and prostate cancer cells undergoing apoptosis 
and reduces BCL-2 mRNA and protein expression in breast cancer cells 
. Moreover, expression of HRK (a BH3 domain-only BCL2 family member) is induced in hematopoietic progenitor cells upon growth factor removal or chemotherapeutic administration 
. In this study, treatment of CEM cells with 7.5 µM DIM rapidly and continuously elevated HRK expression in vitro
, which represents a putative and novel therapeutic target of these dietary indoles.
Bioactive dietary components may be utilized as part of a healthy lifestyle aimed at disease prevention or therapy. The ability of I3C/DIM to target multiple pro-survival pathways in cancer cells, while causing few adverse effects on normal cells, has been explored in a number of cancer models with substantial success. Collectively, our work points to the potential benefit of exposure to these agents at early life stages for chemoprotection, from gestation through adolescence, when leukemia is most prevalent [26, this study]
. Based on available human and animal data 
, the concentrations of DIM used in this study in vitro
are likely achievable in vivo
. In conclusion, our observations suggest that DIM may be a beneficial chemotherapeutic agent or adjunct therapy for T-ALL patients.