3, 3′-Diindolylmethane Exhibits Antileukemic Activity In Vitro and In Vivo through a Akt-Dependent Process

3,3′-diindolylmethane (DIM), one of the active products derived from Brassica plants, is a promising antitumor agent. The present study indicated that DIM significantly induced apoptosis in U937 human leukemia cells in dose- and time-dependent manners. These events were also noted in other human leukemia cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal bone marrow mononuclear cells. We also found that DIM-induced lethality is associated with caspases activation, myeloid cell leukemia-1 (Mcl-1) down-regulation, p21cip1/waf1 up-regulation, and Akt inactivation accompanied by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a constitutively active Akt construct prevented DIM-mediated caspase activation, Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway by pharmacologic or genetic approaches attenuated DIM-induced caspases activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor growth of mouse U937 xenograft, which was related to induction of apoptosis and inactivation of Akt, as well as activation of JNK. Collectively, these findings suggest that DIM induces apoptosis in human leukemia cell lines and primary human leukemia cells, and exhibits antileukemic activity in vivo through Akt inactivation and JNK activation

Melt extrusion deposition (MED™) 3D printing technology – A paradigm shift in design and development of modified release drug products

Three-dimensional printing (3DP) technology offers unique advantages for pharmaceutical applications. However, most of current 3D printing methods and instrumentations are not specifically designed and developed for pharmaceutical applications. To meet the needs in pharmaceutical applications for precision, compatibility with a wide range of pharmaceutical excipients and drug materials without additional processing, high throughput and GMP compliance, an extrusion-based 3D printer based on Melt Extrusion Deposition (MED™) 3D printing technology was developed in this study. This technology can process powder pharmaceutical excipients and drugs directly without the need of preparing filament as required by FDM 3D printing. Six different tablet designs based on compartment models were used to demonstrate the precision and reproducibility of this technology. The designed tablets were fabricated using the GMP-compliant MED™ 3D printer and were evaluated in vitro for drug release and in vivo for selected designs using male beagle dogs. Tablet designs with one or more compartments showed versatile release characteristics in modulating the release onset time, release kinetics, duration of release and mode of release. Multiple drugs or formulations were fabricated into a single tablet to achieve independent release kinetics for each drug or to fine-tune the pharmacokinetic profile of a drug. Building upon the theoretical analysis of models, precision and reproducibility of MED™ 3D printing technology, a novel product development approach, 3D printing formulation by design (3DPFbD®) was developed to provide an efficient tool for fast and efficient pharmaceutical product development. The MED™ 3D printing represents a novel and promising technology platform encompassing design and development of modified drug release products and has potential to impact the drug delivery and pharmaceutical product development

DIM induces apoptosis in U937, Jurkat, and HL-60 cells, and in AML blast samples, but not in normal bone marrow mononuclear cells.

<p>(a) U937, Jurkat, and HL-60 cells were treated with 80 µM DIM for 24 h, after which apoptosis was determined by annexin V/PI staining and flow cytometry. ** Values for cells treated with DIM were significantly increased compared to values in control cells by Student's t-test, <i>p</i><0.01. (b) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (c–d) Blasts from 15 patients with AML and normal bone marrow mononuclear cells were treated with 80 µM DIM for 24 hours, apoptosis was determined by annexin V/PI staining and flow cytometry.</p

<i>In vivo</i> antileukemic activity of DIM in U937 xenografts.

<p>20 NOD/SCID mice were inoculated with U937 cells (2×10⁶ cells/mouse, i.p.) and randomly divided into two groups (10/group) for treatment with DIM (50 mg/kg, i.p., daily, five times per week) or with vehicle control solvent. (a) Average tumor volume in vehicle control mice and mice treated with 50 mg/kg DIM. <i>P</i><0.01, significantly different compared with vehicle control by Student's <i>t</i>-test. (b) body weight changes of mice during the 20 days of study. (c) At the 20 days after DIM treatment, the tumors were excised and subjected to H&E staining for determination of pathological evaluation, TUNEL assay for determination of apoptosis, and immunohistochemical staining to determine Cleavage-caspase-3 immunoreactivity. Original magnification ×400. (d) After treatment with DIM, tumor tissues were sectioned and subjected to immunohistochemistry using anbibodies as indicated.</p

Effects of PI3K inhibitor, LY294002 (LY) and genetic activation of Akt on apoptosis induced by DIM.

<p>U937 cells were pretreated with 20 µM of LY for 1 h, followed by the addition of 40 µM of DIM for 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and LY were significantly greater than those for cells treated with DIM alone by Student's <i>t</i>-test; <i>p</i><0.01. (b–c) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (d) U937 cells were stably transfected with an empty vector (pcDNA3.1), Akt-CA, and Akt-DN. Cells were treated with 80 µM of DIM for 24 h, after which apoptosis was analysed using Annexin V/PI assay. **Values for Akt-CA cells treated with DIM were significantly decreased compared to those for pcDNA3.1 cells by Student's <i>t</i>-test, <i>p</i><0.01. (e–f) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated.</p