Apoptosis is also induced in presence of a pan-caspase inhibitor.

<p>Cells were preincubated with Z-VAD-fmk, a pan-caspase inhibitor, before AMF treatment and apoptosis induction was assessed using Annexin-V and propidiumiodide staining and flow cytometry. n = 4 (number of independent experiments carried out in triplicates).</p

Akacid Medical Formulation Induces Apoptosis in Myeloid and Lymphatic Leukemic Cell Lines <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Akacid medical formulation (AMF) is an oligoguanidine that exerts biocidal activity against airborne and surface microorganisms including bacteria, viruses, fungi, and molds, while showing relatively low toxicity to humans. We have previously shown that AMF exerts antiproliferative effects on a variety of solid tumor cell lines. In this study we raised the question whether AMF could also substantially inhibit cell growth or induce apoptosis in cell lines derived from hematologic malignancies such as leukemia or lymphoma. We found that AMF has antiproliferative effects on various hematologic cell lines derived from human leukemia and lymphoma. Additionally, we show that AMF induces apoptosis in leukemia cell lines not only via the extrinsic and intrinsic pathway, but also in a caspase-independent manner. This effect was found also in G0-arrested cells. Finally, in our animal experiments utilizing male nu/nu Balb/c mice we found a significant growth retardation, which was immunohistochemically associated with a significantly lower number of KI67-positive cells and caspase-3 induction in AMF-treated mice.</p></div

Caspases are activated after AMF treatment.

<p>Capase -8, -9, and -3/-7 activity was measured using a luminometric assay after treatment with various concentrations of AMF for different time periods (4A). n = 3 (number of independent experiments carried out in triplicates). Furthermore Western blot analyses were performed for caspase activation and additionally for cleaved PARP, as a marker for apoptosis induction (4B). n = 3 (number of independent experiments, respresentative blots are shown)</p

Apoptosis is also induced in G1/0 phase arrested cells.

<p>The CEM 6E2 subclone, which expresses tetracycline-regulated p16INK4A (CEM 6E2) and an empty vector transfected control cell line (CEM 2C8) were treated with tetracycline (+) or not (-). Cell viability (trypan blue staining), cell count (manual counting), as well as cell cycle distribution and apoptosis (Annexin-V, propidium iodide) were assessed. % of control for cell viability and cell count refers to the viability / cell number at seeding, which was set to 100%. n = 4 (number of independent experiments carried out in triplicates).</p

AMF induced apoptosis in HL-60 cells.

<p>After incubation with AMF for the indicated time periods Annexin-V and propidiumiodide staining was performed and analysed by flow cytometry. n = 4 (number of independent experiments carried out in triplicates).</p

Jugular vein catheters were implanted in male nu/nu Balb/c mice.

<p>A matrigel CEM C7H2 1:1 mixture was injected into the flanks of male and mice were treated with AMF (5μMol/kg per day) for 13 days. Tumors size was measured regularly and tumors were harvested for immunohistochemistry on day 13. n = 4 per treatment group.</p

AMF induced apoptosis in CEM C7H2 cells.

<p>After incubation with AMF for the indicated time periods Annexin-V and propidiumiodide staining was performed and analysed by flow cytometry. n = 4 (number of independent experiments carried out in triplicates).</p

KI67 positive cells are reduced and caspase 3 is induced after AMF treatment.

<p>After 13 days of AMF treatment mice were killed and tumors were harvested for immunohistochemistry. Representative sections of control and treated tumors (hematoxyline-eosine staining, caspase 3 and KI67 staining) are shown.</p

Apoptosis is also induced in presence of a pan-caspase inhibitor.

<p>Cells were preincubated with Z-VAD-fmk, a pan-caspase inhibitor, before AMF treatment and apoptosis induction was assessed using Annexin-V and propidiumiodide staining and flow cytometry. n = 4 (number of independent experiments carried out in triplicates).</p

Bcl-2 and CrmA overexpression does not significantly inhibit AMF induced apoptosis.

<p>Subclones of CEM C7H2 cells expressing either CrmA (CEM 2E8) or tetracycline-regulated bcl-2 (tet-off, CEM 10E1, - without + with tetracycline) and the respective control subclone (CEM D2.1) were treated with AMF and Annexin-V and propidiumiodide staining was analyzed after 48hours of incubation. n = 4 (number of independent experiments carried out in triplicates).</p