Effect of the PKC-Beta Inhibitor, Enzastaurin, in Acute Leukemia - A Preliminary Study in Cell Culture.

Patients with chemotherapy-resistant acute myeloblastic leukaemia (AML) are rarely cured by non-allogenic transplant therapy. Multiple new investigational agents have become available for treatment of these patients and there are few tools to enable rational drug and clinical trial selection. These new drugs react either with cell surface molecules or signal transduction pathway intermediates. The important role of PKC in processes relevant to neoplastic transformation, carcinogenesis and tumour cell invasion renders it a potentially suitable target for anticancer therapy. The development of a selective inhibitor of PKC-beta agents, such as enzastaurin, with antitumoral activity both alone or in combination with other cytotoxic agents, remains a novel and interesting cancer therapeutic approach that will certainly be relevant in the future.

Our first approach is to evaluate the effect of the PKC-beta inhibitor, enzastaurin, in cancer cell proliferation and death, namely in acute leukaemia, as single agent and/or in combination with conventional chemotherapy.

For this purpose we began to evaluate PKC beta-I and beta-II gene expression in the haematopoietic cell lines, K562 (Chronic Myeloid Leukemia in blast crisis) and HL-60 (Acute Promyelocytic Leukemia) by real-time reverse transcriptase-polymerase chain reactions analysis using SYBR Green assay (1- PKC beta I: forward primer - 5′ GGA GAA ACT TGA ACG CAA AGA G-3′; reverse primer - 5′ AGG CTC AAC GAT GGA GTT TG -3′; 2- PCK beta II: forward primer -5′ GGA GAA ACT TGA ACG CAA AGA G-3′; reverse primer - 5′CGG AGG TCT ACA GAT CTA CTT AGC TCT -3′; GenBank M13975 for PCKB-II and NM_212535 for PCKB-II) (according Chalfant et al., 1998).

After, we incubate the K562 and HL-60 cells, in absence and presence of enzastaurin in different concentrations (rangin from 1 to 200 μM), as single agent and/or with combination with imatinib and All-Transretinoic Acid (ATRA), respectively (both in lower dose than IC50), during 72 h.

Cell growth and viability were evaluated by cell density and viability tests using Trypan blue exclusion, each 24 h during 72 hours to establish a dose-response curve and the IC50 for each drug, as single agent and/or in combination. Cell death analysis were performed by morphological studies using optic microscopy and by flow cytometry, using annexin-V and/or propidium iodine incorporation.

Our preliminary results suggest that Enzastaurin induces a decrease in K562 and HL-60 cells viability, as single agent, in a dose, time and cell line dependent manner (IC₅₀ in a ranging concentration 50–100 mM), inducing cell dead predominantly by apoptosis. On the other hand, when we treated the cells with lower dose of enzastaurin in combination with conventional chemotherapeutic agents, namely imatinib and ATRA (also in lower dose than IC50), respectively, we observe an increase in the cytotoxic effect.

Our preliminary results show that Enzastaurin shows efficacy as single agent and may increase the cytotoxic effect induced by imatinib and ATRA, witch suggests that this new drug may be used as a therapeutic approach in AML.