Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the protein kinase CB isozyme. The objective of this study was to assess the efficacy of enzastaurin in inducing apoptosis in multiple myeloma (MM) cell lines and to investigate possible mechanisms of apoptosis. Cell proliferation assays were done on a variety of MM cell lines with unique characteristics (dexamethasone sensitive, dexamethasone resistant, chemotherapy sensitive, and melphalan resistant). The dexamethasonesensitive MM.1S cell line was used to further assess the effect of enzastaurin in the presence of dexamethasone, insulin-like growth factor-I (IGF-I), interleukin-6, and the pan-specific caspase inhibitor ZVAD-fmk. Enzastaurin increased cell death in all cell lines at clinically significant low micromolar concentrations (1 -3 Mmol/L) after 72 hours of treatment. Dexamethasone and enzastaurin were shown to have an additive effect on MM.1S cell death. Although IGF-I blocked the effect of 1 Mmol/L enzastaurin, IGF-I did not abrogate cell death induced with 3 Mmol/L enzastaurin. Moreover, enzastaurin-induced cell death was not affected by interleukin-6 or ZVAD-fmk. GSK3B phosphorylation, a reliable pharmacodynamic marker for enzastaurin activity, and AKT phosphorylation were both decreased with enzastaurin treatment. These data indicate that enzastaurin induces apoptosis in MM cell lines in a caspase-independent manner and that enzastaurin exerts its antimyeloma effect by inhibiting signaling through the AKT pathway. [Mol Cancer Ther 2006;5(7):1783 -9]
Multiple myeloma (MM) is a malignancy of clonal B-cells that accounts for 10% of all hematologic malignancies. We have shown previously that a novel purine analogue, 8-chloro-adenosine, has significant activity for MM in preclinical studies. Objective: Using MM cell lines, we investigated the molecular mechanism of related congener of adenosine, 8-amino-adenosine (8-NH2-Ado). Methods: We employed biological and biochemical assays in MM cell lines to evaluate the clinical potential of 8-NH2-Ado. Results: In MM cell lines both sensitive and resistant to conventional chemotherapies, 8-NH2-Ado is cytotoxic, with IC50 ranging from 300 nmol/L to 3 μmol/L. A mouse leukemic cell line lacking adenosine kinase activity was resistant to 8-NH2-Ado, indicating that phosphorylation of 8-NH2-Ado to its triphosphate form is required for cytotoxicity. A 4-hour incubation of MM cells with 10 μmol/L analogue resulted in an accumulation of >7 mmol/L 8-NH2-ATP with a parallel decline in the endogenous ATP levels. Accumulation of 8-NH2-ATP was dependent on both exogenous concentration of 8-NH2-Ado and incubation time. The accumulation of 8-NH2-ATP was accompanied by a decrease in both RNA and DNA synthesis. The mechanism of 8-NH2-Ado-mediated cytotoxicity was due to apoptosis as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a sub-G1 DNA content. Conclusion: Based on these results, we conclude that 8-NH2-Ado may hold great potential as a therapeutic agent for the treatment of MM.
The protein kinase C (PKC) family consists of 11 serine/threonine protein kinase isoforms that are involved with cell proliferation and differentiation, gene transcription, and tumor-induced angiogenesis. The PKC pathway has been shown to play a role in the regulation of cell growth in several hematologic malignancies. However, in multiple myeloma (MM), the role of the PKC pathway has not been extensively studied. Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the PKC β isozyme. Enzastaurin has been reported to induce apoptosis and suppress proliferation in the HCT116 colon cancer cell line by inhibiting the AKT pathway. The objective of this study was to assess the efficacy of Enzastaurin in inducing apoptosis in MM cell lines and to investigate possible mechanisms of apoptosis. A spectrum of MM cell lines, with unique characteristics (dexamethasone-sensitive, dexamethasone-resistant, chemotherapy-sensitive, chemotherapy-resistant) were treated with Enzastaurin. There is evidence of cell death in all cell lines at clinically significant concentrations (1–3μM) after 72 hours of treatment. The dexamethasone-sensitive MM1.S cell line was used to further assess the effect of Enzastaurin in the presence of dexamethasone, insulin-like growth factor-1 (IGF-1), and IL-6. IGF-1 and IL-6 are potent growth factors for MM and have been observed to blunt the cytotoxic activity of dexamethasone. Dexamethasone and Enzastaurin appear to have an additive effect in the induction of apoptosis. In addition, while IGF-1 slightly decreases the effect of Enzastaurin, IL-6 has no effect. Enzastaurin treatment also induces a decrease in GSK3β and AKTSerine473 phosphorylation. GSK3β phosphorylation is thought to be a reliable pharmacodynamic marker for Enzastaurin activity. In conclusion, these data indicate that Enzastaurin induces apoptosis in MM cells and suppression of the AKT pathway may be one of the mechanisms by which Enzastaurin exerts its anti-myeloma activity.
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