The Ribonucleoside Analog, 8-Chloro-Adenosine, Inhibits the mTOR Pathway and Induces Autophagy.

Abstract

Abstract In contrast to deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-chloro-adenosine (8-Cl-Ado) possesses a ribose sugar. This unique nucleoside analog is RNA directed and is in a phase I clinical trial for hematological malignancies. Preclinically, it has been also found to be cytotoxic to a number of epithelial and neuronal cell lines. Previously, we demonstrated that in the breast cancer cell lines, MCF-7 and BT-474, 8-Cl-Ado accumulates as a triphosphate reaching its plateau by 6 h of treatment with 10 μM 8-Cl-Ado. This accumulation was coupled with a parallel decrease in the level of the endogenous ATP pool by 60 and 40%. These changes in nucleoside levels then lead to ∼50% inhibition of RNA synthesis. The net effect of these metabolic changes results in a growth inhibition of ∼90% after 5 d treatment with 10 μM 8-Cl-Ado as measured by MTS assay. This growth inhibition was not due to a cell cycle arrest. Additionally, flow cytometry analysis of annexin V staining showed only a modest induction of apoptosis first detected within 24 h of treatment and reached ∼30% by 5 d. Because the low level of apoptosis can not account for high percent of growth inhibition suggests alternative mechanism(s) for the cytotoxic activity of 8-Cl-Ado. Depletion of the cellular ATP pool is often associated with the activation of AMP-activated protein kinase (AMPK) through its phosphorylation on Thr172. Correspondingly, we demonstrated that 8-Cl-Ado treatment of MCF-7 and BT-474 cells induced AMPK Thr172 phosphorylation within 7 h. Once activated, AMPK alters various energy regulating signaling cascades which includes the mTOR pathway. Inhibition of mTOR leads to a decrease of energy depletion by reducing protein translation and promotes nutrient restoration through the induction of autophagy. 8-Cl-Ado treatment of MCF-7 and BT-474 cells was associated with a decrease in the phosphorylation of the mTOR translation targets, 4E-BP1 and p70S6K within 8 h of treatment. Likewise, there was an increase in the conversion of microtubule-associated protein 1 light chain 3B (LC3B)-I to its lipidated form, LC3B-II, which is an autophagosome marker. Consistent with the immunoblot analysis, fluorescence microscopic analysis of 8-Cl-Ado-treated MCF-7 cells and BT-474 cells expressing a GFP-LC3 construct also demonstrated an increase in the formation of autophagosomes. Finally, acridine orange staining of 8-Cl-Ado treated MCF-7 and BT-474 cells demonstrated the formation autophagolysosomes. This was further confirmed in MCF-7 cells by monodansylcadaverine staining. In conclusion, our data indicates that 8-Cl-Ado treatment induces autophagy in breast cancer cells which in part elicits its growth inhibitory effects on these cells. Based on this biological activity, we are planning to test 8-Cl-Ado in the clinic for patients with breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6116.