Abstract #874: The ribonucleoside analog, 8-chloro-adenosine, induces autophagy in breast cancer cells

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 a plateau of ~100 and ~300 \#956;M, respectively, by 6 h of treatment with 10 \#956;M 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 \#956;M 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 at Thr172. Once activated, AMPK then stimulates various energy regulating signaling cascades, most notable of which is the induction of autophagy. Because 8-Cl-Ado readily depletes the ATP pool, we wanted to determine if 8-Cl-Ado affects AMPK activity. Using immunoblot analysis, we demonstrated that while total AMPK protein levels were not perturbed by 8-Cl-Ado treatment, the phosphorylation of Thr172 AMPK was induced in a dose dependent manner (1-100 \#956;M). This was associated with 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, within 8 h of treatment. The levels of LC3B-II were further increased with bafilomycin A1 inhibition of lysosomal proteolysis, signifying lysosomal turnover; a hallmark feature of the final steps of autophagy. 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, monodansylcadaverine staining of 8-Cl-Ado treated MCF-7 cells demonstrated the formation autophagolysosomes. 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: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 874.