ATP directed agent, 8-chloro-adenosine, induces AMP activated protein kinase activity, leading to autophagic cell death in breast cancer cells

Abstract

Background8-chloro-adenosine (8-Cl-Ado) is a unique ribonucleoside analog which is currently in a phase I clinical trial for hematological malignancies. Previously, we demonstrated in breast cancer cells that a 3-day treatment with 10 μM 8-Cl-Ado causes a 90% loss of clonogenic survival. In contrast, there was only a modest induction of apoptosis under these conditions, suggesting an alternative mechanism for the tumoricidal activity of 8-Cl-Ado.MethodsCellular metabolism, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway signaling, as well as autophagy induction was evaluated in breast cancer cell lines treated with 8-Cl-Ado. The effects of knocking down essential autophagy factors with small interfering RNA on 8-Cl-Ado-inhibited cell survival was assessed in breast cancer cells by examining apoptosis induction and clonogenic survival. In vivo efficacy of 8-Cl-Ado was measured in two breast cancer orthotopic model systems.ResultsWe demonstrate that in breast cancer cell lines, the metabolism of 8-Cl-Ado results in depletion of endogenous ATP that subsequently induces the phosphorylation and activation of the energy sensor, AMPK. This was associated with an attenuation of mTOR signaling and an induction of the phosphorylation of the autophagy factor, Unc51-like kinase 1 on Ser555. 8-Cl-Ado-mediated induction of autophagy was evident by increased aggregates of microtubule-associated protein 1 light chain 3B (LC3B) which was associated with its conversion to its lipidated form, LC3B-II, p62 degradative flux, and increased formation of acidic vesicular organelles. Additionally, transfection of MCF-7 cells with siRNA to ATG7 or beclin 1 provided partial protection of the cells to 8-Cl-Ado cytotoxicity as measured by clonogenicity. In vivo, 8-Cl-Ado inhibited growth of both MCF-7 and BT-474 xenograft tumors. Moreover, in 9 of 22 BT-474 tumors treated with 100 mg/kg/day 3 times a week, there was an absence of macroscopically detectable tumor after 3 weeks of treatment.ConclusionsOur data demonstrates that 8-Cl-Ado treatment activates the AMPK pathway leading to autophagy induction of in breast cancer cells, eliciting, in part, its tumoricidal effects. Additionally, 8-Cl-Ado effectively inhibited in vivo tumor growth in mice. Based on this biological activity, we are planning to test 8-Cl-Ado in the clinic for patients with breast cancer.

B-cell Receptor Signaling Regulates Metabolism in Chronic Lymphocytic Leukemia

“…Five technical replicates for each condition were plated. Glycolysis and cell mitochondrial stress tests were performed as described previously (Supplemental Figure 1) (14). Generally for OCR, data were expressed as basal OCR, which is the starting level of OCR (Supplemental Figure 1).…”

“…Perchloric acid was used to extract nucleotide pools, and pools were separated using HPLC as described previously (14). Standard NTPs were used to quantitate nucleotide pools and the concentration was determined based on mean cell volume.…”

“…Cells were washed and protein extracts were probed using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) as described previously (14). p-AKT Thr308, AKT, p-MAPK(44/42), MAPK, GAPDH (Cell Signaling Technology, Danvers, MA) and OxPhos cocktail (Abcam, Cambridge, UK) primary antibodies were used.…”

miR-493-5p attenuates the invasiveness and tumorigenicity in human breast cancer by targeting FUT4

“…Each year more than a half a million new cases of breast cancer are diagnosed in the US and Europe (3), and in China. Breast cancer is a very heterogeneous disease.…”

Strain Promoted Click Chemistry of 8-Azidopurine and 5-Azidopyrimidine Nucleosides and Nucleotides with Cyclooctynes and Applications to Living Cell Imaging

“…20 It is currently being tested for activity against breast cancer [21][22] and is in a Phase I clinical trial for the treatment of chronic lymphocytic leukemia. Sharpless described a set of criteria that a reaction must meet to be considered a click reaction.…”