The online version of this article (doi:10.1186/1756-8722-7-23) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interest.
CMS wrote the manuscript, performed transfections experiments, immunoblot, HPLC, mouse xenograft, flow cytometry and microscopic analyses, analyzed and designed all experiments; HVP performed and analyzed glycolysis and aerobic respiration analysis; XFL and WM provided assistance and guidance in mouse xenograft studies; SS performed immunoblot and mouse xenograft analysis and prepared samples for HPLC and flow cytometry. All authors read and approved the final manuscript.
8-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.
Cellular 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.
We 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.
Our 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.
Additional file 1: Figure S1: Effect of 8-Cl-Ado on the survival of breast cancer cells. Figure S2: Accumulation of 8-Cl-ATP and effects on ATP production. Figure S3: 8-Cl-Ado-induces autophagy. (PDF 101 KB)13045_2014_447_MOESM1_ESM.pdf
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Lange-Carter CA, Vuillequez JJ, Malkinson AM: 8-Chloroadenosine mediates 8-chloro-cyclic AMP-induced down-regulation of cyclic AMP-dependent protein kinase in normal and neoplastic mouse lung epithelial cells by a cyclic AMP-independent mechanism. Cancer Res. 1993, 53: 393-400. PubMed
Van Lookeren Campagne MM, Villalba Diaz F, Jastorff B, Kessin RH: 8-Chloroadenosine 3′,5′-monophosphate inhibits the growth of Chinese hamster ovary and Molt-4 cells through its adenosine metabolite. Cancer Res. 1991, 51: 1600-1605. PubMed
Gandhi V, Ayres M, Halgren RG, Krett NL, Newman RA, Rosen ST: 8-chloro-cAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma cells. Cancer Res. 2001, 61: 5474-5479. PubMed
Stellrecht CM, Rodriguez CO, Ayres M, Gandhi V: RNA-directed actions of 8-chloro-adenosine in multiple myeloma cells. Cancer Res. 2003, 63: 7968-7974. PubMed
Ghias K, Ma C, Gandhi V, Platanias LC, Krett NL, Rosen ST: 8-Amino-adenosine induces loss of phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Akt kinase: role in induction of apoptosis in multiple myeloma. Mol Cancer Ther. 2005, 4: 569-577. 10.1158/1535-7163.MCT-04-0303. CrossRefPubMed
Langeveld CH, Jongenelen CA, Theeuwes JW, Baak JP, Heimans JJ, Stoof JC, Peters GJ: The antiproliferative effect of 8-chloro-adenosine, an active metabolite of 8-chloro-cyclic adenosine monophosphate, and disturbances in nucleic acid synthesis and cell cycle kinetics. Biochem Pharmacol. 1997, 53: 141-148. 10.1016/S0006-2952(96)00593-X. CrossRefPubMed
Langeveld CH, Jongenelen CA, Heimans JJ, Stoof JC: Growth inhibition of human glioma cells induced by 8-chloroadenosine, an active metabolite of 8-chloro cyclic adenosine 3′:5′-monophosphate. Cancer Res. 1992, 52: 3994-3999. PubMed
Krett NL, Zell JL, Halgren RG, Pillay S, Traynor AE, Rosen ST: Cyclic adenosine-3′,5′-monophosphate-mediated cytotoxicity in steroid sensitive and resistant myeloma. Clin Cancer Res. 1997, 3: 1781-1787. PubMed
Halgren RG, Traynor AE, Pillay S, Zell JL, Heller KF, Krett NL, Rosen ST: 8Cl-cAMP cytotoxicity in both steroid sensitive and insensitive multiple myeloma cell lines is mediated by 8Cl-adenosine. Blood. 1998, 92: 2893-2898. PubMed
Cheong J-H, Park ES, Liang J, Dennison JB, Tsavachidou D, Nguyen-Charles C, Wa Cheng K, Hall H, Zhang D, Lu Y, Ravoori M, Kundra V, Ajani J, Lee J-S, Ki Hong W, Mills GB: Dual inhibition of tumor energy pathway by 2-deoxyglucose and metformin is effective against a broad spectrum of preclinical cancer models. Mol Cancer Ther. 2011, 10: 2350-2362. 10.1158/1535-7163.MCT-11-0497. PubMedCentralCrossRefPubMed
Shanmugam M, McBrayer SK, Qian J, Raikoff K, Avram MJ, Singhal S, Gandhi V, Schumacker PT, Krett NL, Rosen ST: Targeting glucose consumption and autophagy in myeloma with the novel nucleoside analogue 8-aminoadenosine. J Biol Chem. 2009, 284: 26816-26830. 10.1074/jbc.M109.000646. PubMedCentralCrossRefPubMed
Bartek J, Iggo R, Gannon J, Lane DP: Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines. Oncogene. 1990, 5: 893-899. PubMed
Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, Vasquez DS, Joshi A, Gwinn DM, Taylor R, Asara JM, Fitzpatrick J, Dillin A, Viollet B, Kundu M, Hansen M, Shaw RJ: Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011, 331: 456-461. 10.1126/science.1196371. PubMedCentralCrossRefPubMed
Stellrecht CM, Shentu S, Gandhi V: In vivo activity of the ribonucleoside analog, 8-chloro-adenosine, in breast cancer mouse models. Proceedings AACR. 2013, 52: 5506-
Stellrecht CM, Krett N, Ayres M, Rosen S, Gandhi V: 8-chloro-cAMP serves as a prodrug for the RNA directed nucleoside analog, 8-chloro-adenosine. Acute leukemia IX: Basic Research, Experimental Approaches and Novel Therapies. Volume 9. Edited by: Hiddemann W, Büchner T, Ritter J, Unterhalt M, Haferlach T. 2003, Heidelberg, Germany: Springer, 193-199. CrossRef
Liang J, Shao SH, Xu ZX, Hennessy B, Ding Z, Larrea M, Kondo S, Dumont DJ, Gutterman JU, Walker CL, Slingerland JM, Mills GB: The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat Cell Biol. 2007, 9: 218-224. 10.1038/ncb1537. CrossRefPubMed
Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I: Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res. 2003, 63: 2103-2108. PubMed
- ATP directed agent, 8-chloro-adenosine, induces AMP activated protein kinase activity, leading to autophagic cell death in breast cancer cells
Christine M Stellrecht
Hima V Vangapandu
- BioMed Central
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