Figure 1.
Adenosine (Ade) increases intracellular ATP contents in multiple cells.
(A) Primary thymocytes were exposed to either vehicle DMSO (DM) or Ade in normal culture medium for 4 h and cells were collected for total ATP assay by HPLC (LC-6AD; Shimadzu). ATP contents (µM) of equal number of cells (2×105) were compared (n = 4). Each column represents the average of independent repeated experiments. Mean ±SD. *p<0.05 compared to controls. (B) K562 cells were treated with indicated doses of Ade or oligomycin (Oli; 1 µg/ml) in the absence of d-glucose in RPMI 1640 medium for 6 h. d-glucose (2 g/L) was used as a positive control. Mean ±SD (n = 4). *p<0.05 vs. control; #p<0.05 vs. Oli treatment alone. (C) K562 was exposed to 2 mM of Ade for 0.5, 2, and 6 h in the absence of d-glucose in the culture medium. Mean ±SD. *p<0.05 vs. 0.5 h treatment. (D) Increase of ATP in multiple cell lines: A549, MCF7, and Hela cells were exposed to either DMSO or 2 mM of Ade for 6 h in the absence (G-) or presence of d-glucose (2 g/L, G+) in the culture medium. ATP contents were detected by HPLC (n = 4) and the increase of ATP after Ade treatment was calculated as: Ade-treated/vehicle-treated. All controls were set as 1.0.
Figure 2.
Ade decreases cell viability and induces cell death.
(A) A549, MCF7, and Hela cells were exposed to Ade as indicated in normal culture medium for 72 h. Cell viability was detected using the MTS assay. Each column represents the average of five replicated experiements. Mean ±SD (n = 5). p<0.05, vs. vehicle control. (B) Ana-1 cells were treated with Ade for 72 h, cell viability was detected as in (A). Mean ±SD (n = 5). *p<0.05, **p<0.01, vs. vehicle control. (C, D, E) Ana-1 cells were incubated with Ade in normal culture medium for 12 h, then cell apoptosis was detected by either flow cytometry (FACScan; BD Biosciences) or Western blot. Representative cell death image and cell death data in Ana-1 cells are shown in (C, D). Mean ±SD (n = 3). *p<0.05, **p<0.01 vs. vehicle control. PARP cleavage is shown in (E). GAPDH was used as a loading control. (F, G) Thymus lymphocytes were incubated with Ade as indicated for 12 h, cell death was detected. Cell death images by PI staining in living cells under an inverted fluorescence microscope were shown in (F) and cell death data by flow cytometry are summarized in (G). **p<0.01 vs. vehicle control. Mean ±SD (n = 3).
Figure 3.
Ade increases cell viability in low ATP states and rescued cell death induced by ATP-depletion.
(A, B) K562 cells were exposed to 2 mM of Ade cultured in RPMI 1640 medium with or without d-glucose for different time points. Cell density was imaged using an inverted microscope (Axio Obsever Z1; Zeiss, Germany). Typical images of cell density were selected from cells treated with Ade in d-glucose-free medium for 24 h (A) or in d-glucose-containing medium for 72 h (B). Scale bar = 50 µm. (C) K562 cells were exposed to Ade for 24 h (left) or 48 h (right) with or without d-glucose; absolute cell numbers were counted using a cell counter. Mean ±SD (n = 3). *p<0.05 vs. d-glucose-containing cells. (D) K562 cells were incubated with Ade with or without d-glucose for 36 h. Cell viability was detected using the MTS assay. Mean ±SD (n = 3). *p<0.05 vs. glucose-containing cells. (E, F) K562 cells were treated with Oli with or without Ade (2 mM) in the d-glucose-free RPMI 1640 medium for 6 h, then cell apoptosis was detected by flow cytometry. Typical flow images are shown in (E) and cell death in (F). Mean ±SD (n = 3). *p<0.05 vs. Ade-treated cells. (G) K562 cells were treated with Oli (1 µg/ml) and Ade (2 mM) for 18 h in the glucose-free medium, and cells were then stained with PI and dynamically recorded under an inverted epi-fluorescent microscope. A typical image is shown. Scale bar = 50 µM.
Figure 4.
Ade transportation, but not Ade receptors, is required for Ade to exert its cytotoxic or cytoprotective effects.
(A) K562 cells cultured in glucose-free culture medium were treated with Oli (0.5 µg/ml), Ade (2 mM) and DP (10 µM) for 3 h, followed by HPLC ATP assay. Mean ±SD (n = 3). *p<0.05 vs. Oli+Ade treatment. (B) K562 cells were cultured and treated in the d-glucose-free medium as indicated for 18 h, and cells were then stained with Annexin V/PI followed by flow cytometry. Viable cells are shown. Mean ±SD (n = 3). *p<0.05 each compared with Oli treatment alone; #p<0.05 each compared with Oli+Ade combination treatment. (C) K562 cells were cultured in normal medium and exposed to 2 mM Ade and DP (10 µM) for 18 h, cell numbers were counted using a cell counter. Mean ±SD (n = 3). *p<0.05 compared with vehicle control; #p<0.05 compared with Ade treatment alone. (D) As treated in (B), typical flow images are shown (Ade: 2 mM, Oli: 1.0 µg/ml, DP: 10 µM). (E) K562 cells were cultured in d-glucose-free medium and treated with the agents as indicated (Oli: 1.0 µg/ml, Ade: 2 mM, 8-SPT: 10 µM) for 4 h followed by ATP assay. Mean ±SD (n = 3). (F) K562 cells were cultured in normal glucose-containing medium and treated as indicated (Ade: 2 mM, 8-SPT: 10 µM) for 18 h, cell numbers were counted and summarized. Mean ±SD (n = 3). (G) K562 cells were cultured in glucose-free medium and treated as indicated (Oli: 1.0 µg/ml, 8-SPT: 10 µM) for 15 h, cell death was detected by flow cytometry. Mean ±SD (n = 3).
Figure 5.
Ade increases the sensitivity to proteasome inhibition-induced cytotoxicity.
(A,B) K562 cells were treated with proteasome inhibitor MG132 (5 µM) for 15 h in the presence of various doses of Ade; cell apoptosis was detected using Annexin V/PI staining by flow cytometry. Typical flow images are shown in (A) and the cell death data summarized in (B). Mean ±SD (n = 3).*p<0.05 vs. MG132 treatment alone; #p<0.05 vs. control. (C, D) As treated in (A) dynamically, cell death was detected with PI staining in live cells under an inverted microscope. Typical images at 12 h and 24 h are shown in (C) and (D) respectively. (E) K562 cells were treated with MG132 (5 µM), Ade (2 mM) and the combination for 12 h, PARP was detected by Western blot. GAPDH was used as a loading control.
Figure 6.
Oligomycin decreases proteasome inhibition-induced cell death in the presence of Ade.
(A, B) K562 cells cultured in d-glucose-free medium were exposed to Ade (0.2 and 2 mM), MG132 (5 µM, or MG262 (1 µM) and their combinations; PI staining was dynamically recorded under a fluorescent microscope, typical images at 12 h are shown in (A) and (B). (C, D) K562 cells were treated with Oli (1 µM), MG132 (5 µM), or MG262 (1 µM) and their combinations in the absence or presence of Ade (2 mM) for 12 h; cell apoptosis was detected using Annexin V/PI staining. Typical images are shown in (C) and a summary of cell death is shown in (D). Mean +SD (n = 3). *p<0.05 vs. proteasome inhibitor treatment alone.