Fig 1.
Clozapine inhibited cancerous growth of MCF-7 cells.
(A) MCF-7 cells were seeded into 24-well plates at a density of 2 × 104 cells/well for 24h, followed by incubating with the indicated concentration of clozapine for 1 to 3 days. Cell proliferation was then measured using an MTT assay. (B) 500 cells were seeded in a 60 mm Petri dish and incubated with clozapine at various concentrations for 14 days. Representative images of colony formation assays were shown. (C) Colonies were stained with crystal violet and quantified. Data were presented as mean ± standard deviation (S.D.) P value below 0.05 was denoted with the asterisks, where * indicates < 0.05; ** means < 0.01; *** signifies < 0.001.
Fig 2.
Clozapine arrested cell cycle of MCF-7 cell.
(A) MCF-7 cells were treated with the indicated concentration of clozapine for 72 h, harvested, stained with propidium iodide, and analyzed using a flow cytometer. Data were presented as mean ± standard deviation (S.D.) (B) MCF-7 cells were treated with 50 μM clozapine for 6, 12, 18, and 24h, after which total protein lysates were collected and analyzed by western blotting using antibodies against cell-cycle-related proteins. β-actin was used as an internal control to confirm equal protein loading. P value below 0.05 was designated with the asterisks, where * indicates < 0.05; ** means < 0.01; *** signifies < 0.001.
Fig 3.
Clozapine modulated apoptosis and autophagy in MCF-7 cells.
MCF-7 cells were seeded into 6-well plates at a density of 105 cells/well and treated with an indicated concentration of clozapine for 72 h. Cells were harvested and stained with (A) 0.25 μg/mL of acridine orange and (C) fluorescein-conjugated Annexin V antibody and propidium iodide for flow cytometric analysis of autophagy and apoptosis, respectively. (B) The protein levels of autophagic and apoptotic modulators and mediators, with or without 50 μM of clozapine treatment, were measured by western blotting. (D) The fragmented DNA resulting from apoptosis was fluorescently imaged using a TUNEL assay. Results were presented as mean ± standard deviation (S.D.) ** means < 0.01; *** signifies < 0.001.
Fig 4.
Clozapine suppressed MCF-7 cell growth via modulation of ROS.
(A) MCF-7 cells were incubated with the indicated concentration of clozapine for up to 72 h and labeled with H2DCFDA (10 μM) to measure the total intracellular ROS content by flow cytometry. (B and C) Cells were treated with or without vitamin E (α-Tocopherol) to determine. (B) cell proliferation by MTT assay or (C) intracellular ROS content by flow cytometry. Cells without treatment were assigned 100% viability to emphasize the proliferation changes following clozapine treatment. Results were presented as mean ± standard deviation (S.D.). P value below 0.05 was denoted with the asterisks, where * indicates < 0.05; ** means < 0.01; *** signifies < 0.001.
Fig 5.
Clozapine enhanced apoptosis and autophagy of MCF-7 cells via modulation of ROS.
MCF-7 cells were incubated with clozapine for 72 h, with or without treatment with vitamin E, and then (A) stained with acridine orange to identify autophagic cells or (B) with fluorescein-conjugated Annexin V antibody and propidium iodide to identify apoptotic cells, followed by flow cytometry analysis. (C) Autophagy- and apoptosis-associated proteins in clozapine-treated MCF-7 cells were collected and measured by western blotting. Data were plotted as mean ± standard deviation (S.D.). P value below 0.05 was denoted with the asterisks, where * indicates < 0.05; ** means < 0.01; *** signifies < 0.001.
Fig 6.
Chloroquine boosted apoptosis induced by clozapine.
MCF-7 cells were treated with 50 μM of clozapine for 72 h, with or without chloroquine, followed by measuring (A) cell proliferation via MTT assay, (B) intracellular ROS content by H2DCFDA uptake assay, and (C) apoptotic percentages by flow cytometry. (D) Total protein lysates from each treatment of clozapine and/or chloroquine were collected to assess cleaved caspase 9 levels after 3 days of incubation. Data were plotted as mean ± standard deviation (S.D.). P value below 0.05 was denoted with the asterisks, where * indicates < 0.05; ** means < 0.01.
Fig 7.
Clozapine exhibited cytotoxicity against MDA-MB-231 cells.
(A) MDA-MB-231 cells were treated with 50 μM of clozapine for the indicated concentration and time points, up to 72 h, followed by an MTT assay to determine the cell proliferation. (B-D) α-Tocopherol (vitamin E) was used to counteract ROS triggered by clozapine after 3 days of incubation, followed by (B) cell proliferation assessed by MTT assay, (C) autophagic cells, and (D) intracellular ROS content, with the latter two assays were conducted by flow cytometry. Data were presented as mean ± standard deviation (S.D.). P value below 0.05 was denoted with the asterisks, where *** means < 0.001.