Fig 1.
Effect of vitamin K2 on the viability of three human bladder cancer cells.
(A). Vitamin K2 dose-dependently reduced the viability of human bladder cancer T24, J82 and EJ cells. Cells were treated different concentration of vitamin K2 for 24 hours, respectively and cell viability was measured by MTT assays. (B). Vitamin K2 time-dependently decreased the viability of T24, J82 and EJ cells. Cells were treated with 100 μM vitamin K2 for 0, 6, 12, 18 and 24 hours, respectively, and cell viability was evaluated by MTT assays. Data represent the mean ± SEM of three different experiments with triplicate sets in each assay. * P<0.05, ** P<0.01 and *** P<0.001 vs vitamin K2-untreated group.
Fig 2.
Vitamin K2 induced apoptotic cell death in human bladder cancer cells.
(A). T24 cells were treated with the indicated concentration of vitamin K2 and the apoptosis was evaluated with Annexin V-FITC/PI dyes and measured by Flow cytometry. (B). The quantification of apoptotic death in vitamin K2-treated T24 cells. (C). Flow cytometry showed that vitamin K2 induced the apoptotic death in another two human bladder cancer J82 and EJ cells. (D). The effect of vitamin K2 on apoptosis in T24 cells was determined by TUNEL method using a detecting kit. Scale bar: 100μm (E). Western blots indicated that vitamin K2 induced activation of caspase-3 and cleavage of PARP in T24 cells. (F) Vitamin K2 inhibited the caspase-3-dependent viability of T24 cells by MTT assays. 10μM Z-DEVD-FMK, a caspase-3 inhibitor, was pretreated for 1 hours before exposure of 100 μM vitamin K2 to T24 cells for 24 hours. (G). Z-DEVD-FMK, a caspase-3 inhibitor, remarkably attenuated the apoptosis in vitamin K2-treated T24 cells. Cell apoptosis was evaluated with Annexin V-FITC/PI dyes and measured by Flow cytometry. * P<0.05, ** P<0.01 and *** P<0.001.
Fig 3.
Vitamin K2 triggered mitochondria-related apoptosis in human bladder cancer cells.
(A). T24 cells were treated with the indicated concentration of vitamin K2 for 24 hours and the disruption of mitochondria membrane potential was measured using a specific mitochondria dye Rhodamine 123 by flow cytometry. M1 stands for the percentage of cells with low mitochondria membrane potential. (B). Quantification of T24 cells with low mitochondria membrane potential. (C). J82 and EJ cells were treated the indicated concentration of vitamin K2 for 24 hours and cells with low mitochondria membrane potential was determined using the Rhodamine 123 dye by flow cytometry. (D). T24 cells were treated by the indicated concentration of vitamin K2 for 24 hours, then cells were harvested and separated into cytosolic and mitochondrial fractions using a commercial kit. The expression of cytochrome C in cytosol and mitochondria was evaluated by western blots. (E) T24 cells were treated with 100μM vitamin K2 for 0, 12, 18, 24 hours respectively, then the total proteins were isolated from the cells and the expression of Bax and Puma were analyzed by western blots. * P<0.05, ** P<0.01 and *** P<0.001.
Fig 4.
Activation of JNK/p38 is required for vitamin K2-triggered apoptosis in human bladder cancer T24 cells.
(A). T24 cells were treated with the indicated concentration of vitamin K2 for 24 hours, then the total proteins were isolated from the cells and the phosphorylation of JNK/p38 was analyzed by western blots. (B). Western blots indicated that vitamin K2 at the concentration of 100 μM induced sustained phosphorylation of JNK/p38 in T24 cells. (C). T24 cells were treated with 40 μM SP600125(SP), a pharmacological inhibitor of JNK activation, for 1 hour before treatment with 100 μM vitamin K2 for 24 hours and cell viability was evaluated by MTT assays. (D). T24 cells were treated 40 μM SP600125(SP) for 1 hour before treatment with 100 μM vitamin K2 for 12 hours and apoptotic death was assessed by flow cytometry. (E). T24 cells were treated with 10 μM SB203580(SB), a pharmacological inhibitor of p38 activation, for 1 hour before treatment with 100 μM vitamin K2 for 24 hours, then cell viability was assessed by MTT assays. (F). T24 cells were pre-treated with 10 μM SB203580(SB) for 1 hour, then treated with 100 μM vitamin K2 for 12 hours and apoptotic death was determined by flow cytometry. (G). T24 cells were treated with 40μM SP600125(SP) for 1 hour prior to treatment with 100 μM vitamin K2 for 24 hours. The total proteins extracted from the cells were assessed by western blots. (H). T24 cells were treated with 10 μM SB203580(SB) for 1 hours before treatment with 100 μM vitamin K2 for 24 hours, the total protein was evaluated by western blots. * P<0.05, ** P<0.01 and *** P<0.001.
Fig 5.
Vitamin K2 induced ROS-mediated apoptosis in human bladder cancer cells.
(A). T24 cells were treated with the indicated concentration of vitamin K2 for 24 hours and the intracellular ROS generation was evaluated using the DCFH-DA probe by flow cytometry. M reflects the positive DCF fluorescence (B). Quantification of the intracellular ROS generation in vitamin K2-treated T24 cells. (C). J82 and EJ cells were treated with the indicated concentration of vitamin K2 for 24 hours and intracellular ROS generation was assessed by flow cytometry. (D and E). T24 cells were treated with 5mM antioxidant N-acetyl cysteine (NAC) for 1 hour before the treatment with or without 100 μM vitamin K2 for 24 hours and the apoptotic death was determined by flow cytometry. (F). T24 cells were treated with 5mM antioxidant NAC for 1 hour before the treatment with or without 100 μM vitamin K2 for 12 hours and intracellular ROS generation was evaluated using the DCFH-DA probe by flow cytometry. (G). Activation of caspase-3 and cleavage of PARP were analyzed by western blots after T24 cells were treated with 5mM NAC for 1 hour before the treatment with or without 100 μM vitamin K2 for 24 hours. * P<0.05, ** P<0.01 and *** P<0.001.
Fig 6.
ROS mediated the mitochondria dysfunction and regulated activation of JNK/p38 in vitamin K2-triggered apoptosis of human bladder cancer T24 cells.
(A). T24 cells were treated with 5mM antioxidant NAC for 1 hour prior to the treatment with or without 100 μM vitamin K2 for 24 hours, then the mitochondria membrane potential was assessed using the Rhodamine 123 dye by flow cytometry. (B). The expression of Bax, Puma and Bcl-2 were changed after treatment with 100 μM vitamin K2 for 24 hours in the present or absent of 5mM antioxidant N-acetyl cysteine (NAC) to human bladder cancer T24 cells. (C). T24 cells were treated 40 μM SP600125(SP) for 1 hour before treatment of 100 μM vitamin K2 for 24 hours, mitochondria membrane potential was evaluated using Rhodamine 123 dye by flow cytometry. (D). T24 cells were treated 10 μM SB203580(SB) for 1 hour before treatment of 100 μM vitamin K2 for 24 hours, mitochondria membrane potential was evaluated using Rhodamine 123 dye by flow cytometry. (E). T24 cells were treated with 5mM NAC for 1 hour before exposure to 100 μM vitamin K2 for 24 hours, then the total proteins were isolated from the cells and activation of JNK/p38 were determined by western blots. ** P<0.01 and *** P<0.001.
Fig 7.
Vitamin K2 inhibited the tumor growth in mouse bearing human bladder cancer cells.
Nude mice with EJ transplant tumors were directly injected with 30 mg/kg vitamin K2 at tumors each day for 21 days. (A). Tumor volume changed after administration with 30 mg/kg vitamin K2 everyday. (B). Measurement of tumor volume in mice after treatment with or without 30 mg/kg vitamin K2 each day for 21 days before sacrificed the nude mice. (C). After 21 days treatments, mice were sacrificed and tumors were excised to sections. Activation of caspase-3 in the sections was measured with the immuno-histo-chemistry method using antibody against caspase-3. Staining TUNEL and HE in the sections was measured by the commercial kits, respectively. Scale bar: 50μm.
Fig 8.
Schematic diagram of pathway involved in vitamin K2-induced apoptosis in human bladder cancer cells.