Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways

<div><p>The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways.</p></div

Vitamin K2 inhibited the tumor growth in mouse bearing human bladder cancer cells.

<p><b>Nude mice with EJ transplant tumors were directly injected with 30 mg/kg vitamin K2 at tumors each day for 21 days</b>. (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.</p

Vitamin K2 triggered mitochondria-related apoptosis in human bladder cancer cells.

<p>(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.</p

Modified Inguinal Microscope-Assisted Varicocelectomy under Local Anesthesia: A Non-randomised Controlled Study of 3565 Cases

Abstract Varicocele is a common abnormality, but the conventional microsurgical subinguinal varicocelectomy (CMSV) has some disadvantages. We invented Modified Inguinal Microscope-Assisted Varicocelectomy (MIMV) under local anesthesia. This study aims to evaluate MIMV by comparing it to CMSV in operating duration, time to return to normal activity, postoperative complications, achievement of natural pregnancy and improvement of semen quality for patients with infertility, pain score for those with scrotal pain, and so on. We enrolled 3089 patients who underwent MIMV and 476 who underwent CMSV in our hospital. Both the operating duration and the time to return to normal activity of MIMV was shorter than that of CMSV (P < 0.001). The recurrence rate (P < 0.001) and injury rate of vas deferens (P = 0.011) after MIMV were lower than that after CMSV. Moreover, patients with MIMV showed higher degree of satisfaction with the surgery experience and outcome than those with CMSV (P < 0.001). However, no statistical difference was found between the two groups in scores of pain due to surgery, postoperative varicose veins diameters, reflux duration, and the postoperative complications of wound infection, hydrocele, atrophy of testis, epididymitis, and scrotal hematoma. In summary, MIMV is a promising varicocelectomy and could be applied more in clinical practice

ROS mediated the mitochondria dysfunction and regulated activation of JNK/p38 in vitamin K2-triggered apoptosis of human bladder cancer T24 cells.

<p>(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.</p

Vitamin K2 induced apoptotic cell death in human bladder cancer cells.

<p>(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.</p

Schematic diagram of pathway involved in vitamin K2-induced apoptosis in human bladder cancer cells.

<p>Schematic diagram of pathway involved in vitamin K2-induced apoptosis in human bladder cancer cells.</p