Effect of DR4 Demethylation to the Proliferation and Apoptosis of Myeloid Leukemia K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of tumor necrosis factor death receptor (DR) 4 demethylation to the proliferation and apoptosis of myeloid leukemia K562 cells.@*METHODS@#The logarithmic phase of K562 cells were treated by desitabine (DCA) at 0, 0.8, 1.6 and 3.2 μmol/L, and the cells were divided into control group, DCA low dose group, DCA medium dose group and DCA high dose group respectively. The cells in control group were treated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 0.5 μg/ml for 24 h, and the cells were divided into TRAIL group. The cells in DCA high dose group were treated by TRAIL 0.5 μg/ml for 24 h, and were divided into DCA high dose + TRAIL group. Methylation-specific polymerase chain reaction (MS-PCR) was used to measure the methylation status of the DR4 gene promoter in the control group and DCA low, medium and high dose groups. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to determine the relative expression of DR4 mRNA and protein in the control group and DCA low, medium and high dose groups. Dime- thylthiazole (MTT) method was used to determine the inhibition rate of cell proliferation of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. Flow cytometry was used to determine the apoptotic rate of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group.@*RESULTS@#The cells in the control group were methylation-positive, the brightness of the methylation bands of the cells in the DCA low, medium, and high dose groups was gradually decreased to disappear, and the DCA high dose group showed negative for methylation. The relative expression of DR4 mRNA and protein in the control group, DCA low, medium and high dose groups was increased sequentially (r=0.624, 0.704). The inhibition rate of cell proliferation of the cells in the control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group was increased sequentially (r=0.653, 0.754, 0.709, 0.725) at 24, 48 and 72 h.@*CONCLUSION@#DCA can reverse the methylation level of DR4 gene promoter in ML K562 cells and up-regulate the expression of DR4, which may enhance the proliferation inhibition and apoptosis promotion effects of TRAIL on K562 cells.

Andrographolide sensitizes prostate cancer cells to TRAIL-induced apoptosis / 亚洲男科学杂志(英文版)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy. The identification of small molecules that can establish the sensitivity of prostate cancer (PCa) cells to TRAIL-induced apoptosis is crucial for the targeted treatment of PCa. PC3, DU145, JAC-1, TsuPr1, and LNCaP cells were treated with Andrographolide (Andro) and TRAIL, and the apoptosis was measured using the Annexin V/PI double staining method. Real time-polymerase chain reaction (PCR) and Western blot analysis were performed to measure the expression levels of target molecules. RNA interference technique was used to down-regulate the expression of the target protein. We established a nude mouse xenograft model of PCa, which was used to measure the caspase-3 activity in the tumor cells using flow cytometry. In this research study, our results demonstrated that Andro preferentially increased the sensitivity of PCa cells to TRAIL-induced apoptosis at subtoxic concentrations, and the regulation mechanism was related to the up-regulation of DR4. In addition, it also increased the p53 expression and led to the generation of reactive oxygen species (ROS) in the cells. Further research revealed that the DR4 inhibition, p53 expression, and ROS generation can significantly reduce the apoptosis induced by the combination of TRAIL and Andro in PCa cells. In conclusion, Andro increases the sensitivity of PCa cells to TRAIL-induced apoptosis through the generation of ROS and up-regulation of p53 and then promotes PCa cell apoptosis associated with the activation of DR4.

The Synergistic Apoptotic Interaction of Indole-3-Carbinol and Genistein with TRAIL on Endometrial Cancer Cells

Induction of apoptosis in target cells is a key mechanism by which chemotherapy promotes cell killing. The purpose of this study was to determine whether Indole-3-Carbinol (I3C) and Genistein in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induce apoptosis in endometrial cancer cell (Ishikawa) and to assess apoptotic mechanism. The MTT assay and flow cytometry were performed to determine cell viability and cell cycle. The induction of apoptosis was measured by caspase-3 activity test, DNA fragmentation assay, annexin V binding assay and western blot analysis. There was no effect in cell growth inhibition and cell cycle progression alone or in two-combination. However, the treatment of I3C and Genistein followed by TRAIL showed significant cell death and marked increase in sub-G1 arrest. Three-combination treatment revealed elevated expression of DR4, DR5 and cleaved forms of caspase-3, caspase-8, PARP. The Flip was found down regulated. Moreover, increase in caspase-3 activity and DNA fragmentation indicated the induction of apoptosis. The results indicate that I3C and Genistein with TRAIL synergistically induced apoptosis via death receptor dependent pathway. Our findings might provide a new insight into the development of novel combination therapies against endometrial cancer.

Bisphosphonate enhances TRAIL sensitivity to human osteosarcoma cells via death receptor 5 upregulation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, is one of the most promising candidates for cancer therapeutics. However, many osteosarcomas are resistant to TRAIL. Bisphosphonates are very effective in the treatment of bone problems associated with malignancies; the antitumor effects are due to the inhibition of protein prenylation that is essential for cell function and survival. The purpose of this study was to determine the effects of bisphosphonates on TRAIL-resistant MG 63 human osteosarcoma cells. The cells showed no response to TRAIL alone; however, pre-treatment with bisphosphonates significantly increased TRAIL-mediated apoptosis and cellular activation of caspase-3. Bisphosphonates significantly induced mRNA and protein expression of the TRAIL receptor, DR5. Bisphosphonates induced protein unprenylation in MG 63 cells; in addition, co-treatment with TRAIL also significantly increased protein unprenylation. Blocking of protein unprenylation using geranylgeraniol attenuated the cellular responses, including cell apoptosis and protein unprenylation induced by bisphosphonates and TRAIL. This is the first study to demonstrate that bisphosphonates markedly enhanced TRAIL-induced apoptosis in human osteosarcoma cells. These findings suggest that bisphosphonates may be a new and effective anticancer treatment with TRAIL proteins for TRAIL-resistant cancer cells.