Expression of caspase-3 and TRAIL receptors in CD4^+ and CD8^+ T cells of SLE patients

Objective: To study the expression of caspase-3 and tumor necrosis factor-related apoptosisinducing ligand (TRAIL) receptors in the CD4+ and CD8+ T cells of systemic lupus enythematosus (SLE) patients. Methods: RT-PCR was used to analyze the expression of caspase-3 and TRAIL receptors in CD4+ and CD8+ T cells of SLE patients and normal subjects. Results: The death domain-containing TRAIL-R1/R2 as well as 'decoy' TRAIL-R3/R4 were co-expressed in majority of CD4+ and CD8+ T cells in both SLE patients and normal subjects. The CD8+ T cells from SLE patients showed significantly higher expression of caspase-3 and TRAIL-R2 than those from normal subjects,and the expression was correlated with the activity of the disease. Conclusion: The TRAIL-R2 signal pathway might contribute to the apoptosis of T cells in SLE.

Targeting androgen receptor and trail： a novel treatment paradigm for breast cancer

OBJECTIVE TNF-related apoptosis-inducing ligand(TRAIL)is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors.However,most breast cancer cells are resistant to TRAIL-induced apoptosis.Our objectives are to investigate the underlying molecular mechanisms and to develop strategies to overcome such resistance.METHODS To identify modulators of TRAIL-induced apoptosis,we carried out a genome wide si RNA screen.To validate the screening result,we either silenced or overexpressed the identified genes in various breast cancer cells and changes in growth and TRAIL-induced cell apoptosis were determined in vitro and in an orthotopic xenograft mouse model.Finally,we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL-therapy.RESULTS We unexpectedly identified androgen receptor(AR)to be responsible for TRAIL resistance.While AR is classically viewed as the key factor in prostate cancer progression,we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple-negative breast cancers(TNBC)that are highly aggressive with poor prognosis.Importantly,breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance.AR overexpression in MDA-MB-231 and MDA-MB-436 cells suppressed the TRAIL sensitivity whereas knockdown of AR rendered MCF-7 and MDA-MB-453 cells sensitive to TRAIL-induced apoptosis.AR overexpression also induced TRAIL resistance in breast tumors in vivo.Further,we observed an upregulation of the TRAIL receptor,death receptor 5(DR5)in breast cancer cells,following the removal or inhibition of AR by its antagonists Casodex and MDV3100.Treatment with AR antagonists also enhanced TRAIL-induced breast cancer cell apoptosis.CONCLUSION AR signaling suppresses TRAIL-induced breast cancer cell apoptosis,in part,by suppressing DR5 expression,and a combination of AR antagonists together with TRAIL may be a novel and effective therapy for TNBC.

Vitamin E Succinic Acid enhances the effect of mDRA-6 to eradicate leukemia cells by inducing apoptosis

Objective: The aim of our study was to detect whether Vitamin E Succinic Acid (VES) could regulate the expression level of DR5 in the cells and further elucidate the potential mechanisms involving that VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. Methods: MTT method was used to detect the growth inhibition of VES and mDRA-6 to Raji and K562 cells. Annexin V-FITC/PI double staining assay was used to analysis the apoptosis of leukemia cell. Flow cytometry was used to detect the cell surface DR5 expression. Immunoblotting technique was used to detect the DR5 protein expression. Results: MTT detection showed that 10 μmol/L mDRA-6 on the cell death rates of Raji and K562 cells were 21.98% and 5.23%, respectively. While increasing concentration of VES (5 μmol/L, 10 μmol/L, 20 μmol/L) and mDRA-6 both on the cell viability of Raji or K562 cells, the mortality of Raji cells elevated to 24.67%, 35.65% (P<0.01) and 40.22% (P<0.01), respectively. Similarly, the mortality of K562 cells increase to 6%, 7.89% (P<0.01) and 8.67% (P<0.01), respectively. To further specify the increased cell death rate induced by mDRA-6 and VES, the treated cells were analyzed by Annexin-V/PI staining assay. As shown in Fig. 1, the apoptosis rates of Raji and K562 cells treated with 2 μg/mL mDRA-6 for 12 h were 20.79% and 7.74%. Compared with this, the proportion of apoptotic cells increased upon exposure to 2 μg/mL mDRA-6 combination with 10 μmol/L VES, the apoptosis rates of Raji and K562 cells were 43.18% and 16.99%, respectively. To examine the anticancer effects of a combination strategy based on mDRA-6 and VES. We analyzed whether VES could elevated the expression level of DR5 on Raji and K562 cytomembrane by FACS. Interestingly, after treated with 10 μmol/L VES for 12 h, the expression level of DR5 on Raji and K562 cell surface increased from 50.66% to 70.08%, and 15.02% to 16.38%, respectively. Immune imprinting technology test showed that, different concentrations of VES could increase Raji and K562 cell DR5 protein expression. Conclusion: VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. The proper mechanism is VES could enhance the Raji and K562 cell membrane expression of DR5, and VES can also enhance the DR5 protein expression of cells.

Human Soluble TRAIL Protein Inducing Apoptosis in Osteosarcoma Cell

This study is to examine the effect of human recombinant soluble TRAIL （TNF-related apoptosis-inducing ligand） protein inducing apoptosis in MG-63 human osteosarcoma cells. The inhibitive rates of TRAIL to MG-63 cells were detected by MTT assay. The apoptosis induced by TRAIL in MG-63 human osteosarcoma cells was analyzed with FACS and TUNEL and the apoptotic bodies were observed by transmission electron microscope. MTT assay showed that the inhibitive rates of 500, 1 000, 2 000 and 4 000 ng/mL TRAIL for 24 h were 10.1%, 24.3%, 50.6% and 97.7% respectively. Flow cytometric analysis showed that after MG-63 cells were treated with 2 gg/mL TRAIL for 6 h, obvious apoptotic peak would immediately appear before diploid peak. Human soluble TRAIL protein can quickly kill MG-63 osteosarcoma cells selectively, and may have potential value for clinical treatment of osteosarcoma.

EGCG Enhances TRAIL-mediated Apoptosis in Human Melanoma A375 Cell Line

Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） is a promising anti-cancer agent. Epigallocatechin-3-gallate （EGCG） is a polyphenolic constituent of green tea. In this study, inhibitory effect of combined use of EGCG and TRAIL on human melanoma A375 cells was examined and the possible mechanism investigated. The cells were divided into 4 groups： control group, EGCG group （EGCG： 10, 20 μg/mL）, TRAIL group （TRAIL： 25 ng/mL） and EGCG＋TRAIL group （combined group）. The growth inhibition was measured in the A375 cells treated with different concentrations of TRAIL （（25, 50, 75, 100, 125, 150 ng/mL） by MTT assay. The apoptosis was assessed by flow cytometry. The expressions of DR4 and DR5 were detected by flow cytometry and western blotting. The activities of caspase-8 and caspase-3 were determined by colorimetric assay. The results showed that TRAIL could dose-dependently inhibit the growth of A375 cells and the IC50 of TRAIL was 150 ng/mL. The apoptosis rate was 11.8% in the TRAIL group, 5%–7% in the EGCG group and 48.9%–59.1% in the combined group. Significant difference was found in the apoptosis rate between the combined group and the EGCG or TRAIL group （P〈0.05 for each）. The expression of DR4 instead of DR5 was significantly increased in the EGCG group. The activity of caspase-3 rather than caspase-8 was substantially enhanced in the EGCG group. These results suggest that EGCG is useful for the TRAIL-based treatment for melanoma.

The flavonoid casticin enhances TRAIL-induced apoptosis of colon cancer cells through endoplasmic reticulum stress-mediated up-regulation of DR5

Objective： The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）-induced apoptosis in colon cancer cells. Methods： Human colon cancer HT-29 cells were treated with TRAIL or casticin. Cytotoxicity was examined by MTT assay, and apoptosis determined by morphological observation and flow cytometric analysis. Death receptor 5 （DRS）, DR4, and endoplasmic reticulum （ER） stress response markers, including glucose regulating protein 78 （GRP78）, activating transcription factor 4 （ATF4） and CHOP （CCAAT/enhancer binding protein homologous protein）, were examined with western blot. Small interfering RNA （siRNA） transfection was employed to knock down CHOP. Results： HT-29 cells were resistance to TRAIL-induced apoptosis, but casticin, at subtoxic concentrations, potentiated HT-29 cells to TRAIL-induced apoptosis. Casticin up-regulated the expression of DR5 time-and dose-dependent manners, but had no effect on the expression of DR4. Also, casticin increased the levels of ER stress response markers （GRP78, ATF4 and CHOP） in a similar way to DR5. Knockdown of CHOP by specific siRNA, or salubrinal, an ER stress inhibitor, abolished the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by casticin. Conclusion： Casticin enhances TRAIL-induced apoptosis of colon cancer cells by ER stress-mediated up-regulation of DR5.