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Objective: Gastric cancer cells are insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To sensitize gastric cancer cells to TRAIL, we treated gastric cancer MGC803 cells with TRAIL and cisplatin., Methods: Cell proliferation was measured using MTT assay. Cell apoptosis was determined by flow cytometry. Expression of proteins was analyzed by Western blot. The distribution of lipid rafts and death receptors was analyzed by immunofluorescence microscopy. MGC803 cells were pretreated with 50 mg/L nystatin for 1 h, and followed by the treatment of cisplatin and TRAIL., Results: 100 µg/L TRAIL resulted in (8.51 ± 3.45)% inhibition of cell proliferation and caused (3.26 ± 0.89)% cell apoptosis in MGC803 cells. Compared with the treatment with cisplatin alone, treatment with TRAIL (100 µg/L) and cisplatin (8.49 mg/L, IC(50) dose of 24 h) led to a dramatic increase in both inhibition of cell proliferation [(52.58 ± 4.57)% vs. (76.43 ± 5.35)%, P < 0.05] and cell apoptosis [(23.10 ± 3.41)% vs. (42.56 ± 4.11)%, P < 0.05]. Moreover, cleavage of caspase-8 and caspase-3 was detected. TRAIL (100 µg/L) did not induce obvious lipid rafts aggregation and death receptor 4 (DR4) clustering, while cisplatin (8.49 mg/L) significantly promoted the localization of DR4 in aggregated lipid rafts. Pretreatment with 50 mg/L nystatin, a cholesterol-sequestering agent, triggered (3.66 ± 0.52)% cell apoptosis after 24 h. Pretreatment with nystatin for 1 h before the addition of 8.49 mg/L cisplatin for 24 h caused a decreased tendency to cell apoptosis [(25.74 ± 3.28)% vs. (22.76 ± 2.97)%]. While, pretreatment with nystatin before the addition of cisplatin and TRAIL, the proportion of apoptotic cells decreased from (43.16 ± 4.26)% to (31.52 ± 3.99)% (P < 0.05)., Conclusion: Cisplatin enhances TRAIL-induced apoptosis in gastric cancer MGC803 cells through clustering death receptors into lipid rafts.

Objective: Glucocorticoid (GC) has occupied a central role in the treatment of acute lymphoblastic leukemia due to its ability to induce apoptosis in neoplastic lymphoid cells. Glucocorticoid resistance is present among 20% initial acute lymphoblastic leukemia, even 80% refractory acute lymphoblastic leukemia. Glucocorticoid resistance has been an important determinant of clinical outcome. Glucocorticoid depends on glucocorticoid receptor (GR) to induce apoptosis. Glucocorticoid receptor, a number of nuclear hormone receptor superfamily, is mediated by many signal transduction systems. The mitogen-activated protein kinases (MAPK) superfamily of serine/threonine kinases has emerged as an important component of cellular signal transduction. Four MAP kinase families, ERK, p38 MAP kinase, JNK, and ERK5, have been well characterized. p38 MAPK usually plays a role in regulating apoptosis, cell cycle arrest and cytokines production, et al. In steroid resistance patients, IL-2 combined with IL-4 can decrease glucocorticoid receptor ligand-binding affinity via p38MAPK. In human alveolar epithelial A549 cells, dexamethasone could inhibit the activation of p38MAPK. It is unclear that the effect of p38MAPK on glucocorticoid receptor function induced by dexamethasone in CEM cells. This study aimed to investigate effect of p38 mitogen-activated protein kinase on glucocorticoid receptor function induced by dexamethasone in CEM cells., Methods: Cell viability was determined by trypan blue dye exclusion. Apoptosis was evaluated by morphology and flow cytometry. Glucocorticoid receptor protein and p-p38MAPK protein were analyzed by Western Blot., Results: When treatment with SB203580 and dexamethasone for 24 h to 72 h, the survival percentage was increased from 62.3%, 35.5% and 11.6% to 82.8%, 54.7% and 48.1%, respectively (P < 0.01). Co-treatment with SB203580 and dexamethasone resulted in the decrease of apoptotic percentage from 26.2% to 7.1% for 36 h (P < 0.01). p38 MAPK activation was apparent at 15 min, peaked at 1 h after dexamethasone treatment, and was sustained for 6 h. The phosphorylation was still observed at 48 h. Treatment with dexamethasone at 5 micromol/L for 12, 24, 36 and 48 h resulted in increase of GR(alpha) protein to 117%, 121%, 122% and 125% respectively. Unbinding to dexamethasone, GR(alpha) is in the cytoplasm. Nuclear-to-cytoplasmic ratio of GR(alpha) is 0.27. Treatment with dexamethasone at the same concentration and time resulted in the nuclear-to-cytoplasmic ratio increase to 0.48, 0.59, 0.95, 2.16 and 4.08 respectively. Combined treatment with SB203580 and dexamethasone resulted in the nuclear-to-cytoplasmic ratio decrease from 4.08 to 0.43 for 48 h (P < 0.05). The total GR(alpha) protein was unaffected., Conclusions: Expression of GR(alpha) protein is upregulated and translocated into nucleus. p38MAPK enhances GR(alpha) protein translocation into nucleus.

Objective: To investigate the expression of mitochondria-mediated apoptosis pathway related genes bcl-2, Bax, survivin and Smac/DIABLO in bufalin induced HL-60 cell apoptosis., Methods: Cell viability was determined by trypan blue exclusion, apoptosis by morphology and flow cytometry, expressions of Bcl-2, Bax, Survivin, Smac/DIABLO and caspase-3 protein by Western blot, and expression of survivin mRNA by RT-PCR., Results: Proliferation of HL-60 cells was inhibited by bufalin and the IC(50) at 24, 48 and 72 h were 25.8, 8.0 and 2.1 nmol/L, respectively. Apoptosis was induced when the cells were treated with bufalin at concentrations of 50.0 nmol/L and higher. Compared with the control, HL-60 cells treated with bufalin at 50.0 nmol/L for 6, 12, 24 and 48 h showed decrease of Bcl-2 protein expression to 88.6%, 53.3%, 19.2% and 9.5%, Bcl-2/Bax ratio (control 2.0) to 1.7, 1.1, 0.4 and 0.2, Survivin protein expression to 75.2%, 54.8%, 37.5% and 20.3%, and survivin mRNA to 85.7%, 39.4%, 12.5%and 0%, respectively. The expression of Smac/DIABLO protein was downregulated to 77.5% (12 h), 21.2% (24 h) and 15.3% (48 h) in mitochondrial fraction and upregulated to 1.4-(12h), 2.0-(24 h) and 3.5- folds (48 h) in cytosolic fraction, respectively. The active subunits of caspase-3 were displayed after treatment for 8 h till 48 h., Conclusion: Apoptosis induced by bufalin is related to downregulation of expressions of bcl-2 and survivin, decrease of Bcl-2/Bax ratio, mitochondrial release of Smac/DIABLO, and activation of caspase-3. The mitochondria-mediated apoptotic pathway may be involved in the apoptosis induced by bufalin in HL-60 cells.