Significant Difference In Apoptosis Rate Research Articles

Glycated nisin enhances nisin's cytotoxic effects on breast cancer cells

Antimicrobial peptides, such as nisin, are proposed as promising agents for cancer treatment. While glycation has been recognized as an effective method for enhancing various physicochemical properties of nisin, its anticancer effects remain unexplored. Therefore, we aimed to assess the anticancer potential of glycated nisin against MDA-MB-231 cells. The MDA-MB cells were treated with increasing concentrations of nisin and glycated nisin for 24, 48, and 72 h. The IC50 values for nisin were higher than those for glycated nisin. Glycated nisin at concentrations of 20 and 40 µg/mL decreased cell viability more than nisin at the same concentrations. The rate of apoptosis in the group treated with 20 µg/mL of nisin was lower compared to other treatment groups, and no significant difference in apoptosis rates was observed at different time points (p > 0.05). However, in the glycated nisin groups with concentrations of 10, 20, and 40 µg/mL, the level of apoptosis was very high after 24 h (73–81% of cells undergoing apoptosis). Overall, our study suggests that glycated nisin exhibits stronger cytotoxic effects on MDA-MB-231 cells, primarily involving the induction of apoptosis. This indicates its potential utilization as an alternative approach to address the issue of drug resistance in cancer cells.

Effect of rhTPO to the Proliferation and Apoptosis of Acute Myeloid Leukemia Cell Lines

To investigate the effects of recombinant human thrombopoietin (rhTPO) to proliferation and apoptosis of acute myeloid leukemia (AML) cell lines. After the treatment of different concentrations of rhTPO (0, 50, 100 ng/ml) for different time (24,48,72 h)，the cell proliferation rates of the AML cell lines (Kasumi-1, Skno-1, HEL, HL-60, THP-1) were determined by CCK-8 method. Apoptosis rate of each cell line cocultured with rhTPO was detected by Annexin V/PI method. The relative expression of TPO receptor c-MPL (myeloproliferative clonal antibody) mRNA in AML cell lines was detected by Q-PCR. The expression of c-MPL protein in each cell line was detected by Western blot. The expression of c-MPL antigen in HL-60 cells treated by different concentrations of rhTPO was detected by Flow cytometry. RhTPO showed no promotion to the proliferation of Kasumi-1, Skno-1, HEL, HL-60, THP-1 cell lines，however，it showed inhibitory effect to cell proliferation (72 h 0 ng/ml vs 100 ng/ml, P= 0.029) and pro-apoptotic (48 h 0 ng/ml vs 50 ng/ml, P=0.0143) in HL-60 cells. In Kasumi-1, Skno-1, HEL and THP-1 cells, there showed no statistically significant differences in apoptosis rate among each groups treated by different concentrations of rhTPO. Each AML cell line showed different levels of c-MPL gene and c-MPL protein expression, but HEL cells showed the highest expression in both of them. After HL-60 cells were treated by different concentrations of rhTPO for 48 hours, there showed no statistical difference in c-MPL antigen expression among each groups. RhTPO can not promote the proliferation of Kasumi-1, Skno-1, HEL, HL-60 and THP-1 leukemia cell lines. On the contrary, rhTPO can inhibit HL-60 cell proliferation and promote its apoptosis, and this effect is not related to c-MPL gene expression or protein expression.

Receptor pathways of glycated basic fibroblast growth factor affecting the proliferation and vascularization of human dermal microvascular endothelial cells

Objective: To investigate the receptor pathways of glycated basic fibroblast growth factor (bFGF) on proliferation and vascularization of human dermal microvascular endothelial cells (HDMECs). Methods: The experimental research method was used. Glycated bFGF stimulating solution was prepared with glucose and bFGF. HDMECs of the third to sixth passages were used in the experiment. Cells were divided into small interfering RNA (siRNA)-positive control group, siRNA-negative control group, siRNA-receptor for advanced glycation end product (RAGE) group, and siRNA-receptor for fibroblast growth factor (FGFR) group and transfected with siRNA-positive control glyceraldehyde-3-phosphate dehydrogenase, siRNA-negative control, siRNA-RAGE, and siRNA-FGFR for 4 to 6 hours, and then were added into HDMEC culture medium for routine culture. The transfection effect of siRNA was identified by reverse transcription polymerase chain reaction. The cells were divided into normal control group, glycated bFGF alone group, siRNA-RAGE alone group, and siRNA-RAGE+ glycated bFGF group, and seeded into 96-well plate and 6-well plate. Cells in siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group were transfected with siRNA-RAGE and then were added into HDMEC culture medium for routine culture. After two days, the original HDMEC culture medium was discarded, and cells in siRNA-RAGE alone group were routinely cultured in HDMEC culture medium, cells in siRNA-RAGE+ glycated bFGF group were routinely cultured in glycated bFGF stimulating solution. Cells in normal control group were routinely cultured in HDMEC culture medium, and cells in glycated bFGF alone group were routinely cultured in glycated bFGF stimulating solution. After transfection with siRNA-RAGE, cells were seeded into 48-well plate and divided into siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group. Another cells were directly seeded into 48-well plate without transfection and divided into normal control group and glycated bFGF alone group. Cells in the 4 groups were conducted with the corresponding treatment as above. Cells were divided into normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group and seeded into 96-, 6-, and 48-well plates, respectively, with the corresponding treatment the same as above. Only siRNA-RAGE was replaced by siRNA-FGFR. Cell counting kit 8 method was used to determine the proliferation of cells after 2 days of culture (sample number was 6), flow cytometry was used to detect the apoptosis of cells after 2 days of culture (sample number was 3), tube forming test was used to detect the angiogenesis of cells after 6 hours of culture (sample number was 4). Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: At the 200 bp band, there were no target genes in siRNA-positive control group, siRNA-RAGE group, or siRNA-FGFR group, but target genes were detected in siRNA-negative control group, indicating the success of siRNA transfection. After 2 days of culture, the absorbance value of cells in glycated bFGF alone group was significantly lower than that of normal control group (t=2.359, P<0.05); absorbance value of cells in siRNA-RAGE+ glycated bFGF group was significantly higher than that of glycated bFGF alone group (t=3.858, P<0.01), which was similar to that of siRNA-RAGE alone group (t=2.148, P>0.05). The absorbance value of cells in siRNA-FGFR+ glycated bFGF group was similar to that of glycated bFGF alone group (t=0.805, P>0.05), but significantly lower than that of siRNA-FGFR alone group (t=4.201, P<0.01). After 2 days of culture, the apoptotic rate of cells in glycated bFGF alone group was significantly higher than that of normal control group (t=2.416, P<0.05). The apoptotic rate of cells in siRNA-RAGE+ glycated bFGF group was significantly lower than the rates in glycated bFGF alone group and siRNA-RAGE alone group (t=3.861, 2.724, P<0.05 or P<0.01). There were no statistically significant differences in apoptosis rate of cells among normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group (F=2.218, P>0.05). After 6 hours of culture, the number of tubules of cells in normal control group (636±5) was significantly more than that of glycated bFGF alone group (580±8, t=10.825, P<0.01), and the number of tubules of cells in siRNA-RAGE+ glycated bFGF group (647±10) was significantly more than those of glycated bFGF alone group and siRNA-RAGE alone group (628±4, t=13.040, 3.641, P<0.01). After 6 hours of culture, the number of tubules of cells in siRNA-FGFR+ glycated bFGF group (619±5) was more than that of glycated bFGF alone group (t=9.000, P<0.01), but less than that of siRNA-FGFR alone group (632±3, t=2.814, P<0.05). Conclusions: Glycated bFGF affects the proliferation and angiogenesis of HDMEC through RAGE pathway, which may be one of the reasons for impaired wound healing of diabetic skin.

Cellular damage of low-dose combined exposure to mercury, lead and cadmium on hippocampal neurons in rats

Objective: To observe the cellular damage of low-dose combined exposure to Hg, Pb and Cd on hippocampal neurons in rat. Methods: SH-SY5Y cells were randomly divided into 8 groups by 2×2×2 factorial design: control group, Pb exposure group, Hg exposure group, Pb+Hg exposure group, Pb+Cd exposure group, Hg+Cd exposure group and Pb+Cd+Hg exposure group. And the cell viabilities were measured. On this basis, an animal model was established. Twenty eight-week-old SD pregnant rats were randomly divided into four groups by random number table, and five in each group: the control group(distilled water), 1-fold metal mixture exposure group (1×MM, poisoning solution containing mercury chloride 0.15 mg/L, lead acetate trihydrate 25 mg/L, cadmium chloride 7.5 mg/L), 5-fold metal mixture exposure group (5×MM, poisoning solution containing mercury chloride 0.75 mg/L, lead acetate trihydrate 125.00 mg/L, cadmium chloride 37.50 mg/L), 10-fold metal mixture exposure group (10×MM, poisoning solution containing mercury chloride 1.50 mg/L, lead acetate trihydrate 250.00 mg/L, cadmium chloride 75.00 mg/L). Pregnant rats drank water until delivery. Twenty male pups were selected and exposed to these metals through breast milk until weaned. The heavy metals dose of poisoning water was adjusted, and then the weaned rats were exposed to heavy metals via drinking poisoning water until adulthood (postnatal day 83). The blood samples and brain hippocampus samples were collected to observe the ultrastructural changes of hippocampus, and to determine the levels of Hg, Pb and Cd in blood. In addition, apoptosis rate and fluorescence intensity of reactive oxygen species and intracellular free calcium concentration ([Ca(2+)](i)) in hippocampal neurons were measured. Results: Cellular factorial design analysis showed that Hg+Pb+Cd (at no observed adverse effect level, 1.0, 0.5 and 0.1 μmol/L, respectively)had a interaction on cell viability after 48 or 72 hours of combined exposure (P<0.05). The results of ultrastructure showed that mitochondria decreased, ridges and matrixes gradually dissolved in rat hippocampal neurons of 5×MM group; nuclear chromatin aggregated, more ridges and matrixes dissolved and the mitochondria also decreased in rat hippocampal neurons of 10×MM group. The concentration of Hg, Pb and Cd in the blood of 1×MM group, 5×MM group and 10×MM group were higher than those in the control group, and the differences were statistically significant (P<0.001). There was no significant difference in apoptosis rate between the 1×MM group and the control group. The apoptosis rate of 5×MM group and 10×MM group was higher than that in the control group, and the differences were statistically significant (P<0.001). There was no statistically significant difference in the fluorescence intensity of reactive oxygen species in hippocampal neurons of the 1×MM group and the control group. The fluorescence intensity of reactive oxygen species in the 5×MM group and the 10×MM group was higher than that in the control group, the difference was statistically significant (P<0.05). There was no significant difference in the fluorescence intensity of [Ca(2+)](i) between the 1×MM group and the control group. The fluorescence intensity values of [Ca(2+)](i) in the 5×MM group and the 10×MM group were higher than the control group, the differences were statistically significant (P<0.001). Conclusion: Low-level combined exposure to Hg, Pb, and Cd caused synergistic neurotoxic damage, and the process may be related to the changes of neuronal apoptosis, reactive oxide species, and [Ca(2+)](i) levels.

In vivo inhibitory effection of Silibinin on bladder cance proliferation by classic and non-classic apoptosis related signals

Objective To investigate the growth inhibition of Silibinin on human bladder cancer cells 5637 subcutaneous tumor and the mechanism of it. Methods The human bladder cancer cells 5637 were cultured in vitro until logarithmic phase, then were inoculated into the right hind dorsal subcutaneous of nude mice to establish the subcutaneous tumor model. Different concentrations of Silibinin were given through the oral method for 32 days, and then executed all the nude mice, removed the tumors and weighed.After that, flow cytometry was used to analyze the tumor cell apoptosis, Western blot and immunohistochemical were engaged to detect the expression of apoptosis related factors Caspase-3, Cleaved PARP, Bax, Bcl-2 and Cytochrome C, also the data were compared and analyzed with statistical methods. Results In fifty nude mice, 42 nude mice were modeled successful, and 40 nude mice were selected into experiment randomly, there was no mice died during the experiment. By measuring the volumes of the tumor, the tumor growth inhibition rates was 10.08%, 47.64%, 63.14%, respectively among the low, medium and high does group(F=52.79, P<0.01). The tumor weights(g) of control, low, medium and high does group was (1.54±0.25), (1.46±0.22), (0.94±0.15) and (0.72±0.16), respectively, and the difference between the three groups was statistically significant(F=794.17, P<0.01). Flow cytometry detected the tumor cells apoptosis rate illustrated that there was significantly difference in tumor cells apoptosis rate among the different does groups(F=794.17, P<0.01). The tumor cells apoptosis rates of control, low, medium and high does group were (6.70±1.34), (8.08±1.19), (26.11±1.91) and (44.87±2.96). Compared with the control group, low dose group had no significant difference in apoptosis rate (P=0.14). All groups’Caspase-3, Cleaved PARP, Bax, Bcl-2 and Cyt-C protein expression were significantly different (P<0.05), but the comparison results in different doses of Silibinin group and with control group were not all P<0.05. Conclusions Silibinin can significantly inhibit bladder cancer growth in vivo, and this effection will more significant accompany with the increase of the Silibinin does.The mechanisim of the inhibition of Silibinin on bladder cancer may associate with caspase-mediated apoptosis pathway, mitochondrial-mediated apoptosis pathway and perhaps also has some relationship with Bcl-2 family proteins down-regulation and the up-regulation of Bax family proteins. Key words: Urinary Bladder Neoplasms; Apoptosis; Silymarin

Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats

To explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Eight Sprague Dawley rats were selected to obtain the L 1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O 2 condition (group A), and under 0.5% O 2 condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O 2 condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B ( t=1.026, P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A ( t=22.672, P=0.015; t=18.396, P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B ( t=0.594, P=0.781; t=1.251, P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C ( t=27.143, P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C ( t=21.097, P=0.015; t=34.829, P=0.002; t=18.673, P=0.022; t=31.949, P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C ( t=37.648, P=0.006; t=16.729, P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C ( t=25.583, P=0.011). HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.

Effects of pioglitazone pre-treating on acinar cells apoptosis induced by caerulein in acute pancreatitis

Objective To investigate the effects of pioglitazone pre-treating on pancreatic acinar cell (AR42J cells) apoptosis induced by caerulein. Methods AR42J cells were divided into blank control group (with normal culture), pioglitazone group (40 μmol/L), caerulein control group (1×10-8 mol/L), pioglitazone+ caerulein group (40 μmol/L pioglitazone+ 1×10-8 mol/L caerulein) and pioglitazone+ GW9662+ caerulein group (40 μmol/L pioglitazone+ 5 μmol/L GW9662+ 1×10-8 mol/L caerulein). Pioglitazone and GW9662 were added 30 minutes earlier than caerulein. Cell proliferation rate of each group was determined by MTT assay at three, six, 12 and 24 hour. The cell apoptosis rate was detected by flow cytometry with Annexin Ⅴ/PI staining and terminal dexynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining. The activity of Caspase 3, 8 and 9 of each group was measured. Mitochondrial membrane potential (MMP) was detected by flow cytometry with JC-1 staining. Single factor analysis of variance and LSD test were performed for data analysis. Results At six, 12 and 24 hour, the cell proliferation rate of pioglitazone group and pioglitazone+ caerulein group was 0.19±0.02, 0.22±0.02, 0.36±0.02 and 0.20±0.04, 0.23±0.02, 0.38±0.02, respectively, which were significantly lower than those of blank control group (0.25±0.04, 0.28±0.03 and 0.46±0.02) and caerulein group (0.23±0.02, 0.29±0.01 and 0.46±0.05, tcontrol group=-3.16, -4.61 and -6.25, tcaerulein group=-1.58, -4.61 and -6.15, all P 0.05), but cell apoptosis of these two groups were higher than those of control group ((5.52±0.64)%, (5.30±0.97)% and (5.47±0.88)%) and caerulein group ((5.98±1.21)%, (7.47±0.58)% and (8.11±1.32)%) respectively, and the differences were statistically significant (tcontrol group=9.81, 4.45 and 10.74, tcaerulein group=4.38, 7.62 and 6.98, all P <0.05). There was no significant difference in apoptosis rate between pioglitazone+ GW9662+ caerulein group ((5.82±0.26)%, (6.05±0.83)% and (9.23±0.90)%) and caerulein group; while significantly higher when compared with those of pioglitazone+ caerulein group (t=-4.63, -10.07 and -5.70, all P<0.05). At 12 hour, the apoptosis rate detected by TUNEL staining of pioglitazone group ((3.93±0.40)%) was significantly higher than that of control group ((2.73±0.68)%), the apoptosis rate of pioglitazone+ caerulein group ((8.43±1.65)%) was significantly higher than that of caerulein group ((2.80±0.56)%), the apoptosis rate of pioglitazone+ GW9662+ caerulein group ((3.87±0.35)%) was lower than that of pioglitazone+ caerulein group (t=7.93, 8.92, -5.35, all P<0.05). At 24 hour, the activity of Caspase 3, 8 and 9 of pioglitazone+ caerulein group (1.28±0.05, 1.38±0.04 and 1.53±0.09) significantly increased compared with those of caerulein group (1.12±0.88, 1.22±0.02 and 0.53±0.07, t=3.20, 8.62 and 1.29, all P<0.05). After treated with GW9662, part of activity of Caspase enzymes recovered. The number of cells with potential change of mitochondrial membrane in pioglitazone group and pioglitazone+ caerulein group was more than that of caerulein group (28.50±0.91)% and (28.20±2.56)% vs (15.00±3.67)%) and part of membrane potential recovered after GW9662 added ((20.67±2.20)%). Conclusions Pioglitazone might promote AR42J cell apoptosis through the activation of caspases enzymes and changing membrane potential. And the antagonist GW9662 would partially inhibit the apoptosis induced by pioglitazone. Key words: Pioglitazone; Caerulein; AR42J cells; Acute pancreatitis

Effect of Paridis Rhizoma total saponins on apoptosis of human gastric cancer cell MKN-45 and Fas/FasL signaling pathway

The study aimed to test if Paridis Rhizoma total saponins (PRTS) could induce apoptosis of human gastric cancer cell MKN-45. Based on the previous researches, PRTS was set by different concentrations to treat human gastric cancer cell for 12 h (5, 10, 20 mg x L(-1)). Fluorescent staining methods were adopted to observe apoptotic morphological changes of MKN-45. The apoptosis rates were analyzed by flow cytometry with Annexin V-FITC/PI staining. The enzymatic activities of caspase-3 and caspase-8 were measured by ELISA. The protein levels of Fas and FasL were detected by Western blotting. Under a fluorescence microscope, MKN-45 treated by PRTS was seen typical apoptotic morphological features. PRTS significantly increased the rate of apoptosis. Compared with the control group, there exsited significant differences in apoptosis rate of PRTS concentration of 20 mg x L(-1) (P < 0.01); besides, the enzymatic activities of caspase-3 and caspase-8 were promoted obviously after the effect of PRTS on MKN-45 cells for 12 h (P < 0.01). The protein levels of Fas and FasL in the MKN-45 were upgraded significantly. PRTS can induce apoptosis of human gastric cancer cell MKN-45 , which is concerned with caspase-3 and caspase-8 and upgraded Fas and FasL.

The effect of heme oxygenase-1 on apoptosis and aquaporin-1 expression in type II primary alveolar epithelial cells in rat

To explore the effect of heme oxygenase-1 (HO-1) on apoptosis and expression of aquaporin-1 (AQP-1) in primary type II alveolar epithelial cells (AECII) in rats with hydrogen peroxide (H₂O₂) induced oxidative damage. Lung tissue of male Sprague-Dawley (SD) rats was collected. Primary AECII were isolated, purified, and cultured for 24 hours, then they were divided into four groups: (1) normal group (treated with normal saline); (2) H₂O₂ injury group (treated with H₂O₂ 0.5 mmol/L); (3) HO-1 control group (treated with HO-1 1 μmol/L); (4) HO-1 protection group (treated with HO-1 1 μmol/L and H₂O₂ 0.5 mmol/L). Cells of each group were cultured for 12 hours after various treatment. The cell suspension was collected before and 1, 3, 6, 12 hours after treatment, the expression of AQP-1 was determined by Western blotting and the apoptosis rate was assessed with flow cytometer. The expression of AQP-1 in H₂O₂ injury group was significantly declined with time, and was lower than that in normal group at each time point after treatment. The expression of AQP-1 in HO-1 control group and HO-1 protection group was significantly increased with time, and was higher than that of other groups at each time point after treatment. The expression of AQP-1 in HO-1 protection group was significantly up-regulated compared with that in H₂O₂ injury group (1 hour: 60.81 ± 5.78 vs. 46.21 ± 4.81, 3 hours: 63.05 ± 9.61 vs. 39.32 ± 4.96, 6 hours: 92.59 ± 8.21 vs. 36.82 ± 4.32, 12 hours: 86.16 ± 14.84 vs. 34.88 ± 2.66, all P<0.05). No significant difference in apoptosis rate was found between normal group and HO-1 control group. The apoptosis rate in H₂O₂ injury group was increased with time, and was significantly higher than that of normal group at each time point. The apoptosis rate in HO-1 protection group was gradually increased within 3 hours after treatment, then decreased and remained stable after 6 hours, while it was significantly lower than that of H₂O₂ injury group at each time point [1 hour: (9.04 ± 2.17)% vs. (15.14 ± 2.47)%, 3 hours: (12.90 ± 2.04)% vs. (22.37 ± 4.84)%, 6 hours: (10.42 ± 1.68)% vs. (27.83 ± 3.93)%, 12 hours: (11.97 ± 1.91)% vs. (33.63 ± 6.61)%, all P<0.05]. A negative correlation was found between AQP-1 and apoptosis rate in H₂O₂ injury group (r=-0.723, P<0.001), and a regression correlation was found [y=672.548(0.914) (x), R²=0.597]. AQP-1 was not correlated with apoptosis rate in HO-1 protection group (r=0.210, P=0.193), but a regression correlation was found [y=e((3.130 - 59.654/x )), R²=0.225]. HO-1 could increase the expression of AQP-1 in H₂O₂ injured AECII of rat, and lower its apoptosis rate. Increase in the expression of AQP-1 may be the underlying mechanism of anti-oxygenation property of HO-1.

PUMA enhanced chemotherapeutic sensitivity of human glioma stem cells against temozolomide

Objective To explore differences of drug resistance of temozolomide(TMZ) on different CD133 immune prototype of glioma cells and study on the changes of their sensitivity to TMZ through increased PUMA. Methods CD133+-U87MG cells sorted by CD133 magnetic beads were cultured in serum-free stem cell medium respectively. The cells were infected with recombinant adenovirus, Ad-PUMA, diluted in cell culture medium with or without TMZ intervention.The inhibitory rate of cell proliferation was detected by MTT assay and 50 ％ inhibition concentration of TMZ was calculated. Apoptosis rates of CD133+-U87MG cells were assessed by flow cytometry (FCM) before and after intervention of exogenous PUMA and TMZ.Results The TMZ IC50 values of CD133+glioma cells were higher than that of CD133- glioma cells. There were significant differences in apoptosis rate between CD133+ glioma cell and CD133- glioma cell (all P＜0.05).Conclusion AdPUMA joint TMZ can promote glioma stem cells apoptosis, thus improve the sensitivity to chemotherapy of glioma. Key words: Glioma ; Apoptosis; Drug resistant; Temozolomide; p53 up-regulated modulator of apoptosis

Effect of mechanical pressure on expression of signal transduction molecule Casaase-3, bcl-2, bax mRNA

Objective To explore the effects of mechanical stimuli on the expression of signal transduction molecule Caspase-3,B-cell lymphoma-2 ( bcl-2),bax mRNA.Methods Rabbit knee articular cartilage cells obtained by enzymatic digestion were cultured and passaged for 3 generations.The chondrocytes were cultivated in a culture plate added with different doses of inhibitor of Caspase-3,bcl-2,bax by a closed container with 90 kPa pressure for 24 h.Chondrocytes apoptosis was detected by flow cytometry (FCM),and the expression of Caspase-3,bcl-2,bax mRNA by semiquantitative analysis of polymerase chain reaction (PCR).Results Mechanical pressure could induce chondrocytes apoptosis.There was significant difference in apoptosis rate between inhibitors-treated groups and contral group ( all P ＜ 0.05 ).There was significant difference in the expression of signal transduction molecule Caspase-3,bcl-2 and bax between inhibitors-treated groups and control group ( all P ＜ 0.05).There were correlations between apoptosis rate and expression of Caspase-3,bcl-2 and bax in Caspase-3,bcl-2 and bax inhibitors-treated groups respectively ( for Caspase-3,r =0.69,t =3.41,P ＜ 0.01 ; for bcl-2,r =0.73,t =3.97,P ＜ 0.O1 ; for bax,r =0.89,t =6.69,P＜0.01).Conclusion The inhibitors of Caspase-3,bcl-2 and bax can influence the apoptosis of chondrocytes,and make the expression of Caspase-3,bcl-2 and bax mRNA change. Key words: Chondrocyte; Mechanical pressure; Apoptosis

Relationship between tumor associated macrophages and cytotoxic T lymphocytes in rat liver carcinoma

Objective To investigate the influence of tumor associated macrophages on cytotoxic T lymphocytes (CTL) infiltration and apoptosis in rat liver carcinoma.Methods Walker-256 Wistar rat hepatoma models were established.Then rats were randomly divided into three groups:group A (clodronatelipsomes by intraperitoneal injection) ; group B (PBS-lipsomes by intraperitoneal injection) ; group C (normal saline by intraperitoneal injection).Animals were killed at the 12th day after treatment.The tumor volume was measured,and the number of positive cells of CD68,CD8,CD163,and Granzyme B was detected by using immunohistochemical method,respectively.The apoptosis of CD8 + T cells in tumor tissues was tested by the method of double staining with immunohistochemistry and TUNEL.Results ( 1 ) There was no significant difference in tumor volume among groups A,B and C ( P ＞ 0.05 ) ; (2) There was significant difference in CD68 positiv cells between groups A and B,CDE163 positive cells between groups A and B,CD8 positive cells between group A and B or between groups A and C; and Granzyme B positive cells between groups A and C (all P ＜0.05).The number of CD68 and CD163 positive cells in group A was less than in groups B and C.The number of CD8 and Granzyme B positive cells in group A was more than in groups B and C ; (3) There was a negative correlation not only between the positive cells of CD68 and CD8,but also between CD163 and Granzyme B positive cells.However,there was no correlation between the positive cells of CD68 and Granzyme B,as well as between the positive cells of CD163 and CD8 ;(4) There was significant difference in apoptosis rate of CD8 T cells between groups A and C group (P ＜0.05).Apoptosis rate of CD8 T cells in group A was lower than in groups B and C.Conclusion Clodronate liposome can deplete macrophages of rat liver carcinoma tissues effectively.After macrophages were depleted,the number of CTL was increased and the apoptosis rate of CTL was decreased in rat liver carcinoma.Tumor associated macrophages can promote apoptosis and inhibit killing activity of cytotoxicity T lymphocyte,and play an important role in tumor development. Key words: Tumor associated macrophage; Cytotoxic T lymphocyte; Infiltration

Relationship between NRAGE and the radioresistance of esophageal carcinoma cell line TE13R120

The mRNA levels of 59 genes, detected by cDNA microarray, were up-regulated in the radioresistant human esophageal cacinoma cell line TE13R120 as compared with its parental cell line TE13 before and after radiation, and the expression of NRAGE gene showed a gradually up-regulating tendency. This study aimed to further detect the differences of NRAGE gene and protein expression and apoptosis between TE13R120 and TE13 cells, and to investigate the relationship between the NRAGE and the radioresistance of TE13R120 cells and its mechanism. The two cell lines were irradiated by ⁶⁰Co γ-ray at different conditions. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry were used to detect the expression of NRAGE. Flow cytometry (FCM) was used to detect the cell apoptosis before and after irradiation. The mRNA level of NRAGE was higher in TE13R120 cells than in TE13 cells before and after irradiation (before radiation: 0.25 ± 0.03 vs. 0.49 ± 0.03; 4 Gy 4 h: 0.31 ± 0.03 vs. 0.53 ± 0.02; 4 Gy 16 h: 0.32 ± 0.04 vs. 0.59 ± 0.04; 4 Gy 24 h: 0.36 ± 0.05 vs. 0.72 ± 0.04; 2 Gy 12 h: 0.32 ± 0.02 vs. 0.64 ± 0.04; 6 Gy 12 h: 0.36 ± 0.02 vs. 0.79 ± 0.05; 10 Gy 12 h: 0.46 ± 0.04 vs. 0.85 ± 0.01; P < 0.01), and the mRNA level of NRAGE was increased gradually with the increase of radiation dose and time in the two cell lines (P < 0.05 and P < 0.01). Western blot results showed no difference of NRAGE protein level in cytoplasm between TE13R120 cells and TE13 cells before and after irradiation, but its level in nuclei was higher in TE13R120 cells than in TE13 cells at different radiation time and dosages. Immunocytochemistry showed similar results as Western blot. FCM showed no significant difference in apoptosis rate between TE13R120 and TE13 cells before and after radiation. NRAGE may play an important role in the radiation responses of the two cell lines, and may participate in the formation of radioresistance of TE13R120 cells by changing its subcellular localization, but its relationship with cell apoptosis has not been confirmed.

Expression and immune effect of toll-like receptor 4 in human trophoblast cells

This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells (1 mg/L LPS, 12 h) was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry (FCM), and the level of TNF-alpha determined by using enzyme linked immunosorbent assay (ELISA) respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells (P<0.01). FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group (P<0.05), or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-alpha in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group (P<0.05). Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-alpha of human term trophoblast cells stimulated by LPS.

Effects of HCV co-infection on apoptosis of CD4+ T-cells in HIV-positive patients

Apoptosis importantly contributes to loss of CD4+ T-cells in HIV infection, and modification of their apoptosis may explain why HIV/HCV (hepatitis C virus)-co-infected patients are more likely to die from liver-related causes, although the effects of HCV on HIV infection remain unclear. In the present study, we studied in a cross-sectional and serial analysis spontaneous ex vivo CD4+ T-cell apoptosis in HIV/HCV-co-infected and HIV-mono-infected patients before and after HAART (highly active antiretroviral therapy). Apoptosis of peripheral blood CD4+ T-cells was measured by both a PARP [poly(ADP-ribose) polymerase] and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay to detect cells with irreversible apoptosis. Although hepatitis C alone did not increase CD4+ T-cell apoptosis, HCV co-infection disproportionately increased elevated rates of apoptosis in CD4+ T-cells from untreated HIV-positive patients. Increased CD4+ T-cell apoptosis was closely correlated with HIV, but not HCV, viral loads. Under HAART, increased rates of CD4+ T-cell apoptosis rapidly decreased both in HIV-mono-infected and HIV/HCV-co-infected patients, without any significant difference in apoptosis rates between the two patient groups after 4 weeks of therapy. Nevertheless residual CD4+ T-cell apoptosis did not reach the normal levels seen in healthy controls and remained higher in HIV patients receiving protease inhibitors than in patients with other antiretroviral regimens. The results of the present study suggest that HCV co-infection sensitizes CD4+ T-cells towards apoptosis in untreated HIV-positive patients. However, this effect is rapidly lost under effective antiretroviral therapy.

Apoptosis of HepG2 cells after transfection with LIGHT gene and interferon-γ.

Objective To investigate apoptosis of HepG2 ceils after transfecfion with LIGHT gene and interferon-γ. Methods LIGHT gene and interferon-γ were transfected into HepG2 cells by liposome mediated method. The HepG2 cells were divided into group A (transfected with LIGHT gene or interferon-γ), group B (transfeeted with LIGHT gene and interferon-γ) and group C (non-transfection group). The apoptosis rate of the HepG2 cells and the expression of Bcl-2 and Caspase-8 were detected 12, 24, 48 hours after transfeetion. Results (1) The apoptosis rates of HepG2 cells at hour 12, 24 and 48 after transfeetion were 18.8% ± 3.5%, 25.7%± 2.8% and 36.4% ±3.6% in group A, 23.8% ±2.4%, 31.1% ±2.1% and42.5% ±4.5% in group B, and 8.7% ± 2.1%, 9.3% ± 1.6% and 10.9% ± 1.2% in group C. There was a significant difference in apoptosis rate among the 3 groups (F = 15.69, 53.33, 48.28, P < 0.01). (2) The expression of Bcl-2 in HepG2 cells at hour 12, 24 and 48 after transfection was 16.4% ± 5.0%, 13.4% ± 3.5% and 8.6% ± 2.3% in group A, 14.7%±3.8%, 9.1% ±2.0% and 4.6% ±2.0% in group B, and 25.3% ±6. 3%, 19.8% ±4.4% and 10.1% ±3.8% in group C. There was a significant difference in the expression of Bcl-2 among the 3 groups (F = 6.19, 12.29, 5.81, P <0.05). (3) The expression of Caspase-8 at hour 12, 24 and48 after transfection were 19.3% ±2.4%, 27.2% ±1.9% and 33.7% ±3.0% in group A, 22.7% ±2.2%, 30.9% ±3.1% and 38.2% ±3.2% in group B, and 1.2% ±0.8%, 1.8% ±0.6% and 3.2% ±1.5% in group C. There was a significant difference in the expression of Caspase-8 among the 3 groups (F =71.54, 112. 78, I01.61, P < 0.01). Condusions LIGHT gene can signiticanfly promote cell apoptosis through regulating the expression of Bcl-2 and Caspase-8. Interferon-γ enhanced the effect of LIGHT gene on the apoptosis of HepG2 cells. Key words: Carcinoma,hepatocellulur; HepG2 cells; LIGHT gene; Intederon-γ; Apoptosis

Effect of target-directed regulation of uncoupling protein-2 gene expression on ischemia-reperfusion injury of hepatocytes

The effect of target-directed regulation of the uncoupling protein-2 (UCP-2) gene expression on the ischemia-reperfusion injury of hepatocytes under different conditions was investigated. The expression plasmid and RNAi plasmid targeting UCP-2 gene were constructed and transfected into normal hepatocytes and fatty liver cells, respectively. The expression of UCP-2 mRNA was detected by real time PCR. The cells were divided into normal cell group (NCG), group of normal cells transfected with empty vector (EVNCG), group of normal cells transfected with expression plasmid (EPNCG), fatty liver cell group (FCG) and group of fatty liver cells transfected with RNAi plasmid (RPFCG). The ischemia-reperfusion model in vitro was established. One, 6, 12 and 24 h after reperfusion, Annexin V/PI flow cytometry was used to measure cell necrosis rate, apoptosis rate and survival rate. Simultaneously, the intracellular ATP, ROS and MDA levels were determined. The results showed that 1, 6, 12 and 24 h after ischemia-reperfusion, the intracellular ROS, MDA and ATP levels and cell survival rate in EPNCG were significantly lower, and cell necrosis rate significantly higher than in NCG and EVNCG, but there was no significant difference in apoptosis rate among NCG, EVNCG and EPNCG (P>005). Six, 12 and 24 h after reperfusion there was no significant difference in ROS, MDA levels and apoptosis rate between FCG and RPFCG (P>0.05), but the ATP level and survival rate of cells in RPFCG were higher than in FCG (P<0.05). It was concluded that down-regulation of the UCP-2 gene expression in steatotic hepatocytes could alleviate the ischemia-reperfusion injury of liver cells.

The relationship between p38MAPK and apoptosis during paclitaxel resistance of ovarian cancer cells

To investigate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and cell apoptosis during the paclitaxel resistance of ovarian carcinoma cell lines, flow cytometry (FCM) and PI staining were employed to determine the effect of p38MAPK inhibitor SB203580 on the apoptosis of A2780/Taxol cells, a drug-resistant human ovarian carcinoma cell line. p38MAPK protein expression in SB203580-treated cells was immunochemically measured. The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/Taxol cells was determined by MTT assay. MDR-1 mRNA, and expression of p38MAPK and phospho-p53 protein were detected by RT-PCR and Western blotting, respectively. The apoptosis rate of A2780/Taxol cells was (19.7+/-1.04)% 24 h after SB203580 treatment. A significant difference in apoptosis rate was found among experiment group, control group and untreated group (P<0.05). The relative reversal rate of A2780/Taxol cells to paclitaxel was (57.18+/-2.01)%. As compared with the control group and the untreated group, p38MAPK protein and MDR-1 mRNA in SB203580-treated cells was substantially decreased. The expression of p53 protein was significantly increased. It is concluded that p38MAPK pathway is related to paclitaxel resistance of ovarian carcinoma, and blockade of this pathway can promote the apoptosis of the drug-resistant cells and reverse the drug-resistance. Moreover, p38MAPK-mediated apoptosis in paclitaxel-resistant ovarian carcinoma cells depends on the activation of p53.