Objective To determine whether lymph node-targeted chemotherapy with carbon nanoparticles absorbing 5-FU affects expressions of bcl-2, bax and caspase-3 in gastric cancer tissues, metastatic lymph nodes and normal gastric mucosa. Methods Twenty-eight patients with gastric cancer in our department were divided into lymph node-targeted chemotherapy (LNTC) group and control group from October 2005 to August 2006. The patients were treated with carbon nanoparticles absorbing 5-FU before operation in LNTC group and those were operated directly in control group. The gastric cancer tissues, metastatic lymph nodes and normal gastric mucosa were collected after operation. The expressions of bcl-2, bax and caspase-3 in those tissues were determined by immunohistochemical technique. Results In LNTC group, the positive expression rate of bcl-2 in gastric cancer tissues and metastatic lymph nodes was significantly lower than those in control group (28.6% vs . 78.6% , 25.0% vs . 70.0% , P ＜ 0.05), the positive expression rate of bax (85.7% vs . 28.6% , 80.0% vs . 30.0% ) and caspase-3 (57.1% vs . 14.3% , 55.0% vs . 15.0% ) in gastric cancer tissues and metastatic lymph nodes was significantly higher than those in control group ( P ＜ 0.05). The positive expression rate of bcl-2, bax and caspase-3 in normal gastric mucosa was not significantly different between two groups ( P ＞ 0.05). Conclusion The lymph node-targeted chemotherapy with carbon nanoparticles absorbing 5-FU can down-regulate the expression of bcl-2 and up-regulate the expression of bax and caspase-3 in gastric cancer tissues and metastatic lymph nodes, and therefore by affecting the expression levels of these apoptosis molecules may be one of the ways to induce tumor cell apoptosis.
Objective To observe the human mononuclear cell releasing TNF-α and the activation of Caspase-3 during apoptosis after stimulated by Co2+ and Cr3+, to discuss the mechanism of artificial joint wear production metal ion on aseptic loosening. Methods CoCl2 powder and CrCl3 powder were dissolved by asepsis inject water, preparing solution for10 mg/L and 500 mg/L, respectively. Mononuclear cells were acquired from peripheral blood, 4 × 106/culture dish. According to the difference of culture solution, the cells were divided into 3 groups. Group A: mononuclear cell was culture with normal sal ine as control; group B: mononuclear cell was cultured with CoCl2 solution; group C: mononuclear cell was cultured with CrCl3 solution. The production of TNF-α was assessed by ELISA, the activation of Caspase-3 was measured by colorimetric assay and the apoptotic cell was detected by TUNEL assays at 12, 24 and 48 hours after co-cultured respectively. Results The concentration of TNF-α and the expression of Caspase-3 in groups B and C were higher than those in group A (P lt; 0.05), and reached the peak level at 24, 48 hours, respetively. The TUNEL positive cells were detected in the all groups, nucleus was pyknotic and darker-staining, cell body was crinkle and cell membrane was integrity. There were significant differences in the apoptosis rate between groups B, C and group A (P lt; 0.05). And the activation of Caspase-3 increased and had the positive correlation with the apoptosis rate (r=0.765). Conclusion Co2+ and Cr3+ ions can stimulate human mononuclear cell to release TNF-α and induce human mononuclear cell apoptosis, which result in periprosethetic osteolysis and related to activation of Caspase-3.
Objective To observe expression of Caspase-3 and apoptosis around the prosthesis and explore the relationship of the expression and the apoptosis with the periimplant osteolysis. Methods From April 2001 to August 2006, 16 patients (10 males, 6 females) underwent the revision total hip arthroplasty surgery, who had the primary total hip arthroplasty at the ages of 45-67 years and had the revision total hip arthroplasty at the ages of 55-78 years, with the implantation duration of 7-13 years. According to their preoperative X-ray films andthe findings during the operation, the patients were divided into two groups: theloose/osteolytic group (n=8) and the loose/non-osteolytic group (n=8). The interface tissues were obtained from the peri-implant region in the patients. The synovial samples were taken from another 6 patients (2 males, 4 females; age, 54-68years; illness course, 9-15 years), who underwent the primary total hip arthroplasty for osteoarthritis. These 6 patients were used as controls. The tissues were prepared for the immunohistochemical assays to determine the expression of Caspase-3. The TUNEL assays were performed to quantify the apoptotic cells. The quantitative analysis on the positive cells and the correlation with the presence of the particulate wear debris and the severity of osteolysis were also performed. Results The level of the expression for Caspase-3 and the apoptosis index inthe loose/osteolytic group were significantly increased when compared with those in the loose/non-osteolytic group and the control group (P＜0.01). The polyethylene particles were surrounded by more positive cells than the metal particles. The positive cells were present at a higher level in the tissue sections where the high-wear status was present when compared with the areas where the low-wear status was present (P＜0.05). Conclusion There is a statistical correlation of the Caspase-3 expression to the apoptosis index and to the presence of the particulate wear debris and the severity of osteolysis, which may be one of the key points for the bone reconstruction inhibition and the bone resorption at the boneimplant interface under the stimulation of the wear debris. The apoptosis is involved in the pathogenesis of the aseptic loosening, which is closely related to the signal transportation of Caspase-3.
Objective To observe the effect of exogenous basic fibrob last growth factor (bFGF) on apoptosis of cultured human retinal pigment epithelial (RPE) cells exposed to visible light,and determine the role of bFGF, fibroblast growth factor receptor 1 (FGFR1),bcl-2 and caspase-3. Methods 2000±500) lx cold white light was used. Exogenous bFGF was utilized during culture. Annexin annexin V-fluoresce in isothiocyanate/propidium iodium (V-FITC/PI) labeling,flow cytometry, Immunocytochemical staining, enzyme associated absorb examing and reverse transcriptional polymerase chain reaction (RT-PCR) were used to determine the apoptosis, the expression levels of bFGF, FGFR1, bcl-2, as well as the activity of caspase-3. Results No protective effect of bFGF was observed under the concentration 5 ng/ml.A significant inhibition of apoptosis was found in 10 ng/ml and 20 ng/ml groups （P＜0.05）. The upregulation of bcl-2 was observed in bFGF (10 ng/ml, 20 ng/ml) protreated groups（P＜0.01）.Compared to no light exposure group,all light exposure groups (including bFGF pro-treated) had higher endogenous bFGF and FGFR1 levels （P ＜0.05）, and the increase was concentration dependent.The bFGF and FGFR1 levels were higher in exogenous bFGF applied (gt;5 ng/ml) groups than light exposure groups（P＜0.05）. The caspase-3 activity was significantly inhibited in bFGF (10 ng/ml) pro-treated groups. Conclusions Human RPE cells exposed to visible light were rescued by application of exogenous bFGF in vitro.The probable protective mechanism of bFGF partly is directly binding to FGFR1 or potentiating endogenous bFGF autocrine loop,to upregulate bcl-2 and to inhibit caspase-3 activation. (Chin J Ocul Fundus Dis，2003，19：24-28)
ObjectiveTo evaluate therapeutic effect of the targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3 on primary hepatocellular carcinoma cells (HepG2) and subcutaneous implanted tumors of nude mice. MethodsHepG 2 cells, breast cancer MDA-MB-231 cells， and normal hepatic L-02 cells were infected with the previously constructed targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3, then morphological change was observed and apoptosis index was detected by cell morphologic assay. Tumor growth and pathological changes were observed after the implanted tumors model of nude mice were injected with the recombinant adenovirus. Results Both apoptosis and obvious apoptotic peak of cells were observed by flow cytometry (FCM) in HepG 2 cell group. The apoptosis index of MDA-MB-231 and L-02 cells was 0 and 7.3%, respectively, which were significantly lower than that of HepG2 cells (48.2%), Plt;0.01. The volume of subcutaneous tumor of nude mice was (0.26±0.31) cm 3 in HepG2 cell group, (0.25±0.15) cm 3 in MDA-MB-231 cell group， and (0.26±0.28) cm 3 in control group on two weeks after implantation of cancer cells, and no statistical difference was found among them (Pgt;0.05). The volume of subcutaneous tumor of nude mice was (0.53±0.12) cm 3 in HepG 2 cell group, (0.49±0.22) cm 3 in MDA-MB-231 cell group， and (0.54±0.13) cm 3 in control group on four weeks after implantation of cancer cells, and the difference was not significant among them (Pgt;0.05). But on eight weeks after implantation of cancer cells, the volume of tumor was (0.65±0.13) cm 3 in HepG 2 cell group, (1.27±0.32) cm 3 in MDA-MB-231 cell group， and (1.43±1.09) cm 3 in control group, which suggested that the growth of tumor in HepG 2 cell group slowed down significantly when compared with MDA-MB-231 cell group and control group (Plt;0.01), although the difference in the latter two groups was not significant (Pgt;0.05). The necrosis regions were found with lymphocytic infiltration and apoptosis cells in HepG2 cell group after injection of recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3, but no pathological change of cells was found in control group after injection. It was found that the cells in MDA-MB-231 cell group were solidly arranged with small glandular lumenlike structure, marked cellular atypia, multinucleated tumor cells and megakaryocytic tumor cells, which was not different from that before injection. ConclusionThe targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3 induces the apoptosis of primary hepatocellular carcinoma in vivo and in vitro, which may provide new ways of targeted gene therapy.
Objective To study the effects on MCF-7 breast cancer cells with combination of tamoxifen（TAM） and antisense oligonucleotide （ASODN） targeting survivin mRNA. Methods MCF-7 breast cancer cells were treated with a 20 mer ASODN targeting survivin mRNA and TAM, which were divided into three groups: TAM group (treated by TAM only), ASODN group (by ASODN only), and TAM+ASODN combined group (by TAM+ASODN combination). The growth inhibition of MCF-7 cells, the changes of cell cycles and apoptotic rate, the positive rate of survivin mRNA expression, and the activity of caspase-3 were tested by MTT, flow cytometry, hybridization in situ, and spectrophotometric method, respectively.Results The rate of growth inhibition of MCF-7 cells in the TAM+ASODN combined group was (62.26±3.92)%, which was significantly higher than that in the TAM group 〔(42.30±6.63)%〕 or ASODN group 〔(54.77±9.99)%〕, Plt;0.05. The apoptotic rate of MCF-7 cells was (28.08±4.32)% in the TAM+ASODN combined group, which was significantly higher than that in the TAM group 〔(18.94±4.01)%〕 or ASODN group 〔(21.12±3.95)%〕, Plt;0.01. The effect of arresting MCF-7 cells in G0/G1 phase in the TAM+ASODN combined group was ber than that in the TAM or ASODN group (Plt;0.05, Plt;0.01). The positive rate of survivin mRNA in the TAM+ASODN combined group was (13.38±3.45)%, which was significantly lower than that in the TAM group 〔(39.67±7.42)%〕 or ASODN group 〔(27.50±5.80)%〕, Plt;0.01. The activity of caspase-3 in the TAM+ASODN combined group （0.93±0.13） was significantly higher than that in the TAM group (0.50±0.09) or ASODN group (0.64±0.08), Plt;0.01. Conclusion The ASODN targeting survivin mRNA can promote the apoptosis of MCF-7 breast cancer cells, and make MCF-7 cells more sensitive to tamoxifen.
【Abstract】ObjectiveTo investigate the protective effects and the impact on the expression of Bcl-2 and Caspase-3 mRNA by Panax notoginseng saponins (PNS) preconditioning in rat liver transplantation. MethodsMale Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT) and were divided into PNS preconditioning group (PNS group) and NS control group (NS group) randomly according to whether PNS was injected by venous (50 mg/kg) 1 h before liver grafts harvesting. The animals were respectively killed 2 h, 6 h and 24 h after reperfusion. Plasma samples were collected for ALT and AST test. Liver tissues were collected to detect histological changes, apoptosis and the expression of Bcl-2, Caspase-3 mRNA. ResultsThe serum levels of ALT and AST and the apoptosis index (AI) of liver tissue in PNS group were lower than those in NS group’s significantly (P＜0.05) at 2 h, 6 h and 24 h after reperfusion. The expression of Bcl-2 mRNA was enhanced significantly in PNS group at 6 h, 24 h after reperfusion and the expression of Caspase-3 mRNA was decreased significantly in PNS group at 2 h, 6 h after reperfusion as compared with NS group’s(P＜0.05). ConclusionPNS preconditioning can attenuate liver grafts ischemia/reperfusion injury and apoptosis of hepatocytes. Affecting expression of Bcl-2 and Caspase-3 genes may be one of the mechanisms of PNS antiapoptotic effects.
ObjectiveTo investigate the influence of sepsis on the expression of apoptotic protease caspase-3 in hippocampus neurons of rats. MethodsModels of rats with sepsis were established by the cecal ligation and puncture (CLP) method. Eighty 30-day-old male Wistar rats were randomly divided into CLP group (n=50) and control group (n=30). In the CLP group, only CLP was performed on the rats. Ten rats in the CLP group and another 10 in the control group were taken at 6, 12, and 24 hours after operation, respectively. Five of them in each group were taken for neurobehavioral score, and the other five were killed and their brains were removed. Then the Western blot and immunohistochemistry staining were used to detect the expression changes of apoptosis protein caspase-3. ResultsIn the control group, there were very low expression of apoptotic protease caspase-3 and high scores of neurological behavior. In the CLP group, the expression of apoptotic protease caspase-3 started to increase at the 6th hour, and reached the peak at the 24th hour after CLP, both of which were significantly higher than the control group (P<0.05). The scores of neurological behavior of the CLP group began to decline at 6h after CLP, and decreased gradually along with the time, and the scores were significantly lower at various time points after CLP in the CLP group than those in the control group (P<0.05). ConclusionThe scores of neurological behavior decrease and the expression of apoptosis protease caspase-3 increase in the rat hippocampus with sepsis, and fluctuate with time change.
ObjectiveTo investigate the effects of celastrol on the growth and apoptosis of huamn hepatoma SMMC-7721 cells, and investigate its preliminary action mechansim. MethodsSMMC-7721 cells were cultured in vitro, CCK-8 assay and Annexin V-FITC/PI staining method were conducted to investigate the effects of celastrol on the growth and apoptosis of huamn hepatoma SMMC-7721 cells after the cells were treated with drugs, and then the Caspase-3 activity and NF-κB protein expression were determined by Caspase-3 activity determination kit and Western blot. Huamn hepatoma SMMC-7721 cells transplantation tumor models in nude mice were established and the effect of celastrol on the growth of transplantation tumor were observed. ResultsCelastrol could inhibit the SMMC-7721 cells growth in a dose and time dependent manner. Annexin-V/PI staining showed that SMMC-7721 cells were induced to death with the concentration increasing of celastrol. Caspase-3 activity was measured after treatment with celastrol and the results indicated that the activity of caspase-3 was significantly enhanced. Western blot experiments showed that the expression of NF-κB protein decreased in a time-dependent manner after treatment with celastrol. Celastrol could inhibit SMMC-7721 cells transplantation tumor growth in nude mice. ConclusionsCelastrol could inhibit the proliferation of human hepatoma SMMC-7721 cells and induces apoptosis, and inhibit SMMC-7721 cell transplantation tumor growth in nude mice. Celastrol induce apoptosis of SMMC-7721 cells might through activating Caspase-3 pathway and NF-κB pathway.
ObjectiveTo investigate the inhibitory effect of T lymphocyte transplantation of EphrinAl-Caspase-3 on the growth of breast cancer.MethodsSix-week-old BALB/c nude mice were used to inoculate breast cancer cells to construct a nude mouse model of breast cancer. They were randomly divided into 3 groups according to random number table: PBS group received intratumoral injection of 10 μL PBS, and negative control group received intratumoral injection of 1×106 T lymphocytes uninfected with adenovirus, 1×106 EphrinAl-Caspase3-T lymphocytes were injected intratumorally into the infected group, and the tumors size (0, 3, 6, 9, 12 and 15 d) were measured with vernier calipers every 3 days until end of experiment. The content of EphrinAl-Caspase-3 in the tissues of the nude mice was measured. The presence of T lymphocytes expressing green fluorescent protein and the ratio of Caspase-3-positive and Ki-67-positive cell were observed by pathological examination.ResultsOn the day 0 and day 3, there were no significant difference in tumor volume between the 3 groups (P>0.05). On the 6th day and later, the difference between the infected group and the PBS group/negative control group were statistically significant (P<0.05), but there were no significant difference in tumor volume between the PBS group and negative control group at each time point (P>0.05). The presence of scattered green fluorescent protein-labeled EphrinAl-Caspase-3-T lymphocytes was observed in the tumor tissues of the infected group, while the presence of green fluorescent protein were not detected in the PBS group and the negative control group. In the infected cells, ratio of Caspase-3-positive cell was up-regulated and ratio of Ki-67-positive cell was down-regulated. The expression of EphrinAl-Caspase-3 could be detected on the 3rd day in the infected group, and at the peak on the 6-day, then the amount of secretion gradually decreased. The expression of EphrinAl-Caspase-3 were not detected in the PBS group and the negative control group at each time point.ConclusionEphrinAl-Caspase-3 can significantly inhibit the growth of breast cancer cells and promote apoptosis.