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 review the relationship between the expression levels of bcl-2, bax and bad gene and other biological factors of breast cancer in the growth and development of breast cancer. Methods Related literatures were summarized and reviewed. Results The expression level change of antiapoptosis gene bcl-2 was still under research and the expression levels of apoptosis gene bax and bad were down-regulated progressively in the evolution from benign breast tissue to breast cancer tissue. The expression level of bcl-2 had positive correlation with some positive factors in breast cancer such as estrogen receptor （ER） and progesterone receptor (PR), while it had negative correlation with some negative factors such as p53, EGFR, c-erbB-2 and lymph node metastasis. The levels of ER, PR and the expression level of p53 of breast cancer had no relationship with the expression level of bax. Up to now there was no report about the relationship between the expression level of bad and other biological factors of breast cancer. Conclusion The role of altered expression level of bcl-2, in the treatment and prognosis of breast cancer is still controversial, and the relationship between the expression of bad and the prognosis of breast cancer is still unknown, but expression level of bax is correlated positively with the prognosis of breast cancer. Research on these genes can provide us some new index to evaluate the prognosis of breast cancer and new ideas on treatment of breast cancer including gene therapy.
Objective To study the inhibitory effect of RNA interference (RNAi) on bcl-2 expression of vascular smooth muscle cells （VSMCs) in rabbit. Methods The expression vector of bcl-2 gene-targeting small interference RNA (pshRNA-bcl-2) was constructed and was transfected into VSMCs by lipofectamine, and the unloaded vector was used as control. The expression of bcl-2 mRNA was identified by RT-PCR and Western blot, respectively. The growth of the transfected VSMCs was examined by MTT. Results The pshRNA-bcl-2 may inhibit the expression of bcl-2 gene at the levels of transcription and translation. There were significant differences (P＜0.01) of the expressions of bcl-2 mRNA between the VSMCs that were transfected with pshRNA-bcl-2 and the ones in plasmid transfected group and control group, respectively. There was a significant difference (P＜0.01) in the growth of VSMCs between the plasmid transfected and the control groups. Conclusion The plasmid containing the small interference RNA of bcl-2 may have an inhibitory effect on the cell growth and endogenous expression of bcl-2 gene at the levels of transcription and translation in VSMCs.
Objective To investigate the growth inhibition and the apoptosis inducement effects of polysaccharide of trametes robiniophila murr (PS-T) on human rectal cancer cell line HR8348 and elucidate the possible mechanisms. Methods After treatment with PS-T, the growth inhibition rate of human rectal cancer cell strain HR8348 was studied by MTT method. The apoptotic index was detected by methyl green and pyronine Y staining and by terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling (TUNEL). The bcl-2, bcl-xl, bax, bak and p53 gene expressions of HR8348 cells were examined by immunohistochemical method.Results PS-T induced a dosedependent inhibition of HR8348 cells in vitro. The maximum percentage of growth inhibition was 71.1% by 36 h after administration of PS-T at a concentration of 4.0 mg/ml. There was no significant difference in the inhibitory rate as compared to the positive control (5-FU 10 μg/ml, P gt;0.05). The typical apoptosis phenomenons were observed under the light microscope by both staining methods. The apoptotic index increased parallelling with the increase of concentration of PS-T. When the PS-T concentration was 4.0 mg/ml, the apoptotic index determined by methyl green and pyronine Y staining and by TUNEL method increased rapidly to 0.1620±0.0128 and 0.2612±0.0158, respectively, which was greater than that of the positive control (5-FU 10 μg/ml, Plt;0.05). The expression of bcl-2, bcl-xl, bak and p53 was increased in the PS-Ttreated HR8348 cells by 36 h, while the bax remained unchanged. Expression index of bak/bcl-2 and bak/bcl-xl was increased significantly as compared with the control (Plt;0.05). Conclusion PS-T can significantly inhibit growth and induce apoptosis of human rectal cancer cell strain HR8348 in vitro. The apoptosis induced by PS-T might be related to the increase of the ratio of bak to bcl-2 and to bcl-xl and upregulation of the expression of p53 gene.
Objective To study the effect of 5-fluorouracil (5-FU) induced apoptosis of the rectal carcinoma cell line HR8348 in vitro and the relationship between apoptosis induced by 5-FU and the expression of bcl-2，bcl-xl，bax and p53,and to investigate the possible mechanism of apoptosis of rectal carcinoma cell line HR8348 induced by 5-FU.Methods After treatment with 5-FU for 24 h,the apoptotic index was detected by methyl green and pyronine Y staining and by terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL).The bcl-2,bcl-xl,bax and p53 gene expression of HR8348 cells were examined by immunohistochemical method.Results After treatment with 5-FU,the apoptotic index of experiment group was significantly increased,there was significant difference as compared with the control.Exposed to 5-FU for 12 h,24 h and 36 h,the expression of bcl-2 of HR8348 cell line remained unchanged,but the expression of bcl-xl slightly diminished,while the expression of bax was remarkly increased,the expression of p53 was not detected in both experiment and control groups.Conclusion This results indicate that 5-FU may induce apoptosis of rectal carcinoma cell line HR8348 and the possible mechanism of apoptosis induction is through upregulation of bax expression and the change of bax to bcl-xl ratio.
To elucidate bcl-2 protein expression in hepatic carcinogenetic process and its relationship with apoptotic changes. bcl-2 protein was evaluated immunohistochemically while apoptosis was approached with terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL) technique in 8 normal livers (NL), 17 liver cirrhosises (LC) and 77 hepatocellular carcinomas (HCC). The results showed that bcl-2 protein was expressed in 3 of 8 NLs(37.5%), 5 of 17 LCs(29.4%) and 7 of 77 HCCs(9.1%) with significant differences between group NL and HCC and between LC and HCC (Ｐ＜0.05). Apoptosis rates of 1.18±0.42%, 4.85±2.78%, 12.89±2.33% in NL, LC, HCC group respectively were demonstrated with significant differences among them (Ｐ＜0.01). Compared the bcl-2 expression with the apoptosis rate in this hepatocarcinogenetic process, reversed trends were presented. Conclusion: bcl-2 expression could be detected in NL, LC and HCC, and its decreasing expression was related to the inhibition attenuation of hepatocellular apoptosis in the process of hepatocarcinogenesis.
Objective To investigate the temporal and spatial expression pattern of Caspase3、Bax and Bclxl in NmethylNnitrosourea (MNU) damaged rat retina. Methods Twenty-four 50 dayold female Sprague-Dawley rats (n=24) received single intraperitoneal injection of MNU 40 mg/kg and were examined at 1, 3, 7 and 10 days after MNU treatment (6 rats sacrificed at each timepoint). As control, six rats were injected with saline (5 ml/kg) and sacrificed 3d after injection. Expressions of Caspase-3 and bax and bcl-xl were detected by RTPCR and immunofluorescence assays, photoreceptor cell apoptosis was measured by terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatedigoxigenin nick-end labeling (TUNEL). Results Animal models were successful established and confirmed by pathological studies. RTPCR results indicated that caspase3 and bax upregulated at 1 d (caspase-3 RA =83.23plusmn;8.11，P= 0.009; bax-RA=72.73plusmn;9.46，P=0.004) and peaked at 3 d （caspase-3 RA=140.48plusmn;18.40，P=0.000；bax-RA=102.36plusmn;13.97，P=0.001）compared with control (caspase-3 RA=62.45plusmn;7.65; bax-RA =46.53plusmn;4.41). Bcl-xl expression increased and peaked at 3d (3d RA=79.83plusmn;5.58, P=0.000 vs control 45.98plusmn;3.06). It was noted that the ratios of bax / bclxl expression at 1 d, 3 d and 7 d after MNU injection were enhanced (1 d 1.15plusmn;0.14, P= 0.143; 3 d 1.28plusmn;0.16, P=0.001; 7 d 1.17plusmn;0.08, P= 0.079, vs control 1.01plusmn;0.09), and at 3 d the ratio reached the peak, whereas at10 d bax / bcl-xl ratio (0.73plusmn;0.07, P= 0.001) was decreased compared with the control. Immunofluorescence assays demonstrated that the changes of bax, bclxl and caspases3 protein expressions coincided with their RTPCR results respectively. The Bax positive cells were detected in the outer nuclear layer; while caspase3 and bclxl positive cells emerged in several layers of retina included the pigment epithelium layer, the photoreceptor cell inner segments, the outer nuclear layer, the outer plexiform layer, the inner plexiform layer and the ganglion cell layer. Photoreceptor cell apoptosis was only detected in the outer nuclear layer and peaked at 3 d in MNU treated rats (AI= 76.97plusmn;5.83, P= 0.000 vs control 0.00 plusmn; 0.00). Conclusions These data suggest that bax and bcl-xl and caspases3 may involve in the MNUinduced rat photoreceptor cell apoptosis.
Objective To explore the expression of survivin gene in retinoblastoma (RB) and its relationship with the stages and histodifferentiation degree of RB and the expression of p53、bcl-2 proteins. Methods Expression of survivin, p53 and bcl-2 proteins in 38 RB conventional paraffin samples were detected with survivin, p53 and bcl-2 monoclonal antibodies respectively by immunohistochemical assay. The expression of survivin of normal retina in 6 control samples was observed. Results In 38 cases of RB, positive expression of survivin was found in 20 (52.6%); while none of the 6 normal retinal tissue expressed survivin, which had significant difference between the two group (P＜0.05). The positive expression of survivin did not correlate with sex of patient, disease stages and histological type (P＞0.05). In 38 RB cases, positive expression of p53 was in 25 with the rate of 65.8%, and of bcl-2 in 18 with the rate of 47.4%. The positive-expressed rates were much higher in positive-expressed p53 and bcl-2 group than those in the negative-expressed p53 and bcl-2 group(P＜0.05). Conclusion The increase of the expression of survivin implies that it may take part in the occurrence and development of RB; the interaction among survivin, p53 and bcl-2 may participate in the access and the course of RB. (Chin J Ocul Fundus Dis，2004，20：215-217)
Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
Objective To study the effect of hypoxia on proliferation of cultured bovine retinal pigment epithelium (RPE) cells and expression of the antiapoptotic protein bcl-2. Methods The bovine RPE cells were cultured under normal and hypoxic chamber respectively. After 24 hours, the proliferation of RPE cells was evaluated by［3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide, MTT］test. At the same time, anti-bcl-2 protein antibody was examined by immuno-histochemistry method. Results The A value in the hypoxia group was higher than that in the normal group after 24 hours (P＜0.05 )in MTT-test. Positive staining for anti-bcl-2 protein antibody was seen in 72.6% cells in hypoxia group and 38.64% in normal group. The positive staining was more obvious near the nucleus, and fine granules scattered in cytoplasm of some cells. Conclusion Hypoxia can stimulate the proliferation of RPE cells and expression of antiapoptotic protein bcl-2. The results indicate that bcl-2 may play an important role in mediating the proliferation activity of RPE cells. (Chin J Ocul Fundus Dis, 2002, 18: 293-295)