Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

AIM： To investigate anti-tumor activities and apoptosisregulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.
METHODS： BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups （n = 15）. Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg （BF1）, 1 mg/kg （BF2） and 0.5 mg/kg （BF3） for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues.
RESULTS： The tumor volumes in each group of bufalin were reduced significantly （35.21±12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P 〈 0.01, respectively）, and the survival times were prolonged in group BF1-2 （31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P 〈 0.05, respectively）, and necrosis was mainly in severe or moderate degree in group BF1-2. No morphological changes were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA     

Anticancer activity of genistein on implanted tumor of human SG7901 cells in nude mice

AIM： To investigate genistein-induced apoptosis of implanted tumors of SG7901 cells in nude mice, and the relationship between this apoptosis and expression of Bcl-2 and Bax. METHODS： Establishing a transplanted tumor model by injecting human SG7901 cells into subcutaneous tissue of nude mice. Genistein （0.5, 1 and 1.5 mg/kg） was directly injected adjacent to the tumor, six times at 2-d intervals. Then, changes in tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphological alterations by transmission electron microscopy （TEN）, measured the apoptotic rate by the TUNEL staining method, and detected the expression of apoptosisregulated gene Bcl-2 and bax by immunohistochemical staining and RT-PCR. RESULTS： Genistein 0.5, 1 and 1.5 mg/kg significantly inhibited carcinoma growth when it was injected near the tumor by 10.8%, 29.9% and 39.6%, respectively. Genistein induced implanted tumor cells to undergo apoptosis, with apoptotic characteristics seen by TEM. The apoptosis index was increased progressively with increasing genistein dose （28.9% ± 1.2%, 33.8% ±1.6% and 37.7% ±1.2%）. The positive rate of Bcl-2 protein was decreased progressively （11.9%± 0.9%, 5.9%± 0.7% and 4.2% ±0.6%）, and the positive rate of bax protein was increased progressively （0.9% ±1.7%, 24.9% ±0.8% and 29.6% ± 1.7%） by immunohistochemical staining, with increasing dose of genistein. The density of Bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR. CONCLUSION： Genistein was able to induce apoptosisof transplanted tumor cells. This apoptosis may be mediated by down-regulation of the apoptosis-regulated gene Bcl-2 and up-regulation of apoptosis-regulated gene bax.     

Resveratrol induces apoptosis in human esophageal carcinoma cells

AIM: To investigate the apoptosis in esophageal cancer cells induced by resveratrol, and the relation between this apoptosis and expression of Bcl-2 and Bax. METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of esophageal cancer cell line EC-9706 before and after the resveratrol treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax. RESULTS: Resveratrol inhibited the growth of esophageal cancer cell line EC-9706 in a dose-and time-dependent manner. Resveratrol induced EC-9706 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. TUNEL assay showed that after the treatment of EC-9706 cells with resveratrol (10 mmol/L) for 24 to 96 hours, the AIs were apparently increased with treated time (P<0.05). Immunohistochemical staining showed that after the treatment of EC-9706 cells with resveratrol (10 mmol/L) for 24 to 96 hours, the PRs of Bcl-2 proteins were apparently reduced with treated time (P<0.05) and the PRs of Bax proteins were apparently increased with treated time (P<0.05). CONCLUSION: Resveratrol is able to induce the apoptosis in esophageal cancer. This apoptosis may be mediated by down-regulating the apoptosis-regulated gene Bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.     

Antitumor and immunomodulatory activity of resveratrol onexperimentally implanted tumor of H22 in Balb／c mice

AIM: To study the antitumor and immunomodulatory activity of resveratrol on experimentally implanted tumor of H22 in Balb/c miceMETHODS: The cytotoxicity of peritoneal macrophages (M φ ) against H22 cells was measured by the radioactivity of [3H]TdR assay, mice with H22 tumor were injected with different concentrations of resveratrol, and the inhibitory rates were calculated and IgG contents were determined by single immunodiffusion method. the plaque forming cell (PFC) was measured by improved Cunningham method, the levels of serum tumor necrosis factor-α (TNF-α) were measured by cytotoxic assay against L929 cells.implanted tumor of H22 in mice. The inhibitory rates were 31.5 %, 45.6 % and 48.7 %, respectively (P＜0.05), which could raise the level of serum IgG and PFC response to production of serum TNF-α in mice H22 tumor. However,the effect of resveratrol was insignificant (P ＞0.05).CONCLUSION: Resveratrol could inhibit the growth of H22tumor in Balb/c mice. The antitumor effect of resveratrol might be related to directly inhibiting the growth of H22cells and indirectly inhibiting its potential effect on nonspecific host immunomodulatory activity.     

白藜芦醇对实验性兔骨关节炎软骨细胞凋亡及bcl-2 bax表达的影响

目的 研究白藜芦醇对兔实验性骨关节炎(OA)软骨细胞凋亡及凋亡调控基因bcl-2、bax表达的影响,探讨其治疗OA的机制.方法 30只新西兰大白兔随机平分为5组,A组(健康对照组)、B组(模型对照组)、C组(白藜芦醇高剂量干预组)、D组(白藜芦醇中剂量干预组)、E组(白藜芦醇低剂量干预组).除A组外,其他各组均以Hulth法复制膝OA模型.术后第4周开始,A组、B组每天以5 ml含0.1%二甲基亚砜的蒸馏水灌胃;白藜芦醇干预各组每天以相应剂量白藜芦醇溶液(浓度为60 mg/ml)灌胃,C、D、E组日剂量分别为120、60、30mg·kg-1·d-1,连续6周.第10周处死大白兔,取右膝关节股骨内髁内侧软骨,软骨组织切片用脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法观察软骨细胞凋亡,免疫组织化学法观察软骨细胞bcl-2和bax的表达.结果 ①模型对照组关节软骨细胞凋亡率明显高于健康对照组;白藜芦醇干预各组可不同程度地降低OA软骨细胞凋亡率,差异有统计学意义(P＜0.05),且呈剂量依赖性.②与健康对照组相比,模型对照组关节软骨细胞bcl-2和bax阳性率均升高(P＜0.01),bcl-2/bax的比值降低;白藜芦醇干预后,bcl-2阳性率升高更为明显(P＜0.01);而bax阳性率有不同程度降低(P＜0.01);bcl-2/bax的比值均不同程度提高(P＜0.01).结论 白藜芦醇通过上调bcl-2的表达,下调bax的表达,提高bcl-2/bax的比值,以抑制兔实验性OA中软骨细胞的过度凋亡,达到保护软骨,防治OA的目的 .     

Anticancer effect and apoptosis induction of gambogic acid in human gastric cancer line BGC-823

AIM: To investigate the anticancer effect of a traditional Chinese medicine gambogic acid (GA) in human gastric cancer line BGC-823 and further study the mechanism of apoptosis induction of GA. METHODS: Low differential human gastric cancer line BGC-823 were treated with GA at different doses and different times, the inhibitory rates were detected by MTT assay. Apoptosis induced by GA in BGC-823 cells was observed by Annexin-V/PI doubling staining flow cytometry assay. And T/C (%) was chosen to detect the inhibition of GA on human gastric adenocarcinoma BGC-823 nude mice xenografts. Apoptosis on nude mice xenografts was observed by Annexin-V/PI doubling staining flow cytometry assay and DNA fragmentation assay. To further determine the molecular mechanism of apoptosis induced by GA, the changes on the expression of bcl-2 and bax genes were detected by RT-PCR. RESULTS: After incubation with GA, low differential human gastric cancer line BGC-823 was dramatically inhibited in a dose-dependent manner. After these cells were exposed to GA for 24, 48 and 72 h, the IC50 value was 1.02±0.05, 1.41±0.20 and 1.14±0.19 μmol/L, respectively. Apoptosis in BGC-823 cells induced by GA was observed by Annexin V/PI doubling staining flow cytometry assay. The apoptotic population of BGC-823 cells was about 12.96% and 24.58%, respectively, when cells were incubated with 1.2 μmol/L GA for 48 and 72 h. T/C (%) of human gastric carcinoma adenocarcinoma BGC-823 nude mice xenografts was 44.3, when the nude mice were treated with GA (8 mg/kg). Meanwhile, apoptosis induced by GA was observed in human gastric carcinoma adenocarcinoma BGC-823 nude mice xenografts. The increase of bax gene and the decrease of bc1-2 gene expressions were found by RT-PCR. CONCLUSION: The inhibition of GA on human gastric cancer line BGC-823 was confirmed. This effect connects with the inducing apoptosis in BGC-823 cells and the molecular mechanism might be related to the reduction of expression of apoptosis-regulated gene bcl-2, and the improvement of the expression of apoptosis-regulated gene bax. The result was also confirmed in vivo.     

三氧化二砷抗肝癌作用的实验研究

目的 研究三氧化二砷的抗肝癌作用,并探讨其作用机制。方法 建立小鼠ＨｅｐＡ肝癌实体型及腹水型移植瘤模型,静注三氧化二砷,观察三氧化二砷对荷瘤鼠的作用。结果 三氧化二砷２．０ｍｇ．ｋｇ＾－１．ｄ＾－１和３．５ｍｇ．ｋｇ＾－１．ｄ＾－１连续静脉注射７ｄ,实体型荷瘤瘤鼠皮下肿瘤的生长受到明显抑制,抑瘤率分别为３１．６３％和４２．１３％;实体瘤标本在电镜下见有部分细胞发生了典型的凋亡形态学改变;原位末端标     

Effect of resveratrol and in combination with 5-FU on murine liver cancer

AIM: To study the anti-tumor effect of resveratrol and in combination with 5-FU on murine liver cancer. METHODS: Transplantable murine hepatoma22 model was used to evaluate the anti-tumor activity of resveratrol (RES) alone or in combination with 5-FU in vivo. H22 cell cycles were analyzed with flow cytometry. RESULTS: Resveratrol could inhibit the growth of murine hepatoma22, after the mice bearing H22 tumor were treated with 10 mg/kg or 15 mg/kg resveratrol for ten days, and the inhibition rates were 36.3% (n = 10) and 49.3% (n = 9), respectively, which increased obviously compared with that in control group (85+/-22 vs 68+/-17, P<0.01). RES could induce the S phase arrest of H22 cells, and increase the percentage of cells in S phase from 59.1% (n = 9) to 73.5% (n = 9) in a dose-dependent manner (P<0.05). The enhanced inhibition of tumor growth by 5-FU was also observed in hepatoma22 bearing mice when 5-FU was administered in combination with 10 mg/kg resveratrol. The inhibition rates for 20 mg/kg or 10 mg/kg 5-FU in combination with 10 mg/kg resveratrol were 77.4% and 72.4%, respectively, compared with the group of 20 mg/kg or 10 mg/kg 5-FU alone, in which the inhibition rates were 53.4% and 43.8%, respectively (n = 8). There was a statistical significance between the combination group and 5-FU group. CONCLUSION: RES could induce the S phase arrest of H22 cells and enhance the anti-tumor effect of 5-FU on murine hepatoma22 and antagonize its toxicity markedly. These results suggest that resveratrol, as a biochemical modulator to enhance the therapeutic effects of 5-FU, may be potentially useful in cancer chemotherapy.     

二硫代氨基甲酸吡咯烷联合苦参碱对人肝癌裸鼠移植瘤生长的抑制作用

目的 探讨二硫代氨基甲酸吡咯烷(PDTC)抑制核因子-κ B(NF-κ B)活化后对苦参碱抑制人肝癌裸鼠移植瘤生长的影响.方法 建立人肝癌细胞HepG2裸鼠皮下移植瘤模型,随机分为对照组(灭菌等渗盐水)、苦参碱组(35 mg/kg)、PDTC组(120 mg/kg)和PDTC(120 mg/kg)+苦参碱(35 mg/kg)联合组,腹腔注射用药.绘制肿瘤生长曲线,测定肿瘤生长抑制率;TdT介导的dUTP缺口末端标记法检测肿瘤细胞凋亡情况;电泳迁移率变动分析法检测细胞核内NF-κB的活化水平;免疫组织化学法检测肿瘤组织bcl-2和bax蛋白表达水平;RT-PCR法检测肿瘤细胞NF-κB、bcl-2和bax的mRNA表达水平.多组间比较用SNK-q检验,单独效应比较采用LSD法,相关分析采用Pearson法进行分析.结果 PDTC增强了苦参碱对肿瘤增殖的抑制作用(P＜0.05);苦参碱在诱导肿瘤细胞凋亡的同时激活NF-κB;PDTC能显著抑制苦参碱诱导的NF-κB活化,NF-κB活性的灰度值由93.64±2.95降至65.78±5.65(F=124.754,P＜0.01),同时促进苦参碱诱导肿瘤细胞凋亡,细胞凋亡指数由55.9%±2.8%升高至74.3%±4.8%(P＜0.05).NF-κB的mRNA表达水平与bcl-2的mRNA表达水平呈正相关(r=0.983,P＜0.01).结论苦参碱诱导皮下移植瘤细胞凋亡的同时激活NF-κB;PDTC可通过抑制NF-κB的活化而下调bcl-2的表达,改变bcl-2与bax的比值,增强苦参碱诱导肿瘤细胞凋亡的作用.

Abstract：

Objective To investigate the relationship between activation of nuclear factor-κ-gene binding (NF-κ B) and apoptosis induced by matrine(MT) in transplanted tumor of human hepatocellular carcinoma in nude mouse. Methods Tumors were established by injection of hepatocellular carcinoma cell line HepG2 into the back of nude mice. The mice were divided randomly into four groups: Control group, MT group (35 mg/kg), PDTC group (120 mg/kg) and Combination group: PDTC+MT group (120 mg/kg+35 mg/kg), the reagents were injected peritoneally. The tumor growth curve of nude mice bearing transplanted tumor were observed and the inhibition ratios were evaluated. Apoptosis of carcinoma cells was analyzed by TUNEL. The DNA-bingding activity of NF-κ B was determined by electrophoretic mobility shift assay (EMSA). Expression of bcl-2 and bax in carcinoma tissue were detected by immunohistochemical method.NF-κ B mRNA, bcl-2 mRNA and bax mRNA in carcinoma tissue were detected by RT-PCR. Results Pyrrolidine dithiocarbamate (PDTC) could enhance the inhibition of matrine on carcinoma proliferation (P＜0.05). The apoptosis and activation of NF-κB in carcinoma cells could be induced by matrine. PDTC significantly suppressed NF-κ B activation induced by matrine in carcinoma cells from 93.64±2.95 to 65.78±5.65 (F=124.754, P＜0.01). Meanwhile, PDTC increased the apoptosis induced by matrine from 55.9%±2.8%to 74.3%±4.8% (P＜0.05).A positive correlation observed between the expressions of NF-κ B and of bcl2 (Pearson correlation coefficient=0.983,P＜0.01). Conclusions Matrine could induce apoptosis and activation of NF-κ B in transplanted tumor. PDTC could increase apoptosis in hepatocellular carcinoma cells might be due to the suppression of NF-κ B activation and the enhancement of bcl-2 expression.     

Effect of resveratrol on cartilage protection and apoptosis inhibition in experimental osteoarthritis of rabbit

To observe the effect of resveratol on cartilage,chondrocyte apoptosis, and nitric oxide in experimental osteoarthritis (OA) of rabbit and to study the mechanism of resveratol in the treatment of osteoarthritis. Thirty New Zealand rabbits were randomly divided into 5 groups: group A (normal control group), group B (model control group), group C (resveratrol intervention high-dosage group), group D (resveratrol intervention middle dosage group), and group E (resveratrol intervention low-dosage group). The model of OA of the knee was established using Hulth technique in groups B, C, D, and E. After 4?weeks, group A and group B rabbits were administered daily a knees injection of dimethylsulfoxide (DMSO), whereas groups C, D, and E were administered daily a knees injection of resveratrol in DMSO in different dosages for 2?weeks. Daily dosage for rabbits of groups C, D, and E was fixed at 50, 20, and 10?μmol/kg, respectively. Then, the rabbits were killed, and the lateral cartilage sections of right femoral medial condyle were evaluated using a histological scoring system (H&E and safranin-O staining) and analyzed by TUNEL for apoptosis. Nitric oxide (NO) in synovial fluid was measured by nitrate reduction method. Histological evaluation of cartilage tissue revealed a significantly reduced cartilage destruction; the evaluation also revealed the loss of matrix proteoglycan content in cartilage in resveratrol intervention groups compared to the model control. Resveratrol reduced the apoptosis rate of chondrocyte and level of NO in the synovial fluid. In the above experiments of OA rabbits, the protective effects of resveratrol were enhanced with increased resveratrol dosage. Resveratrol controls the progression of experimental OA. One of the mechanism(s) responsible for this effect would include lowering of the apoptosis rate of chondrocyte and reducing the production of NO in experimental OA.     

Effect of resveratrol on microcirculation disorder and lung injury following severe acute pancreatitis in rats

AIM: To investigate the mechanism of resveratrol underlying the microcirculation disorder and lung injury following severe acute pancreatitis (SAP). METHODS: Twenty-four rats were divided into 3 groups (SAP, sham and resveratrol groups) randomly. SAP model was established by injecting 4% sodium taurocholate l mL/kg through puncturing pancreatic ducts. Sham (control) group (8 rats) was established by turning over the duodenum. Resveratrol was given at 0.1 mg/kg b.m. intraperitoneally. Rats were sacrificed 9 h after SAP was induced. Blood samples were obtained for hemorrheological examination. Lung tissues were used for pathological observation, and examination of microvascular permeability, dry/wet ratio and myeloperoxidase (MPO) activity. Gene expression of intercellular adhesion molecule-1 (ICAM-1) was detected by RT-PCR. RESULTS: Compared with SAP group, resveratrol relieved the edema and infiltration of leukocytes in the lungs. Resveratrol improved markers of hemorrheology: high VTB (5.77±1.18 mPas vs9.49±1.34 mPas), low VTB (16.12±3.20 mPas vs30.91±7.28 mPas), PV (4.69±1.68 mPas vs 8.00±1.34 mPas), BSR (1.25±0.42 mm/h vs50.03±0.03 mm/h), VPC (54.67±3.08% vs 62.17±3.39%), fibrinogen (203.2?7.8 g/ L vs 51.3±19.1 g/L), original hemolysis (0.45±0.02 vs 0.49±0.02), and complete hemolysis (0.41±0.02 vs 0.43±0.02) (P<0.05). Resveratrol decreased the OD ratio of ICAM-1 gene (0.800±0.03 vs 1.188±0.10), dry/wet ratio (0.74±0.02 vs 0.77±0.03), microvascular permeability (0.079±0.006 vs 0.112±0.004) and MPO activity (4.42±0.32 vs 5.03±0.51) significantly (P<0.05). CONCLUSION: Resveratrol can improve the microcirculation disorder of the lung by decreasing leukocyte-endothelial interaction, reducing blood viscosity, improving the decrease of blood flow, and stabilizing erythrocytes in SAP rats. It may be a potential candidate to treat SAP and its severe complications (ALI).     

Anti-hepatoma activity of resveratrol in vitro

AIM: To study the anti-tumor effect of resveratrol alone and the synergistic effects of resveratrol with 5-FU on the growth of H22 cells line in vitro. METHODS: The number of cells was measured by MTT method the morphological changes of H22 cells were investigated under microscopy and electron microscopy examination. RESULTS: Resveratrol inhibited the growth of hepatoma cells line H22 in a dose- and time-dependent manner, IC50 of the resveratrol on H22 cells was 6.57mg x L(-1),The synergistic anti-tumor effects of resveratrol with 5-FU increased to a greater extent than for H22 cells treated with 5-FU alone (70.2% vs 28.4%) P<0.05 .Under microscope and electron microscope, characteristics of apoptosis such as typical apoptotic bodies were commonly found in tumor cells in the drug-treated groups. CONCLUSION: Resveratrol can suppresses the growth of H22 cells in vitro,its anti-tumor activity may occur through the induction of apoptosis.     

Effects and mechanisms of somatostatin analogs on apoptosis of pancreatic acinar cells in acute pancreatitis in mice

AIM: To investigate the effects of somatostatin analogs (SSa) on apoptosis of pancreatic acinar cells and apoptosis-regulated gene bax, and p53 in treating acute pancreatitis in mice. METHODS: In cerulein-induced pancreatitis, with or without treatment of somatostatin, analogs (Octreotide) in CD-1 (BALB/c x DBetaAlpha/1) mice, apoptosis of pancreatic acinar cells was detected by using the TdT-mediated dUTP nick-end labeling (TUNEL) method, and the expression of apoptosis-regulated gene bax and p53 was determined by using the streptavidin-peroxidase immunohistochemical technique and the RT-PCR method, respectively. RESULTS: On HE staining, acinar cells in the pancreas showed pyknotic nuclei and the formation of apoptotic bodies, which are the typical morphological features of apoptosis. Regarding TUNEL use, the apoptotic index of pancreatic acinar cells in the non-treated group at 5 and 14 h after induction of acute pancreatitis was significantly lower than those of the SSa-treated group, respectively (P < 0.01). On immunohistochemistry and RT-PCR, there was an expression of neither bax nor p53 in normal pancreatic tissues. The expression of bax in the SSa-treated group at 5 and 14 h after treatment of SSa was markedly higher than those of the non-treated group, respectively (P < 0.01), but there was no significant difference in the expression of p53 between the SSa-treated group and the non-treated group. CONCLUSIONS: The induction of apoptosis in pancreatic acinar cells injury to reduce inflammatory reaction might be one of the mechanisms of SSa in treating acute pancreatitis in mice, and the mechanisms of apoptosis probably correlated with the expression of apoptosis-regulated gene bax, but have no relationship with the expression of p53.     

Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML)

The inability to undergo apoptosis is a crucial mechanism of multidrug resistance in acute myeloid leukemia (AML), and the analysis of mitochondrial apoptotic proteins may represent a significant prognostic tool to predict outcome. Bcl-2 and Bax oncoproteins were evaluated in 255 de novo AML patients (pts) by flow cytometry using an anti-bcl-2 monoclonal antibody (MoAb) and an anti-bax MoAb. The results were expressed as an index (bax/bcl-2) obtained by dividing bax mean fluorescence intensity (MFI) and bcl-2 MFI. Lower bax/bcl-2 ratio was associated with French-American-British (FAB) M0-M1 classes (P =.000 01) and CD34 more than 20% (P <.000 01). There were striking inverse correlations between CD34 or CD117 MFI and bax/bcl-2 values (r = -.40, P <.000 001 and r = -.29, P =.000 002), confirming that immaturity is consistent with this index. Moreover, lower bax/bcl-2 levels were correlated with poor-risk cytogenetics (P =.0002). A significant higher complete remission (CR) rate was found in pts with higher bax/bcl-2 levels (79% versus 45%; P =.000 01). Also, both a longer overall survival (OS) and disease-free survival (DFS) were observed in pts with higher bax/bcl-2 levels (P =.000 01 and =.019). Noteworthy, bax/bcl-2 levels accurately predicted the clinical response and outcome of pts with normal or unknown cytogenetics. Indeed, within this subset of 147 pts, higher bax/bcl-2 ratio was significantly associated both with a higher CR rate (86% versus 42%; P <.000 01) and a longer OS (P =.0016). The independent prognostic value of bax/bcl-2 ratio was confirmed in multivariate analysis. Therefore, mitochondrial oncoproteins, such as bcl-2 and bax, represent both sensitive indicators of clinical outcome and potential targets of novel proapoptotic molecules in order to circumvent chemoresistance.     

苦参素注射液联合顺铂对人肝癌SMMC-7721细胞凋亡相关基因c-myc、bcl-2和bax表达的影响

目的探讨苦参素注射液(MI)联合顺铂(DDP)对人肝癌SMMC-7721细胞凋亡相关基因c-myc、bcl-2和bax表达的影响。方法分别采用MI、顺铂及MI联合顺铂干预SMMC-7721细胞。TUNEL法检测细胞凋亡率,半定量RT-PCR法检测c-myc、bcl-2和bax mRNA表达,二步法免疫组化检测c-myc、bcl-2和bax蛋白表达。结果苦参素组、顺铂组和联合用药组细胞凋亡率显著上升,与对照组(不干预)比较差异有统计学意义(P<0.05或P<0.01),联合用药具有单纯相加或协同作用;联合用药组的细胞凋亡率显著增加,bax mRNA和蛋白表达显著升高,c-myc、bcl-2 mRNA和蛋白表达显著减少,与DDP单药组比较差异有统计学意义(P<0.05或P<0.01)。结论苦参素注射液联合顺铂具有单纯相加或协同诱导肝癌SMMC-7721细胞凋亡作用,其机制可能与bax基因表达上调和c-myc、bcl-2基因表达下调有关。     

Apoptosis of human primary gastric carcinoma cells induced by genistein

AIM: To investigate the apoptosis in primary gastric cancer cells induced by genistein, and the relationship between this apoptosis and expression of bcl-2 and bax. METHODS: MTT assay was used to determine the cell growth inhibitory rate in vitro. Transmission electron microscope and TUNEL staining were used to quantitatively and qualitatively detect the apoptosis of primary gastric cancer cells before and after genistein treatment. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-associated genes bcl-2 and bax. RESULTS: Genistein inhibited the growth of primary gastric cancer cells in dose-and time-dependent manner. Genistein induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the apoptotic rates of primary gastric cancer cells increased time-dependently. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the positivity rates of Bcl-2 proteins were apparently reduced with time and the positivity rates of Bax proteins were apparently increased with time. After exposed to genistein at 20 micromol/L for 24, 48, 72 and 96 respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time. CONCLUSION: Genistein is able to induce the apoptosis in primary gastric cancer cells. This apoptosis may be mediated by down-regulating the apoptosis- associated bcl-2 gene and up-regulating the expression of apoptosis-associated bax gene.     

Correlation between expression of gastrin, somatostatin and cell apoptosis regulation gene bcl-2／bax in large intestine carcinoma

AIM: To explore the correlation between expression of somatostatin (SS), gastrin (GAS) and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma. METHODS: Sixty-two large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma. Immunohistochemical staining for bcl-2, bax, GAS, SS was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. According to the semi-quantitative integral evaluation, SS and GAS were divided into three groups as follows. Scores 1-3 were defined as the low expression group, 4-8 as the intermediate expression group, 9-16 as the high expression group. Bax and bcl-2 protein expressions in different GAS and SS expression groups of large intestine carcinoma were assessed. RESULTS: The positive expression rate of bax had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, x2SS = 9.246; P<0.05, x2GAs = 6.981). The positive expression rate of bax in SS high (80.0%, 8/10) and intermediate (76.5%, 13/17) expression groups was higher than that in low expression group (40.0%, 14/35) (P<0.05, X2high vslow = 5.242; P<0.05, x2middle vs low = 6.097). The positive expression rate of bax in GAS high expression group (27.3%, 3/8) was lower than that in low expression group (69.4%, 25/36) (P<0.05, x2 = 4.594). However, bax expression in GAS intermediate expression group (46.7%, 7/15) was lower than that in low expression group, but not statistically significant. The positive expression rate of bcl-2 had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, x2ss = 7.178; P<0.05, x2GAS = 13.831). The positive expression rate of bcl-2 in GAS high (90.9%, 10/11) and intermediate (86.7%, 13/15) expression groups was higher than that in low expression group (44.4%, 16/36) (P<0.05, x2high,vslow = 5.600; P<0.05, x2middle vs low = 7.695). However, the positive expression rate of bcl-2 in SS high (40. 0%, 4/10) and intermediate (47.1%, 8/9) expression groups was lower than that in low expression group (77.1%, 27/35) (P<0.05, x2high vs low = 4.710; P<0.05, x2middle vs low = 4.706). There was a significant positive correlation between the integral ratio of GAS to SS and the integral of bcl-2 (P<0.01, r = 0.340). However, there was a negative correlation between the integral ratio of GAS to the SS and bax the integral of (P<0.05, r= -0.299). CONCLUSION: The regulation and control of gastrin, somatostatin in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of bcl-2, bax.     

β1-AR阻断剂联合β2-AR激动剂对心肌梗死大鼠心功能的影响及机制

目的 观察美托洛尔与特布他林联用对心梗大鼠心功能的影响,从心肌细胞凋亡及氧化应激角度探讨其机制.方法 结扎冠状动脉前降支制备心梗大鼠模型,随机分为美托洛尔组(M 组)、特布他林组(T 组)、联合治疗组(L 组)和安慰剂组(A 组),另选一组为假手术组(S 组),每组8 只.术后1 周灌胃给药,M 组给予美托洛尔20 mg/kg,T 组给予特布他林2 mg/kg,L 组给予美托洛尔和特布他林,剂量同上,A 组给予等量生理盐水,S 组不予处理.生理盐水及药物皆为2 次/d 灌胃.8 周后行超声心动图、血流动力学、心肌组织GSH/GSSG、bax/bcl-2 蛋白测定.结果 ①用药组LVEDD、LVESD 较A 组明显降低(P＜0.05),EF、FS 明显升高(P＜0.05),T 组与L 组较M 组进一步改善(P＜0.05).②用药组LVEDP 较A 组明显降低(P＜0.05),LVSP、±dp/dt 明显升高(P＜0.05),L 组较M 组进一步改善(P＜0.05),T 组LVEDP 较M 组明显降低(P＜0.05).③用药组谷光甘肽(GSH)较A 组明显升高(P＜0.01),氧化型谷光甘肽(GSSG)明显降低(P＜0.01),L 组较单药治疗组进一步改善(P＜0.05),T 组与M 组相比,GSH 明显升高(P＜0.05).④用药组bcl-2 较A 组明显升高(P＜0.01),bax 明显降低(P＜0.01),T 组与L 组bax 水平接近(P=0.141),两组较M 组明显降低(P＜0.05),L 组bcl-2 水平与S 组接近(P=0.453),两单药治疗组bcl-2 差异无统计学意义(P=0.132),均显著低于L 组,L 组bcl-2/bax 明显高于T 组,T 组bcl-2/bax 明显高于M 组.结论 特布他林对心梗后大鼠心功能及心室重构的改善作用优于美托洛尔,二者联用改善作用进一步增强.其相关机制可能与改善心肌组织氧化应激、明显减轻心肌细胞凋亡有关.     

基质细胞衍生因子-1及其受体对大鼠主动脉血管平滑肌细胞增殖与凋亡的影响

目的研究基质细胞衍生因子-1(SDF-1)对氧化低密度脂蛋白(ox-LDL)诱导的血管平滑肌细胞(VSMC)增殖与凋亡的影响。方法选用体外培养的大鼠主动脉VSMC,并分为正常对照组、ox-LDL组[动脉粥样硬化(AS)模型]、SDF-1组(AS模型+SDF-1)、SDF-1+12G5组(AS模型+SDF-1+CXCR4单克隆抗体)和12G5组(AS模型+CXCR4单克隆抗体),应用MTT法测VSMC细胞增殖,TUNEL法测细胞凋亡率,RT-PCR法测凋亡基因bax、bcl-2mRNA的表达。结果ox-LDL组的增殖率0.38±0.01,明显高于正常对照组0.28±0.02(P<0.01),明显低于SDF-1组0.44±0.02(P<0.01),与SDF-1+12G5组和12G5组比较差异无显著性(P>0.05);ox-LDL组的凋亡率24.2±2.4,高于正常对照组19.8±2.7和SDF-1组20.7±2.8(P<0.05),而与SDF-1+12G5组和12G5组比较差异无显著性(P>0.05);ox-LDL组bcl-2和bax的比值明显低于正常对照组和SDF-1组(P<0.01),而与SDF-1+12G5组和12G5组差异无显著性(P>0.05)。结论SDF-1可明显促进ox-LDL诱导的VSMC增殖,并抑制细胞凋亡;SDF-1及其受体CXCR4构成的生物学轴可能通过bcl-2/bax途径影响VSMC凋亡。     

肿瘤坏死因子α、白细胞介素1β对牛肺动脉内皮细胞凋亡的影响及意义

目的　研究肿瘤坏死因子α(TNF α)、白细胞介素 1β(IL 1β)对牛肺动脉内皮细胞 (BPEC)损伤的机制及在急性肺损伤 (ALI)发病过程中的作用。方法　建立BPEC的体外培养 ,采用流式细胞仪膜联蛋白V 异硫氰酸荧光素 (Annexin VFITC)、碘化吡啶 (PI)染色检测TNF α、IL 1β对BPEC凋亡的影响以及抗TNF α单克隆抗体、AC DEVD CHO (caspase 3的竞争性抑制剂 )的保护效应。结果　 (1)TNF α作用 2 4h ,随着其浓度的增加 (浓度为 50 0、10 0 0、2 0 0 0U/ml) ,BPEC凋亡率逐渐增加 [分别为(8 2 1± 0 70 ) %、(9 63± 0 71) %、(17 43± 1 99) % ] ,与对照组 [(3 0 9± 0 0 8) % ]比较差异有显著性(P均 <0 0 5) ;(2 )TNF α (2 0 0 0U/ml)培养时间延长 (分别为 6、12、2 4、3 6h) ,BPEC凋亡率逐渐增加 [分别为 (6 72± 0 3 8) %、(7 72± 1 66) %、(12 95± 0 3 2 ) %、(17 70± 1 79) % ,P均 <0 0 5] ;(3 )加入抗TNF α单抗、AC DEVD CHO的TNF α组的BPEC凋亡率 [(7 78± 0 2 1) %、(7 3 2± 0 11) % ]显著高于单纯TNF α(2 0 0 0U/ml)组 [(10 59± 0 49) % ,P均 <0 0 1] ,而加入IL 1β的TNF α组的凋亡率 [(10 73±0 60 ) % ]与单纯TNF α组比较差异无显著性 (P >0 0 5)。结论　ALI过程中是TNF