Effect of NF-κB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand

AIM: To study the effect of NF-κB, survivin, Bd-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells. METHODS: Gastric cancer cells of SGC-7901, MKN28, MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis indudng ligand (TRAIL) with a flow cytometer. The expression of NF-κB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot. RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45,AGS and SC-C-7901cells respectively. Western blot revealed that the expressions of NF-EB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells. CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines.The anticancer potency of TRAIL is associated with the decreased expression of NF-κB and survivin and increased expression of Caspase3 of gastric cancer cells.     

Protective effects of tumor necrosis factor α antibody and ulinastatin on liver ischemic reperfusion in rats

AIM: To study the protective effects of tumor necrosis factor α (TNFα) antibody and ulinastatin on liver ischemic reperfusion in rats.METHODS: One hundred and twenty male SD rats were randomly divided into four groups: normal control group, ischemic group, TNFα antibody group and TNFα antibody + ulinastatin group. The animals were killed at 0, 3, 6, 9, 12 h after ischemia for 60 min and followed by reperfusion. Serum alanine aminotransferase (ALT), malondialdehyde (MDA) and liver histopathology were observed.RESULTS: After ischemic reperfusion, the serum ALT and MDA were remarkably increased, and the hepatic congestion was obvious. Treatment of TNFα antibody and ulinastatin could significantly decrease serum ALT and MDA levels, and relieve hepatic congestion.CONCLUSION: Ulinastatin and TNFα antibody can suppress the inflammatory reaction induced by hepatic ischemic reperfusion, and have protective effects on rat hepatic ischemic reperfusion injury.     

The mRNA expression patterns of tumor necrosis factor-α and TNFR-I in some vital organs after thermal injury

AIM: To investigate changes of tumor necrosis factor-α (TNF-α and TNFR-I expression in vital organs and their significance in the pathogenesis of multiple organ damage associated with endogenous endotoxin following major burns.METHODS: Wistar rats subjected to a 35% full-thickness scald injury were sacrificed at 12h, 24h, 48h, and 72 hpostburn, respectively. Meanwhile, eight rats were taken as normal controls. Tissue samples from liver, spleen, kidney,lung and intestine were collected to assay tissue endotoxin levels and measure TNF-α and TNFR-I expression. In addition, blood samples were obtained for the determination of organ function parameters.RESULTS: Endotoxin levels in liver, spleen and lung increased markedly after thermal injury, with the highest level in liver. The gene expression of TNF-α in liver, lung and kidney was up-regulated after thermal injury, while the TNFR-I mRNA expression in liver, lung, kidney and intestine was shown decreased throughout the observation period. Thus, the mRNA expression ratio of TNF-α to TNFR-I was significantly increased postburn, particularly in pulmonary tissue (67-fold). In addition, the significant correlations between the expression of TNFR-I or the expression ratio of TNF-α/TNFR mRNA in liver tissue and serum aspartate aminotransferase levels were noted (P<0.05-0.01). Similar results were also obtained between pulmonary TNF-α mRNA expression and myeloperoxidase activities (P<0.01), whereas there was a highly negative correlation between levels of renal TNFR-I mRNA expression and serum creatinine.CONCLUSION: Burn injury could result in the translocation of gut-derived endotoxin that was mainly distributed in the liver, spleen and lung. The translocated endotoxin then made the expression of TNF-α and TNFR-I mRNA up-regulated and down-regulated respectively in various organs, which might be involved in the pathogenesis of multiple organ damage following burns.     

Defensive nature of Sargassum polycystum （Brown alga） against acetaminophen-induced toxic hepatitis in rats： Role of drug metabolizing microsomal enzyme system, tumor necrosis factor-α and fate of liver cell structural integritv

AIM: To assess the defensive nature of Sargassum polycystum (S. polycystum) (Brown alga) against acetaminophen (AAP)-induced changes in drug metabolizing microsomal enzyme system, tumor necrosis factor (TNF-alpha) and fine structural features of the liver during toxic hepatitis in rats. METHODS: Male albino Wistar strain rats used for the study were randomly categorized into 4 groups. Group I consisted of normal control rats fed with standard diet. Group II rats were administered with acetaminophen (800 mg/kg body weight, intraperitoneally). Group III rats were pre-treated with S. polycystum extract alone. Group IV rats were orally pre-treated with S. polycystum extract (200 mg/kg body weight for 21 d) prior to acetaminophen induction (800 mg/kg body weight, intraperitoneally). Serum separated and liver was excised and microsomal fraction was isolated for assaying cytochrome P450, NADPH Cyt P450 reductase and b(5). Serum TNF-alpha was detected using ELISA. Fine structural features of liver were examined by transmission electron microscopy. RESULTS: Rats intoxicated with acetaminophen showed considerable impairment in the activities of drug metabolizing microsomal enzymes, such as cytochrome P450, NADPH Cyt P450 reductase and b(5) when compared with the control rats. The rats intoxicated with acetaminophen also significantly triggered serum TNF-alpha when compared with the control rats. These severe alterations in the drug metabolizing enzymes were appreciably prevented in the rats pretreated with S. polycystum. The rats pretreated with S. polycystum showed considerable inhibition in the elevation of TNF-alpha compared to the rats intoxicated with acetaminophen. The electron microscopic observation showed considerable loss of structural integrity of the endoplasmic reticulum, lipid infiltration and ballooning of mitochondria in the acetaminophen-intoxicated rats, whereas the rats treated with S. polycystum showed considerable protection against acetaminophen-induced alterations in structural integrity. CONCLUSION: These observations suggest that the animals treated with S. polycystum extract may have the ability to protect the drug metabolizing enzyme system and mitochondrial functional status from free radical attack, thereby showing its defense mechanism in protecting hepatic cells from acetaminophen toxic metabolite N-acetyl-para-benzoquinone-imine (NAPQI).     

Suppression of FasL expression in tumor cells and preventing TNF-induced apoptosis was better for immune cells survival

OBJECTIVE: To elucidate if Fas/FasL signal pathway participates in the immune escape of tumor cells, and if contemporarily preventing Fas/FasL and TNF-induced apoptosis is better for immune cells survival than just blocking Fas/FasL-induced apoptotic signal. METHODS: Suppression of FasL expression in mouse H22 hepatocellular cancer cells by siRNA technique. Wild-type Ad5 14.7K gene was amplified by PCR and transduced into Jurkat T cells. Detecting apoptosis of target Jurkat cells by Flow Cytometry. Detection of TNF-alpha in the culture supernatant of H22 cells by ELISA. FasL and 14.7K gene expression in stably transfected or transduced clones were determined by western blotting. RESULTS: FasL expression in H22 cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression in H22 cells had no effect on tumor growth in vitro. There was an apparent decrease in the number of apoptotic Jurkat T cells following coculture with transfected H22 cells, relative to coculture with FasL-expressing untransfected cells. Compared with untransduced Jurkat cells, apoptotic rates in 14.7K transduced Jurkat cells were significantly reduced in three different E/T ratios (P < 0.01), respectively. CONCLUSIONS: Fas/FasL signal pathway participated in the immune escape of tumor cells by inducing immune cells apoptosis. Reducing the expression of FasL in tumor cells can decrease the apoptotic rate of immune cells. Further blocking of apoptotic signal pathway of immune cells by preventing TNF-induced apoptosis can increase the survival of immune cells.     

肺癌组织中肿瘤坏死因子相关凋亡诱导配体受体的表达及其临床意义

目的　探讨不同类型肿瘤坏死因子相关凋亡诱导配体 (TRAILR )在非小细胞肺癌(NSCLC)中的表达及其临床意义。方法　采用RT PCR检测 40例肺癌组织、对应癌旁非癌肺组织以及非小细胞肺癌细胞株A5 49不同类型TRAILR的表达 ,并结合临床资料进行分析。结果　 40例肺癌组织表达死亡受体DR4,3 5例肺癌组织表达死亡受体DR5 ;40例癌旁肺组织均表达死亡受体DR5和DR4,3 3例肺癌组织不表达诱捕受体DcR1,2 5例肺癌组织不表达DcR2 ,而 40例癌旁肺组织均表达DcR。肺癌细胞株A5 49中有DR5、DR4、DcR2的表达 ,但DcR1表达缺失。肺癌组织中DR的表达水平与肿瘤的分期有关 ,Ⅲ期肿瘤DR的表达显著低于Ⅰ /Ⅱ期DR表达 (P <0 .0 5 )。DR的表达与病人的性别、年龄、病理学分型无关。结论　肺癌存在TRAILR类型的表达差异 ,诱捕受体 (特别是DcR1)的表达缺失为TRAIL治疗肺癌提供了靶点     

肿瘤坏死因子相关凋亡诱导配体对肝癌耐药细胞株阿霉素化疗敏感性的影响

目的 研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合阿霉素(ADM)处理肝癌耐药细胞株HePG2／ADM对化疗敏感性的影响。方法 通过培养液中ADM的浓度梯度增加法长期筛选培养，建立肝癌HepG2／ADM耐药细胞株，荧光定量PCR检测多药耐药(MDR)1的表达，TRAIL联合化疗药物ADM处理HePG2／ADM，MTT比色法检测细胞增殖，细胞凋亡采用流式细胞仪和TUNEL法检测观察HePG2／ADM对化疗药物的敏感性变化。结果 HepG2／ADM细胞是一个明确的多药耐药细胞模型，联合TRAIL(100ng／L) ADM(0．1mg／L)后，MTT显示HepG2／ADM细胞的增殖明显抑制，流式细胞术、TUNEL法检测TRAIL联合ADM处理HePG2／ADM细胞诱导的凋亡，与对照组相比，凋亡指数显著增加。结论 MDR1不参与TRAIL耐受。TRAIL可部分逆转HepG2／ADM对ADM的耐药，增加其对化疗药物的敏感性。联合TRAIL和亚毒剂量化疗药物可望克服肿瘤细胞中存在的化疗耐药和TRAIL耐受。     

New mechanism of transforming growth factor‐β signaling in hepatoma: Dramatic up‐regulation of tumor initiating cells and epidermal growth factor receptor expression

Aim: Transforming growth factor‐β (TGF‐β) has dual activity in tumor cells. We studied the effect of TGF‐β on tumor‐initiating cells (TICs), which are similar in self‐renewal and differentiation features to normal adult stem cells. Methods: We used side population (SP) cells that exclude DNA binding dye Hoechst 33342 to obtain TICs, studied the differences in the kinetics of the SP cell response to TGF‐β treatment between hepatic tumor cell lines, and performed gene analysis. Results: SP cells from all cell lines have higher proliferative ability compared to non‐SP cells and they are drug resistant. TGF‐β treatment increased the percentage of SP cells (%SP) and the survival rate; chemotherapeutic drug resistance developed only in K‐251 SP cells. Gene analysis showed that TGF‐β up‐regulated epidermal growth factor receptor (EGFR) only in K‐251 cells. There were no EGFR mutations in K‐251, which had been reported in lung cancer. Knockdown of Smad4 using the small‐interfering RNA technique in K‐251 cells inhibited EGFR overexpression and significantly decreased the %SP. In contrast, the JNK inhibitor had little effect on EGFR expression or the %SP. Conclusion: TGF‐β treatment of K‐251 cells causes tumor progression and the anti‐cancer drug resistant phenotype by increasing SP.