Function of apoptosis and expression of the proteins Bcl-2,p53 and C-myc in the development of gastric cancer

INTRODUCTIONIn China ,the incidence and mortality of gastric cancer rank the second among all cancers. Recent development of cancer [1-20].The aim of this study was investigat the insight of apoptosis and bcl-2, p53 and C-myc protein expression in the development of gastric cancer .

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Paclitaxel induces apoptosis in human gastric carcinoma cells

AIM;To investigate the apoptosis in gastric cancer cells induced by paclitaxel,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 gastric cancer cell line SGC-7901 before and after the paclitaxel treatment.Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax. RESULTS:Paclitaxel inhibited the growth of gastric cancer cell line SGC-7901 in a dose-and time-dependent manner. Paclitaxel induced SGC-7901 cells to undergo apoptosis with typically apoptotic characteristics,including morphological changes of chromatin condensation,chromatin crescent formation,nucleus fragmentation and apoptotic body formation.Paclitaxel could reduce the expression of apoptosis-regulated gene Bcl-2,and improve the expression of apoptosis-regulated gene Bax. CONCLUSION:Paclitaxel is able to induce the apoptosis in gastric cancer.This apoptosis may be mediated by down- expression of apoptosis-regulated gene Bcl-2 and up- expression of apoptosis-regulated gene Bax.

Changes of NF-kB,p53,Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells:role of reactive oxygen species

AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. CONCLUSION: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the 'ladder pattern' revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

Protective effect of estradiol on hepatocytic oxidative damage

AIM: To examine the protective effect of estradiol on the cultured hepatocytes under oxidative stress. METHODS: Hepatocytes of rat were isolated by using perfusion method, and oxidative stress was induced by a serum-free medium and FeNTA. MDA level was determined with TBA method. Cell damage was assessed by LDH assay. Apoptosis of hepatocytes was assessed with cytoflowmetric analysis. Expression of Bcl-xl in cultured hepatocytes was detected by Western blot. The radical-scavenging activity of estradiol was valued by its ability to scavenge the stable free radical of DDPH. RESULTS: Oxidative stress increased LDH from 168 +/- 25 x 10(-6)IU.cell(-1) to 780 +/- 62 x 10(-6)IU.cell(-1) and MDA(from 0.28 +/- 0.07 x 10(-6)nmol.cell(-1) to 1.35 +/- 0.12 x 10(-6)nmol.cell(-1)) levels in cultured hepatocyte, and estradiol inhibited both LDH and MDA production in a dose dependent manner. In the presence of estradiol 10(-6)mol.L(-1), 10( -7 )mol.L(-1) and 10(-8)mol.L(-1),the LDH levels are 410 +/- 53 x 10(-6)IU.cell(-1) (P【0.01 vs oxidative group), 530 +/- 37 X 10(-6)IU.cell(-1 ) (P【0.01 vs oxidative group), 687+/-42 x 10(-6)IU.cell(-1) (P【0.05 vs oxidative group) respectively, and the MDA level are 0.71+/-0.12 x 10(-6)nmol.cell(-1) (P【0.01 vs oxidative group),0.97+/-0.11 x 10(-6)nmol.cell(-1 )(P【0.01 vs oxidative group) and 1.27+/-0.19 x 10(-6)nmol.cell(-1) respectively. Estradiol suppressed apoptosis of hepatocytes induced by oxidative stress, administration of estradiol(10(-6)mol/L)decreased the apoptotic rate of hepatocytes under oxidative stress from 18.6 +/- 1.2% to 6.5 +/-2.5%, P【0.01. Bcl-xl expression was related to the degree of liver cell damage due to oxidative stress, and estradiol showed a protective action. CONCLUSION: Estradiol protects hepatocytes from oxidative damage by means of its antioxidant activity.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Alanyl-glutamine dipeptide inhibits hepatic ischemiareperfusion injury in rats

AIM： To investigate the protective effect and mechanism of alanyl-glutamine dipeptide （Ala-GIn） against hepatic ischemia-reperfusion injury in rats. METHODS： Rats were divided into group C as normal control Group （/7=16） and group G as alanyl-glutamine pretreatment 07=16）. Rats were intravenously infused with 0.9% saline solution in group C and Ala-GIn -enriched （2% glutamine） 0.9% saline solution in group G via central venous catheter for three days. Then all rats underwent hepatic warm ischemia for 30 min followed by different periods of reperfusion. Changes in biochemical parameters, the content of glutathione （GSH） and the activity of superoxide dismutase （SOD） in liver tissue, Bcl-2 and Bax protein expression and morphological changes of liver tissue were compared between both groups. RESULTS： One hour after reperfusion, the levels of liver enzymes in group G were significantly lower than those in group C （P〈0.05）. Twenty-four hours after reperfusion, the levels of liver enzymes in both groups were markedly recovered and the levels of liver enzyme in group G were also significantly lower than those in group C （P〈0.01）. One and 24 h after reperfusion, GSH content in group G was significantly higher than that in group C （P 〈0.05）. There was no statistical difference in activities of SOD between the two groups. One and 24 h after reperfusion, the positive expression rate of Bcl-2 protein was higher in group G than in group C （P〈0.05） and the positive expression rate of Bax protein was lower in group G than in group C （P〈0.05）. Histological and ultrastructural changes of liver tissue were inhibited in group C compared to group G. CONCLUSION： Our results suggest that Ala-GIn pretreatment provides the rat liver with significant tolerance to warm ischemia-reperfusion injury, which may be mediated partially by enhancing GSH content and regulating the expression of Bcl-2 and Bax proteins in the liver tissue.

Neutron-induced apoptosis of HR8348 cells in vitro

INTRODUCTIONTo date ,the major therapy for rectal carcinoma is extensive abdomino-perineal resection[1]. Unfortunately ,after resection of rectal carcinoma ,many patients still die of blood-borne metastases ,usually in the liver or lungs ,or local prlvic recurrence[2,3],which is the major cause of morbidity and mortality in patients with rectal carcinoma .Pre-or postoperative radiotherapy can reduce the incidence of local rdcurrence[4-7].

Detection of BCL2-IGHrearrangement on paraffin-embedded tissue sections obtained from a small submucosal tumor of the rectum in a patient with recurrent follicular lymphoma

A 59-year-old woman was admitted to our hospital because of recurrent follicular lymphoma(FL).Colonoscopic examination revealed a rectal submucosal tumor(SMT)without any erosions and ulcers.In this patient,it was difficult to distinguish non-Hodgkin's lymphoma(NHL)invasion from other disorders of the colon including carcinoid tumor merely based on endoscopic findings.Histopathologic and immunohistochemical studies on biopsy specimens showed an infiltration of atypical lymphocytes that were positive for CD20 and BCL2 but negative for UCHL-1.Fluorescence in situ hybridization on paraffin-embedded tissue sections (T-FISH)identified a translocation of BCL2 with IGHgene. Based on these findings,the tumor was defined as an invasion of FL.T-FISH method is useful for the detection of a monoclonality of atypical lymphocytes in an SMT of the gastrointestinal tract,and particularly for the detection of chromosomal translocations specific to lymphoma subtypes.

Effects of External Application of Chinese Medicine on Diabetic Ulcers and the Expressions of β-catenin,c-myc and K6

Objective：To observe the clinical efficacy of Chinese medicine（CM） treatment of Hongyou Ointment（红油膏） and Shengji Powder（生肌散） on diabetic ulcers,and to observe the influence of CM treatment on the expressions of proteins associated with the Wnt signaling pathway,such asβ-catenin,c-myc and K6.Methods：sixty-two patients fitting the registration standards were randomly divided into the CM group（31 patients） and the Western medicine（WM） group（31 patients） by a random number table.The patients in the CM group were treated with Hongyou Ointment and Shengji Powder externally.The patients in the WM group were treated with mupirocin ointment,growth factor（bFGF）,and Vaseline gauze for external use and with basic therapies.Wound-healing time and four-week healing rate were recorded.The wounds were measured by digital photography and ImageJ software.Skin biopsies were obtained from 24 patients before CM treatment and 20 patients after CM treatment.Immunohistochemical tests and semi-quantitative imaging with NIH ImageJ 1.42 software were used to analyze the changes in protein expression ofβ-catenin,c-myc,and K6.Results：Fifty-three patients completed the trial;four patients in the CM treatment group and five patients in the WM group dropped out.Among them,four were dissatisfied with the treatment process,two could not continue because of their jobs,two failed to complete the course of follow-up appointments,and one was diagnosed with squamous cell carcinoma during treatment.The comparison of ulcer healing rates between the two groups showed insignificant differences（P=0.77）.The ulcer healing rates were 33.33%（9/27） in the CM group and 26.92%（7/26） in the WM group.However,the effective rate was significantly higher in the CM group（81.48%,22/27） than in the WM group（57.69%,15/26,P=0.04）.The mean wound healing time was shorter in the CM group（22.71±5.46 days） than in the WM group（26.56±7.56 days,P=0.04）.CM treatment was well tolerated,and there was no withdrawal due to adverse reactions.Immunohistochemical analysis in the refractory wound indicated higher expressions ofβ-catenin,c-myc and K6 compared with the normal skin.β-catenin was abnormally expressed in the nuclei of the keratinocytes and fibroblasts at the wound margins,and the expressions of c-myc and K6 were highly expressed in the full hyperplastic epidermis,especially in the granular layer（P0.05）.The expressions of these proteins decreased after CM treatment.The expression levels ofβ-catenin,c-myc,and K6 proteins before and after the treatment were 101.88±10.76 vs.140.42±8.45;113.27±16.75 vs.153.79±8.32; 90.39±11.07 vs.151.29±7.39,respectively.Conclusions：CM treatment using Hongyou Ointment and Shengji Powder was efficient in the management of diabetic skin ulcers.The mechanism of action might be related to the inhibition of the Wnt signaling pathway.

Mcl-1 mediates cytokine deprivation induced apoptosis of human myeloma cell line XG-7

OBJECTIVE: To investigate apoptosis in XG-7, a human myeloma cell line, induced by IL-6 deprivation and the function of three anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-kappa(L), Mcl-1) in the apoptotic process. METHODS: Apoptosis in XG-7 myeloma cells induced by IL-6 withdrawal was determined by flow cytometry with propidium iodide (PI) nuclear staining. Expressions of three Bcl-2 proteins in XG-7 cells were monitored by immunoblotting assay. RESULTS: In the absence of IL-6 for a certain time, a significant percentage of apoptiotic XG-7 cells can be observed, as well as down-regulated expression of one of the three anti-apoptotic proteins (Mcl-1) in XG-7 cells. IL-6 re-stimulation in XG-7 cells following cytokine removal up-regulated the expression of Mcl-1 and inhibited cell apoptosis. CONCLUSION: Mcl-1,instead of Bcl-2 and Bcl-kappa(L), plays an important role in IL-6 deprivation induced apoptosis in XG-7 human myeloma cells.

Molecular mechanism of damage and repair of mouse thymus lymphocytes induced by radiation

OBJECTIVE: To investigate the role of apoptosis in radiation-induced mouse thymus lymphocyte damage and repair and provide the basis for understanding the molecular mechanism of radiation-induced lymphocyte damage and repair as well as the prevention and treatment of acute radiation sickness. METHODS: We studied the dynamic changes of apoptosis of mouse thymus lymphocytes and the expression of bax and bcl-2 gene products after 2, 4, 6 and 8 Gy of whole body gamma-irradiation using in situ terminal labeling, DNA electrophoresis and immunohistochemical techniques. RESULTS: At the early stage after irradiation, the percentage of apoptotic lymphocytes increased rapidly in accordance with the increasing of radiation doses, while the counts of the thymus and peripheral lymphocytes decreased sharply, showing an opposite change to lymphocyte apoptosis. After 6 Gy gamma-irradiation, typical morphological characteristics of thymus apoptotic lymphocytes in early, middle and late stages were found by transmission electron microscopy. The thymus lymphocytes displayed characteristic DNA ladders 4 hr and 8 hr after 2-6 Gy gamma-irradiation,using DNA gel electrophoresis techniques. Abnormal expression of bcl-2 and bax gene products were shown in irradiated lymphocytes. CONCLUSIONS: Apoptosis plays an important role in the process of radiation-induced mouse thymus lymphocyte damage and repair. Bcl-2 and Bax proteins may regulate the process of lymphocyte apoptosis.

Effect of arsenic trioxide on inhibition of restenosis after rabbit vascular injury and its mechanism

OBJECTIVE: To investigate the effect and mechanism of arsenic trioxide (As(2)O(3)) on the prevention of restenosis after vascular injury. METHODS: Apoptosis induction of As(2)O(3) on cultured rabbit vascular smooth muscle cells (VSMCs) in vitro was observed. Thirty-two New Zealand white rabbits were randomly divided into 2- and 4-wk study groups, and their controls. 10% As(2)O(3) at 2.5 mg x Kg(-1) x d(-1) or 0.9% sodium chloride was intraperitoneally infused for 3 days before left common carotid arteries were denudated with a balloon. After denudation 2- and 4-wk animals were sacrificed for morphometry and immunohistochemical studies on carotid arteries, and for histopathology on liver and kidney. RESULTS: It was shown via cellular morphology and DNA fragments in electrophoresis that promotion of As(2)O(3) on cultured vascular smooth muscle cell apoptosis was dependent upon its concentration and duration. Compared with the control animals, the mean vascular intimal proliferation areas were reduced in 2-wk study animals (P 0.05), while the mean vascular luminal areas were all enlarged in both study groups (all P

Clinical pathological analysis and immunohistochemical study of ten solitary fibrous tumors

OBJECTIVE: To study the clinical and pathological characteristics of solitary fibrous tumor (SFT). METHODS: Clinical pathological analysis and immunohistochemical studies were performed on ten patients with SFT. RESULTS: The SFTs located variously and showed different histological features. All cases showed positive staining for CD(34), VM (vimentin) and Bcl-2, but negative staining for Desmin, S-100, CK (cytokeratin) and EMA (epithelial membrane antigen). CONCLUSIONS: SFT is described as a 'patternless' growth pattern. According to clinical pathological features and immunohistochemistry, it is different from other soft tissue tumors. Long-term clinical follow-up is necessary for this kind of tumor.

Overexpression of Bcl-2 partly inhibits apoptosis of human cervical cancer SiHa cells induced by arsenic trioxide

OBJECTIVE: To study the biological effect of arsenic trioxide (As2O3) on human cervical cancer SiHa cells and SiHa cells overexpressing bcl-2 gene. METHODS: SiHa cells with overexpression of Bcl-2 (SiHa-Bcl2 cells) were established by transfecting SiHa cells with Bcl-2 expression vector. The sensitivities of SiHa and SiHa-Bcl2 cells to As2O3 were determined using MTT (Thiazolyl blue) reduction and colony forming ability assay, morphological analysis, flow cytometric analysis, DNA agarose gel electrophoresis, in situ cell death detection (TUNEL), Northern blot, RT-PCR and Western blot. RESULTS: As2O3 inhibited the growth of SiHa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR and Western blot analysis revealed that As2O3 induced SiHa cell apoptosis possibly via inhibiting the expression of HPV16 E7 and decreasing the expression of c-myc. However, we found that SiHa-Bcl2 cells partly resisted As2O3 induced apoptosis, which might be related to the prevention of the down-regulation of HPV16 E7 and c-myc gene expression. Nevertheless, As2O3 at a high concentration could still induce apoptosis of SiHa-Bcl2 cells mainly via decreasing Bcl-2 expression and slightly inhibiting viral gene expression. CONCLUSION: As2O3 is an inducer of the apoptosis of human cervical carcinoma cells and the cells overexpressing Bcl-2 can partly resist As2O3 induced apoptosis, but the exact mechanism is unclear.

Diazoxide preconditioning plus subsequent hypothermia increased resistance of rat cultured hippocampal neurons against hypoxia-reoxygenation injury

Background Cerebral ischemia-reperfusion/hypoxia-reoxygenation insult triggers lots of pathophysiological and biochemical events that separately affect the evolution of cerebral damage. Accordingly, all known effective neuroprotective measures should be taken to get the optimal efficacy of therapy. This study was undertaken to investigate whether diazoxide （DZ） preconditioning combined with the following hypothermia could contribute to synergistic neuroprotection compared with either hypothermia or DZ preconditioning alone. Methods Cultured for 9-10 days in vitro, the hippocampal neurons of SD rats were preconditioned with DZ 0 pmol/L or DZ 250 pmol/L for 1 hour per day and this treatment lasted for 3 days. Subsequently, neurons were subjected to deprivation of oxygen for 4 hours at 37°0, 34°C, 30℃ and 22℃, respectively. This experiment consisted of 8 groups （4 temperature groups and 4 combination groups） and each group contained 12-well or 2-dish cells. Survival rate, expression of Bcl-2, fluorescence magnitude of intracellular calcium, and concentration of malondialdehyde （MDA） were determined at 24 hours after reoxygenation. Results The survival rate and expression of Bcl-2 were both increased in individually hypothermic conditions compared with those at 370G （P〈0.05）, whereas intracellular calcium and MDA did the opposite exhibition simultaneously （P〈0.05）. 22℃ contributed to a higher survival rate and greater expression of Bcl-2 in comparison with other hypothermia （P〈0.05）. Preceding administration of 250 pmol/L DZ took the similar effects on the neurons like hypothermia. Moreover, compared with individual hypothermia or DZ preconditioning, the neuronal survival rate and expression of Bcl-2 in the combination group were increased significantly （P〈0.05）, whereas the calcium fluorescence density and concentration of MDA were reduced further （P〈0.05）. 250 Iamol/L DZ preconditioning combined with 22℃ provided a maximal neuroprotection. Conclusions Compared with either individual hypothermia or DZ preconditioning, the combination of both treatments conferred synergistic protection for cultured hippocampal neurons in vitro against hypoxia- reoxygenation insult.